Cell suspensions

Am J Clin Pathol, 1991 Oct, 96(4), 479 - 90
The role of gene rearrangements for antigen receptors in the diagnosis of lymphoma obtained by fine-needle aspiration . A study of 63 cases with concomitant immunophenotyping; Katz RL et al.; To assess the efficacy of performing genotyping in addition to immunophenotyping as an adjunct to cytologic diagnosis, 63 consecutive patients with fine-needle aspirates of lymphoproliferative lesions who had concurrent immunophenotyping and genotyping performed on fine-needle aspirate cell suspensions were studied . Thirty-nine of 63 specimens (62%) that appeared to contain non-Hodgkin's lymphoma and that proved to be of B-cell lineage by genotyping were accurately phenotyped and shown to be monotypic for immunoglobulin light chains by cell suspension immunocytochemistry . Genotyping facilitated lineage assignment and/or confirmed clonality in 17 of 63 specimens (27%) that were difficult to determine based on morphologic data . These include cases of atypical lymphoid proliferations with polyclonal or inconclusive markers (n = 6), peripheral T-cell lymphoma (n = 3), extracutaneous mycosis fungoides (n = 1), lymphoblastic lymphoma (n = 4), null cell lymphoma (n = 1), and specimens with equivocal or technically unsatisfactory markers (n = 2) . Based on these results, it is proposed that genotyping for lineage assignment and/or clonality be performed to include cases of atypical lymphoid proliferations, T-cell malignant neoplasms, lymphoid malignant neoplasms with equivocal markers, and differentiation of lymphoid from nonlymphoid neoplasms . Genotyping by antigen-receptor gene rearrangement appears to be redundant in cases with mature B-cell phenotypes that demonstrate monoclonality by immunophenotyping.

J Anat, 1991 Oct, 178, 101 - 13
The uterine response in pregnant inbred and non-inbred rats; Matthews J et al.; Comparisons were made between fetal, placental and metrial gland weights and the cellular composition of the placentae and metrial glands of pregnant inbred (Agus), outbred (Agus x Wistar) and random-bred (Wistar) rats . Fetal and placental weight differences between inbred and outbred rats provided evidence of hybrid vigour . Metrial gland weight differences between inbred and outbred rats showed that the Agus mothers responded to hybrid (Agus x Wistar) fetuses by producing heavier metrial glands than if they were carrying inbred fetuses, up to and included Day 18 of pregnancy . Histological examination revealed some differences between the 3 groups of rats at corresponding stages of pregnancy . Granulated metrial gland (GMG) cells formed a larger proportion of cells in the metrial gland in Agus rats carrying hybrid fetuses than in Agus rats carrying inbred fetuses up to Day 14 of pregnancy, but the situation was reversed after Day 14 . Degenerative changes were more apparent in the metrial glands from Agus rats carrying hybrid fetuses than in rats from the other 2 groups from Day 15 of pregnancy onwards . Clusters of lymphocytes were a prominent feature of the placental labyrinths of Agus rats (mated with Agus or Wistar males) around Day 15 of pregnancy . Examination of single cell suspensions of metrial gland showed variations in the proportion of immunoglobulin G (Fc gamma) receptor bearing cells during pregnancy and between rats in the 3 groups of corresponding stages of pregnancy . The significance of the differences occurring in the metrial gland during pregnancy and between the groups at corresponding stages of pregnancy may be related to the functional involvement of GMG cells in cytotoxic activity directed against placental trophoblast.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8656 - 60
Human bone marrow non-B, non-T cells produce interleukin 4 in response to cross-linkage of Fc epsilon and Fc gamma receptors; Piccinni MP et al.; Human bone marrow (BM) cells lacking T- and B-cell markers expressed RNA encoding interleukin (IL) 4 and secreted detectable amounts of IL-4 in supernatants in response to Fc epsilon or Fc gamma receptor (Fc epsilon R or Fc gamma R) cross-linking . In some experiments, IL-5 RNA expression in response to Fc epsilon R cross-linkage could also be detected . In contrast, RNA transcripts for, and secretion of, IL-2, IL-6, and interferon gamma were never observed . The presence of IL-3 in the cultures was essential for IL-4 production by non-B, non-T BM cells in response to Fc gamma R cross-linking and enhanced IL-4 RNA expression in response to Fc epsilon R cross-linking . Under the same experimental conditions, BM T and B lymphocytes, as well as peripheral blood T, B, and non-B, non-T cells, did not express IL-4 RNA . Prolonged incubation of non-B, non-T cells in IgE-free medium followed by extensive washing did not inhibit IL-4 production induced by anti-IgE antibodies, suggesting that the Fc epsilon R involved in the response has the characteristics of a high-affinity receptor . The Fc epsilon R+ cells were separated from the Fc epsilon R- cells by sorting non-B, non-T BM cell suspensions with fluorescein isothiocyanate-conjugated IgE and then assessed for both IL-4 RNA expression and alcian blue staining . Both IL-4-producing and alcian blue-positive cells segregated with the Fc epsilon R+ fraction . These data suggest that human BM cells, probably belonging to the mast cell and/or basophil lineage, are capable of producing IL-4 in response to Fc epsilon R or Fc gamma R cross-linkage.

Anal Biochem, 1991 Oct, 198(1), 200 - 2
Use of a mutant strain of the cyanobacterium Synechococcus R2 for the determination of nitrate; Madueno F et al.; A sensitive procedure for the determination of nitrate within the 1- to 50-microM range is described . The method is based on the photoreduction of nitrate to nitrite by whole cells of a nitrite reductase-less mutant strain of the unicellular cyanobacterium Synechococcus R2 . Cell suspensions of this cyanobacterium retain high levels of nitrate photoreduction activity for at least 2 months when maintained in the presence of dimethyl sulfoxide at -70 degrees C.

J Biochem Biophys Methods, 1991 Oct-Nov, 23(3), 237 - 48
Fluorimetric quantification of cell death in monolayer cultures and cell suspensions; Ruiz MC et al.; A fluorimetric assay using ethidium bromide (EB) was employed to quantify cell death in monolayer cell cultures (MA-104 cells) in situ and isolated cell suspensions (isolated colonic cells and Leishmania) . Fluorescence of EB stained cells was measured with a photometer coupled to an inverted microscope for cell monolayers or in a spectrofluorometer for cell suspensions . Dead cells stained with trypan blue were fluorescent with EB in all preparations studied, but the latter gave an unequivocal signal . Staining with EB and fluorescein diacetate was mutually exclusive . The relationship between the number of EB fluorescent cells and the intensity of fluorescence measured in the microphotometer was linear for a large range of cell numbers (1-14000) from different types of preparations . Applicability of the method for measuring living and dead cells in two different time scales (minutes and hours) is shown using MA-104 cell monolayers infected with rotavirus and Leishmania suspensions treated with amphotericin B . The method is fast, simple, sensitive and reliable, enabling quantification of living and dead cells in monolayers and suspensions.

Bone Marrow Transplant, 1991 Oct, 8(4), 301 - 5
Alpha-interferon broadens the difference between surviving fractions of normal and leukemic progenitor cells in vitro by heat: its application to marrow purging; Moriyama Y et al.; Alpha-interferon (IFN) may inhibit the proliferation of human leukemic progenitor cells (L-CFU) in vitro and enhance the anti-tumor effects by heat . In this study, the combined effects of IFN and hyperthermia on the growth of L-CFU and human granulocyte-macrophage progenitors (CFU-GM) were examined to determine if this combination resulted in a greater selective killing of L-CFU than that obtained by heat treatment alone . The survival of normal CFU-GM without IFN decreased at elevated temperatures (42-44 degrees C) . However, IFN added during heating (42 and 43 degrees C) appeared significantly to protect against the hyperthermic killing of CFU-GM in vitro leaving over 50% of CFU-GM surviving . The optimal dose to protect CFU-GM in vitro dropped to a rather low dose (100 U/ml) . On the other hand, the addition of IFN to leukemic cell suspensions enhanced the hyperthermic killing of myeloid leukemic cell lines (HEL and KG-1) as well as a T lymphoblastic cell line (CEM) in a dose-related manner . In addition, similar results were observed in the study of L-CFU from patients with acute myelogenous leukemia . These results suggest that IFN can be used to broaden the difference between surviving fractions of CFU-GM and L-CFU by heat . Thus, this combination could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous remission marrow graft before autologous bone marrow transplantation.

J Neurosci Res, 1991 Oct, 30(2), 359 - 71
Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia; Engele J et al.; Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons . To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum-free conditions . To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons was determined . This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10-fold lower at the plating density of 80,000 cells/cm2 . Cell density had only a minimal effect on {3H}dopamine uptake per TH-IR neuron . Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH-IR neurons 160-330% . These effects were dose dependent and heat sensitive . All CM stimulated neurite elongation of TH-IR neurons, while only the B49-CM increased {3H}dopamine uptake . The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50-100% . Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)-immunocytochemistry in combination with {3H}thymidine-autoradiography . By contrast, medium conditioned by the pheochromocytoma cell line, PC12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons . Inhibition of glial proliferation reduced the neurotrophic effects of the B49-, R33-, and JS1-CM by 40-80% . These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors . The partial decrease of the survival-promoting effects of these CM on dopaminergic neurons in glial-free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line-derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.

Int J Radiat Biol, 1991 Oct, 60(4), 635 - 46
Trypsin-induced changes in cell shape and chromatin structure result in radiosensitization of monolayer Chinese hamster V79 cells; Kapiszewska M et al.; Trypsin is the enzyme commonly used to prepare single cell suspensions from monolayer and spheroid cultures, both to determine survival and to assay DNA damage . Trypsin induces rounding, dissociation and radiosensitization of anchorage-dependent cells . Radiosensitivity and chromatin structure were compared between trypsin-treated (0.05%) round V79 cells from monolayers and spheroids vs . untreated spread monolayer cells in situ . The fluorescent halo technique was used to measure the changes in DNA supercoiling in nucleoids isolated from control and irradiated round and spread cells . Maximal halo diameters, the amount of initial and residual radiation-induced DNA damage (estimated from nucleoid halo diameter changes), and the radiosensitivity were higher in round cells than in spread monolayer V79 cells . The effects on cellular radiosensitivity and maximal halo diameter of other agents which also round and dissociate cells, e.g . 0.25% trypsin, pronase E and a non-enzymatic cell-dissociation solution, were similar to those of 0.05% trypsin . In LY-S cells, which are anchorage-independent, DNA loop size, the initial amount of DNA damage and radiosensitivity were not affected by trypsin . We suggest that the higher radiosensitivity of anchorage-dependent cells under immediate trypsinization and plating conditions, compared to cells with postirradiation in situ repair incubation, is due to correlated changes in cell shape and chromatin structure.

J Electron Microsc (Tokyo), 1991 Oct, 40(5), 364 - 7
Electron microscopic observation of recombinant Escherichia coli cells overproducing human tumor necrosis factor-alpha mutant as inclusion bodies; Moriya H et al.; Escherichia coli C600 r-m- carrying plasmid pTNF483 (E . coli {pTNF483}) produces a tumor necrosis factor-alpha (TNF-alpha) mutant protein in an insoluble form . A swollen region was observed in the SEM images to encircle the outside of most of the E . coli {pTNF483} cells just like a bandage . On the other hand, inclusion bodies of the TNF-alpha mutant as large as the short axis of the cell were observed in TEM images . This position was regarded as coinciding with the swollen region of SEM images . The inclusion bodies revealed in the swollen region of the cell envelopes were clearly observed in SEM images of isolated insoluble structures obtained by centrifugal sedimentation of a sonicated cell suspension.

Fiziol Zh SSSR Im I M Sechenova, 1991 Oct, 77(10), 114 - 9
{The effect of aldosterone on the second messenger systems in the rat kidney}; Svitasheva NG et al.; The effect of aldosterone on the cAMP level, the state of inositol-phosphate pool and the intracellular free Ca2+ activity in the rat kidney, were studied . The cAMP level in the kidney cortex homogenate was not changed after i.p . injection of aldosterone . Reliable changes in the content of inositol-phosphates in the kidney cell suspension was not revealed after aldosterone treatment . A short-term enhancement of the intracellular Ca2+ activity in the isolated distal convoluted and cortical collecting tubules under the influence of aldosterone, was revealed . The participation of the second messenger systems in the realisation of aldosterone effects in kidney and the role of Ca2+ in the mechanism of aldosterone action, are discussed.

Biophys J, 1991 Oct, 60(4), 804 - 11
Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis; Klenchin VA et al.; Simian Cos-1 cells were transfected electrically with the plasmid pCH110 carrying the beta-galactosidase gene . The efficiency of transfection was determined by a transient expression of this gene . When the plasmid was introduced into a cell suspension 2 s after pulse application, the transfection efficiency was shown to be less than 1% as compared with a prepulse addition of DNA . Addition of DNAase to suspension immediately after a pulse did not decrease transfection efficiency, thus the time of DNA translocation was estimated to be less than 3 s . The use of electric treatment medium, in which the postpulse colloid-osmotic cell swelling was prevented, did not affect the transfection efficiency . These results contradict both assumptions of free DNA diffusion into cell through the long-lived pores and of involvement of osmotic effects in DNA translocation . Transfection of cells in monolayer on a porous film allowed creation of the spatial asymmetry of cell-plasmid interaction along the direction of electric field applied . A pulse with a polarity inducing DNA electrophoresis toward the cells resulted in the 10-fold excess of transfection efficiency compared with a pulse with reverse polarity . Ficoll (10%) which increases medium viscosity or Mg2+ ions (10 mM) which decrease the effective charge of DNA, both reduced transfection efficiency 2-3-fold . These results prove a significant role of DNA electrophoresis in the phenomenon considered . The permeability of cell membranes for an indifferent dye was shown to increase noticeably if the cells were pulsed in the presence of DNA . This indicates a possible interaction of DNA translocated with the pores in an electric field, that results in pore expansion.

Infect Immun, 1991 Oct, 59(10), 3801 - 10
Roles of human peripheral blood leukocyte protein kinase C and G proteins in inflammatory mediator release by isogenic Escherichia coli strains; Konig B et al.; The signal transduction pathway (protein kinase C {PKC}, calcium influx, and G protein involvement) was studied with isogenic Escherichia coli strains expressing different types of adhesins (MSH+/- MS-Fim+/-, P-MRH+/- P-Fim+/-, and S-MRH+/- S-Fim+/-) or varying only in the expression of E . coli alpha-hemolysin . As target cells, human polymorphonuclear granulocytes (PMN) and a lymphocyte-monocyte-basophil (LMB) cell suspension were used . The alpha-hemolysin-producing (Hly+) strain E . coli K-12(pANN5211) induced calcium influx in a dose-dependent manner in both cell types . No calcium influx was detected after stimulation with the hemolysin-negative (Hly-) E . coli bacteria independent of the type of fimbriae . With Hly+ bacteria, a dose-dependent activation of PKC was observed in both cell types . The Hly- E . coli K-12 induced PKC to a lesser degree, expressing kinetics different from those of E . coli K-12(pANN5211) (Hly+) . E . coli MSH+ MS-Fim+ was the most potent activator for PKC . Membrane preparations from leukocytes stimulated with Hly+ E . coli K-12(pANN5211) showed increased binding of {3H}guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and increased GTPase activity compared with leukocytes stimulated with Hly- E . coli K-12 . The amounts of GTPase activation and {3H}guanylylimidodiphosphate binding were similar for all Hly- E . coli bacteria in human PMN as well as in human LMB; no activation was obtained for E . coli bacteria without any type of fimbriae . GTP-gamma-S, a nonhydrolyzable GTP analog, inhibited the leukotriene B4 (LTB4) generation from human PMN by Hly- bacteria, unlike E . coli K-12(pANN5211) . However, in the presence of NaF, a predominant activator of Gi, LTB4 generation by Hly+ and by Hly- bacteria was significantly enhanced . For LMBs only LTB4 generation by Hly+ bacteria was increased in the presence of GTP-gamma-S . NaF decreased the chemiluminescence induced by all E . coli strains . Our results thus indicate that (i) Hly+ and Hly- bacteria induce the activation of distinct G proteins, e.g., Gi, to different degrees, (ii) LTB4 generation and chemiluminescence response are differently regulated, and (iii) in comparison with PMN, a different signal transduction pathway is activated by E . coli bacteria in LMBs.

Toxicol Appl Pharmacol, 1991 Sep 15, 110(3), 435 - 49
Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells; Mortensen AM et al.; Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins . The association of the Ca(2+)-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis . HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis . Phenylhydrazine (4 mM) increased the rate of leucine, lysine, and histidine release by approximately 12-, 7-, and 5-fold, respectively . N-acetylcysteine (20 mM), dithiothreitol (50 mM), and dimethylthiourea (50 mM) decreased the rate of phenylhydrazine-stimulated amino acid release by approximately 30-50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 mM) and dimethyl sulfoxide (50 mM) were without significant effect . The calcium chelator, EGTA (10 mM) inhibited phenylhydrazine-stimulated proteolysis by approximately 30% . Phenylhydrazine (4 mM) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of approximately 28 kDa . Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE . Immunoblot analysis of spectrin confirmed these results . Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control . Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed . Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control . The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms . In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain . In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control . These forms were absent when EGTA (10 mM) was included in the incubation and the level of proenzyme was decreased . These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca(2+)-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).

Cancer Res, 1991 Sep 15, 51(18), 4937 - 41
Growth and metastasis of fresh human melanoma tissue in mice with severe combined immunodeficiency; Hill LL et al.; Cryopreserved cell suspensions of freshly excised melanoma metastases from nine patients were injected s.c . into C.B-17 severe combined immunodeficiency (SCID) mice . All 9 tumors grew as s.c . masses and six of nine were successfully transplanted into other SCID mice . Transplant inocula as low as 5 x 10(5) cells resulted in 100% tumor incidence . Moreover, seven of nine tumors metastasized, five from the original s.c . implants and two from transplanted s.c . tumors . Metastases were detected mainly in the lungs but also were found in abdominal viscera (liver, spleen, and pancreas) and thoracic lymph nodes . Flow cytometric analysis showed that expression of a panel of melanoma antigens, melanoma-associated proteoglycan, ganglioside GD3, and ganglioside GD2, was maintained with SCID passage . The original tumor inocula contained a variable percentage of tumor-associated lymphocytes (1-76%) . Flow cytometry analysis indicated that these were mainly CD3+ T-cells . However, there was no correlation between the percentage of tumor-associated lymphocytes and the time required for development of a palpable tumor after s.c . injection or the ability to metastasize . These results demonstrate the growth and spontaneous metastasis of fresh human melanoma in SCID mice and suggest that this model could be important for therapeutic and basic biological studies.

J Immunol Methods, 1991 Sep 13, 142(2), 215 - 22
Simultaneous flow cytometric detection of antibodies against platelets, granulocytes and lymphocytes; Sintnicolaas K et al.; We present a time-saving and objective flow cytometric immunofluorescence assay for the simultaneous detection of antibodies against platelets, granulocytes or lymphocytes using a reconstituted mixture of these cell populations . Platelets, granulocytes and lymphocytes could be distinguished on the basis of their forward (FSC) and sideways (SSC) light scattering properties plotted on scales of 4 log orders . After setting FSC/SSC gates around the platelets, granulocytes and lymphocytes, the reactivity of the sera with the cell populations was determined by histogram analyses of immunofluorescence for each gate . The flow cytometric assay of reconstituted cell mixtures showed a strong, positive correlation with a reference microscopic immunofluorescence assay of separate cell suspensions . The reproducible procedures for the isolation and staining of the cells and the electronic stability of the flow cytometer permitted the use of the same gate and marker settings throughout the experiments . Consequently, the entire analysis of data stored in list mode could be performed using a keystroke, so that time consuming and subjective manual analyses were avoided.

J Immunol Methods, 1991 Sep 13, 142(2), 199 - 206
A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation; Blaheta RA et al.; Measuring the incorporation of radioactive thymidine into the cell nucleus gives important information as to cell activation and proliferation . In this study the DNA-intercalating fluorochromes, Hoechst 33342 and Hoechst 33258, were tested as an alternative to the classical {3H}thymidine assay . Mitogen and alloantigen stimulated lymphocytes as well as FK 506 and CsA inhibited lymphocytes were treated with the two dyes, and the cell number and proliferation rates by means of measured fluorescence values . Of these tested fluorochromes H33342 appears to be an appropriate alternative to the {3H}thymidine assay . It mirrors the cell number in a fast and convenient manner without any pretreatment of the cell suspension which can remain in the culture plates . The complete assay procedure including data analysis can be performed rapidly and the standard deviations are small . This dye may also prove to be of value in other assay procedures, e.g., adhesion experiments.

Brain Res, 1991 Sep 6, 558(2), 251 - 63
Regional changes of striatal dopamine receptors following denervation by 6-hydroxydopamine and fetal mesencephalic grafts in the rat; Gagnon C et al.; Young adult female rats received a 6-hydroxydopamine lesion in the left substantia nigra and, 3 weeks later, some of them were grafted with a cell suspension from the ventral mesencephalon of rat embryos (14-15 days old) . Six months after transplantation, some grafted rats, following injection of amphetamine, had switched to turning only toward the intact side (type 1), whereas others turned toward the intact side only during the first half of the test (type 2) . Levels of dopamine, dihydroxyphenylacetic acid and homovanillic acid were, respectively, 2%, 15% and 35% of the intact side in the denervated striatum of 6-hydroxydopamine rats . Dopamine concentrations were restored to 13% and 10% of the intact side in the grafted striatum of type 1 and type 2 animals, respectively . Levels of homovanillic acid were unchanged following grafts whereas those of dihydroxyphenylacetic acid increased by 209% and 247% in the grafted striatum of type 1 and type 2 animals, respectively . The ratios of dihydroxyphenylacetic acid/dopamine as well as homovanillic acid/dopamine were low in the intact striatum whereas they increased in the denervated striatum with or without graft . The tyrosine hydroxylase immunoreactivity decreased by about 80% in the denervated striatum of 6-hydroxydopamine rats . In type 1 rats, tyrosine hydroxylase immunoreactivity revealed that the graft was localized in the dorsomedial part of the denervated striatum, whereas in type 2 animals, it was also in the medial striatum but it overlapped the dorsal and ventral parts of it equally . D1 as well as D2 dopamine receptors were measured throughout the striatum (9.0-7.6 rostral-caudal coordinates), by autoradiography, using {3H}SCH 23390 (D1 antagonist) and {3H}spiperone (D2 antagonist) binding . Supersensitive D2 receptors were normalized in the dorso- and ventromedial parts of the grafted striatum . D2 receptor density was higher in type 2 than in type 1 rats, more specifically at 8.6-8.2 rostral-caudal coordinates, where the graft was . D1 receptor supersensitivity was modest compared to D2 receptors in the striatum of 6-hydroxydopamine rats and decreased following grafts . DA receptors changes in the striatum, following fetal mesencephalic grafts, may explain the behavioral recovery seen in grafted rats.

Transfus Med, 1991 Sep, 1(3), 155 - 8
The pH, conductivity and osmolality of low ionic strength solutions used within the U.K . for the antiglobulin test; Phillips PK et al.; Low ionic strength solutions (LISS) for use in the antiglobulin test were obtained from 356 U.K . laboratories . Of the 324 laboratories using LISS to suspend the test red cells and who returned details of their LISS technique, 15 used unequal proportions of red cell suspension and serum despite the LISS being formulated for use with equal proportions . Of the 22 laboratories mixing LISS with serum and red cells suspended in a normal ionic strength medium, four used a LISS preparation formulated for a LISS-suspension technique and three used a commercially available LISS-addition preparation using proportions of red cells, serum and LISS not recommended by the manufacturer . The mean (standard deviation) pH, conductivity and osmolality of the 334 LISS preparations for LISS-suspension was 6.9 (0.2), 4.1 (0.4) mS/cm and 298 (15) mmole/kg, respectively . It is suggested that attention should be paid to the osmolality and, particularly, conductivity during the preparation of LISS because values were observed that were clearly outside the acceptable range cited in the Guidelines for the Blood Transfusion Services in the United Kingdom, i.e . pH 6.7 +/- 0.2, conductivity 3.7 +/- 0.3 mS/cm and osmolality 295 +/- 5 mmole/kg.

Int J Hyperthermia, 1991 Sep-Oct, 7(5), 785 - 93
Thermal sensitivity of the murine CFU-S-12: role of environmental cells; Wierenga PK et al.; The hyperthermic sensitivity of the CFU-S-12 in bone marrow from normal and anaemic mice was determined . The terminal slope of the survival curves, demonstrated by the T0 values, does not significantly differ in the resting and active cycling stem cells . In the active cycling stem cells the initial shoulder region was less dominant compared with the resting stem cells . The difference in heat sensitivity between resting and active proliferating CFU-S-12 might be explained by a difference in the accumulation of damage before lethality becomes manifest . The difference in heat sensitivity appears to be independent of the environmental accessory cells, demonstrated by a similar hyperthermic effect of the purified stem cells from bone marrow and spleen and the stem cells in the total cell suspensions . Therefore the heat sensitivity of the haemopoietic stem cell is not mediated by a release of injurious substances from environmental heat-damaged cells . The heat treatment does not result in a selection of macroscopic detectable colonies 12 days after inoculation, as is demonstrated by the same morphology of the spleen colonies from the stem cells before and after the hyperthermic treatment.

Immunology, 1991 Sep, 74(1), 121 - 6
Reciprocal haematopoietic cell transfers between C57BL/6 mice differing at the lpr locus; Montecino-Rodriguez EM et al.; Reciprocal transfers of spleen and bone marrow cell suspensions have been performed between mice of the C57BL/6 (B6) genetic background, differing at the lymphoproliferation (lpr) locus . These immune system chimaeras were followed for almost one year after sublethal irradiation and cell reconstitution . In addition to the survival of the chimaeras, the major lymphoid organs (bone marrow, spleen, thymus and lymph nodes) were examined for cell numbers, percentages of membrane immunoglobulin-positive cells and responses to mitogenic stimulations with concanavalin and lipopolysaccharide . The {lpr----lpr} chimaeras were similar to untreated lpr mice . The {wild----lpr} did not develop the lpr-induced syndrome and remained similar to {wild----wild} chimaeras . Therefore, B6 wild haematopoietic stem cells could rescue sublethally irradiated B6 lpr mice from the lpr-induced autoimmune pathology . The radioresistant lpr environment alone was not sufficient to induce the lpr syndrome . It may however be required for its development since {lpr----wild} chimaeras displayed a profound aplasia of their lymphoid organs, together with a normal cellularity of their bone marrow . In contrast to chimaeras constructed with MRL mice, the {lpr----wild} B6 chimaeras did not die following the lpr haematopoietic stem cell transfer . Therefore, the lymphoid aplasia of {lpr----wild} radiation chimaeras does not result from an lpr graft-versus-host-like syndrome . More likely is that a normal, non-lpr, haematopoietic environment may not allow the differentiation of the lpr haematopoietic stem cells into the lymphoid lineages.

Arthritis Rheum, 1991 Sep, 34(9), 1116 - 24
Human synovial mast cell involvement in rheumatoid arthritis and osteoarthritis . Relationship to disease type, clinical activity, and antirheumatic therapy; Bridges AJ et al.; Mast cells were isolated by enzymatic digestion of synovium obtained from 48 patients with rheumatoid arthritis (RA) and 42 patients with osteoarthritis (OA) . A significantly lower percentage of stainable synovial mast cells was obtained by tissue digestion from patients with clinically active RA compared with those with less active disease . The 54 patients treated with nonsteroidal antiinflammatory drugs had a significantly lower percentage of stainable synovial mast cells in cell suspension than did the other 36 patients . When anti-IgE antibody was used as a secretagogue in vitro, significantly greater histamine release was observed from synovial mast cells of RA patients compared with OA patients . Greater histamine release in response to anti-IgE was observed in the RA patients with more clinically active disease and those who were treated with prednisone, compared with RA patients without these features . Synovial mast cells of RA patients treated with a disease-modifying antirheumatic drug had a significantly lower mean histamine content than did cells from patients not receiving such treatment . Our data suggest that there are differences between synovial mast cells from tissues of patients with RA and OA and suggest that synovial mast cells may be activated in clinically active RA . In addition, the data indicate an effect of systemic antirheumatic therapy on mast cells isolated from synovium of patients with arthritis.

Clin Exp Metastasis, 1991 Sep-Oct, 9(5), 485 - 97
Organ-specific growth of a murine lymphoma of spontaneous origin in nude mice; Tomasoni A et al.; The MOC-25 tumour arose spontaneously in a female nude mouse and was established as a continuous line intraperitoneally in nude mice, where it reproduces the topological features of its origin, growing preferentially in the uterus, ovaries and liver . Karyotype analysis showed that MOC-25 cells are hyperdiploid . Tumorigenicity and malignant behaviour were studied by transplanting tumour cells into different sites in nude mice . The comparison of tumour take after i.p . and s.c . injections of scaled concentrations of MOC-25 cell suspension showed preferential growth in the peritoneum . Regardless of the route of implantation (s.c., i.v., i.p.), this tumour rapidly and preferentially disseminated to the liver, uterus, ovaries, spleen and bone marrow . No significant differences in tumour growth and metastatic behaviour were observed when MOC-25 was injected in ovariectomized nude mice or in male nude mice . Morphology studies using light and electron microscopy, immunophenotyping and molecular analysis indicated a B-lymphoid origin of the MOC-25 tumour.

Invest Ophthalmol Vis Sci, 1991 Sep, 32(10), 2700 - 10
Analysis of immunosuppressive properties of iris and ciliary body cells and their secretory products; Streilein JW et al.; The anterior chamber of the eye is an immunosuppressive microenvironment as shown experimentally by immune privilege, anterior chamber-associated immune deviation, and inability to display local delayed-type hypersensitivity responses . It recently was reported that both the aqueous humor and the cells of the iris and ciliary body (I-CB) have immune inhibitory properties in vitro, suggesting that these components of the anterior segment might contribute to the unique properties of this microenvironment . To explore the cellular sources of immunosuppressive factors in the anterior chamber, cultures of I-CB cells were established from normal eyes of BALB/c mice . Supernatants were harvested from these cultures and assayed in vitro for their ability to inhibit T-lymphocyte activation . It was found that I-CB cell-derived supernatants profoundly suppressed alloantigen-driven T-cell proliferation (mixed lymphocyte response) and interleukin-2 production by a T-cell hybridoma that responds to stimulator cells bearing I-Ad . The inhibitory activity of I-CB supernatants did not appear to be related to prostaglandins; supernatants of I-CB cells cultured with indomethacin retained their suppressive properties, as did supernatants to which neutralizing antiprostaglandin E2 antibodies had been added . Moreover, suppression by I-CB supernatants was not relieved by antibodies specific for transforming growth factor-beta, even though this cytokine is known to be present in normal aqueous humor . Thus, the identity of the suppressive factor(s) in cultured I-CB cell supernatants remains elusive . Finally, by separating I-CB cell suspensions into bone marrow-derived (T-200-positive) and those not derived from bone marrow (parenchymal) subpopulations with a fluorescence-activated cell sorter, it was determined that the inhibitory activity of I-CB cell suspensions was produced by parenchymal, rather than hematogenous, cells . It is proposed and discussed that inhibitory factors and cytokines secreted by parenchymal I-CB cells contribute to the immunosuppressive qualities of the anterior chamber.

Eur J Immunol, 1991 Sep, 21(9), 2281 - 4
Negative signaling by surface IgM on B cells isolated from ileal Peyer's patch follicles of sheep; Griebel PJ et al.; Lymphoid follicles from the sheep ileal Peyer's patch (PP) were used to prepare a cell suspension consisting of 98% surface IgM-positive (sIgM+) B cells and 1% T cells . Co-stimulation of follicular cells with pokeweed mitogen and either recombinant bovine interleukin 1 (IL 1) or IL 2 resulted in a marked proliferative response . In contrast, the addition of soluble F(ab')2 rabbit anti-sheep Ig completely inhibited the proliferative response induced by pokeweed mitogen and IL 1 or IL 2 co-stimulation . Anti-Ig inhibition of B cell proliferation was specific for ileal PP follicular cells and was not observed with mesenteric lymph node cells or splenocytes . Furthermore, suppression of ileal PP follicular B cell proliferation required at most divalent cross-linking of sIg was independent of Fc receptors, but was dependent on the concentration of anti-Ig and required 48 h for maximal effect . Negative signaling by sIgM indicates that ileal PP follicular B cells are functionally distinct from B cells in other secondary lymphoid tissues . Also, the present observations are consistent with previous reports indicating that B cell proliferation in ileal PP follicles is antigen independent.

Radiat Res, 1991 Sep, 127(3), 297 - 307
Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells; Freyer JP et al.; There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase . In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting . This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels . We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses . For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells . Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase . For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells . None of these age fractions showed a dependence of OER on survival level . Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells . These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations . For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.

J Invest Dermatol, 1991 Sep, 97(3), 454 - 60
T-lymphocyte-activating properties of epidermal antigen-presenting cells from normal and psoriatic skin: evidence that psoriatic epidermal antigen-presenting cells resemble cultured normal Langerhans cells; Demidem A et al.; Fresh and cultured human Langerhans cells display disparate functional programs, based on their capacities to activate autologous and allogeneic T cells, and with respect to their susceptibility to inhibition by transforming growth factor-beta (TGF beta) . We have compared the functional properties of epidermal antigen-presenting cells (APC) procured from uninvolved and involved skin of patients with psoriasis with fresh and cultured normal epidermal cells . Freshly obtained psoriatic epidermal APC resembled cultured normal epidermal cells in their superior capacity to activate syngeneic and allogeneic T cells; fresh normal epidermal cells failed to activate syngeneic T cells, and induced only modest proliferation among allogeneic T cells . The modest T-cell--activating properties of fresh, normal epidermal cells were not suppressed by TGF beta, whereas the T-cell--activating potential of psoriatic epidermal cells, cultured normal epidermal cells, and blood APC was inhibited approximately 50% by TGF beta . Thus, fresh psoriatic epidermal APC resemble cultured normal epidermal cells functionally . Because these properties are already evident in cells obtained from uninvolved psoriatic skin, the "cultured" functional phenotype of epidermal APC in this disease may precede the appearance of active psoriatic skin lesions . Surface marker analysis of normal and psoriatic epidermal cell suspensions revealed that virtually all of the bone marrow--derived cells in normal epidermal cell suspensions were conventional (CD1+) Langerhans cells, whereas CD1+ cells comprised only a minority of bone marrow--derived (CD45+) cells in psoriatic epidermis . It is speculated that some of the CD1-, CD45+ cells in psoriatic epidermis may be Langerhans cells that have lost their "fresh" phenotype . These data indicate that an abnormality in epidermal APC function exists in psoriatic skin--even before clinical lesions develop, and we speculate that the abnormal capacity of psoriatic epidermal APC to activate syngeneic T cells may be important in the expression of keratinocyte pathology . Because psoriatic epidermal APC functions were profoundly inhibited in vitro by treatment with cyclosporin A, the effectiveness of this drug in psoriasis may be due in part to its ability to inhibit epidermal antigen-presenting cell function in vivo.

Br J Cancer, 1991 Sep, 64(3), 508 - 12
Distribution of Photofrin between tumour cells and tumour associated macrophages; Korbelik M et al.; Photofrin levels in cells derived from SCCVII tumours, excised from mice that previously received the drug, were measured using a fluorescence activated cell sorter (FACS) . Concomitantly, in the same cells the FACS was used to measure fluorescein isothiocyanate (FITC) fluorescence that originated from FITC-conjugated antimouse IgG added to the cell suspension before sorting . This later measurement enabled discrimination between IgG negative tumour malignant cells and IgG positive host cells (primarily macrophages) . In addition, cellular Photofrin content in 'tumour' and 'host' cells sorted by FACS was determined by chemical extraction . The measurements were performed for the time intervals 1-96 h post Photofrin administration . The data showed consistently higher Photofrin levels in the 'host cells', i.e., tumour associated macrophages (TAM), than in 'tumour' cells . On a per cell basis, at any time point studied there was a minimum of 1.7 times more Photofrin in 'host' than in 'tumour cells', while at 4-12 h postadministration, ratios of up to 3.0 times were observed . This corresponds to ratio values greater than 9, when based on Photofrin content per micrograms cell protein.

Int J Dev Biol, 1991 Sep, 35(3), 177 - 89
Development of separated germ layers of rodent embryos on ectopic sites: a reappraisal; Levak-Svajger B et al.; The method of separation of germ layers of rodent embryos by treating the embryonic shields with proteolytic enzymes and by microsurgery with the subsequent transplantation to ectopic sites has helped to gain a more detailed insight into what is going on during gastrulation in mammals . The space under the kidney capsule of adult animals seems to be the most appropriate ectopic site for transplantation of early postimplantation rat embryos or separated germ layers . After transplantation the grafts develop into teratomas whose complex histological structure reflects the initial developmental capacities of the graft . At the pre-primitive streak and the early primitive streak stages the primitive ectoderm differentiates into tissue derivatives of all three definitive germ layers, often in complex organotypic combinations . This is indirect evidence that all cells of the embryonic body originate from the primitive embryonic ectoderm . Halves of the primitive ectoderm obtained by a longitudinal or transverse cut through the egg cylinder give the same result . At the head fold stage the capacity for differentiation of the ectoderm is restricted to ectodermal and mesodermal derivatives . One day before gastrulation the isolated primitive ectoderm is not able to differentiate as renal isograft . The mesoderm isolated at the head fold stage and at later stages when its segmentation occurs, differentiates almost exclusively into the brown adipose tissue . The embryonic endoderm differentiates only in combination with the mesoderm . After transplantation the embryonic ectoderm loses its epithelial organization and breaks up into a mass of mesenchyme-like cells in which epithelial structures subsequently appear and differentiate in a way reminiscent of the reaggregation of cells in mixed cell suspension in vitro.

Appl Biochem Biotechnol, 1991 Sep, 30(3), 273 - 84
Production of exocellular polysaccharide by Azotobacter chroococcum; De la Vega MG et al.; Environmental conditions affect the production of extracellular polysaccharide by Azotobacter chroococcum ATCC 4412 . Production of exocellular polymer from a variety of carbon sources depended on the air flow rate . A high sucrose concentration in medium (8%) markedly favored expopolysaccharide production, which reached 14 g/L in about 72 h . In cell suspensions incubated in the presence of 8% sucrose in a nitrogen-free medium, biopolymer final concentration of 9 g/L corresponds to 68 g/g biomass . Maximum efficiency of sucrose conversion into exopolysaccharide peaked at 70% for initial disaccharide concentration of 6% . High performance liquid chromatography and gas liquid chromatography of acid hydrolysates of the exopolymer revealed the presence of mannuronosyl, guluronosyl, and acetyl residues, but not neutral sugars . The infrared spectrum corroborated the presence of carboxylate anions and O-acetyl groups in the exopolymer . Though the presence of more than one kind of polysaccharide cannot be ruled out, these data suggest that, under the experimental conditions used in this work, only a type of alginate-like exopolysaccharide is produced by A . chroococcum ATCC 4412.

Shi Yan Sheng Wu Xue Bao, 1991 Sep, 24(3), 181 - 7
{Purification and survival of retinal ganglion cell}; Zhao LP et al.; We had used a specific anti-Thy 1.1 antibody binding method and a nylonmembrane sieve method to isolate and purify retinal ganglion cells from neonatal rats in order to compare the effect of tectal extract on these purified cells retinal ganglion cells . Isolated retinal cell suspension with retinal ganglion cells retrograde-prelabelled with Fast Blue were seeded on culture dishes coated with the specific anti-Thy 1.1 antibody for 30 minutes before nonadherent cells were removed . The percentage purity of the adherent retinal ganglion cells determined microscopically to be 95% . However, the percentage purity of the Fast Blue-labelled retinal ganglion cells recovered using the nylon membrane of pore size 15 microns was only 60 +/- 5% . Retinal ganglion cells purified by both methods could survive and grow into large, active neurons with neurite outgrowths in the presence of tectal extract . A MTT colorimetric microassay was used to quantify the survival growth activity of these purified retinal ganglion cells after culture for 24 hours . The result showed that the optical density ratio (+Te/-Te) of the retinal ganglion cells purified by anti-Thy 1.1 antibody binding method was 12.3 (0.111/0.009) and by the nylon membrane method was 6.4 (0.102/0.016), and the optical density ratio of the non-purified retinal cells was 3.8 (0.095/0.025), p less than 0.01 for all 3 sets of results . It was concluded that in the absence of other cells, the purified retinal ganglion cells responded specifically to the trophic activity in tectal extract, the purer the retinal ganglion cells and the clearer the effect.

Nihon Kyobu Shikkan Gakkai Zasshi, 1991 Sep, 29(9), 1119 - 25
{Gene analysis of pulmonary lymphoproliferative disorders}; Shiota T et al.; The authors performed gene analysis of pulmonary lymphoproliferative disorders . Cell suspensions were obtained from tissues of malignant lymphoma or pseudolymphoma in Cases 1 to 3 . High-molecular-weight DNA was extracted from these specimens, digested with restriction endonucleases, size-fractionated by agarose-gel electrophoresis and transferred by the Southern procedure to nitrocellulose . Hybridization to nick-translated 32P DNA probes of the immunoglobulin JH, C kappa, C lambda, regions, and T cell receptor beta 1 region . In case 1 and 2, which were diagnosed as B cell lymphoma, cells from tumor had rearranged heavy chain genes, clearly establishing the clonal nature . In Case 3, which was diagnosed as pseudolymphoma, the tumor contained clonal immunoglobulin gene rearrangements as detected with both the JH heavy chain and C kappa light chain gene probes . It was concluded that gene analysis is an effective procedure for establishing a diagnosis of lymphoma in neoplastic disorders of uncertain cell type and for detecting clonal T cell or B cell populations with atypical lymphofollicular hyperplasia.

Cell Tissue Res, 1991 Sep, 265(3), 527 - 34
Growth of enteric neurones from isolated myenteric ganglia in dissociated cell culture; Saffrey MJ et al.; Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods . The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro . The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro . Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum-supplemented medium without mitotic inhibitors . Glial cell numbers appeared to decline in serum-free medium after the first week in vitro . Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media . This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall.

J Neurocytol, 1991 Sep, 20(9), 732 - 45
Immunocytochemical analysis of glial cells in the hypomyelinated optic nerve of the BW mutant rat; Chan CL et al.; The Browman-Wyse (BW) rat is a mutant with structural defects of the visual system, including a failure of the proximal (retinal) end of the optic nerve to myelinate . This latter abnormality is correlated with an absence of CAII+ oligodendrocytes, but we have previously shown that astrocytes are normally distributed, as judged by morphological characteristics of GFAP+ cells in vivo . We have further examined in vitro the immunohistochemical characteristics of macroglia isolated from the BW optic nerve, either as cell suspensions or after 4 days in culture . Cell cultures derived from the hypomyelinated proximal segment of BW optic nerves contained very few 0-2A progenitor cells (from which oligodendrocytes and cells with the GFAP+/A2B5+ phenotype develop), whereas over 90% of the glia were Schwann cells . A proportion of these few 0-2A progenitor cells differentiated normally after 4 days in vitro into both progeny phenotypes in appropriate media . Accordingly, we conclude that the myelination deficiency in the BW optic nerve could be explained as a failure of 0-2A progenitor cells to populate fully the proximal extremity of the nerve during development . Since most glia isolated from adult optic nerves did not adhere to the culture substrate, we analysed the phenotypes of freshly isolated cells in suspension . Comparing optic nerves of normal adult rats with those of BW mutants, a significantly higher fraction of the GFAP+ cells reacted with A2B5 in cell suspensions of the latter . The double-labelled cells which are present in abnormally high numbers may be the differentiated progeny of 0-2A progenitors in the hypomyelinated segment of nerve . One explanation for these findings is that Schwann cells within the BW nerve induce the differentiation of 0-2A progenitor cells to the GFAP+/A2B5+ phenotype . We investigated this possibility using conditioned medium from cultured Schwann cells which increased tenfold the frequency of GFAP+/A2B5+ cells in normal neonatal rat optic nerve cultures . Oligodendrocyte numbers showed a concomitant decline with increasing concentration of Schwann cell conditioned medium . Hypomyelination in the BW rat optic nerve may therefore arise because Schwann cells, present in the proximal segment of the nerve, not only impede the migration of 0-2A progenitor cells but also release a factor which induces those 0-2A progenitor cells which arrive in the proximal segment of the nerve to differentiate into GFAP+ cells at a critical stage in oligodendrocyte development.

Am J Clin Pathol, 1991 Sep, 96(3), 351 - 9
Leukemia and lymphoma immunophenotyping in cell smears with immunogold-silver staining; De Waele M et al.; The potential of the immunogold-silver staining (IGSS) technique for immunophenotyping leukemia and lymphoma cells in cell smears was examined . Peripheral blood, bone marrow aspirates, lymph node biopsy specimens, fine-needle aspirates, and biologic fluids of 83 patients with acute or chronic leukemias, non-Hodgkin's lymphomas, or Hodgkin's disease were labeled . Cell smears, cytocentrifuge preparations, or imprints were fixed, incubated with the reagents, and counterstained with May-Grunwald-Giemsa . Stable immunostaining and good morphologic characteristics allowed accurate cell identification and rapid enumeration of the positive cells . The immunophenotypes obtained with the use of 35 monoclonal antibodies with different specificities were similar to those determined by flow cytometry or immunohistochemical studies on the same samples . This IGSS method was especially useful for the examination of poor samples or complex cell suspensions with rare malignant cells . It could be an alternative to the immunoenzyme methods that generally are used for this purpose.

Curr Eye Res, 1991 Sep, 10(9), 811 - 6
Human retinal pigment epithelial cells possess V1 vasopressin receptors; Friedman Z et al.; Membrane preparations of cultured human retinal pigment epithelial (RPE) cells were incubated with various concentrations of {3H}arginine vasopressin (AVP) in the presence and absence of 10 microM nonradioactive AVP . Saturable, specific binding to a single site with a Kd of 6.2 nM and Bmax of 111 fmol/mg protein was detected . Vasopressin had no effect on RPE cyclic AMP levels measured by radioimmunoassay . Intracellular calcium fluxes were measured by spectrofluorometry of RPE cell suspensions preloaded with quin 2 . The baseline cytosolic calcium level was 217 +/- 20 nM, and AVP caused a concentration-dependent increase in this level with a 3.5-fold maximal response at 10(-6) M and an EC50 of 120 nM . The production of inositol phosphates was measured in RPE preloaded with {3H}myoinositol, and AVP caused a concentration-dependent increase in their production with a 2.1-fold maximal response at 10(-5) M and an EC50 of 80 nM . A specific vasopressin receptor antagonist, SKF 101926, prevented the AVP-induced increase in calcium mobilization and inositol phosphate production in RPE . These data suggest that RPE cells possess V1 AVP receptors coupled to calcium mobilization and inositol phosphate metabolism.

Clin Chim Acta, 1991 Aug 30, 200(2-3), 175 - 81
Immunological assay of erythrocyte acetylcholinesterase; Jones JW et al.; An immunoassay for the quantitation of erythrocyte surface acetylcholinesterase is described; using a red cell suspension, bound mouse monoclonal acetylcholinesterase antibody is detected by an alkaline phosphatase conjugated rabbit anti mouse IgG . Extraction is not required . In addition, the activity of erythrocyte surface acetylcholinesterase using dithiobisnitrobenzoate to detect released thiocholine has been measured . The coefficient of variation for each method is 7% . Reference ranges have been established for healthy adults and cord blood.

Cancer Res, 1991 Aug 15, 51(16), 4287 - 94
Cellular glutathione and thiol measurements from surgically resected human lung tumor and normal lung tissue; Cook JA et al.; Cellular glutathione (GSH) levels were measured from 27 human lung tumor biopsies, enzymatically disaggregated, and compared with cells isolated from normal lung of the same patients . GSH levels from normal lung were similar among patients with a mean value of 11.20 +/- 0.58 (SEM) nmol GSH/mg protein (24 patients) with a range from 6.1 to 17.5 nmol GSH/mg protein . GSH levels varied considerably within and across histological tumor types with the following values: adenocarcinomas, 8.83 +/- 0.96 nmol/mg protein (8 patients); large cell carcinomas, 8.25 +/- 2.51 nmol/mg protein (3 patients); and squamous cell carcinomas, 23.25 +/- 5.99 nmol/mg protein (8 patients) . The cyclic GSH reductase assay gave only average GSH values and could not distinguish possible GSH variation among subpopulations of cells isolated . Cell volume measurements and microscopic evaluation of cells isolated from both tumors and normal lung revealed heterogeneity with respect to cell types present . To determine the extent of thiol variation among tumor cell subpopulations, tumor cell suspensions were stained with the thiol-specific stain, monochlorobimane (MCB) . The accuracy of MCB staining was tested by flow cytometric analysis of 12 in vitro human tumor cell lines and 3 rodent cell lines . A linear relationship was found between the bimane cellular fluorescence and the cyclic GSH reductase assay for cell lines having less than 80 nmol GSH/mg protein (R2 = 0.82) . Above 80 nmol GSH/mg protein the rate of change of the bimane fluorescence intensity with respect to increasing GSH concentrations was much reduced . However, by labeling cells with MCB it was possible to distinguish between cell lines with low versus high GSH content . MCB staining of tumor samples revealed multiple populations of cells with respect to thiol levels . In particular, 2 of 8 squamous cell carcinomas had a proportion of cells with elevated fluorescence intensities (from 10 to 35% of the population) suggesting the presence of cells with greatly elevated thiol levels . These findings underscore the complexity of quantitating intracellular GSH levels from tumor biopsies . The combined use of MCB with flow cytometry and conventional GSH assays may help to delineate subpopulations of cells within tumors with different thiol levels.

Gastroenterology, 1991 Aug, 101(2), 303 - 11
Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth; Evers BM et al.; The authors have established a long-term tissue culture cell line (BON) derived from a metastatic human carcinoid tumor of the pancreas . The cells have been in continuous passage for 46 months . Tissue culture cells produce tumors in a dose-dependent fashion after SC inoculation of cell suspensions in athymic nude mice . BON tumors, grown in nude mice, are histologically identical to the original tumor; they possess gastrin and somatostatin receptors, synthesize serotonin and chromogranin A, and have a doubling time of approximately 13 days . The antiproliferative effects of the long-acting somatostatin analogue, SMS 201-995 (300 micrograms/kg, t.i.d.), and 2% alpha-difluoromethylornithine on BON xenografts in nude mice were examined . Tumor size was significantly decreased by day 14 of treatment with either agent and at all points of analysis thereafter until the animals were killed (day 33) . In addition, tumor weight, DNA, RNA, and protein contents were significantly decreased in treated mice compared with controls . Establishment of this human carcinoid xenograft line, BON, provides an excellent model to study further the biological behavior of carcinoid tumors and the in vivo effect of chemotherapeutic agents on tumor growth.

Biochim Biophys Acta, 1991 Aug 2, 1059(1), 99 - 105
Extracellular generation of active oxygen species catalyzed by exogenous menadione in yeast cell suspension; Yamashoji S et al.; Luminol chemiluminescence was observed by addition of menadione to yeast cell suspension and was amplified 1000-fold by further addition of Fe-complex . Catalase, superoxide dismutase and ceruloplasmin had inhibitory effects on luminol chemiluminescence, indicating the extracellular generation of active oxygens (H2O2 and O2-) and reduction of Fe-complex . The generation of H2O2 and reduction of Fe-complex were mainly dependent on the activity of NADH: menadione oxidoreductase in the plasma membrane and cytosol fractions . Both luminol chemiluminescence and H2O2 production were sensitive to the inhibitory effects of proton conductor, ionophorous antibiotics and ATPase inhibitor rather than the inhibitors of the mitochondria electron transport system . The incubation of glucose with yeast cells caused a parallel increase in luminol chemiluminescence, H2O2 production and intracellular NADH concentration . These facts suggest that menadione-catalyzed H2O2 production and chemiluminescence are used as the indicators of cell activity to keep the NADH concentration and NADH: menadione oxidoreductase activity which may be sensitive to the change in pH and ion concentrations.

Exp Cell Res, 1991 Aug, 195(2), 443 - 57
Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface; Schafer IA et al.; Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis . These morphogenetic events occur in a serum-free environment in the absence of fibroblasts . Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum . The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules . The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes . Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel . These proteins are not organized into a cytological basement membrane . Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure . Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins . The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel . This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.

Cell Immunol, 1991 Aug, 136(1), 122 - 32
Factors determining the ability of cytokine-activated killer cells to lyse human ovarian carcinoma targets; Hirte HW et al.; Lysis of human ovarian carcinoma cells by natural killer (NK) cells, interferon-alpha activated NK cells (alpha-NK) and lymphokine-activated killers cells (LAK) was studied using both fresh tumor cells and a cell line (HEY) as targets . A clonogenic assay to measure cell kill was more sensitive than a 4-h 51Cr release assay . Both assays showed that single cells were more effectively lysed than were tumor clumps (spheroids) . Freshly isolated tumor cells studied in the 51Cr release assay appeared equally susceptible to lysis by LAK cells whether in the form of clumps or single cells, but NK and alpha-NK effectors appeared much less effective in lysing susceptible target cells when they were in clumps . Tumor cells from some patients showed marked resistance to lysis by NK and alpha-NK cells in fractions enriched for clonogenic cells, even when tested in a single cell-suspension, whereas LAK cells were always cytolytic . These data suggest that intrinsic resistance of ovarian carcinoma to lysis by LAKs is unlikely to explain failure of LAK + IL-2 therapy to eradicate tumor in vivo.

Respir Physiol, 1991 Aug, 85(2), 231 - 43
Bohr shift by lactic acid and the supply of O2 to skeletal muscle; Boning D et al.; Conditions simulating changes during physical exercise were induced in erythrocytes to determine the resulting Bohr effect . Lactic acid was added to red cell suspensions and whole blood with initial 25 and 60% SO2, at 42 Torr PCO2, and temperatures of 20 and 37 degrees C . Changes in pH, PO2 and SO2 were measured . CO2 liberation from buffering lactic acid in the extracellular fluid and its diffusion into erythrocytes resulted in an exaggerated Bohr shift, if the gas could not escape from the liquid phase (closed system, 'muscle' conditions) . PO2 at constant SO2 increased by up to 11.7%.mmol-1.L lactic acid . After reequilibration to initial PCO2 values (open system, 'lung' conditions) the Bohr shift decreased (remaining PO2 increase 0.7-1.4%.mmol-1.L) mainly caused by the reduced acidification . In addition, the Bohr coefficients (BC) under closed conditions were larger (-0.36 to -0.50) than after reequilibration (-0.20 to -0.38) . This difference is attributed to a larger CO2 BC than fixed acid BC . These effects might be enhanced in vivo by temperature differences between muscle and lung, lowered nonbicarbonate buffering of blood and counter-current blood flow in muscle.

Cryobiology, 1991 Aug, 28(4), 391 - 9
Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures; McCaa C et al.; Monocytes were isolated from fresh whole human blood and resuspended in Hanks balanced salt solution; a portion of the cells was mixed with an equal volume of 2M dimethyl sulfoxide (DMSO) to form a 1 M solution . Microliter volumes of cell suspension were placed directly onto a computer-controlled cryostage and cooled to a predetermined subzero temperature . Ice was nucleated in the extracellular medium and a continuous video record was made of the subsequent osmotically induced volume changes of individual cells owing to exposure to the concentrated extracellular solutes . Selected micrographs emphasizing the initial transient data were digitized for computer analysis with an interactive boundary tracing algorithm to determine metric parameters of specific cells, and apparent volume changes were measured as a function of elapsed time after nucleation . The Kedem-Katchalsky-coupled transport equations were fit to the data using a network thermodynamic model implemented on a microcomputer to determine values for the permeability properties Lp, omega, and sigma . Experiments were performed over the temperature range from -7 degrees to -10 degrees C . Cells pre-equilibrated with DMSO had a lower Lp and a higher activation energy, delta E, than without additive, although the statistical significance of the difference could not be substantiated . It was found that the movement of DMSO across the plasma membrane in response to extracellular freezing was apparently so much smaller than the water flux that values for omega and sigma could not be determined from the data base.

Thymus, 1991 Aug, 18(1), 15 - 31
Murine CD4-CD8- thymocytes are stimulated by interleukin-2 to proliferate in vitro in chemically defined medium; Wood GW et al.; The ability of fetal and young adult CD4-CD8- thymocytes to proliferate in chemically defined (serum-free) medium in the presence and absence of IL-2 was examined . Dissociated thymocytes from day 15 and older fetal mice proliferated in vitro in the presence but not the absence of IL-2 . The degree of proliferation was increased by including IL-1 with the IL-2 . Inclusion of IL-1 in cultures of fetal thymocytes was associated with an increase in the number of IL-2 receptor positive cells, relative to cultures containing IL-2 alone . Although unfractionated thymocytes failed to proliferate in chemically defined medium, CD4-CD8- cells purified from thymic cell suspensions from young adult mice from several inbred strains proliferated to a limited extent in the absence of added cytokines . Proliferation was augmented 40-100 fold by inclusion of IL-2 in cultures . IL-1 stimulated some proliferation by young adult CD4-CD8- cells, but, unlike the effect of IL-1 and IL-2 on fetal thymocytes, combination of IL-1 with IL-2 did not have a notable additive effect on IL-2 induced proliferation . Proliferation stimulated by both IL-1 and IL-2 was completely abrogated by inclusion of anti-IL-2 receptor antibody in the cultures . Thymocytes from F1 progeny of inbred strains of mice proliferated to a greater extent in the absence of IL-2 than did thymocytes from either parent strain, although the response to IL-2 was not significantly different . The data demonstrate that both fetal and adult CD4-CD8- thymocytes area capable of proliferating in response to IL-2 in vitro, suggesting that, as is the case during antigen specific responses by mature T cells, IL-1 and IL-2 cooperate to stimulate T cell proliferation during development in vivo.

Arch Biochem Biophys, 1991 Aug 1, 288(2), 552 - 7
Coordinate- and elicitor-dependent expression of stilbene synthase and phenylalanine ammonia-lyase genes in Vitis cv . Optima; Melchior F et al.; The mechanisms controlling the induction of stilbene synthase and phenylalanine ammonia-lyase (PAL), two putative key regulatory enzymes of the biosynthetic pathway to stilbene phytoalexins, have been investigated . The induction was studied in cell suspension cultures of grape (Vitis cv . Optima) by treatment with fungal cell wall . Several independent cDNA clones for PAL and stilbene synthase were isolated from a cDNA library of fungal cell wall-induced grape cells and identified by sequence analysis . The stilbene synthase cDNA sequence of pSV21 predicted a protein of 392 amino acids and Mr 42,791, similar in size to that observed experimentally for immunodetected stilbene synthase . The cDNA sequences of pSV21 and pSV25 differed in 76 bp in the coding region . The sequences of grape stilbene synthase cDNAs exhibited significant homology to the sequence reported for the peanut stilbene synthase cDNA . Both PAL and stilbene synthase mRNA, measured by RNA blot hybridizations, were induced within 1 h of addition of fungal cell wall preparations to the cell cultures, rose to a maximum by the sixth hour, then declined slowly over the next 20 h . The activities of PAL and stilbene synthase were also induced in parallel, but reached their maximum at different times after fungal cell wall addition to the cell cultures . The induction patterns of stilbene synthase and PAL in grape and peanut are discussed.

Eur J Surg Oncol, 1991 Aug, 17(4), 338 - 41
Evaluation of resection margins after breast conservative surgery with monoclonal antibodies; Veronesi U et al.; An immunocytochemical method for the detection of cancer cells, in the cell suspension obtained by scraping the surface of the surgical resection margins is described and its sensitivity compared to the conventional histology performed on random biopsies from the same margins . The reactivity of the cells with a pool of monoclonal antibodies (Mab) directed against epithelial markers indicated that in 80% of the 42 cases tested, the scraping method was adequate for the gathering of cells from the margins . The analysis of the samples using B72.3 Mab specific for tumor cells revealed that, among B72.3-positive tumor cases, 31% of breast margins contained tumor cells, whereas only 12% were histologically positive . Our results indicate that the immunocytological methodology is therefore more sensitive and should be used alongside histological examination to detect the tumor contamination in the surgical resection margins.

J Surg Res, 1991 Aug, 51(2), 128 - 32
Changes in blood rheologic factors following coronary artery surgery; Kato T et al.; Blood rheologic parameters were examined in 16 patients without multiple organ failure 2 weeks after coronary artery surgery . The whole blood viscosity (shear rate = 94.5 sec-1) was unchanged (4.59 +/- 0.53 to 4.56 +/- 0.58 mPa.s) despite a significant decrease in hematocrit (39.8 +/- 4.3 to 37.1 +/- 3.8%, P less than 0.05) . The oxygen delivery index (the ratio of hematocrit to whole blood viscosity) was significantly decreased (8.67 +/- 0.35 to 8.18 +/- 0.59%/mPa.s, P less than 0.05) . Plasma viscosity (shear rate = 94.5 sec-1) increased significantly (1.60 +/- 0.13 to 1.72 +/- 0.10 mPa.s, P less than 0.01), as did serum levels of globulin (2.8 +/- 0.4 to 3.0 +/- 0.4 g/dl, P less than 0.01), alpha 1-antitrypsin (232 +/- 41 to 308 +/- 60 mg/dl, P less than 0.001), and fibrinogen (381 +/- 81 to 479 +/- 117 mg/dl, P less than 0.001) . Total protein and globulin levels showed a good correlation with plasma viscosity, but the fibrinogen concentration demonstrated no correlation . The passage time of a 40% red blood cell suspension (0.5 ml) was shortened significantly from 10.2 +/- 1.1 to 9.2 +/- 1.0 sec (P less than 0.05) . These results indicate that an increase in plasma viscosity is important in determining blood rheologic properties 2 weeks after coronary artery surgery.

Appl Environ Microbiol, 1991 Aug, 57(8), 2283 - 6
Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences; Pillai SD et al.; Bacterial cells can be differentially separated from soil colloids on the basis of their buoyant densities . By using this principle, a modified sucrose gradient centrifugation protocol has been developed for separating bacterial cells from most of the soil colloids . Since the bacterial cell suspension still contained some colloidal soil particles, which inhibited polymerase chain reaction amplification, a new "double" polymerase chain reaction method of analysis was adopted for amplification of Tn5-specific gene sequences . This new protocol allowed rapid detection of small numbers (1 to 10 CFU/g) of bacterial cells present in soil samples.

Brain Res, 1991 Jul 19, 554(1-2), 30 - 7
Hippocampal grafts into the intact brain induce epileptic patterns; Buzsaki G et al.; Spontaneous hippocampal EEG activity and evoked field potentials were investigated in intact rats and in animals with fetal hippocampal grafts . Pieces of hippocampal grafts, derived from 15- to 16-day-old fetuses, were used to prepare cell suspensions and grafted directly into the intact hippocampus . Control animals received suspension grafts of the cerebellum derived from fetuses of identical age . Host hippocampal electrical patterns were monitored with chronic single electrodes or with a 16-microelectrode probe from 7 to 10 months after grafting . In contrast to previously reported high survival rates of fetal grafts in studies with damage to the host brain prior to grafting, survival of both hippocampal (60%) and cerebellar grafts (20%) was very poor in the intact hippocampus . In animals with cerebellar transplants or without surviving grafted neurons the electrical activity of the host hippocampus was indistinguishable from normal controls . In rats with hippocampal grafts short duration, large amplitude EEG spikes (up to 10 mV) were recorded, predominantly during immobility . When the EEG spikes (putative interictal spikes) were of large amplitude and contained population spikes, test evoked responses delivered to the perforant path were suppressed after the spontaneous events . In contrast, evoked responses were facilitated by interictal spikes without population spikes . The threshold of electrically induced afterdischarges did not differ significantly between groups of intact rats and animals with or without hippocampal grafts . However, in three rats with hippocampal grafts the evoked afterdischarges were associated with behavioral seizures . In two of these rats spontaneously occurring seizures were also observed . Synaptophysin-immunoreactivity demonstrated growth of the host mossy fibers into the graft.(ABSTRACT TRUNCATED AT 250 WORDS)

J Leukoc Biol, 1991 Jul, 50(1), 57 - 68
Development, differentiation, and maturation of macrophages in the chorionic villi of mouse placenta with special reference to the origin of Hofbauer cells; Takahashi K et al.; In blood vessels of the chorionic villi of mouse placenta, primitive macrophages first emerged at 10 days of gestation, then differentiated and matured into fetal macrophages . After emigration into the chorionic villous mesenchymal stroma, they ingested fluid-like stromal materials and transformed into Hofbauer cells . In our observation of their differentiation and maturation, no promonocytes or monocytes were detected . In the culture of cell suspensions from the placenta with LP3-conditioned medium, CFU-GM was confirmed, but in the culture with the mouse bone marrow stromal cell line (ST2) the primitive/fetal macrophage population occurred predominantly before the development of the monocyte/macrophage population . Proliferative potential of the primitive and fetal macrophages is slight . With the progress of gestation, the monocyte/macrophage population appeared in the chorionic villous stroma, forming a heterogeneous population of placental macrophages in the late fetal stage.

Cancer, 1991 Jul 1, 68(1), 169 - 77
Multi-parameter flow cytometric quantitation of the expression of the tumor-associated antigen SM3 in normal and neoplastic ovarian tissues . A comparison with HMFG1 and HMFG2; van Dam PA et al.; SM3 is a monoclonal antibody that reacts with a peptide epitope in the core protein of polymorphic epithelial mucin . Multi-parameter flow cytometry was used to characterize the expression of SM3 and compare it with two related tumor-associated antigens, HMFG1 and HMFG2, in cell suspensions of 44 malignant ovarian tumors, 15 benign ovarian tumors, and 16 normal ovaries . Tumor-associated antigen expression was significantly higher in malignant ovarian neoplasms than in benign neoplasms (P less than 0.001 for all three antigens) . SM3 was expressed more specifically in malignant than benign tumors but had a lower affinity than HMFG1 and HMFG2 . Multi-parameter flow cytometric evaluation of a panel of monoclonal antibodies can be used to help in choosing the best antibody for immunohistochemistry, imaging, and eventually treatment of ovarian tumors.

Cancer, 1991 Jul 1, 68(1), 1 - 8
Continuous interleukin-2 and tumor-infiltrating lymphocytes as treatment of advanced melanoma . A national biotherapy study group trial; Dillman RO et al.; Melanoma metastases were harvested from 82 patients for the purpose of growing and expanding tumor-infiltrating lymphocytes (TIL) . Tumor tissue cell suspensions were incubated with interleukin-2 (IL-2), followed by repeated exposure to tumor antigen with or without OKT3 monoclonal antibody (MoAb) . Initial growth success was achieved in 56 of 82 cultures (72%) . Efforts were made to expand 26 of these 56 cultures for therapeutic TIL; 23 of 26 early cultures (88%) were successfully expanded for in vivo therapy . It took a mean of 78.5 +/- 25.4 days to grow sufficient TIL for treatment . Therapy included cyclophosphamide (1 g/m2) on day 1, followed by a 96-hour continuous infusion of IL-2 (18 x 10(6) IU/m2/d) on days 2 to 5, and approximately 10(11) (mean 1.49 +/- 0.93 x 10(11)) TIL on day 2 . Patients who responded received monthly IL-2 as a 96-hour infusion . Median patient age was 45 years of age . Sixty-seven percent of the patients were men . Performance status was 0 to 1 in 77% of patients . Thirty-four percent of the patients had liver metastases . The usual IL-2 toxicities were seen . Response rate for 21 patients was 24% (95% confidence interval, 10% to 49%) . One complete response was achieved with cells 98% CD4+; four partial responses were achieved with cells 80%, 94%, 98%, and 98% CD8+, respectively . Four of eight patients who received TIL, which had never been stimulated with OKT3, had tumor response . The authors conclude that a treatment plan for IL-2/TIL is technically difficult, costly, and effective for only a minority of patients . Overall, clinical results are not clearly superior to those obtained with other IL-2 regimens.

Vet Pathol, 1991 Jul, 28(4), 259 - 66
Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa; Hooser SB et al.; The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes . Male Sprague Dawley rats were used for all studies . For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation . Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg . Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin . In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes . In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments . These alterations were dose and time dependent . Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR . In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing . These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.

Respir Physiol, 1991 Jul, 85(1), 1 - 14
Gas exchange in isolated perfused frog skin as a function of perfusion rate; Pinder AW et al.; Patches of skin were removed from bullfrogs and perfused with a red cell suspension through the cutaneous artery to define the gas exchange characteristics of frog skin without complications caused by in vivo regulatory mechanisms . Oxygen uptake was primarily perfusion limited at low perfusion rates but primarily diffusion limited at perfusion rates that were similar to cutaneous blood flows previously reported in vivo . Diffusing capacity (DO2) increased only slightly as perfusion rate increased . Because the DO2 in isolated skin, in which DO2 should be maximal, was not significantly higher than estimates of DO2 in vivo, there seems to be little opportunity for increasing cutaneous DO2 or oxygen uptake in vivo.

Mol Endocrinol, 1991 Jul, 5(7), 949 - 58
Spontaneous and agonist-induced calcium oscillations in pituitary gonadotrophs; Iida T et al.; Basal and receptor-regulated changes in cytoplasmic calcium concentration ({Ca2+}i) were monitored by fluorescence analysis in individual rat pituitary gonadotrophs loaded with the calcium-sensitive dye indo-1 . Most gonadotrophs exhibited low amplitude spontaneous oscillations in basal {Ca2+}i that were interspersed by quiescent periods and abolished by removal of extracellular Ca2+ or addition of calcium channel blockers . Such random fluctuations in {Ca2+}i, which reflect the operation of a plasma membrane oscillator, were not coupled to basal gonadotropin secretion . The physiological agonist GnRH induced high amplitude {Ca2+}i oscillations; when a threshold {Ca2+}i level was reached, a cytoplasmic oscillator began to generate extremely regular Ca2+ transients . The time required to reach the threshold {Ca2+}i level was inversely correlated with agonist dose; the frequency, but not the amplitude, of agonist-induced Ca2+ spiking increased with agonist concentration . The duration of the latent period decreased and the frequency of Ca2+ spiking increased with the increase in ambient temperature . At high GnRH concentrations, the calcium transients merged into biphasic responses similar to those observed in cell suspensions at all GnRH concentrations . The presence of spontaneous fluctuations in basal {Ca2+}i did not significantly change the patterns of agonist-induced {Ca2+}i responses . Also, removal of extracellular Ca2+ did not interfere with the frequency or amplitude of Ca2+ spikes, but caused the loss of the plateau phase . Blockade of intracellular Ca(2+)-ATPase pumps by thapsigargin was usually accompanied by a subthreshold increase in {Ca2+}i . In such cells the agonist-induced oscillatory pattern was transformed into the biphasic response . In about 10% of the cells, however, high thapsigargin concentrations induced coarse {Ca2+}i oscillations; subsequent stimulation of such cells with GnRH was ineffective . The cytoplasmic oscillatory and biphasic responses may represent a mechanism for differential activation of Ca(2+)-dependent enzymes and their dependent cellular processes, including hormone secretion . The membrane oscillator is probably responsible for refilling of agonist-sensitive pools during and after agonist stimulation.

Mod Pathol, 1991 Jul, 4(4), 503 - 13
Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers; Hopman AH et al.; An improved protocol for in situ hybridization (ISH) to routinely processed, paraffin-imbedded tissue sections from transitional bladder carcinoma (TCC) is presented . The protocol to detect numerical chromosome aberrations involved treatment of sections with thiocyanate prior to proteolytic digestion, resulting in reproducible ISH reactions . It was used to explore the influence of nuclear truncation in the detection of numerical chromosome aberrations and the detection of tumor cells among stromal and inflammatory cells, to compare the flow cytometric DNA index with chromosome copy number, and to study chromosome heterogeneity within tumors . For this study, a DNA probe for the chromosome region 1q12 was used . Hybridization of model systems with known chromosome numbers, such as sections of paraffin-embedded lymph nodes, paraffin-embedded human peripheral lymphocytes, T24 and Molt-4 cells with two, three, and four chromosomes 1, respectively, showed in at least 50% of the cells the proper number of chromosome hybridization signals in standard 6-microns-thick sections . Depending on the size of the nucleus, a certain percentage of the cells showed lower copy numbers as a result of truncation . In four cases of normal urothelium in paraffin sections, the percentage of nuclei with more than two chromosome spots did not exceed 5% . Comparison of the number of ISH signals, as detected in ethanol-fixed single cell suspensions of 11 TCCs {five flow cytometric (FCM) diploid, three FCM aneuploid, and three FCM tetraploid}, with ISH results obtained in paraffin sections of the same tumors showed that typical numerical chromosome aberrations, such as trisomy and tetrasomy up to nonasomy, could be detected . However, the real chromosome copy number is underestimated, especially in tumors with high copy numbers, as detected in the single cell suspensions of the same tumors . Hybridization of a TCC with extremely large nuclei (DNA index = 3.2) containing six to nine ISH signals as detected in the isolated tumor cells, showed that an indication of these real chromosome copy numbers could be obtained in 6-microns paraffin sections . The accuracy for the detection of the chromosome copy number was even higher in cases where hybridization signals were counted in the mitotic cells . Furthermore, chromosome heterogeneity was detected by ISH using centromeric probes for chromosomes 7, 9, and 18, even though nuclei are truncated in the section . The surplus value of ISH on paraffin sections, as compared with ISH on isolated tumor cells, can be summarized as follows . (a) The focal tumor cell areas with chromosome aberrations can be recognized in the sections and be correlated with the histologic appearance.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroendocrinology, 1991 Jul, 54(1), 7 - 13
Regulated production and secretion of immunoreactive neuropeptide Y by aggregating fetal brain cells in cultures; Barnea A et al.; The aim of this study was to establish a culture system of fetal brain cells that could serve as a model for the study of the developmental regulation of the neuropeptide Y (NPY) neuron . Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation in serum-free medium under constant rotation . Aggregate formation was complete within 24-48 h, and cultures were maintained for up to 23 days . The content of immunoreactive (IR) NPY in the medium and in the aggregates increased progressively with time in culture and at each time point, the medium contained 5- to 10-fold more NPY-IR . A 48-hour exposure to forskolin resulted in a 2-fold increase in the accumulation of NPY-IR in the aggregates and in the medium, indicating that both production and secretion of NPY are regulated by the cAMP intracellular pathway . Sephadex gel filtration revealed the presence of proNPY- and NPY-size substances . The ratio of NPY- to proNPY-size substances increased progressively with age of the aggregates as well as in tissues obtained from perinatal rats of comparable age . Thus, production and secretion of NPY-IR in the cultured aggregates are regulated processes and hence, this culture system can serve as a model to study regulatory processes in the developing NPY neuron.

Arch Biochem Biophys, 1991 Jul, 288(1), 157 - 62
Inhibition of a plant sesquiterpene cyclase by mevinolin; Vogeli U et al.; The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined . Exogenous mevinolin inhibited {14C}acetate, but not {3H}mevalonate incorporation into free sterols . In contrast, mevinolin inhibited the incorporation of both {14C}acetate and {3H}mevalonate into capsidiol, an extracellular sesquiterpene . Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by microM mevinolin, while squalene synthetase was insensitive to even 600 microM mevinolin . Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 microM . Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor . The results suggest that the mevinolin inhibition of {3H}mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.

J Histochem Cytochem, 1991 Jul, 39(7), 905 - 14
Production and characterization of osteoclast-selective monoclonal antibodies that distinguish between multinucleated cells derived from different human tissues; James IE et al.; Osteoclastoma-derived giant cells were used to produce 11 mouse monoclonal antibodies (MAb) reactive against human osteoclasts on undecalcified sections of adult human bone . All exhibited unique reactivities across a wide range of human tissues . Three in particular demonstrated distinctive reactivities; C35 was highly selective for bone osteoclasts, C27 showed selective reactivity for osteoclasts, tissue macrophages and blood-borne monocytes, and C22 showed selective membrane staining of osteoclasts . Consequently, C22 was used to coat Dynabeads to affinity-purify viable human osteoclasts from osteoclastoma-derived cell suspensions . Immunocytochemical staining of inflammatory osteoarthritic synovium/granulation tissue demonstrated positivity in the majority of giant cells with MAb C22 and C27 . In contrast, C35 reacted with only very occasional giant cells . Furthermore, multinucleated cells formed in long-term human bone marrow cultures demonstrated similar selective staining . C27 stained all giant cells and the majority of mononuclear cells . C22 detected only a small proportion of giant cells . In contrast to its staining on bone osteoclasts, C22 demonstrated granular cytoplasmic staining in cultured giant cells . C35 stained no cells at all in these cultures . These MAb can therefore distinguish between giant cells of various origins and authentic mature osteoclasts . Alternatively, they can recognize antigens expressed at different stages of osteoclast differentiation and therefore provide an excellent tool for the study of the human osteoclast lineage.

Kardiologiia, 1991 Jul, 31(7), 56 - 8
{Effect of vasorenal hypertension and neodicoumarin on the rheological properties of erythrocytes and the balance of blood electrolytes, heart and abdominal aorta wall in rats}; Pustovalov AP et al.; It has been shown that with vasorenal hypertension, erythrocyte suspension viscosity coefficient increases, the abdominal aorta transmural potential difference decreases with lower levels of Na+, K+, Ca2+, and Mg2+ in plasma, cardiac and abdominal aorta tissue and higher red blood cell Na+ levels . Neodicumarinum, 3 mg/kg, modified Na+ and K+ imbalance in the red blood cell-plasma-abdominal aorta wall system, which had been caused by vasorenal hypertension . At the same time the changes in Ca2+ and Mg2+ levels in the abdominal aorta and heart tissue, as well as the value of red blood cell suspension viscosity coefficient were demonstrated to be effectively abolished with neodicumarinum in a dose of 30 mg/kg.

J Dermatol, 1991 Jul, 18(7), 377 - 92
Interaction of human epidermal Langerhans cells with HIV-1 viral envelope proteins (gp 120 and gp 160s) involves a receptor-mediated endocytosis independent of the CD4 T4A epitope; Dezutter-Dambuyant C et al.; The CD4 molecule is known to be the preferential receptor for the HIV-1 envelope glycoprotein . Epidermal Langerhans cells are dendritic cells which express several surface antigens, among them CD4 antigens . To clarify the exact role of CD4 molecules in Langerhans cell infection induced by HIV-1, we investigated the possible involvement of the interactions between HIV-1 gp 120 or HIV-1 gp 160s (soluble gp 160) and Langerhans cell surface . We also assessed the expression of CD4 molecules on Langerhans cell membranes dissociated by means of trypsin from their neighbouring keratinocytes . The cellular phenotype was monitored using flow cytometry and quantitative immunoelectron microscopy . We reported that human Langerhans cells can bind the viral envelope proteins (gp 120 or gp 160s), and that this binding does not depend on CD4 protein expression . This binding is not blocked by anti-CD4 monoclonal antibodies . We show that a proportion of gp 120/gp 160s-receptor complexes enters Langerhans cells by a process identified as a receptor-mediated endocytosis . The amount of surface bound gp 120/gp 160s is not consistent with the amount of CD4 antigens present on Langerhans cell membranes . Gp 120/gp 160s binding sites on Langerhans cell suspensions appeared to be trypsin resistant, while CD4 antigens (at least the epitopes known to bind the HIV-1) are trypsin sensitive . A burst of gp 120 receptor expression was detected on 1-day cultured Langerhans cells while CD4 antigens disappeared . These findings lead to the most logical conclusion that binding of gp 120/gp 160s is due to the presence of a Langerhans cell surface molecule different from CD4 antigens.

Atherosclerosis, 1991 Jul, 89(1), 25 - 34
Isolation of cell populations from arterial tissue, using monoclonal antibodies and magnetic microspheres; Mattsson L et al.; In the present study we describe a method for the isolation of cell populations from human and rabbit atherosclerotic tissue, using monoclonal antibodies against cell surface antigen and magnetic microspheres . Atherosclerotic tissue was digested with proteolytic enzymes . The heterogeneous cell suspensions from human tissue were incubated with monoclonal antibodies: anti-Leu-4 (T lymphocytes), anti-Leu-M3 (macrophages) and anti-HLA-DR (HLA-DR expressing cells) . The rabbit aortic cells were incubated with RAM11 (rabbit macrophages) antibody . The rosetting procedure was carried out by mixing antibody treated cells with magnetic monodisperse particles coated with a secondary antibody (goat anti-mouse IgG) . Morphologically, homogeneous foam cell populations were isolated with anti-Leu-M3 and RAM11 . From rabbit atherosclerotic aorta about 2 X 10(5) RAM11 positive cells were recovered/g tissue . The specificity was tested comparing with FACS analysis . A high degree of specificity was obtained while the FACS detected about 30% more cells than were isolated by immune depletion . The lipids of isolated macrophage derived foam cells from rabbit aorta were dominated by cholesterol ester (42%) and smaller amounts of unesterified cholesterol (17%) or triglycerides (3%) . These experiments indicate that immunomagnetic fractionation of cells will be a useful method for studies of the composition and metabolism of different cell populations of atherosclerotic tissue.

Zhonghua Yi Xue Za Zhi, 1991 Jul, 71(7), 362 - 5, 26
{Localization of hepatocellular carcinoma with monoclonal antibodies}; Liu Y; We prepared monoclonal antibodies (MAbs) against hepatocellular carcinoma using cell suspensions isolated from surgical fresh hepatoma specimens as antigen . Totally we got 6 strains of hybridoma cell lines stably secreting MAbs for more than 2 years . Immunocytochemically they stained positively most of the paraffin embedded hepatoma tissues (63.1 to 91.1%) without reaction to the normal liver tissues . Localization of human hepatoma with 125I or 131I labelled MAbs in nude mice was done by IV injection, which showed clear tumor image by ECT radioimmunodetection and autoradiography of tissues . The T/N ratios of different MAbs were 3.1, 3.6, 5.15 and that of HAb 18-F (ab')2 was 14.4 . Among 15 patients suspected to have hepatoma and given the labelled MAb, 13 proved pathologically to be hepatocellular carcinoma.

Bone Marrow Transplant, 1991 Jul, 8(1), 35 - 40
Purging of small cell lung cancer cells from bone marrow using immunomagnetic beads and a flow-through device; Ball ED et al.; Immunomagnetic beads can be used to remove subpopulations of cells from a mixed cell suspension in a flow-through system . One application of this process is the removal of tumor cells from bone marrow prior to its use in autologous bone marrow transplantation (ABMT) . Based on preliminary data showing that three monoclonal antibodies (MoAb) (SCCL 175, HNK-1, and TFS-4) gave optimal separation in small-scale experiments, we have designed a large-scale separator suitable for clinical use . In our separator, the cell suspension flows through a 150 ml baffled transfer pack which is held over an array of permanent magnets . Direct (one MoAb only) and indirect (MoAb and anti-mouse antibody) methods of binding beads to cells were investigated as were the effects of temperature, bead to cell ratio, and medium additives on tumor cell removal and normal cell recovery . We determined the optimal separation conditions to be the indirect method of binding at 22 degrees C using a bead to tumor cell ratio of 25:1 . Testing of the device on DMS-273 small cell lung cancer (SCCL) cells mixed with normal human bone marrow mononuclear cells resulted in a mean tumor cell removal of 3.64 logs (99.977%) with a concomitant mean normal granulocyte-monocyte colony forming unit (CFU-GM) recovery of 61.3% . These experiments form the basis to use the immunomagnetic beads to purify bone marrow from patients with SCCL for use in ABMT.

J Gen Virol, 1991 Jul, 72 ( Pt 7), 1667 - 75
Parameters influencing the attachment of hepatitis A virus to a variety of continuous cell lines; Zajac AJ et al.; We have investigated the interactions of purified radiolabelled hepatitis A virus (HAV) with a variety of continuous cell lines . Virus labelled either in vitro with radiolabelled iodine or in vivo with radiolabelled uridine bound to cells with similar efficiency . Attachment to BS-C-1 cells was calcium ion-dependent and this correlated with infectivity assay results . The cell tropism of HAV attachment was examined using cell suspensions and confluent cell monolayers at both 4 degrees C and 37 degrees C . The maximum level of attachment was observed at 4 degrees C with cells in suspension, but was severely inhibited by 2% foetal calf serum; these results again correlated with infectivity assays . The components of serum which inhibit attachment have been characterized by gel filtration chromatography, sucrose density gradient analysis, immunoprecipitation and Western blotting . The data show that such components are of high Mr and that the serum glycoprotein, alpha 2-macroglobulin, can partly mimic the inhibitory effect of whole serum.

Am J Pathol, 1991 Jul, 139(1), 1 - 6
Detection of herpes simplex virus using the polymerase chain reaction followed by endonuclease cleavage; Rogers BB et al.; The polymerase chain reaction (PCR) was used to amplify herpes simplex virus DNA using a single set of primers that amplify both herpes simplex virus I (HSVI) and II (HSVII) . The viruses can be differentiated by a single restriction enzyme cleavage . Virus from dilutions of HSV-infected A549 cell suspensions were amplified and the infectivity endpoints of cell culture were compared with the PCR, and with another direct detection method, the enzyme-linked immunosorbent assay (ELISA) . The PCR was capable of detecting virus at a 10(-4) dilution for both HSVI and HSVII, when the corresponding TCID50 endpoints were 10(-5.9) and 10(-5.7), respectively . The ELISA detected virus only down to the 10(-1) dilution . The amplification procedure showed the greatest sensitivity when an initial protease digestion was followed by filtration . The PCR may have use in detection of HSV in clinical situations in which a rapid result is desirable.

Cytotechnology, 1991 Jul, 6(3), 209 - 17
Extended expression of liver functions of hepatocytes in collagen-contained cell aggregates (cell packs); Hirai Y et al.; The functions of hepatocytes under the collagen-contained cell aggregate (cell pack) conditions were studied using liver-specific protein synthesis . Freshly isolated murine hepatocytes were suspended in the medium containing collagen and centrifuged, and the resultant cell masses were cultured on the porous membranes floating on the medium . In these cultures cells were attached to each other three-dimensionally with collagen present in the intercellular spaces . Cultured hepatocytes in the cell pack maintained high and stable activity in the expression of their functions for more than 2 weeks, even when cultured with the medium lacking any hormones and serum, whereas hepatocytes in monolayer cultures lost their functions within a week . Similarly, when the cell packs of rat hepatocytes were transplanted into rat spleens, they could retain viability in the form of cell aggregate with the expression of liver-specific albumin mRNA at a higher level than in the transplanted cell suspensions . The lifespan and the initial expression level of hepatocellular functions in culture were similar to that of the cell pack in cell aggregates without collagen and in cellular monolayers on the collagen gel respectively . It was concluded that the condition where cells are in contact with each other has an important role in the expression of hepatocellular functions and collagen present in the intercellular spaces enhances the functional levels.

J Biotechnol, 1991 Jul, 19(2-3), 145 - 57
Dielectric spectroscopy as a novel and convenient tool for the study of the shear sensitivity of plant cells in suspension culture; Markx GH et al.; Plant cell suspensions of different species and different age were subjected to hydrodynamic stress while following the decline in the volume fraction of intact cells by measuring the permittivity of the cell suspension at radio frequencies . Results were compared with the fresh weight, dry weight, packed cell volume and cell number of the suspensions . At first a rapid decline is seen as the most shear-sensitive cells are broken up, followed by a slower decline as less sensitive cells are broken up . The sensitivity of the cells to shear stress depended strongly on the cell line used but only slightly on their age, older cells being more sensitive . The dependence of the shear sensitivity on the cell line might be an effect of the species investigated, the culturing conditions of the cell line, or both . It was found that cells that grow in a finely dispersed suspension are much less prone to shear stress than is often assumed.

Biochim Biophys Acta, 1991 Jun 18, 1065(2), 135 - 44
Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature; Lelkes G et al.; Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value . While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature . In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval . The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins . Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory . Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6 . We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton . We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.

Eur J Pharmacol, 1991 Jun 18, 199(1), 77 - 91
Relation of anion secretory activity to intracellular Ca2+ in response to lysylbradykinin and histamine in a cultured human colonic epithelium; Pickles RJ et al.; A cultured human epithelial cell line, Colony 29, has been used to investigate the relation between anion secretion and intracellular Ca2+ concentration (Cai) in response to the secretagogues, lysylbradykinin (LBk) and histamine . Anion secretion was measured as short-circuit current (SCC) responses in epithelia cultured on previous supports . Cai was measured both in cell suspensions and epithelial monolayers using Fura-2 fluorescence . While it is concluded that raised Cai is responsible for anion secretion the relationship is complex . For both secretagogues there is a receptor reserve, that is the maximal Cai increase is greater than that required to cause a maximal secretory response . By examining the interactions between maximally effective concentrations of LBk and histamine it was shown that neither the SCC nor Cai responses behaved additively . From observations in the absence of external Ca2+ it was concluded that both secretagogues cause Ca2+ release from the same intracellular source, but that in normal conditions Ca2+ derived from intracellular and extracellular sources is responsible for the full effect.

Cancer Res, 1991 Jun 15, 51(12), 3153 - 8
Infiltration and accumulation of precursor cytotoxic T-cells increase with time in progressively growing ocular tumors; Ksander BR et al.; Precursors of cytotoxic T-cells (pTc) infiltrate P815 tumors growing progressively within the immunologically privileged anterior chamber (AC) of BALB/c mouse eyes, but directly cytotoxic T-cells cannot be detected in these eyes . To determine if the failure to reject these tumors is due to a relative inability of tumor-specific pTc to gain access to, or be retained by, the tumor-containing eye, we have assayed through time the frequency of pTc in eyes that received P511 tumor cells in the AC or subconjunctival space (SC; a site where the tumors are rejected) . P511 tumor cells, a hypoxanthine-amethopterin-thymine medium-sensitive derivative of P815 cells, were selected for these studies because P511 tumor cells can be eliminated from in vitro lymphocyte cultures containing hypoxanthine-amethopterin-thymine medium, permitting us to make accurate estimates of pTc frequencies . To ensure that P511 cells are similar biologically and immunologically to P815 tumor cells, we demonstrated that both P511 and P815 cells form progressively growing tumors when injected into the AC of BALB/c eyes and that recipients of both tumor cell lines develop DBA/2-specific anterior chamber-associated immune deviation . Using cell suspensions harvested from eyes of mice bearing AC or SC P511 tumors, we found that tumor-specific pTc appeared first (day 8) in SC tumor-bearing eyes, compared to their appearance in AC tumor-bearing eyes (day 11) . Thereafter, however, the number of pTc detected was significantly greater in eyes bearing progressively growing AC tumors than in SC tumor-injected eyes . The number and frequency of pTc we found in these eyes appeared to correlate directly with the size of the ocular tumor burden . We conclude that failure to reject P511 tumor from the AC can be ascribed neither to a quantitative deficiency in infiltrating tumor-specific pTc nor to an inability to retain pTc at the site . Our findings suggest that immune acceptance of allogeneic ocular tumor grafts may result from failure of infiltrating pTc to differentiate terminally in situ into cytotoxic effector cells.

Echocardiography, 1991 Jul, 8(4), 423 - 33
The effects of the microbubble suspension SH U 454 (Echovist) on ultrasound-induced cell lysis in a rotating tube exposure system; Williams AR et al.; Human erythrocytes resuspended at different hematocrits in autologous plasma at 37 degrees C were exposed to the therapeutic intensities of continuous-wave 0.75-MHz ultrasound in vitro in a rotating tube exposure apparatus designed to maximize the destructive effects of cavitational activity . Provided that large numbers of additional gas bubbles had not been introduced during the various preparative and manipulatory procedures, the addition of Echovist at final concentrations comparable with those currently being used for clinical investigations resulted in a statistically significant increase in the amount of cell lysis in vitro in those samples having hematocrits less than 2% . The amount of cell lysis produced at any given ultrasound intensity decreased with increasing hematocrit in both the controls and the cell suspensions containing Echovist until it was virtually zero in both cases at hematocrits of 5.5% or greater . The addition of Echovist to samples that already contained large numbers of stabilized gas bubbles and/or had hematocrits greater than 5.5% produced no detectable cell lysis even at ultrasonic intensities as high as 3 W/cm 2 spatial average, temporal average (SATA) . It is therefore unlikely that Echovist would cause appreciable amounts of cell lysis when the gas bubbles were being exposed to ultrasound under the conditions used for clinical investigations in vivo.

J Bacteriol, 1991 Jun, 173(11), 3488 - 91
Induction of manganese-superoxide dismutase by membrane-binding drugs in Escherichia coli; Zhang QM et al.; Treatment of exponentially growing cells of Escherichia coli with membrane-binding drugs such as chlorpromazine (CPZ) and procaine resulted in an induction of manganese-superoxide dismutase (Mn-SOD) . A slight decrease was observed in the amount of Fe-SOD . The induction of Mn-SOD required de novo synthesis of this enzyme, since it was suppressed by rifampin . The treatment did not cause the induction of Mn-SOD when performed under anaerobic conditions . In E . coli cells with a sodA-lacZ operon fusion, CPZ and procaine induced beta-galactosidase in the presence of oxygen, whereas it was not expressed and was not induced by CPZ and procaine under anaerobic conditions . Although CPZ reduced the ability of cell suspensions to take up oxygen, it increased the cyanide-resistant fraction of the total respiration . Therefore, it appeared likely that the induction of the sodA gene was a response to an increase in superoxide radical production mediated by these membrane-binding drugs in E . coli cells, possibly by disruption of the electron transport systems in the cell membranes.

J Bacteriol, 1991 Jun, 173(11), 3303 - 10
NarK enhances nitrate uptake and nitrite excretion in Escherichia coli; DeMoss JA et al.; narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium . Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain . Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed . Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present . These results demonstrate that E . coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed . Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake . The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.

Arch Pathol Lab Med, 1991 Jun, 115(6), 558 - 62
DNA ploidy of spindle cell soft-tissue tumors and its relationship to histology and clinical outcome; Agarwal V et al.; With the use of fresh cell suspensions, the DNA ploidy of 11 benign and 27 malignant spindle cell soft-tissue lesions was determined by flow cytometry (37 cases) and/or image cytometry (35 cases) . Of the 27 malignant lesions, 10 were low- or intermediate-grade sarcomas, and 17 were high-grade sarcomas . In the malignant lesions, the DNA ploidy was correlated with the histologic grade and the clinical outcome . Of the 11 benign lesions, six had a diploid DNA ploidy pattern, and five were nondiploid by either flow cytometry or image cytometry . All benign cases had a favorable outocome regardless of ploidy . Eight of the 10 low- or intermediate-grade malignant lesions were diploid, whereas two were nondiploid . Of the 17 high-grade sarcomas, 15 were nondiploid by either method of measurement and nine by both . Of the 10 diploid malignant tumors, only one had an unfavorable outcome (malignant mesothelioma with biopsy only), whereas of the 17 malignant lesions that were nondiploid, five had no evidence of recurrent disease, three cases recurred, eight patients died of disease, and one patient died of an unrelated cause . We concluded that (1) ploidy does not differentiate benign from malignant spindle cell soft-tissue tumors, (2) the nondiploid DNA pattern is more common in high-grade than in low- or intermediate-grade sarcomas, and (3) there is a statistically significant relationship between nondiploidy and a worse clinical outcome in sarcomas.

Circ Res, 1991 Jun, 68(6), 1660 - 8
Ethanol acutely and reversibly suppresses excitation-contraction coupling in cardiac myocytes; Danziger RS et al.; We used adult rat cardiac myocytes to examine the acute effects of 0.1-5.0% (vol/vol) ethanol (ETOH) on 1) the cytosolic {Ca2+} (Cai) transient measured as the change in indo 1 fluorescence at 410/490 nm and contraction elicited by electrical stimulation of single cells and 2) the sarcoplasmic reticulum (SR) Ca2+ content in cell suspensions . During stimulation at 1 Hz, clinically relevant ETOH correlations (0.1-0.15% {vol/vol}) caused a 10-15% decrease in the contraction amplitude, measured by myocyte edge tracking, without decreasing the Cai transient that initiates contraction . At higher ETOH concentrations (1-5% {vol/vol}), ETOH caused profound contractile depression and also reduced the magnitude of the Cai transient . These effects were reversed within minutes of ETOH washout . Addition of norepinephrine (10 microM) to the bathing solution or an increase in bathing {Ca2+} in the continued presence of ETOH could also reverse its effects . The relation of the amplitude of the Cai transient to the contraction amplitude measured across a range of bathing {Ca2+} was shifted by ETOH, such that for a given Cai transient a marked reduction in contraction amplitude occurred . In unstimulated myocyte suspensions, ETOH (1-5% {vol/vol}) caused a concentration-dependent depletion of SR Ca2+ content, manifested as a diminution in the Cai increase elicited by caffeine in the presence of extracellular EGTA and no added Ca2+ . Thus, in rat cardiac myocytes a reduction in the myofilament Ca2+ response, possibly due to a decrease in myofilament Ca2+ sensitivity, is a mechanism for contractile depression due to clinically relevant ETOH concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Biokhimiia, 1991 Jun, 56(6), 1113 - 22
{Lipoxins: study of various biosynthesis pathways}; Sud'ina GF et al.; Lipoxins A4 and B4 were obtained by using soybean lipoxygenase and blood cells as a source of enzymatic activity . The conditions facilitating lipoxin biosynthesis from arachidonic acid catalyzed by soybean 15-lipoxygenase were selected . A comparative analysis of lipoxin biosynthesis with the use of cell suspensions containing only granulocytes and of mixed suspensions (platelets + granulocytes and platelets + total fraction of blood leucocytes) was carried out.

Biol Chem Hoppe Seyler, 1991 Jun, 372(6), 411 - 8
A non-invasive technique for cell volume determination in perfused rat liver; vom Dahl S et al.; 1) In isolated perfused rat liver, the intracellular ({14C}urea-accessible minus {3H}inulin accessible) water space was determined from the washout profiles of simultaneously infused {3H}inulin and {14C}urea . The washout profile of infused {14C}urea was indistinguishable from that of infused tritiated water . During normotonic perfusions and without hormones or amino acids in influent, the intracellular water space was 548 +/- 10 microliters/g liver wet weight (n = 44) . Use of {3H}raffinose instead of {3H}inulin as marker for the extracellular space yielded almost identical values for the intracellular water space (i.e . 98.9 +/- 0.2% of that found with {3H}inulin/{14C}urea) . When volume-regulatory K+ fluxes were completed following hypo- and hypertonic exposure of perfused rat livers and a steady state was reached, the intracellular water space was found to be increased and decreased, respectively . The extent of anisotonic exposure was linearly related to the change of intracellular water space . 2) Anisotonicity-, glutamine- and glycine-induced liver mass changes were almost fully explained by the simultaneously occurring alterations of the intracellular water space, indicating that cell volume changes in perfused rat liver under these conditions are not accompanied by significant changes of the extracellular space . Volume-regulatory K+ (plus accompanying anion) efflux following hypotonic perfusion accounted for about 70-85% of regulatory cell volume decrease, which occurred during the first 10 min of hypotonic exposure . 3) Cell volume of isolated hepatocytes was determined as the "hepatocrit" after gentle centrifugation of the cell suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1991 Jun, 287(2), 372 - 9
Purification and characterization of S-adenosyl-L-methionine: caffeic acid 3-O-methyltransferase from suspension cultures of alfalfa (Medicago sativa L.); Edwards R et al.; Caffeic acid O-methyltransferase (COMT) is one of a group of proteins present in alfalfa cell cultures which can be photoaffinity labeled with S-adenosyl-L-{methyl-3H}methionine . The enzyme was purified to homogeneity from elicitor-treated suspension cultures and shown to exist as an active monomer of subunit Mr 41,000 . COMT could be separated into two forms on the basis of their isoelectric points and relative affinities for S-adenosyl-methionine and S-adenosylhomocysteine . Both forms had equal affinities for caffeic acid, were highly specific for the 3-hydroxyl group of substituted cinnamic acids, and exhibited negligible activity toward flavonoid substrates . An antiserum raised against COMT from aspen immunoprecipitated alfalfa COMT activity . Peptide mapping studies indicated that the two forms of COMT and an isoflavone O-methyltransferase from alfalfa are closely related proteins . The extractable activity of COMT doubled over a 48-h period following exposure of alfalfa cell suspensions to a yeast elicitor preparation, and this was associated with a small change in the relative proportions of the two forms of the enzyme.

Histopathology, 1991 Jun, 18(6), 495 - 504
Bone marrow diagnosis in lymphoproliferative disorders: comparison of results obtained from conventional histomorphology and immunohistology; Thaler J et al.; In this study we have investigated 313 bone marrow biopsies from 280 patients with lymphoproliferative disorders . Trephines were sectioned transversely to obtain one cylinder for cryostat sectioning and immunostaining and a second for histomorphological evaluation using a plastic-embedding technique . The results obtained by histomorphological and immunohistological evaluation were compared for their contribution to staging and classification . Using both techniques, bone marrow involvement was seen in 3/43 (7.0%) biopsies from patients with Hodgkin's disease and in 193/270 (71.5%) cases with non-Hodgkin's lymphoma, including multiple myeloma and acute lymphocytic leukaemia . Immunohistology proved superior in detecting minimal mainly interstitial bone marrow infiltration in 15 leukaemia/lymphoma cases . Biopsies showing infiltration with both methods (n = 157) were re-examined for classification of lymphomatous infiltrates . Whereas immunohistology did not provide additional information in cases with Hodgkin's disease and myeloma, this method was crucial for establishing the definitive diagnosis in a number of cases with acute lymphocytic leukaemia and non-Hodgkin's lymphoma . In all of six leukaemia cases, in which no or inadequate material was available for immunophenotyping of cell suspensions, immunohistology clearly defined the subtype . In the 140 cases of non-Hodgkin's lymphoma the majority of cases (76.4%) were identically classified . In some cases, with important prognostic and therapeutic implications, immunohistology alone provided the definitive diagnosis: T-cell lymphoma (n = 2), hairy cell leukaemia (n = 2) and centrocytic non-Hodgkin's lymphoma (n = 3) . Bone marrow immunohistology is, therefore, an important supplement for classical lymphoma/leukaemia diagnosis . The differences observed between histomorphology and immunohistology emphasize the importance of lymph node biopsy in lymphoma classification.

Plant Mol Biol, 1991 Jun, 16(6), 983 - 98
Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression; van der Zaal EJ et al.; In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells . The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment . In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35 . To further study the regulation of the expression of these genes their 5' regions were translationally fused with the beta-D-glucuronidase reporter gene (GUS) . The GNT1 5' region led to GUS expression only in the root tips of transgenic plants . By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5' regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity . The homology of the 103-like genes with other auxin-regulated genes is evaluated.

Indian J Biochem Biophys, 1991 Jun, 28(3), 219 - 21
Electroporation of red blood cell membrane and its use as a drug carrier system; Mangal PC et al.; Application of external electric field to cell suspension maintained at ice temperature induces pores in the cell membrane . At this stage, a drug added to the cell suspension equilibrates across the membrane . On raising the temperature to 37 degrees C, the pores appear to be sealed as the drug is retained in the cells . This method was used for encapsulation of Co-57 labelled cynocobalamin in rabbit erythrocytes . It was seen from the in vivo biokinetic study of the drug-loaded erythrocytes that the rate of elimination of the drug was considerably reduced as compared to that of the free drug, indicating that drug delivery by electroencapsulation can give a sustained release of the drug.

Cell Biophys, 1991 Jun, 18(3), 203 - 15
Attenuation of spontaneous pseudopod formation in human neutrophils by pentoxifylline; Wong PM et al.; The effects of pentoxifylline (PTX) on spontaneous pseudopod formation in neutrophils in response to the tripeptide formyl-Met-Leu-Phe (fMLP), endotoxin, human complement C5a, and leukotriene B4 (LTB4) were examined in autologous plasma . Unseparated supernatant leukocyte suspensions from fresh heparinized venous human blood were incubated with PTX (0-5 mM) for 25 min and then stimulated for 5-25 min within a range of concentrations of fMLP, endotoxin, complement C5a, and LTB4 . The cell suspensions were fixed with glutaraldehyde and stained with crystal violet in acetic acid; the percentage of neutrophils with pseudopods was determined under high-resolution light microscope . The results show that PTX significantly decreases formation of pseudopods in the presence of all four stimulators . The mechanism of pseudopod suppression appears to be independent of the adenosine receptor . PTX and its analogues, HWA 138 and HWA 448, decreased pseudopod formation by similar amounts when stimulated with 10(-8)M fMLP . These results suggest that PTX may improve microvascular perfusion and attenuate neutrophil-mediated injury by reducing the degree of neutrophil pseudopod formation in free suspension and microvascular entrapment.

J Comp Neurol, 1991 Jun 1, 308(1), 66 - 78
Dopaminergic innervation of substance P-containing striatal neurons by fetal nigral grafts: an ultrastructural double-labeling immunocytochemical study; Mendez I et al.; Evidence for survival and growth of fetal substantia nigra grafts in host striatum and partial reversal of behavioural and biochemical deficits in the host animal is well documented . Afferent synaptic connections arising from the graft and contacting host structures have also been reported; however, the properties of the neurons receiving this input is less clear . The purpose of this study was to determine if substance P-containing neostriatal neurons receive a dopaminergic input from nigral grafts . Fetal substantia nigra cell suspensions were stereotaxically implanted in the deafferented neostriatum of Wistar rats 2 weeks after a unilateral 6-hydroxydopamine (6-OHDA) lesion in the ipsilateral substantia nigra or medial forebrain bundle . The ultrastructural features of the graft-host synaptic interactions were analysed by employing an electron microscope immunocytochemical double-labeling technique . Tyrosine hydroxylase (TH) and substance P-immunoreactive structures were simultaneously demonstrated by means of the peroxidase-antiperoxidase method using two different chromogens with distinct reaction products easily differentiated at the light and electron microscope levels . TH-immunoreactive sites were first demonstrated using 3,3'-diaminobenzidine tetrahydrochloride (DAB); then substance P immunoreactivity was localized using benzidine dihydrochloride (BDHC) . TH-immunoreactive terminals of axons originating from the graft made synaptic contacts with substance P-positive cell bodies and dendrites from the host . These results indicate that at least partial restoration of the normal nigrostriatal circuitry can be achieved following nigral grafts . The demonstration of specific synaptic input on host substance P neurons provides an anatomical basis for direct functional modulation of the deafferented host neostriatum by the nigral graft.

Am J Respir Cell Mol Biol, 1991 Jun, 4(6), 544 - 54
Properties of rat tracheal epithelial cells separated based on expression of cell surface alpha-galactosyl end groups; Randell SH et al.; We used Griffonia (bandeiraea) simplicifolia I (GS I) lectin and flow cytometry to isolate subsets of rat tracheal epithelial cells based on the presence or absence of cell surface alpha-galactosyl end groups . These fractions were designated GS I-positive and -negative, respectively . Ninety-eight percent of the cells in the GS I-positive fraction expressed cell surface alpha-galactosyl end groups; 95% had immunocytochemically detectable keratin 14-related protein (a basal cell marker) and 98% lacked alcian blue-periodic acid-Schiff (AB-PAS)-stained cytoplasmic granules . More than 90% of the GS I-positive cells had a high nuclear-to-cytoplasm ratio, had tonofilaments, and lacked organelles characteristic of other differentiated cell types; they were thus classified as basal cells . In bioassays, the GS I-positive fraction had a colony-forming efficiency greater than or equal to that of native tracheal cell suspensions, and the cells were able to repopulate denuded tracheal grafts with ciliated, secretory, and basal cells . More than 99% of the cells in the GS I-negative fraction lacked cell surface alpha-galactosyl end groups, 98% did not stain for keratin 14-related protein, 54% had significant numbers of AB-PAS-stained cytoplasmic granules, and 16% were identified as ciliated cells . The GS I-negative fraction had a lower colony-forming efficiency than the GS I-positive fraction but, it too, was able to repopulate denuded tracheal grafts with a complete mucociliary epithelium . These results show that both GS I-positive and -negative cells had the potential to proliferate and differentiate into the major tracheal cell types.

Biokhimiia, 1991 Jun, 56(6), 1011 - 7
{Phosphorylation of proteins in collecting tube cells under the effect of vasopressin}; Dzgoev SG et al.; Endogenous phosphorylation of proteins in cell suspensions of collecting tubes was studied . Using SDS disc electrophoresis in polyacrylamide gel with subsequent autoradiography, it was shown that vasopressin increases the 32P incorporation into two proteins with molecular masses of 15 kDa and 33 kDa, which serve as endogenous substrates for cAMP-dependent protein kinase . The hormone-dependent phosphorylation of these proteins was typical of the membrane fraction of collecting tube cells but was absent in the cytosolic fraction . The results obtained are suggestive of the direct involvement of vasopressin in the regulation of membrane protein phosphorylation by cAMP-dependent protein kinase which may increase the permeability of cells for H2O.

J Electron Microsc Tech, 1991 Jun, 18(2), 197 - 202
Ultrastructural localization of gold particles within neural grafts labeled with gold-filled Sendai viral envelopes; Kott JN et al.; The ability to discriminate between host and donor cells is required to interpret the organization of neural grafts at the electron microscopic (EM) level . Using light microscopy, Ardizzoni et al . (Ardizzoni, S.C., Michaels A., and Arendash, G.W . {1988} Science 239:635-637) described a method, using gold-filled Sendai viral envelopes, for labeling cell suspensions prior to grafting . As the colloidal gold used in this procedure is especially attractive for use with EM, we have examined the ultrastructural distribution and character of this label with transplanted cells . Cell suspensions taken from the nucleus basalis of fetal rats were labeled using gold-filled Sendai viral envelopes and grafted into the dorsal neocortex of adult host rats with nucleus basalis lesions . After varying survival times ranging from 1 to 14 months, grafts and surrounding host tissue were examined using standard EM techniques . Within the graft site, gold particles ranging from 10-200 nm were found associated with various membranes throughout the cytoplasm of both neurons and glia . Gold particles of similar size were also found within the nuclei of neuronal and non-neuronal cells . Host cells near the graft site contained some small gold particles (10-40 nm) . Control injections of non-viable, gold-labeled cells or colloidal gold alone resulted in similar patterns of small gold particles which were readily discriminable from the larger virally inserted gold particles found in viable labeled donor cells . We conclude that this method allows discrimination between closely associated host and donor cells.

Mol Pharmacol, 1991 Jun, 39(6), 762 - 70
Calcium signaling and secretory responses in endothelin-stimulated anterior pituitary cells; Stojilkovic SS et al.; Endothelin (ET) receptors are present in pituitary cells and stimulate hormone release through the phosphoinositide/Ca2+ signaling system . In pituitary cell suspensions, ET caused {Ca2+}i elevations of much higher amplitudes than those induced by other vasoactive hormones, including angiotensin II, vasopressin, and noradrenalin . The action of ET was coupled to rapid and transient activation of exocytosis in gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs . In contrast, angiotensin II did not stimulate luteinizing hormone release, and luteinizing hormone responses to vasopressin and noradrenalin were very small . Single gonadotrophs exhibited three types of {Ca2+}i responses to increasing doses of ET, (a) subthreshold responses, with amplitude modulation; (b) threshold-oscillatory responses, with frequency modulation; and (c) threshold-biphasic responses, as the summation of single Ca2+ spikes . The same {Ca2+}i patterns were also seen in gonadotropin-releasing hormone (GnRH)-stimulated cells . In the presence of {Ca2+}e, the amplitudes of the Ca2+ spikes progressively decreased during continuous stimulation with ET or GnRH, reaching the nonoscillatory plateau level after 200-400 sec of stimulation . In cells stimulated with GnRH, subsequent exposure to ET, GnRH, or ionomycin during the plateau phase did not elicit further increases in {Ca2+}i, whereas cells stimulated with ET responded partially to all three agents . In addition, cells exposed to ET or GnRH for 30 min, followed by a 30-min recovery period, were able to mount a full {Ca2+}i response to GnRH, but not to ET-1 . Similarly, both peptides elicited rapid increases in LH release, with comparable potencies, but the response to ET decreased much more rapidly during sustained stimulation and gonadotrophs became refractory to further ET stimulation . This is in part attributable to rapid endocytosis of ET receptors during continuous agonist stimulation . These data indicate that ET exerts potent but transient secretory actions in several pituitary cell types and is a potential regulator of gonadotropin release . The initial receptor-coupling events in both ET- and GnRH-stimulated cells are similar, but the differences observed during continuous or repetitive stimulation indicate that the ET receptor pathway undergoes rapid desensitization that is critical in determining the distinct cellular responses to the two peptides.

Blood, 1991 Jun 1, 77(11), 2379 - 88
Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: the activity of cathepsin G is not prevented by antiproteinases; Evangelista V et al.; Human polymorphonuclear leukocytes (PMN) activated by fMLP (in the presence of CaCl2, fibrinogen, and cytochalasin B) were able to induce aggregation, cytoplasmic Ca2+ increase, and thromboxane A2 production in coincubated autologousplatelets . Cell-free supernatants prepared from n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN were able also to induce platelet activation . Antibodies against cathepsin G and different serin protease inhibitors completely suppressed the activity of PMN-derived supernatants, indicating that cathepsin G is the major platelet activator released by PMN in our system . However, antiproteinases only partially affected platelet activation induced by PMN in mixed cell suspensions . Superoxide dismutase and catalase added to the cell suspension did not affect platelet activation nor potentiated serin protease inhibitors, making a role for short-lived oxygen radicals in our experimental system unlikely . Electron microscopic observation of stirred mixed cell suspensions preincubated for 2 minutes at 37 degrees C before stimulation showed a close PMN-platelets contact without any morphologic or biochemical event suggesting platelet activation . Preincubation of the cells without stirring to minimize PMN-platelet interaction before stimulation did not modify subsequent aggregation and platelet cytoplasmic Ca2+ increase in control samples . However, in this condition trypsin inhibitor from soybean completely prevented PMN-induced platelet activation . In samples preincubated without stirring in the presence of the antiproteinase, activated PMN stuck together but platelets preserved their discoid shape and did not appear significantly activated . We propose that membrane-to-membrane contact could create a microenvironment in which cathepsin G, discharged from stimulated PMN on adherent platelets, is protected from antiproteinases.

Endocrinology, 1991 Jun, 128(6), 3005 - 12
Divalent cations modulate PTH-dependent 3',5'-cyclic adenosine monophosphate production in renal proximal tubular cells; Mathias RS et al.; The kidney and parathyroid gland play key roles in calcium (Ca++) homeostasis . Recent data suggest that the kidney, in addition to being a primary target for PTH, also recognizes changes in the concentration of extracellular Ca++, thereby modulating hormone-dependent cAMP production, 1,25-dihydroxyvitamin D synthesis, and renin secretion . In this study, we examined: 1) the effects of varying concentration of divalent cations on PTH-dependent cAMP production in renal proximal tubular cells; and 2) the mechanisms by which extracellular Ca++ exerts its inhibitory effects on cAMP production . Single cell suspensions composed of 80-90% proximal tubular cells were prepared from cortical homogenates by collagenase digestion and sieving . In the presence of 1 mM isobutylmethylxanthine, cAMP content was measured by RIA in 5-15 min incubations and showed a 5- to 6-fold increase in response to PTH (10(-11) -10(-6) M) . Increasing extracellular Ca++ and magnesium (Mg++) from 0 and 0.5 mM, respectively, to 5.0 mM inhibited PTH-dependent (3 x 10(-9) M) cAMP production by 54 +/- 4% and 47 +/- 6%, respectively . The half maximal inhibitory concentration for both Ca++ and Mg++ was 0.9 mM . In addition, increasing extracellular barium (Ba++) or strontium (Sr++) from 0-10 mM inhibited PTH-dependent (3 x 10(-9) M) production by 54 +/- 7% and 62 +/- 6% with half of the maximal observed inhibition at 2.2 and 2.7 mM, respectively . The inhibition of PTH-dependent cAMP production by 2.5 mM Ca++ was not reversed by the calcium channel blockers diltiazem or verapamil (10(-4) M) . However, changes in intracellular calcium may play some role in the inhibitory effects of Ca++ on cAMP production, since ionomycin (10(-6)-10(-5) M) lowered PTH-dependent cAMP production by 25-36% . Our data suggest that the proximal tubular cell can sense physiologically relevant changes in Ca++, providing a potential mechanism for the modulation of 1,25-dihydroxyvitamin D production or other tubular functions relevant to fluid and mineral homeostasis.

J Comp Neurol, 1991 May 22, 307(4), 695 - 706
Intracerebral grafting of cultured autologous skin fibroblasts into the rat striatum: an assessment of graft size and ultrastructure; Kawaja MD et al.; To identify a suitable donor cell population for gene therapy applications to the central nervous system, primary fibroblasts isolated from skin biopsies and maintained in culture are employed as autologous cells for intracerebral grafting within the adult rat striatum . Results from the present investigation reveal that cultured primary skin fibroblasts cease to proliferate once they reach confluence; these cells are thus contact inhibited in vitro . Following implantation within the striatum, the volume of the primary fibroblast grafts, stained immunohistochemically for fibronectin, does not differ significantly at 3 and 8 weeks . The graft size is dependent on the density of the cell suspension, but not dependent on either the number of passages the cells are taken through in culture prior to grafting or on the postoperative survival period . Ultrastructural evidence reveals that at 8 weeks the grafts are composed primarily of collagen and fibroblasts with rough endoplasmic reticulum and vesicles . Reactive astrocytic processes and phagocytic cells are also present in the grafts . The grafts are extensively vascularized with capillaries composed of nonfenestrated endothelium; intercellular junctions are evident at sites of apposition between endothelial cells . It is concluded that primary skin fibroblasts are able to survive for at least 8 weeks following intracerebral implantation and continue to synthesize collagen and fibronectin in vivo . Also, the grafts maintain a constant volume between 3 and 8 weeks, thereby indicating that primary skin fibroblasts do not produce tumors . Finally, dynamic host-to-graft interactions--including phagocytic migration, astrocytic hypertrophy and infiltration within the grafts, and angiogenesis--are features that constitute the structural integration of primary skin fibroblasts grafted within the adult rat central nervous system.

J Immunol Methods, 1991 May 17, 139(1), 25 - 30
Identification and quantification of human peripheral blood lymphocytes and monocytes following migration into nitrocellulose filters; Schell-Frederick E et al.; Previously, in order to evaluate the migration of specific types of lymphocytes (e.g., T or B cells, CD4 or CD8 subsets) using the Boyden chamber technique, the relevant cell populations have first been purified . We report here a method which permits identification of the lymphocytes following migration into the nitrocellulose filters . After fixation, the filters are exposed to specific anti-lymphocyte monoclonal antibodies and the reaction is visualized via a second gold-linked antibody . Monocyte-depleted lymphocytes isolated from human peripheral blood are routinely used for migration but mixed mononuclear cell preparations can also be used . This was shown by comparing lymphocyte migration in monocyte-containing and monocyte-depleted cell suspensions isolated from the same donor . The presence of monocytes did not influence the migration of normal resting T lymphocytes . With human peripheral blood as starting material the method is most suitable for the evaluation of T cell migration since, in most instances, T cells are the predominant lymphocyte population . When the B cell count is normal (5-10% total lymphocytes), quantification of B cell migration requires enrichment but not complete purification of this population . Migrating monocytes can also be identified . However, the antibody staining is less intense on the spread monocytes and, therefore, quantification must be performed by eye rather than with the Optomax image analyser.

Cancer Res, 1991 May 15, 51(10), 2649 - 54
Biological and antitumor effects of recombinant human macrophage colony-stimulating factor in mice; Bock SN et al.; The administration of recombinant human macrophage colony-stimulating factor (M-CSF) i.p . in doses of 25 or 100 micrograms twice daily for 5 consecutive days to non-tumor-bearing C57BL/6 mice resulted in a dose-dependent infiltration of mononuclear cells in the livers but not the lungs of these treated animals . Immunohistochemical examination of fixed liver tissue with the murine macrophage-specific monoclonal antibody, F4/80, revealed a greater than 5-fold increase in the number of hepatic macrophages . Quantification of F4/80-positive cells in a mononuclear single cell suspension derived from liver revealed a greater than 25-fold expansion in the number of hepatic macrophages compared to control mice . These cells were then tested in 18-h 51Cr release assays for tumoricidal activity, after an 18-h incubation with or without gamma-interferon, against cultured P815 targets . Significant tumor cell lysis by these liver-associated mononuclear cells occurred, which was enhanced by gamma-interferon preincubation . The systemic administration of M-CSF alone at high dose had no antitumor impact in vivo against 3-day pulmonary metastases from the MCA-203 sarcoma and B16 melanoma or hepatic metastases from the B16 melanoma or MCA-105, -203, or -207 sarcomas . Although the systemic administration of M-CSF in combination with tumor-specific monoclonal antibody had no effect on 3-day pulmonary metastases from the B16 melanoma, significant reductions in liver metastases were seen . These murine studies demonstrate the biological activity of recombinant human M-CSF in vivo and suggest that the administration of this cytokine in combination with specific monoclonal antibody may be useful in the treatment of patients with metastatic disease at sites of monocyte/macrophage accumulation.

In Vitro Cell Dev Biol, 1991 May, 27A(5), 369 - 80
Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro; Messiaen L et al.; The lens of the eye is one of the rare organs in which tumors do not occur spontaneously . It therefore appeared to us that lens cells would not present the background of spontaneous transformation toward malignancy found with many other cell cultures . We have cultured C3H/HeA mouse lens explant (MLE) cells for 70 wk and analyzed changes in malignancy-related phenotypes in function of the number of passages . In vitro, we studied morphology, colony forming efficiency on tissue culture plastic substrate (CFEtc) and in soft agar, population doubling time, saturation density, and invasiveness into precultured chick heart fragments . In vivo, tumorigenicity, invasion, and metastasis were analyzed after injection of cell suspensions subcutaneously and intraperitoneally, after implantation of cells aggregated to collagen sponges under the renal capsule and after implantation of cell aggregates subcutaneously into the tail and into the pinna . The CFEtc, population doubling time, and saturation density increased as the number of passages of culture in vitro increased, but colony formation in soft agar was never observed . MLE cells till passage 16 were not invasive in vitro, but hereafter consistently were found to be invasive . After about 17 passages, corresponding to 25 wk of culture, MLE cells acquired the capacity to form tumors in syngeneic mice . These tumors were invasive but metastases were not observed . We concluded that MLE cells acquired in an apparently spontaneous way a number of malignancy-related phenotypes, without, however, reaching the stage of metastasis.

Scand J Immunol, 1991 May, 33(5), 579 - 84
Effect of the in vitro ageing on the restriction of sheep erythrocyte lysis by horse serum; Assis AI et al.; Fresh sheep erythrocytes are resistant to lysis by horse serum but gradually became susceptible to this source of complement after storage (ageing) in the blood at 4 degrees C . This occurs faster when the fresh cells are stored in isotonic buffer . The supernatant of the buffer/red cell suspension stored at 4 degrees C for 10-15 days restrict lysis by horse serum when incubated back at 37 degrees C or 43 degrees C with the 'aged' sheep cells or fresh guinea pig red cells . In assays using fresh guinea pig erythrocytes this effect is described by reincubation of the cells in buffer, and is specific to horse serum when compared to human serum.

Lab Invest, 1991 May, 64(5), 664 - 74
The role of Kupffer cells in glucan-induced granuloma formation in the liver of mice depleted of blood monocytes by administration of strontium-89; Naito M et al.; In order to elucidate the role of Kupffer cells in granuloma formation in the liver of mice under a condition of severe monocytopenia induced by administration of strontium-89, granulomas were produced by particulate glucan injection and examined histopathologically, immunohistochemically, by {3H}thymidine autoradiography, and in culture experiments . Hepatic granulomas were smaller, less numerous, and more irregularly shaped in the monocytopenic mice than in the control mice . The granulomas were composed of multinuclear giant cells, epithelioid cells, Kupffer cells, and T lymphocytes, but not monocytes or granulocytes . Kupffer cells were heavily labeled with {3H}thymidine in the monocytopenic mice, particularly just before the stage of granuloma formation, and then clustered in the liver sinusoids . At 8 days, they formed granulomas, transformed into epithelioid cells, and transformed further into multinuclear giant cells . Although the culture of liver cell suspensions prepared from the livers of monocytopenic mice sustained diffuse proliferation of macrophages on a monolayer of mouse stromal cell line (ST2), no monocyte/macrophage colonies were formed . From these results, it is reasonable to conclude that Kupffer cells alone are activated in a condition without a supply of monocytes from peripheral blood; proliferate and cluster in the hepatic sinusoids; transform into peroxidase-negative macrophages, epithelioid cells, and multinuclear giant cells; and participate in granuloma formation in loco together with T lymphocytes.

J Exp Med, 1991 May 1, 173(5), 1039 - 46
Reentry of T cells to the adult thymus is restricted to activated T cells; Agus DB et al.; To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions . With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells . By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers . Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells . Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer . Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts . Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus . These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase . The physiological significance of blast cell entry to the thymus is unclear . The possibility that these cells participate in intrathymic tolerance induction is discussed.

Tohoku J Exp Med, 1991 May, 164(1), 51 - 8
Simultaneous analysis of the patterns of nuclear DNA and cell protein contents in pancreatic carcinoma with reference to prognosis; Kakizaki K et al.; Simultaneous analysis of the patterns of nuclear DNA and cell protein contents was performed in 13 patients with pancreatic carcinoma to evaluate their prognostic significance . The patients were divided into two groups according to clinical outcome . Namely, six died of the primary disease within one year (group A), and six survived longer than 16 months and one died of other cause (liver abscess) at 9 months after operation (group B) . The cell suspensions prepared from paraffin-embedded specimens were stained with diamidino-phenylindole and hematoporphyrin for nuclear DNA and cell protein, respectively . Fluorescence intensities of 20 lymphocytes as control and 200 carcinoma cells of each case were measured . The nuclear DNA pattern was mainly diploid or tetraploid in group B . Group A showed aneuploid and polyploid patterns . Furthermore, mean DNA content of group A was significantly higher than that of group B . Cell protein content was widely scattered and higher in group A than group B . Analysis of nuclear DNA ploidy, relative value of DNA and cell protein content may be a reliable tool to predict prognosis of patients with carcinoma of the pancreas.

Nippon Hifuka Gakkai Zasshi, 1991 May, 101(6), 609 - 13
{Inhibitory effect of arbutin on melanogenesis--biochemical study using cultured B16 melanoma cells}; Akiu S et al.; Inhibitory effect of arbutin (hydroquinone-beta-D-glucopyranoside) on the melanogenesis was studied biochemically using cultured B16 melanoma cells . The maximum arbutin concentration lacking an inhibitory effect on cell growth was 5 X 10(-5) M . At this concentration, melanin content per cell was decreased significantly to about 39%, compared with that of arbutin untreated cells . Also, tyrosinase activity of arbutin treated cells was decreased significantly . When arbutin was added to B16 melanoma cell suspension, arbutin was not hydrolyzed to liberate hydroquinone . Further, tyrosinase activity in crude preparations from B16 melanoma cells was inhibited by arbutin . From these results, it is suggested that arbutin can inhibit the melanogenesis by affecting not only the synthesis but also the activity of tyrosinase rather than by killing melanocytes B16 melanoma cells . Also, it is suggested that hydroquinone is not responsible for the inhibitory effect of arbutin on the melanogenesis.

Int J Hyperthermia, 1991 May-Jun, 7(3), 499 - 510
Cytotoxic effect of 1,3 bis (2-chloroethyl)-N-nitrosourea at elevated temperatures: Arrhenius plot analysis and tumour response; Urano M et al.; The effect of hyperthermia on the cytotoxicity of 1,3-bis-(2-chloroethyl)-N-nitrosourea (BCNU) was investigated in vitro and in vivo . Tumour cells were early-generation isotransplants of a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II . For in vitro studies, single cell suspensions containing 1.0 x 10(6) cells/ml were treated in a water bath where a desired temperature was maintained by a constant-temperature circulator . Cell survival was determined by lung colony assay immediately thereafter . For in vivo studies the tumour cell suspensions were transplanted into the dorsal site of the C3Hf/Sed mouse foot . Tumours were treated by immersing animal feet into a constant-temperature water bath when tumours reached an average diameter of 4 mm (35 mm3) . The tumour growth (TG) time or the time for one-half of the treated tumours to reach 1000 mm3 from initial treatment day was used as an endpoint . BCNU dose-cell survival curve at 37 degrees C was exponential with a D0 of 1.1 microgram/ml . Dose-cell survival curves at 37-43 degrees C were determined as a function of treatment time at pH 6.7 and 7.4 . BCNU of 1 microgram/ml was added immediately before treatment . The slope of the survival curve became steeper with increasing temperature, indicating that the cytotoxic effect of BCNU was enhanced by hyperthermia . The Arrhenius plot analysis showed that activation energies at pH 6.7 and 7.4 were 53 and 51 kcal/M, respectively (no significant difference) . Of interest in this analysis was that the Arrhenius plot did not show a breaking point which has been observed for other agents . Further investigation demonstrated that the decomposition of BCNU, which has been reported to be essential for production of reactive intermediates, occurred in aqueous medium at elevated temperatures . The magnitude of this decomposition depended on treatment temperature . As a result, preheated BCNU became less cytotoxic with an increase in preheating temperatures . The activation energy for this decomposition was about one-half of the activation energy for BCNU cytotoxicity . Studies in vivo indicated that the effect of BCNU was enhanced with increasing temperatures, and the enhancement was greatest when BCNU was administered i.p . immediately before hyperthermia . A glucose dose of 5 g/kg administered i.p . 60 min before hyperthermia further enhanced the antitumour effect of BCNU.

Agents Actions, 1991 May, 33(1-2), 33 - 6
The role of mast cells in redistribution of tele-methylhistamine in the body; Florjanc TI et al.; The basis of this study was the assumption that mast cells can take up tele-methylhistamine (MeHi), as is known for other biogenic amines . The influence of extracellular MeHi on its uptake (active or passive) in isolated rat mast cells and in different rat tissues was studied in vitro . Rat peritoneal mast cells were incubated for 15 or 30 min at 37 or 1 degree C with different concentrations of MeHi . Submandibular gland, skeletal muscle, lung and heart of the rat (200-500 mg of each) were chopped and incubated for 20 min at 37 or 1 degrees C in buffer containing MeHi . Histamine (Hi) and MeHi in the cells and in the tissues were then determined by HPLC assay . Mast cells were capable of taking up MeHi in a time- and dose-dependent manner . MeHi levels increased from 5-10 ng per ml of incubated cell suspension (control) to 60-100 ng/ml after 15 min incubation . It can be assumed that the accumulation of MeHi in mast cells is due to a simple diffusion process since no significant change was noticed at 1 degree C . The preincubation of the cells with serotonin reduced MeHi accumulation, thus indicating MeHi competes with the same binding sites in mast cells as Hi and serotonin . Tissues showed high capacity for MeHi accumulation and MeHi surmounted endogenous Hi levels . Uptake was reduced at 1 degree C, yet the accumulation of MeHi was still high . The results indicate that mast cells can take up a smaller portion of free MeHi and they can have a function in its micro-regulation whereas other tissue cells have a predominant role in the removal of free MeHi from the blood.

Res Vet Sci, 1991 May, 50(3), 308 - 10
Cross-reactivity between Actinobacillus pleuropneumoniae serotypes comparing different antigens and serological tests; Gutierrez CB et al.; Several antigens were prepared from suspensions of reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae (saline extract {SE}, capsular extract {CE}, whole cell suspension {WCS}, boiled extract {BE}, and autoclaved cell antigen {ACA}) . The cross reactions between each antigen and the antisera against reference strains of the other 11 serotypes were compared by using the complement fixation test, ELISA and the indirect haemagglutination test . ACA produced the most cross reactions, which, in some serotypes, took place in all the antisera tested . BE produced fewer cross reactions, but these were more abundant than those obtained with SE, CE or WCS . The least cross reactivity occurred with SE in the indirect haemagglutination test . This test is, therefore, the most reliable method for serotyping field strains of A pleuropneumoniae.

Photochem Photobiol, 1991 May, 53(5), 633 - 7
Psoralen-photosensitized damage of rat peritoneal exudate cells; Kyagova AA et al.; Psoralen-sensitized photodamage (PUVA) of rat peritoneal exudate cells was investigated . Quartz-activated luminol-dependent chemiluminescence (ChL) was registered and the amount of trypan-positive cells was determined . Irradiation of peritoneal exudate cells in the presence of psoralen resulted in a dose-dependent monotonous inhibition of ChL . The reciprocity law of irradiation intensity and duration of irradiation was not valid for the observed inhibition of ChL: the inhibition increased with higher intensity . When psoralen previously photooxidized in ethanol (POP) was added to peritoneal exudate cell suspension, a double-phase response depending on psoralen irradiation dose was obtained: ChL activation was observed at low doses of UVA, ChL inhibition at high doses . Chemiluminescence inhibition correlated well with the increase in the number of trypan-positive cells . It may be supposed that the observed effects of PUVA or POP treatment are caused by cell cytoplasmic membrane damage.

Anal Chem, 1991 May 1, 63(9), 890 - 3
Determination of boron in tissues and cells using direct-current plasma atomic emission spectroscopy; Barth RF et al.; We have developed a safe, simple, and efficient method for boron determination by means of direct-current plasma atomic emission spectroscopy . Tissues were solubilized by using concentrated sulfuric acid and 70% hydrogen peroxide to digest the samples without the need of high temperatures and pressures . Boron cluster compounds could be measured with sensitivity, precision, and accuracy similar to those of boric acid standards . Results obtained with {(C2H5)3NH}2B12H12, Cs2B12H11SH.H2O, and C15H32B10O6 show that this analytical method is applicable to a variety of compounds with different chemical structures . A sensitivity of 0.1 ppm has been obtained with known standards alone and in a variety of tissue matrices including tumor, blood, liver, skin, and cell suspensions . The measurement of total boron by direct-current plasma atomic emission spectroscopy (DCP-AES) has been achieved with as little as 50 mg of tissue or as few as 5 x 10(7) cells . The procedure is applicable to the analysis of boron in the ppm range with a high degree of precision and accuracy.

Poult Sci, 1991 May, 70(5), 1261 - 4
Developmental ability of twin embryos produced by microinjection treatment into chick blastoderm; Naito M et al.; Twins and triplets of chick embryos were produced by microinjection treatment of cell suspension into the blastoderm . One pair of the twin embryos had an ability to develop normally and survived up to Day 21 of incubation, but did not hatch . The other twins and triplets died within 7 days.

J Surg Res, 1991 May, 50(5), 480 - 4
Anti-CD3-enhanced interleukin-2 immunotherapy of pulmonary metastases; Kim B et al.; A newly developed monoclonal rat IgG 2b antibody which in vitro can activate both helper and cytolytic T-lymphocytes by binding to the CD3 epsilon subunit of the T-cell receptor complex was tested alone and in combination with Interleukin-2, a growth factor for activated T-cells, for ability to reduce established pulmonary metastases in a murine model . C57BL/6 mice injected iv with a tumor cell suspension of a weakly immunogenic fibrosarcoma, MCA106, were randomly assigned to 1 of 15 treatment groups for intraperitoneal injections with YCD3 (0, 0.1, 1, 10, or 100 micrograms) on Days 3, 5, 7, 10, 12, 17, and 19 or with IL-2 (0, 1000, or 50,000 units bid) on Days 3 through 7, 10 through 12, and 17 through 19 . On Day 21 all mice were sacrificed for enumeration of metastases . Pooled splenocytes of three randomly selected mice from each group were assayed for surface expressions of T-cell markers Thy-1, Ly2, and L3T4 . Results: High-dose IL-2 (50,000 units bid) in combination with low-dose YCD3 (1 microgram) reduced metastases 60% (P less than 0.005) . YCD3 or IL-2 alone was ineffective . Combined high-dose IL-2 (50,000 units) and high-dose YCD3 (100 micrograms) resulted in 100% mortality . Phenotypically, YCD3 induced a dose-dependent depletion of T-cells from 25 to 2.4% (0.1 to 100 micrograms, respectively) . These results suggest potential clinical applicability of low-dose anti-CD3 monoclonal antibody to enhance antitumor efficacy of high-dose IL-2 . However, the toxicity of high-dose anti-CD3 and high-dose IL-2 cautions for care in selection of dose.

J Neurochem, 1991 May, 56(5), 1587 - 93
Characterisation of distinct inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive calcium stores in digitonin-permeabilised adrenal chromaffin cells; Robinson IM et al.; The effect of inositol 1,4,5-trisphosphate {Ins-(1,4,5)P3} and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor {Ca2+} . Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in {Ca2+} in the cell suspension . Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine . The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated {Ca2+} . The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores . Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine . The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses . These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.

Agents Actions, 1991 May, 33(1-2), 8 - 12
A novel source of mast cells: the human placenta; Purcell WM et al.; The presence of moderate amounts of histamine in the human placenta was confirmed (0.72 +/- 0.10 microgram/g wet weight), and the hitherto unknown storage site of this biogenic amine was elucidated . Mast cells were identified by their characteristic morphology, staining reactions and secretory activity measured in terms of histamine release . Human placental tissue contains 7.6 x 10(5) mast cells/g wet weight, identified by staining with toluidine blue or alcian blue, and these cells were positive for chloro-acetate-esterase . Light microscope studies of placental tissue stained with HRP-conjugated anti-human IgE demonstrated cells with a typical 'halo' effect indicating cell-bound IgE, and electron microscopy revealed cells containing membrane-bound electron dense granules . A single mast cell was calculated to contain approximately 1 pg of histamine . Enzymatic digestion of placental tissue with collagenase (1.5 mg/ml) yielded viable cell suspension . containing mast cells in a purity of 0.6% which exhibited a low spontaneous output of histamine (12%) . Placental mast cells released histamine in a concentration dependent manner upon challenge with anti-human IgE and the calcium ionophore A23187 . Also, unlike other human mast cells so far studied . with the exception of skin, those dispersed from human placenta were responsive to the polybasic secretagogue compound 48/80 . These findings represent a novel source of human mast cells and, since placentas are readily available in quantity, such tissue is proposed as an ideal source of mast cells for biochemical and pharmacological use.

Agents Actions, 1991 May, 33(1-2), 26 - 9
In vitro and in vivo studies of radiographic contrast media-induced histamine release in pigs; Ennis M et al.; Routine clinical use of radiographic contrast media (RCM) causes adverse reactions in some patients . To elucidate the mechanisms of these reactions both in vitro and in vivo studies are necessary . In this study, RCM-induced histamine release from isolated mast cells was compared with the in vivo release of histamine and cardiovascular symptoms using a porcine model . The 2 non-ionic preparations examined (Solutrast and Ultravist) released little or no histamine from the 4 cell types tested (porcine pulmonary, cardiac, hepatic, and renal mast cells) . The 4 ionic preparations (Angiographin, Hexabrix Rayvist, and Telebrix) caused histamine release from most of the cell suspensions . In almost all cases, the cardiac mast cells were the most sensitive followed by the hepatic mast cells . All 4 RCM tested in vivo produced elevated plasma histamine levels in some animals . The highest incidence was observed using the ionic, high osmolal Rayvist (6 of 12 animals), followed by the non-ionic RCM with the lowest osmolality Ultravist (4 of 12 animals) . In vivo, mechanisms in addition to direct histamine release may also be involved in RCM-induced adverse reactions, since low osmolal, non-ionic RCM can cause elevated plasma histamine levels without in vitro release . The susceptibility of cardiac mast cells to RCM-induced histamine release suggests that patients undergoing e.g . coronary angiography may be especially at risk for an adverse reaction.

Agents Actions, 1991 May, 33(1-2), 20 - 2
Histamine release induced by opioid analgesics: a comparative study using porcine mast cells; Ennis M et al.; Studies in vitro have demonstrated histamine release induced by opioid analgesics from rat peritoneal mast cells and human skin mast cells . In humans, elevated plasma histamine levels have also been found following intravenous or oral administration . This study compared the reactivity of five opioid analgesics on mast cell suspensions obtained from porcine heart, kidneys, liver and lungs . The five compounds investigated were hydromorphone, levomethadone, morphine, pethidine and oxycodone . Both morphine and hydromorphone produced little histamine release from all cell types tested . The other drugs demonstrated clear differences between the different cell populations . Thus, the cardiac cells responded most strongly to oxycodone but were the least responsive to pethidine . These differences, if confirmed in vivo, could indicate which drugs may be more suitable for patients with different underlying disease states.

Anal Biochem, 1991 May 1, 194(2), 316 - 25
Simultaneous determination of intracellular calcium concentration and histamine secretion in rat basophilic leukemia cells (RBL-2H3); Maeyama K et al.; In rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mast cells, the aggregation of IgE receptors initiates increase in the intracellular concentration of calcium ({Ca2+}i), monitored with the fluorescent Ca probe fura-2, and finally results in histamine secretion . In cell suspensions, however, the fluorescence gradually increases due to leakage and exocytosis of the dye . A superfusion system was developed to overcome these problems and {Ca2+}i was calculated from the ratio of fluorescence intensities at 505 nm of fura-2 excited at 340 and 380 nm . Histamine and beta-N-acetylglucosaminidase in granules are released during exocytosis, and both substances in the superfusates were determined simultaneously . This system is useful for studies on the relationships of cell stimulation, changes in second messengers, and final responses.

J Protozool, 1991 May-Jun, 38(3), 229 - 33
Effects of osmotic pressure on the oxidative metabolism of Leishmania major promastigotes; Blum JJ; Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer . The rate of oxidation of 14C-labeled substrates was then measured as a function of osmolality . An acute decrease in osmolality (achieved by adding H2O to the cell suspension) caused an increase in the rates of 14CO2 production from {6-14C}glucose and, to a lesser extent, from {1,(3)-14C}glycerol . An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of 14CO2 production from {1-14C}alanine,{1-14C}glutamate, and {1,(3)-14C}glycerol . The rates of 14CO2 formation from {1-14C}laurate,{1-14C}acetate, and {2-14C}glucose (all of which form {1-14C}acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality . These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress . Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.

Int J Radiat Biol, 1991 May, 59(5), 1207 - 16
Repair of potentially lethal damage in rat mammary clonogens following irradiation in organoid culture; Kamiya K et al.; In the course of investigations of radiogenic neoplasia in mammary cells in vivo, post-irradiation repair of potentially lethal damage (PLDR) was previously observed in mammary epithelial clonogens when they were irradiated and left in their tissue environment for 4-24 h after exposure . This type of PLDR increases the initial shoulder of the survival curve without significantly affecting its terminal slope . It has since been described in similarly treated thyroid and hepatic clonogens, and significantly exceeds that generally seen in most mammalian cells in culture . In this study, a ten-fold increase in the concentration of mammary clonogens which could be assayed was achieved by the isolation and short term culture (2-4 days) of mammary organoids (ductal and end-bud fragments) . Assays of clonogen concentrations were carried out with cell suspensions prepared immediately following irradiation of 4-day organoid cultures or 24 h after irradiation of such organoid cultures . A near four-fold increase in clonogen survival was observed with the delay in assay after irradiation; this is comparable to the PLDR seen in mammary clonogens irradiated and left in situ for 4-24 h after exposure.

Chem Pharm Bull (Tokyo), 1991 May, 39(5), 1352 - 4
Formation of superoxide by benzofurazans in Escherichia coli under aerobic incubation; Takabatake T et al.; The superoxide (O2-.) production in Escherichia coli through the action of benzofurazans (BZs) was examined using the cytochrome c (cyt . c) reduction method . Adding BZs to E . coli cell suspensions caused the cyt . c reduction, which was completely inhibited by superoxide dismutase (SOD) . The effects of BZs on cyt . c reduction was in the order of benzofurazan (1) approximately 4,7-dimethylbenzofurazan (2) approximately 4,7-dibromobenzofurazan (3) less than 4-bromo-6-cyanobenzofurazan (4) less than 4,7-dicyanobenzofurazan (5) . This was correlated with the toxicity of BZs against E . coli growth (1 approximately 2 approximately 3 less than 4 less than 5) and with the redox potentials of BZs (1 approximately 2 less than 3 less than 4 less than 5) . The formation of compound 5 anion radical in the cell suspensions in the absence of dioxygen (O2), was determined using ESR spectrum . The ESR signal of the anion radical disappeared with the addition of O2 . The BZs effected the O2- . production in E . coli cells.

J Endocrinol, 1991 May, 129(2), 291 - 9
Thyrotrophin, but not forskolin, increases intracellular free calcium and calmodulin in pig thyroid cells; Ye QL et al.; The effects of TSH and forskolin were examined on intracellular free calcium ({Ca2+}i) and calmodulin in normal pig thyroid cells in culture . TSH was found to produce acute increases in {Ca2+}i in pig cells . Responses were seen at concentrations of TSH between 0.01 and 10 mU/ml . Sensitivity to TSH was greater in adherent monolayers of cells than in cell suspensions and was also greater in subconfluent rather than confluent monolayers of cells . The increase in {Ca2+}i in response to TSH represented just over a doubling in {Ca2+}i whether examined at 22 degrees C or 37 degrees C . Forskolin failed to affect {Ca2+}i . TSH increased {Ca2+}i in the absence of extracellular calcium . TSH, but not forskolin, produced a significant increase in intracellular calmodulin after 3 days of culture of cells with TSH . The increase in calmodulin was of the order of 60% and did not relate to any effect of TSH on thyroid cell number.

Cell Immunol, 1991 Apr 15, 134(1), 191 - 204
Identification of murine natural killer cell subsets with monoclonal antibodies derived from 129 anti-C57BL/6 immune spleen cells; Lemieux S et al.; Two hybridomas producing monoclonal antibodies reactive with natural killer cells were selected after fusion of 129 anti-C57BL/6 immune spleen cells with P3X63-Ag8.653 myeloma cells . Treatment of normal or stimulated cells with the 4LO3311 or the 4LO439 mAb and rabbit complement inhibited natural killer and antibody-dependent cellular cytotoxicities, whereas cell lysis mediated by natural cytotoxic cells, cytotoxic T lymphocytes, or activated macrophages was unaffected . Lymphokine-activated killer activity was reduced after complement-mediated treatment of interleukin-2-stimulated spleen cells with the 4LO3311 mAb but not after treatment with the 4LO439 mAb . Similar treatment of spleen cells with either mAb had no effect on the mitogen-induced proliferation of T and B lymphocytes and did not alter the frequency of antibody plaque-forming cells in immune spleen cell suspensions . The 4LO3311 and 4LO439 mAbs thus appear to be specific for NK cells and their progeny . Flow cytometry analysis confirmed that 4LO3311+ and 4LO439+ cells are phenotypically identical to NK-1.1+ cells . The epitope recognized by the 4LO3311 mAb has the same strain distribution as the NK-2.1 alloantigen previously detected with NZB anti-BALB/c antiserum, whereas the 4LO439 mAb appears to identify a new NK cell marker exclusively expressed in mice of C57BL lineage . The relationship of the molecules detected with either the 4LO3311 or the 4LO439 mAb to polymorphic antigens of the Ly series is discussed.

FEBS Lett, 1991 Apr 9, 281(1-2), 43 - 6
Rapid aggregation and tight junction formation in single cell suspensions of tumor cells after very low dose trypsin treatment; Kemmner W et al.; The possible participation of tight junction formation in intercellular adhesion of tumor cells was studied in single cell suspensions of HT29 adenocarcinoma cells . Very low dose trypsin treatment (0.15 micrograms/ml, 30 min, 37 degrees C) induced rapid intercellular adhesion of suspended HT29 colon carcinoma cells . Intercellular adhesion was independent of the presence of divalent cations . Electron microscopy of freeze-fractured membrane fragments of trypsin-treated HT29 cells revealed a dramatic increase of typical tight junction structures during aggregation.

Monatsschr Kinderheilkd, 1991 Apr, 139(4), 216 - 9
{Impulse cytophotometry determination of the DNA content of paraffin-fixed neuroblastoma}; Berger K et al.; The aim of the study was to develop a method to obtain a single cell suspension from paraffin fixated neuroblastoma tissue for measuring the DNA content by flow cytometry, and to detect possible correlation between the histological grading and the DNA stemline, and to relate these findings to the patients' prognosis . 51 samples of the fixated neuroblastoma tissue were measured . It was possible to determine the DNA content in 38 probes of the material . 79% of the evaluated samples showed aneuploid DNA stemlines, 21% showed euploid stemlines . Despite the small number of probes it was found, that in relation to the histological grading of neuroblastoma a lower grade of malignancy correlated with an euploid DNA content and a higher grade of malignancy correlated with an aneuploid DNA content . A correlation between DNA stemlines and the patients' prognosis was not found.

Radiol Med (Torino), 1991 Apr, 81(4), 532 - 6
{Influence of radiotherapy on lymphocyte subpopulations}; Ceschia T et al.; The authors investigated the effects of radiation therapy on the immune system by studying lymphocyte subsets and other parameters in 32 patients undergoing radiation therapy for solid cancer . With monoclonal antibody techniques, we studied both T- and B-lymphocytes; cell suspensions were analyzed by means of a Facs Spectrum III Ortho (Ortho-Diagnostic) unit . The first control was performed right after the beginning of radiotherapy, when the dose to the patients was 50 Gy or higher . The second control was performed at 40 Gy because all patients received this dose . 30% of the patients exhibited lymphopenia from the beginning of the study; at 40 Gy the number of T-lymphocytes was low and helper/suppressor ratio was altered . A variable response of B-cells was observed, although all patients exhibited restoration of normal values at 6 months . Four patients only suffered from side-effects: a patient with tongue cancer presented oral mycosis, and a woman--treated for breast cancer--presented vaginal mycosis . Two cases of cystitis were also observed, after 18 Gy, in patients with uterine carcinoma undergoing pelvic irradiation . Disease progression was observed in 2 patients with head and neck cancer, while 3 patients died from lung cancer progression . Another one, with head and neck cancer, died because of heart failure.

Exp Neurol, 1991 Apr, 112(1), 1 - 28
Allografts of CNS tissue possess a blood-brain barrier . II . Angiogenesis in solid tissue and cell suspension grafts; Broadwell RD et al.; Angiogenesis and patency of blood vessels were analyzed qualitatively in solid CNS and peripheral tissue syngeneic, allogeneic, and xenogeneic grafts and in individual cell suspension grafts of astrocytes, fibroblasts, PC12, and three additional tumor cell lines placed intracerebrally in adult host mice . Postgrafting survival times were 1 day through 4 weeks . The patency of graft vessels was determined in sections from immersion-fixed tissues incubated to reveal the endogenous peroxidase activity of host red cells trapped within the lumen of blood vessels . Additionally, horseradish peroxidase (HRP) was administered intravenously to live hosts; HRP labels host brain and graft vessels on the luminal surface and reveals the presence or absence of a blood-brain barrier (BBB) within the grafts . The origins of blood vessels supplying solid tissue xenografts were identified immunohistochemically with primary antibodies against host (athymic AKR mice) and donor (fetal Lewis rats) major histocompatibility complex (MHC) class I . Blood vessels supplying solid CNS grafts at 1-7 days post-transplantation were identified ultrastructurally and possessed interendothelial tight junctional complexes; however, they were not perfused with either host blood or blood-borne HRP prior to 8 days . Graft vessels at 10 days were outlined consistently by peroxidase-positive red cells in immersion-fixed material and labeled with blood-borne HRP . These vessels provided a BBB to the circulating HRP and exhibited interendothelial tight junctions . Evidence of angiogenesis within solid anterior pituitary grafts and the variety of cell suspension grafts was obtained prior to 3 days post-transplantation in immersion-fixed preparations; the vessels, with the notable exception of those supplying astrocyte cell suspensions, failed to present a BBB to blood-borne peroxidase . Endothelia in the solid pituitary allografts and the PC12 cell grafts were highly fenestrated and exhibited open interendothelial junctions; those in the tumor and fibroblast cell grafts, for the most part, appeared nonfenestrated, and many possessed open interendothelial junctional complexes . Immunostaining for host and donor MHC class I revealed that donor blood vessels predominate over host vessels in CNS xenografts and supply pituitary xenografts exclusively; in both preparations, donor vessels were not identified within the host CNS . Because cell suspension grafts were derived from endothelia-free preparations grown in culture, blood vessels supplying these grafts were necessarily of host CNS origin and manifested a morphological transformation from a BBB to a non-BBB endothelium . The data suggest that angiogenesis in solid CNS grafts placed into the adult host CNS, compared to similarly placed solid peripheral tissue/cell suspension grafts, is not rapid.(ABSTRACT TRUNCATED AT 400 WORDS)

Am J Kidney Dis, 1991 Apr, 17(4), 436 - 44
High-energy shock waves: in vitro effects; Clayman RV et al.; To date, there have been multiple studies on the effects of high-energy shock waves (HESW) on benign and malignant cells under in vitro conditions . The major problem in comparing and contrasting these studies has been the wide range of mechanical, biological, and analytical variables facing the investigator . However, if one takes the time to select out only those studies in which a wide range of experimental variables have been defined or controlled, then it becomes possible to begin to understand the effects of HESW on cells in vitro . With this in mind, the literature has been thoroughly reviewed . It would appear that HESW do cause cellular damage regardless of the cell's doubling time . The cell damage is likely due to the impact of cavitation and the attendant shear forces and jets that are produced by the shock waves as it passes through the cell suspension . The damage occurs both at the cell membrane and within the cell itself . With regard to the latter, it would appear that the mitochondria are most sensitive to HESW; however, damage also occurs within the nucleus and along the endoplasmic reticulum and in other cell organelles (eg, lyosomes) . Applications of HESW to other clinical situations are currently being studied . One example of interest is the in vitro combination of chemotherapeutic agents and HESW to enhance the effect of a specific chemotherapeutic regimen on a given tumor cell line . Several investigators have noted a beneficial effect of this combination therapy in vitro; however, similar favorable results have not been obtained when the same or similar tumor system was studied in vivo.

J Immunol, 1991 Apr 1, 146(7), 2305 - 9
Tumor necrosis factor-alpha enhances platelet activation via cathepsin G released from neutrophils; Renesto P et al.; In this paper we show that TNF-alpha enhances platelet activation . Experiments were performed on a human polymorphonuclear neutrophil (PMN)-platelet cooperation system in which PMN, stimulated by FMLP, release cathepsin G (Cat.G), a serine proteinase responsible for the activation of nearby platelets . Pretreatment of the mixed cell suspension with 5 ng/ml TNF-alpha resulted in a strong platelet activation (37.7 +/- 3.2% aggregation; 46.0 +/- 14.4% serotonin release) in response to a weak concentration of FMLP (1.25 x 10(-8) M) inducing by itself only 7.7 +/- 4.0% of aggregation and 3.8 +/- 4.1% of serotonin release (mean +/- SD; n = 10) . This effect was concentration dependent (maximum between 5 and 10 ng/ml) and was optimal for a brief preincubation time (5 min) . Under these experimental conditions the target of TNF-alpha was PMN, as shown by beta-glucuronidase release . The observed potentiation was modified neither by 0.1 mM acetyl salicylic acid (a cyclo-oxygenase inhibitor) nor by 0.1 mM BN 52021 (a platelet-activating factor antagonist), while such a phenomenon was fully inhibited by 20 micrograms/ml eglin C, a strong and specific inhibitor of the human granulocytic proteinases, elastase and Cat.G . In fact, full inhibition was also observed with 300 nM alpha-1-antichymotrypsin, a specific inhibitor of Cat.G . This clear-cut evidence of Cat.G involvement was substantiated by the enhancement of Cat.G release from FMLP-activated PMN primed with TNF-alpha . These results demonstrate that the priming of PMN by TNF-alpha may modulate the activation of other inflammatory cells, particularly of platelets . It is hypothesized that this phenomenon could contribute to pulmonary pathologies, and more specifically to the adult respiratory distress syndrome, a disease for which PMN, platelet and TNF-alpha involvement has been proposed.

Cancer Res, 1991 Apr 1, 51(7), 1959 - 67
Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes; Poddighe PJ et al.; Numerical chromosome aberrations were detected in hematological cancers by nonradioactive in situ hybridization (ISH) procedures, using centromere specific probes for chromosomes 1, 7, 8, 9, 10, 11, 16, 17, 18, X, and Y . All 15 cases could be evaluated by ISH for these 11 probes . Our experiments show that in seven of these randomly selected leukemia bone marrow cell suspensions numerical aberrations for one or two chromosomes could be detected by this method . The results of ISH on interphase nuclei and in some cases on metaphase preparations were compared with karyotyping data . Seven cases of chromosomal aberrations observed with ISH (three for monosomy and four for trisomy) were confirmed by this classical cytogenetic technique, whereas in five instances an aberration was found only with ISH (twice for monosomy, twice for trisomy, and one disomy for the Y-probe) . One case of a trisomy for chromosome 1 observed by ISH on interphase nuclei could be explained by a marker chromosome, a finding that was further substantiated by ISH on metaphase spreads . In this case double-target ISH on interphase cells with the probes for chromosomes 1 and 16 strongly suggested a translocation between these chromosomes . Also, in one case a marker chromosome could be characterized as a translocation between chromosomes 7 and 17 . In this latter case the cytogenetic examinations revealed only monosomy for chromosomes 7 and 17 in addition to noncharacterized marker chromosomes . Our results indicate that the nonradioactive ISH procedure in combination with chromosome specific repetitive centromeric probes is a powerful tool for studying both numerical and structural chromosomal aberrations in interphase nuclei of leukemias . It may therefore become a valuable and routine diagnostic tool in addition to the existing karyotyping procedures.

Cancer, 1991 Apr 1, 67(7), 1865 - 73
Hodgkin's disease in association with human immunodeficiency virus infection . Pathologic and immunologic features; Pelstring RJ et al.; Six patients with Hodgkin's disease (HD) and demonstrable serum antibodies to human immunodeficiency virus (HIV) and two additional patients with HD belonging to HIV-associated high-risk groups but with negative HIV serology were studied . All patients were men and ranged in age from 21 to 45 years . The HIV risk factors included homosexuality (6), intravenous drug abuse (2), and hemophilia A (1) . All patients had high pathologically determined stage (one Stage III and seven Stage IV), and bone marrow involvement was observed in five patients with the initial diagnosis of HD based on marrow biopsy in two cases . Four cases were histologically subclassified as mixed cellularity (MC) and three as nodular sclerosis (NS); one patient underwent only bone marrow biopsy and was not subclassified . Histologically all cases were characterized by numerous Reed-Sternberg cells and variants, and with the exception of one case, all had a distinctive decrease in the proportion of reactive background lymphocytes compared with what is usually expected in MC or NS Hodgkin's disease (relative lymphocyte depletion) . Flow-cytometric immunophenotypic studies done on cell suspensions from diagnostic lymph node biopsies in four cases showed decreased CD4:CD8 ratios (mean = 1.4) compared with expected values of 4 to 6 . The relative lymphocyte depletion observed histologically is probably a reflection of the decreased tissue CD4:CD8 ratios, and this impairment of host immune response may be related to the observed high stage in all eight cases . Patients with high stage HD and the described histologic and immunologic features should be evaluated for the presence of HIV infection.

Kaibogaku Zasshi, 1991 Apr, 66(2), 114 - 29
{Morphological and functional features of endodermal cells in rat yolk sac, with special reference to the fetal macrophage differentiation}; Dai J et al.; Morphological and functional features of the yolk sac endodermal cells with special reference to the fetal macrophage differentiation were investigated morphologically under the light and electron microscopes and immunologically with the antigen phenotypic analysis and the phagocytic activity-test, using the syngeneic DA rat-embryos from 8 to 16 days of gestation . Based on the staining property with toluidin blue and the ultrastructural features, the endodermal layer from day 8 to 16-yolk sacs has been known to consist of two kinds of cell type; 10% "clear" cells with clear cytoplasm and 90% "dark" cells with dark cytoplasm . Numerous primary lysosomes, phagolysosomes, lipid droplets and coated vesicles distributed preferentially in the supranuclear portion of endodermal cells . A broad intercellular space was found between "clear" cells and "dark" cells, indicating the loose intercellular binding . It was often found that "clear" cells tend to migrate from the endodermal layer into the mesenchymal layer, where the poor development of basement membrane was seen between them . Cells phagocytosing red blood cells, that resemble morphologically "clear" cells, were also observed in the fetal liver . At ten hours after latex-injection into the yolk sac cavity of 14 days embryos, some cells which phagocytosed latex beads in their cytoplasm were found in the endodermal layer, and also in the liver tissue and loose connective tissue of fetus . These cells were stained positively with monoclonal antibody Mar3 which recognizes preferentially rat-mononuclear phagocyte system . In vitro-latex uptake of separated endodermal cells was also demonstrated by the culture-study of endodermal cell suspension . The present findings indicate that the yolk sac-endodermal layer derived from the proximal endoderm consists of at least two kinds of cell-population with a great similarity to tissue macrophages in morphological and functional senses, and support the concept that some cell-populations of endodermal layer may migrate into fetal tissue and are closely related to the differentiation of fetal macrophages and their precursors.

Nippon Hoigaku Zasshi, 1991 Apr, 45(2), 128 - 37
{Metabolism and toxicity of n-pentane and isopentane}; Chiba S et al.; n-Pentane and isopentane have a wide range of use, for example, for cleaning precision machinery, extracting essence and oil, and as liquid fuel for now very popular disposable lighters . They are contained in liquefied petroleum gas and natural gas as trace constituents . In our present experiments, we studied the metabolism and toxicity of these n-pentane and isopentane metabolites . Male mice of ICR strain were exposed to about 5% n-pentane for one hour while the oxygen in the environmental air was maintained at about 20% . Then their blood and liver tissue were collected and analyzed by means of GC and GC-MS . The metabolites thus obtained were 2-pentanol, 3-pentanol and 2-pentanone . The same procedure was repeated with isopentane; 3-methyl-2-butanol, 2-methyl-2-butanol and 3-methyl-2-butanone were detected as the resultant metabolites . In the presence of the NADPH-generating system liver microsomes were made to react to the substrate of saturated n-pentane or isopentane aqueous solution at 37 degrees C for one hour . As a result, the same metabolites were produced as obtained in the exposure experiment . It was therefore suggested that n-pentane and isopentane were metabolized chiefly by liver microsomes . Male mice of ICR strain were fed with 80 mg/kg b.w . of phenobarbital for consecutive four days and exposed to n-pentane or isopentane for one hour . This resulted in an increase in the amount of 2-pentanol and 2-pentanone in the n-pentane inhalation and 2-methyl-2-butanol in the isopentane inhalation experiment . The toxicity of each metabolite was studied on cultured cells . The metabolites were individually mixed with HeLa S3 cell suspension, incubated for three days, and their concentration which inhibited the growth of cells by 50% (IGC 50) were compared . It was demonstrated as a result that the IGC 50 for any of the metabolites was lower than that for methanol, ethanol or acetone used as control.

Curr Eye Res, 1991 Apr, 10(4), 363 - 72
Inhibition of lymphocyte proliferation by resident ocular cells; Hooper P et al.; The mechanisms by which the eye maintains an immunosuppressive environment has been the subject of recent investigations . In this report we investigated the ability of resident ocular cells from the iris, choroid, and retina to inhibit lymphocyte responses in vitro . Our results demonstrate that single cell suspensions derived from iris and choroid to inhibit alloantigen induced lymphocyte proliferation . We show that this inhibition was mediated by soluble factors which are low (less than 10,000) and intermediate (10,000-30,000) molecular weight molecules . This capacity is limited to iris and choroid and is not demonstrable in cell preparations derived from the retina . We conclude from our studies that cells derived from iris and choroid are capable of regulating immune responses and suggest that these cells (or their soluble products) may play a role in the immunosuppressive environment of the eye.

Am J Physiol, 1991 Apr, 260(4 Pt 1), L349 - 55
Synthesis of eicosanoids by isolated guinea pig tracheocytes; Prie S et al.; Epithelial cells from the guinea pig trachea (tracheocytes) have been separated from the lamina propria by incubation with EDTA . Contaminating eosinophils were removed by plating on guinea pig immunoglobulin G (IgG)-coated plates . Purity of the cell suspension was assessed by electron microscopy . Enzyme immunoassay analyses of supernatants of guinea pig tracheocytes incubated with 10 microns arachidonic acid (AA) in the presence or absence of 2 microns ionophore A23187 showed that the cells released up to eight times more thromboxane, prostaglandin E2, and 6-ketoprostaglandin F1 alpha than control cells . Ionophore A23187 by itself stimulated to a smaller extent the release of these eicosanoids and did not potentiate the effect of AA . The release of cyclooxygenase products by these cells was highly modified by cell densities in the incubation media . The release of leukotrienes (LT) by stimulated epithelial cells was also investigated using reverse-phase high-performance liquid chromatography . Our results showed that the cells could not release LT on incubation with ionophore A23187 and/or AA . However, the cells released LTB4 in the medium on incubation with 2 microns LTA4 . Peptido-leukotrienes were not detected . These results indicated that guinea pig tracheocytes have the cyclooxygenase and the LTA4 hydrolase but not the 5-lipoxygenase . It is postulated that tracheocytes may participate in transcellular metabolism of LTA4 generated by other cell types.

Gan To Kagaku Ryoho, 1991 Apr, 18(4), 547 - 54
{Growth chamber assay, a novel chemosensitivity test which eliminates normal stromal cells}; Tetsuya T et al.; The chemosensitivity test with growth chamber (GC), a semi permeable polymer matrix, was conducted using human tumor xenografts, comparing the results with those of in vivo nude mouse system . Xenografts used were MX-1, St-4, Co-3, and Co-4 . Normal stromal cells, SM-74, a cell line derived from human adult skin fibroblast, and Clone-A-31, a cell line from BALB/c nu/nu nude mice were used as the control . Dissociated tumor cell suspension in 200 microliters of Medium 199 was plated into GC (80,000 cells, chamber) and incubated with a various concentration of mitomycin C(MMC), cisplatin (DDP), 5-fluorouracil (5-FU), and adriamycin (ADM) at the various concentrations . After incubation of 1 wk, the activity of hexosaminidase was measured with ELISA assay using p-nitrophenyl-N-acetyl-glucosaminide . The antitumor activity of the agents against human tumor xenografts was dose dependent, and the antitumor spectra obtained by GC assay was essentially identical to in vivo nude mouse system . On the other hand, no evaluable optical density could be obtained with normal stromal cells, SM-74 and Clone-A-31 . The optimal cutoff concentration of each drugs to predict the in vivo results was estimated to be 10 micrograms/ml for MMC, 15 micrograms/ml for DDP and 5-FU, and 0.7 microgram/ml for ADM . The predictability of GC assay was 77%, including 89% sensitivity and 70% specificity . Since GC assay could eliminate the normal stromal cells because of the characteristic of chamber surface, this assay was thought to be useful for the clinical chemosensitivity test of human cancers containing a large number of normal stromal cells.

Arch Biochem Biophys, 1991 Apr, 286(1), 226 - 32
Purification, characterization, and subcellular localization of an acid phosphatase from black mustard cell-suspension cultures: comparison with phosphoenolpyruvate phosphatase; Duff SM et al.; An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (mumols orthophosphate produced/min)/mg protein and near homogeneity . The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a pI of 4.5 . The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable . The final preparation hydrolyzed a wide variety of phosphate esters . The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PPi, and phosphoenolpyruvate (PEP) . The enzyme was activated 1.4-fold by 4 mM Mg2+ or Mn2+, but was strongly inhibited by Mo, Pi, F, and several phosphorylated compounds . Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall . By contrast, B . nigra PEP phosphatase appeared to be localized in the cell vacuole . Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases . Their respective CNBr cleavage patterns were dissimilar, suggesting that B . nigra acid and PEP phosphatases are distinct polypeptides . Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B . nigra cell-suspension cultures to nutritional phosphate deprivation.

Nippon Hifuka Gakkai Zasshi, 1991 Apr, 101(5), 533 - 8
{Nuclear DNA contents in the cells of squamous cell carcinoma . III . Separation and analysis of polyploid cells}; Toyoshima H et al.; In order to reveal the cytological nature of polyploid cells, the cell suspension of squamous cell carcinoma was separated into low density (1.050 greater than), intermediate density (1.050 to 1.088), and high density (1.988 greater than) fractions, by density gradient centrifugation . The DNA content of the tumor cells in each fraction were measured on the smear specimens prepared by Giemsa's staining and Feulgen's stainings . As the results, it was found that the cells showing high NC ratio and having high DNA content were observed in the high density fraction . However, there was no specific relationship between the nuclear contour index and density of the tumor cell.

Cell Biol Toxicol, 1991 Apr, 7(2), 129 - 43
Biochemical changes in isolated hepatocytes exposed to tert-butyl hydroperoxide . Implications for its cytotoxicity; Buc-Calderon P et al.; When isolated hepatocytes were exposed to tert-butyl hydroperoxide (tBOOH) they lost their cellular membrane integrity . Decreased levels of GSH, increased phosphorylase a activity (an indirect index of the amount of free cytosolic Ca2+), and increase in the formation of malondialdehyde (MDA)-like products (an index of lipid peroxidation) preceded the release into the culture medium of the cytosolic enzyme lactate dehydrogenase (LDH), indicating that this later process was the consequence of the former intracellular events . While ATP levels were not modified during the incubation of cells with increasing concentrations of tBOOH, protein synthesis was decreased in a concentration-dependent manner . The glycogen content decreased at the same time as the increase in LDH leakage . The addition of promethazine (PMZ) an antioxidant molecule, prevented the lipid peroxidation, but did not protect cells against the oxidative effects of tBOOH, including loss of membrane integrity . Nevertheless, the addition of GSH to cell suspensions incubated with tBOOH, decreased the formation of MDA-like products, restored the protein synthesis rate, prevented partially the activation of phosphorylase a and preserved cell viability . On the basis of these results, we postulate that both GSH depletion and modification in phosphorylase a activity (Ca2+ levels) were the most relevant intracellular events to explain the cytotoxicity of tBOOH.

Respir Physiol, 1991 Apr, 84(1), 93 - 104
Blood oxygen carriage in the marsupial, tammar wallaby (Macropus eugenii), at prenatal and neonatal stages; Tibben EA et al.; The measurements reported here are the first to be made on oxygen carriage of a prenatal marsupial . The oxygen equilibrium curves (OECs) of tammar wallaby blood 1-2 days before the due date of birth showed a high P50 (mean = 44 Torr at 36 degrees C at a PCO2 of 34 Torr), more than 1.5 times that of the mother . This was confirmed by measurements in red cell suspensions at controlled pH . The finding of a higher P50 than in adult is in contrast to the general finding in eutherian (placental) mammals . Also they showed interaction between O2 and CO2 carriage (expressed as delta log P50/delta log PCO2 between 34 and 64 Torr PCO2) about half the magnitude of that in adults . At high PCO2 this effect reversed in the lower part but not in the upper part of the OEC . The Hill plot of the OECs showed a bend in the middle range of saturation: in nearly all cases the Hill coefficient (nH) was greater than 4.0 above about 50% saturation suggesting aggregation of haemoglobin tetramers . These results are similar to those previously reported for neonatal tammars and confirmed by further measurements in this study . The prenatals all had four haemoglobin types, identical with those found in the neonates.

Mol Cell Endocrinol, 1991 Apr, 76(1-3), 35 - 44
Function of prolactin cells in the individual rat pituitary gland is location dependent; Mukherjee P et al.; Anterior pituitary glands from individual ovariectomized (ovx) or ovx-estrogen (E2) treated rats were sectioned into 1/8 cubes . Each section was incubated for four consecutive 15 min periods in order to measure the release of immunoreactive and bioactive prolactin (PRL); each individual section was then trypsinized into a single cell suspension for determination of PRL cell numbers in that section . Hormone release (ng PRL/1000 PRL cells) was not uniform throughout the gland; the consistency of the secretory patterns demonstrated that the amount of PRL release from the gland was location-dependent . Statistical analysis of the data showed that the most active cells were in the gland's left lobe, while the least active were in the right lobe . Within these lobes, the dorsal-caudal and ventral-rostral left lobe areas released the most hormone in vitro while those in the dorsal-rostral, dorsal-caudal and ventral-rostral right lobe areas were least active . Injection of ovx rats with E2 for 2 days altered these secretory patterns . This sectioning procedure should prove useful in future studies addressing issues of cell-cell interaction and geographic location as they relate to pituitary cell function.

Cell Biophys, 1991 Apr, 18(2), 123 - 43
Transglutaminase stabilizes melanoma adhesion under laminar flow; Menter DG et al.; To resist substantial wall shear stress (WSS) exerted by flowing blood, metastatic melanoma cells can form adhesive contacts with subendothelial extracellular matrix proteins, such as fibronectin (FN) . Such contacts may be stabilized by transglutaminase catalyzed-cross-linkage of cell focal adhesion proteins . We analyzed human melanoma cell adhesion under flow by decreasing the flow (WSS) of melanoma cell suspensions and allowing them to adhere to immobilized wheat germ agglutinin or FN . At the wall shear adhesion threshold (WSAT), cell adherence was rapid with no rolling . Following cell adherence, we increased the flow and determined the wall shear detachment threshold (WSDeT) . Cells spread and remained adherent on immobilized FN at high WSDeTs (greater than or equal to 32.5 dynes/cm2) . The high resistance of adherent cells to shear forces suggested that transglutaminase-mediated crosslinking might be involved . Transglutaminase inhibitors monodansylcadaverine and INO-3178 decreased WSAT, and at low concentrations completely inhibited tumor cell spreading and promoted detachment at low WSDeTs (0.67 dynes/cm2) . In static adhesion assays, transglutaminase inhibitors decreased cell adhesion to immobilized-FN in a dose-dependent manner and prevented the formation of crosslinked 125I-FN complex that failed to enter a SDS-polyacrylamide gradient gel . The data suggest that transglutaminase-catalyzed crosslinking, particularly in the presence of WSS, may be important in stabilizing cellular adhesive contacts during adhesion to immobilized-FN.

J Pineal Res, 1991 Apr, 10(3), 122 - 9
Beta-adrenergic stimulation increases cAMP and melatonin production in ovine pinealocyte cultures; Howell HE et al.; An ovine pinealocyte suspension culture method, using glands from young sheep as a source of cells, has been developed to study mechanisms of pineal regulation in this species . Cell suspensions are obtained by enzymatic digestion (preliminary trypsinization followed by collagenase/pronase treatment) of pineal glands and these cells may be usefully retained in culture for up to 48 hr . Initial characterization of cyclic adenosine monophosphate and melatonin production responses to adrenergic stimuli (norepinephrine, phenylephrine, isoproterenol) in the absence and presence of antagonists (propranolol, prazosin) by pinealocytes in suspension culture indicate that a beta-adrenergic receptor-mediated mechanism is primarily involved in transduction of the adrenergic signal to melatonin synthesis . This is in agreement with data from earlier work on the sheep using short-term tissue incubations but contrasts with in vivo evidence suggesting a predominant alpha 1-adrenergic receptor-mediated mechanism.

Int J Radiat Biol, 1991 Apr, 59(4), 927 - 39
Comparison of DNA double-strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation and non-unwinding filter elution in irradiated plateau-phase CHO cells; Iliakis G et al.; The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis . The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC) . Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation . Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t 1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure to 50 Gy . The initial rate of rejoining of chromatin breaks was slower than that of DNA dsb and occurred with t 1/2 of 87 min . The results suggest that all major techniques currently used for assaying rejoining of DNA dsb give similar results despite their widely different biophysical basis, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established.

FEBS Lett, 1991 Mar 25, 280(2), 397 - 9
Actin depolymerization in the cyclic AMP-stimulated toad bladder epithelial cell, determined by the DNAse method; Hays RM et al.; Previous studies with the rhodamine phalloidin binding assay have shown that antidiuretic hormone and 8-Br-cAMP rapidly depolymerize F-actin in toad bladder epithelial cells . We have extended these studies with the DNAse inhibition assay and have found that in isolated epithelial cell suspensions, G-actin increases from 37 to 56% of total actin following 8-br-cAMP stimulation . The G-actin concentration in the epithelial cell greatly exceeds its critical concentration, indicating the requirement for a G-actin sequestering protein or proteins in this system.

J Immunol Methods, 1991 Mar 21, 137(2), 193 - 7
Flow cytometric immunophenotyping of leukemia cells in clotted blood and bone marrow; De Vis J et al.; Immunophenotyping of leukemia cells is generally performed on cells in suspension . These suspensions are usually prepared from anticoagulated peripheral blood or bone marrow samples but when anticoagulation is suboptimal clotted samples may reach the laboratory . In this study cell suspensions were prepared from clotted blood and bone marrow samples of leukemia patients by lysis of the clots with streptokinase . The cell suspensions were then labeled with monoclonal or polyclonal antibodies and examined by flow cytometry or fluorescence microscopy . Enough cells could be isolated from small volumes of clotted blood or bone marrow aspirate to determine the immunophenotype of the cells . The morphology of the cells was well preserved and accurate identification of the cells was possible . The immunophenotypes determined on clotted samples from four patients with acute myeloblastic leukemia, five with acute lymphoblastic leukemia and two with chronic lymphocytic leukemia were identical to those established using EDTA anticoagulated samples of the same patients . When no unclotted samples are available this approach may avoid the necessity for a new sample and the concomitant delay in treatment of the patient.

Proc Natl Acad Sci U S A, 1991 Mar 15, 88(6), 2407 - 11
Protamine transcript sharing among postmeiotic spermatids; Caldwell KA et al.; Sharing of cytoplasmic constituents through intercellular bridges connecting postmeiotic spermatids can allow for functional equivalence of genetically nonequivalent spermatids . The technique of in situ hybridization was used to study postmeiotic distribution of transcripts from the mouse protamine 1 (Prm-1) gene among spermatids of mice with chromosomally unbalanced gametes . The Prm-1 gene is located on chromosome 16 and is expressed exclusively in haploid spermatids . Mice doubly heterozygous for two Robertsonian translocations involving chromosome 16 were used for the study of postmeiotic accumulation of transcripts of the Prm-1 gene in spermatogenic cells . The meiotic segregation pattern of chromosomal homologues in these mice produces some spermatids that are chromosomally unbalanced; some spermatids lack chromosome 16 while others have two . In situ hybridization with a cDNA probe for the Prm-1 gene transcript performed on both whole testis sections and spermatogenic cell suspensions showed that there was no statistical difference in distribution of grains over step-5 to step-10 spermatids from Robertsonian-translocation heterozygous mice and from control mice of normal karyotype . These results are consistent with sharing of transcripts of the Prm-1 gene among spermatids within a syncytium.

Biochim Biophys Acta, 1991 Mar 12, 1082(2), 195 - 203
Biochemical demonstration of endocytosis and subsequent resecretion of high-density lipoprotein by rat peritoneal macrophages; Rahim AT et al.; To investigate the post-binding events of high-density lipoprotein (HDL), rat peritoneal macrophages were enriched by collagen gel-coated plates and incubated in a cell-suspension system with fluorescein isothiocyanate-labeled HDL (FITC-HDL), followed by fluorescence spectroscopic analyses . Upon incubation with FITC-HDL at 37 degrees C for 30 min, the microenvironmental pH of the cell-associated FITC-HDL was 6.50, whereas a 0 degree C-incubation gave a corresponding pH of 7.15 . Carbonyl cyanide m-chlorophenylhydrazone, a protonophore known to dissipate the proton gradient, restored the former acidic pH (pH 6.40) to pH 7.20, but had no effect on the latter . This indicates that cell surface-bound HDL is internalized and exposed to an acidic compartment . When cells were incubated with FITC-HDL at 37 degrees C for 30 min and the cell-associated FITC-HDL was chased at 37 degrees C, the fluorescence intensity at 490 nm showed a time-dependent increase . This increase was explained by a release of endocytosed FITC-HDL into the extracellular medium but not by a simple outward dissociation of the cell-associated FITC-HDL . The microenvironmental acidic pH of the cell-associated FITC-HDL changed to a less acidic pH during the chase whereas that of FITC-HDL became constant after released into the medium, indicating that endocytosed HDL was resected back into the extracellular medium . This resection process was temperature-dependent and accelerated by HDL itself . These results provide the biochemical evidence for the presence of an endocytic-exocytic pathway for HDL in rat peritoneal macrophages.

Photochem Photobiol, 1991 Mar, 53(3), 415 - 8
A new method of determining intracellular free Ca2+ concentration using Quin2-fluorescence; Miyoshi N et al.; We determined intracellular free Ca2+ concentration by fluorescence spectroscopy and the time-resolved measurements of 2-{(2-amino-5-methylphenoxy) methyl}-6-methoxy-8-aminoquinoline-N,N,N',N'-tetraacetic acid, tetrapotassium salt (Quin2) incorporated in suspended mouse leukemia L1210 cells . The paper reports the following two points . (1) Various fluorescence spectrum patterns in cell suspensions dissolved with Quin2 acetoxy methylester were compared with those of the complex in buffer solution containing esterase . (2) The fluorescence lifetime of Quin2 bound to Ca2+ was approx . 4.5-11 times longer (10 +/- 1 ns) than that (1.5 +/- 0.5 ns) of Quin2 . The fraction of the long lifetime component was plotted against the concentration of CaCl2 in buffer solution . From the results obtained, it was found that approx . 35 nM Ca2+ was contained in each L1210 cell.

Pathol Biol (Paris), 1991 Mar, 39(3), 182 - 4
{Analysis of tumor DNA by flow cytometry on prostate cyto-aspiration product . Value of routine cytologic evaluation}; Piaton E et al.; In this prospective study, flow cytometry DNA profile of 169 stage D2 prostatic carcinomas were compared with conventional cytologic data . Two transrectal fine-needle aspiration biopsies were performed in each patient . Aspirates were immediately fixed in 2% polyethylene glycol 1500* alcoholic solution (Merck) . Suspensions were mixed and studied by a conventional cytologic technique (Papanicolaou) and by flow cytometry (propidium iodide stain on isolated nuclei) . A highly significant correlation was found between the DNA profile and the cytologic grade (p less than 0.001) . The DNA profile was bimodal in 14% (3/21) of aspirates containing benign or atypical cells, 18% (3/17) of grade I aspirates, 30% (13/43) of grade II aspirates and 71% (24/34) of grade III aspirates . Routine cytologic evaluation of cell suspensions evaluated by flow cytometry is important in clinical practice since both falsely unimodal and falsely bimodal profiles occur . Leukocytes or benign epithelial cells can interfere with the tumor cell population . In fine-needle aspirates, the broad range of non-malignant contaminating cells limits the value of immunocytochemistry and emphasizes the usefulness of routine conventional cytologic evaluation.

J Exp Biol, 1991 Mar, 156, 399 - 406
Effect of pregnancy and temperature on red cell oxygen-affinity in the viviparous snake Thamnophis elegans; Ingermann RL et al.; The oxygen affinity of red cell suspensions from fetal garter snakes was higher than that of cell suspensions from their mothers . This difference appeared to be due to different concentrations of nucleoside triphosphate (NTP, primarily adenosine triphosphate) . NTP concentrations were significantly higher, and oxygen affinities were significantly lower, in red cell suspensions from pregnant females compared with those from nonpregnant females or males; there is no precedent for such a pronounced effect of pregnancy on the oxygen affinity of maternal blood . These data indicate that pregnancy may result in an enhanced ability of adult blood to deliver oxygen to the fetus . Since the binding of organic phosphates and oxygen to hemoglobin is sensitive to temperature, and since these animals experience diurnal changes in temperature, we examined the influence of relatively low (20 degrees C) and high (34 degrees C) temperatures on red cell oxygen-affinity . The temperature increase of 14 degrees C resulted in a lowered oxygen-affinity of all red cell suspensions examined . However, this increase in temperature lowered the affinity of maternal red cells to a greater extent than it did the affinity of fetal red cells . This suggests that daytime temperatures may further enhance the ability of maternal blood to deliver oxygen to the fetus at times when fetal oxygen demand is probably greatest.

Br Poult Sci, 1991 Mar, 32(1), 79 - 86
Production of quail-chick chimaeras by blastoderm cell transfer; Naito M et al.; 1 . Quail-chick chimaeras were produced by injecting dissociated quail blastoderm cells into chick embryos . 2 . Quail blastoderms were removed from the yolk and the cells were dispersed by trypsin treatment or pipetting . The cell suspension (1 to 5 microliters) was injected into the subgerminal cavity of unincubated chick embryos . The chick embryos were then cultured in recipient eggshells . 3 . Quail blastoderm cells injected into the chick embryos adhered to the chick embryonic cells . The rates of hatching were 8.6% (38 chicks from 441 eggs) and 40.3% (48 chicks from 119 eggs) when the volumes of the cell suspension injected were 3 to 5 microliters and 1 microliter, respectively . 4 . Seven out of 86 hatched birds were clearly identified as being chimaeric because part of the feather colouring was of quail specificity . In addition to these chimaeric birds, there were 8 chimaeric embryos which died before hatching . The distribution patterns of the quail feathers were varied among the chimaeric birds and embryos . 5 . This technique provides a basis for the investigation of chick embryo cryopreservation, genetic transformation and analysis of cell lineage of chickens.

Mod Pathol, 1991 Mar, 4(2), 196 - 200
Flow cytometric analysis of DNA ploidy and S-phase fraction in breast cancer using cells obtained by ex vivo fine-needle aspiration: an optimal method for sample collection; Eliasen CA et al.; A total of 203 primary invasive breast cancers were sampled by ex vivo fine-needle aspiration (FNA), directly yielding adequate single cell suspensions for flow cytometric DNA analysis in 194 (96%) . Labor-intensive and time-consuming steps of mechanical and enzymatic cellular disaggregation required by the use of fresh, frozen, or paraffin-embedded tissue were avoided, thereby minimizing preparation time . Conservation of tumor tissue allowed for the sampling of very small breast cancers . DNA ploidy and S-phase fraction data were comparable to flow cytometric data reported in other breast cancer studies using various sampling methods . Ex vivo FNA is the easiest and fastest method for sampling breast cancers for flow cytometric DNA analysis.

J Bone Miner Res, 1991 Mar, 6(3), 263 - 71
Coexistence of reduced function of natural killer cells and osteoclasts in two distinct osteopetrotic mutations in the rat; Popoff SN et al.; Recent evidence suggesting that immune cells and their products (cytokines) play an important role in the regulation of skeletal development and function, particularly of the osteoclast, implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders . The mammalian osteopetroses are a pathogenetically heterogeneous group of skeletal disorders characterized by skeletal sclerosis resulting from reduced osteoclast-mediated bone resorption . Using a 51Cr-release microcytotoxicity assay we demonstrated that splenic natural killer (NK) cell activity was significantly reduced in two distinctly different osteopetrotic mutations in the rat, osteopetrosis (op) and toothless (tl) . To determine whether this reduction in NK cell-mediated cytotoxicity is caused by decreased cell number and/or function in these osteopetrotic mutants, we quantitated NK cells by analyzing mononuclear cell suspensions labeled for two-color fluorescence with OX8 and OX19 monoclonal antibodies in a fluorescence-activated cell sorter . Flow cytometry of these double-labeled cells revealed that the percentage of NK cells (OX8+/OX19- subset) in op and tl spleens was not significantly different from that of normal spleens . These results suggest that NK cells in these osteopetrotic mutants are functionally defective . Thus aberrations in osteoclast and NK cell function coexist in these mutations, and their developmental relationships deserve further study.

J Acoust Soc Am, 1991 Mar, 89(3), 1394 - 401
Polydisperse scattering theory and comparisons with data for red blood cells; Berger NE et al.; Recent results for low-frequency scattering by polydisperse distributions of correlated low-refracting particles averaged over orientation are analyzed numerically . The roles of shape and correlations (parameterized by c) and polydispersity (specified by the normalized variance d in size governed by the gamma probability density) are investigated . The key variable is the net volume fraction w occupied by the particles . The incoherent scattering is determined by delta = PS(c,d;w) with P as a particle population factor that is independent of w, and S as the fluctuation-correlation function of w . Earlier applications of monodisperse (d = 0) theory emphasized the influence of c on the peak delta = delta and its location w = w in order to invert ultrasonic scattering data of Shung and his associates for red blood cell suspensions under different flow conditions . For d greater than 0, comparable curves for delta (w) decrease more gradually with w increasing past w (because of additional scattering arising from polydispersity) and thereby provide better fits to data for the more controlled flows over broader ranges of hematocrit.

Res Virol, 1991 Mar-Jun, 142(2-3), 119 - 21
Latent infection of epidermal Langerhans cells in HIV-positive individuals; Braathen LR et al.; Epidermal cell suspensions obtained from 3 symptom-free HIV-positive individuals were cultured and marked with monoclonal antibodies for the HIV proteins p15, p24 and gp120 in the alkaline phosphatase anti-alkaline phosphatase staining technique . For 2 individuals, cells were positive after 3 days in culture, and for the third, after 4 days . Supernatant from one of the cultures infected allogeneic peripheral blood mononuclear cells . We conclude that epidermal Langerhans cells from symptom-free HIV-positive individuals are latent-infected and are able to produce and release HIV.

J Reprod Fertil, 1991 Mar, 91(2), 617 - 25
Characterization of rat epithelial epididymal cells purified on a discontinuous Percoll gradient; Finaz C et al.; A method for the purification of epithelial cells from the three anatomical regions of the rat epididymis (corpus, caput and cauda) is described . An enzymic digestion followed by sedimentation of crude cell suspension on discontinuous Percoll gradient yielded quite pure active epithelial cell population as judged by morphological and functional studies . Electron microscopy analysis showed that cells from bands corresponding to densities 1.055 and 1.06 g/ml the gradient preserved a morphology compatible with their epithelial origin and their absorptive and secretory functions . Moreover, they stained positively with anticytokeratin antibody (95-97%) and were negative for antidesmin antibody . They selectively bound L-carnitine through a time-dependent and saturable system and differences in the rate of binding were apparent according to the three anatomical regions of the epididymis.

J Immunol Methods, 1991 Mar 1, 137(1), 55 - 63
Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency; Menegazzi R et al.; Biochemical assays for peroxidase activity do not usually distinguish between different peroxidases . The guaiacol assay, for example, which is one of the most commonly used assays for peroxidase activity, is sensitive to both eosinophil peroxidase (EPO) and the peroxidase of neutrophils, i.e., myeloperoxidase (MPO), thus preventing distinction of the two peroxidases in mixed neutrophil-eosinophil populations . In this paper we describe a simple and sensitive method for selective assays of EPO in EPO-MPO mixtures or mixed populations of neutrophils and eosinophils . The method is based on the peroxidase-mediated oxidation of homovanillic acid (HVA) under appropriate assay conditions in which EPO is still very active in catalyzing the reaction whilst MPO-mediated HVA oxidation is almost undetectable . Optimal assay conditions were as follows: pH 10.5, 10 microM hydrogen peroxide, 0.8 mM HVA and an incubation time of 120 min at 37 degrees C . Under these conditions the assay permits EPO activities as low as 0.025 guaiacol U/ml to be measured even in the presence of 0.175 guaiacol U/ml of MPO . In mixed neutrophil-eosinophil cell suspensions the test permits the detection of as few as 5 X 10(3) eosinophils even in the presence of about 700 X 10(3) neutrophils (eosinophils: neutrophils ratio 1:140) with no appreciable interference by the latter cells . The method described here has been applied to studies of human EPO deficiency and proved to be successful in the identification of individuals with partial EPO deficiency, which is not feasible with non quantitative methods (for example, cytochemistry) or unselective biochemical assay of peroxidase activity.

Haematologica, 1991 Mar, 76 Suppl 1, 41 - 3
CD34 or S313 positive cells selection by avidin-biotin immunoadsorption; Tassi C et al.; A column immunoadsorption method, based on the high affinity between the protein Avidin and the vitamin Biotin has been used to obtain positive CD34+ or S313+ marrow cells selection . Cell suspensions from marrow samples of six healthy subjects were incubated either the monoclonal antibody S313 or HPCA 1 (My 10-like) and a biotinilated goat anti-mouse Immunoglobulins antiserum, passed over a column containing an Avidin coated Polyacrylamide matrix, at the flow rate of 1 ml/min . The phenotype and the clonogenic efficiency of the recovered adherent cell fraction were studied by cytofluorimetric analysis and haemopoietic progenitors short term cultures . The results obtained show a mean CD34+ and S313+ cells recovery greater than 50% with a lower stem cells enrichment . Although these data could not considered optimal for clinical application in haematologic neoplasias, these preliminary studies demonstrate the possible use of the method for autologous bone marrow transplantation.

Radiat Res, 1991 Mar, 125(3), 243 - 7
A method for the selective measurement of the radiosensitivity of quiescent cells in solid tumors--combination of immunofluorescence staining to BrdU and micronucleus assay; Masunaga S et al.; C3H/He mice bearing the SCC VII tumor were irradiated after being given 10 injections of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells in the tumors, and the tumors were then excised and trypsinized . The tumor cell suspensions were incubated with cytochalasin-B (which blocks cytokinesis), and the micronucleus frequency in unlabeled cells was determined using immunofluorescence staining to BrdU . The micronucleus frequency was then used to calculate the surviving fraction of the unlabeled cells, using the regression line relating the micronucleus frequency to the surviving fraction determined separately for the total tumor cell population . Using this technique, a cell survival curve could be determined for the unlabeled cells, which were regarded as the quiescent cells . Assays performed both immediately after and 24 h after irradiation of normally-aerated tumors showed that unlabeled cells were more radioresistant and had a greater capacity for repair of potentially lethal damage than the tumor cell population as a whole . Moreover, when the assay was performed immediately after the irradiation of both normally-aerated and hypoxic tumors, it was found that unlabeled cells had a much higher hypoxic fraction than the tumor cell population as a whole . This appears to be a useful method for determining the responses of quiescent cells in solid tumors to various treatments.

Haematologica, 1991 Mar, 76 Suppl 1, 55 - 7
Collection, processing and storage of peripheral blood stem cells (PBSC); Magrin S et al.; Eight patients with hematological malignancies were treated with autologous blood stem cell transplantation (ABSCT) . Hemopoietic precursor cells were mobilized into the peripheral blood (PB) by chemotherapeutic induction of transient myelosuppression followed by an overshooting of blood stem cell concentration . PB CFU-GM showed a 15-20 fold increase on days 15-20 after chemotherapy . Peripheral blood stem cells were collected by 5-8 continuous flow leukaphereses using a Fenwall CS 3000 blood cell separator when platelet and WBC count was rising rapidly (platelet greater than 50 x 10e9/L and WBC greater than 1 x 10e9/L) . Mean CFU-GM collected per run by leukapheresis were 10.85 x 10e4/Kg (procedure 3), 10.03 x 10e4/Kg (modified procedure 1) . Mononuclear cell suspension in 10% DMSO was frozen at controlled rate freezer (-1 degree C to -4 degrees C) and stored in the liquid phase of nitrogen . After thawing CFU-GM recoveries ranged from 13.8% to 81.5%; 55-70% of recovered cells excluded trypan blue dye.

Haematologica, 1991 Mar, 76 Suppl 1, 3 - 6
Rationale for large scale bone marrow hemopoietic stem cell manipulation; Menichella G et al.; Many years have passed since the first attempt in marrow grafting was performed (1939) . During this period several techniques have been developed in marrow processing and manipulation to overcome bone marrow transplant complications: the ABO barrier in case of major incompatibility between donor and recipient, the graft-versus-host disease due to the presence of allogeneic mature T-lymphocytes in cellular suspension and the neoplastic cell residue in autografts . At the end, the final volume of autologous mononuclear cell suspension must be frozen and an optimized cryopreservation allows a cell viability and subsequently an adequate medullar repopulating capacity.

J Pharm Pharmacol, 1991 Mar, 43(3), 200 - 3
Effect of chronic administration of nicotine or cocaine on steroidogenesis in rat adrenocortical cells; Krueger RJ et al.; Rats were implanted subcutaneously with osmotic mini-pumps containing either 0.9% NaCl, nicotine (1.5 or 4.5 mg kg-1 day-1), or cocaine (30 mg kg-1 day-1), for 14 days . Neither nicotine nor cocaine treatment significantly altered the maximal rate of steroidogenesis in adrenocortical cell preparations from the animals . However, pretreatment with cocaine increased the sensitivity of the preparation to stimulation by ACTH, the ED50 was 5 pM compared with 10 pM from control animals . Addition of nicotine or cocaine at concentrations up to 100 microM to adrenal cell suspensions from naive rats did not stimulate steroidogenesis or increase the sensitivity of cells to ACTH stimulation . These results suggest that the primary chronic effect of nicotine on steroidogenesis is exerted at the level of the hypothalamus and/or pituitary and not directly on adrenocortical cells . On the contrary, pretreatment with cocaine causes persistent changes in adrenocortical cells.

Brain Res, 1991 Feb 22, 542(1), 147 - 50
Cerebrovascular responsiveness to hypercapnia in intracerebral tissue transplants; Sharkey J et al.; Cerebral blood flow was measured using {14C}iodoantipyrine quantitative autoradiography in rats which had previously undergone unilateral ibotenate-induced nucleus basalis lesion followed by intracortical implantation of foetal basal forebrain cell suspensions . Transplants had no effect upon host cortical blood flow, although within the transplant itself, blood flow was significantly lower than the contralateral site . Both the transplant and host cortex exhibited a similar degree of hyperaemia in response to hypercapnia.

J Chromatogr, 1991 Feb 15, 563(2), 435 - 42
Quantification of mitoxantrone in bone marrow by high-performance liquid chromatography with electrochemical detection; de Vries AJ et al.; An analytical method for the determination of mitoxantrone in bone marrow was developed using high-performance liquid chromatography with electrochemical detection . The extraction procedure was optimized by investigating several factors which potentially could influence the recovery of mitoxantrone from bone marrow cells . The mean recovery of mitoxantrone from rat bone marrow was found to be 81.7% with a coefficient of variation 3.8% . High-performance liquid chromatography was carried out to quantitate mitoxantrone using ametantrone as internal standard . The detection limit of our analytical method amounts to 100 pg on-column, corresponding to 1 ng/ml of cell suspension containing 2 x 10(7) cells and a day-to-day variation of maximally 8% . Storage of bone marrow samples, containing mitoxantrone, for one to fourteen days resulted in a mean recovery of 94%, as compared to freshly analysed samples . Subsequently we studied the pharmacokinetics of mitoxantrone in rat bone marrow . It appeared that after an intravenous bolus injection of mitoxantrone (2.5 mg/kg) in rats, the drug accumulated in the femoral bone marrow for about four days, and thereafter gradually declined.

Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1541 - 5
Microglial progenitors with a high proliferative potential in the embryonic and adult mouse brain; Alliot F et al.; Single cell suspensions, prepared from brain stem, cerebellum, and forebrain parenchyma of embryonic and adult mice, were plated on monolayers of an astroglial cell line derived from a spontaneously immortalized mouse cerebellar culture, the D19 clone . A few of the brain cells adhering to the D19 monolayers were immunoreactive to the Mac-1 antibody, which labels all cells of the monocytic and granulocytic lineages . The Mac-1-positive cells proliferated vigorously and later most of them acquired the F4/80 epitope specific for macrophages and microglia cells . Studies in clonal conditions allowed development of large colonies of about 2 x 10(5) cells that expressed typical microglia markers . Bone marrow Mac-1-positive cells cocultured on D19 monolayers were also induced to proliferate, whereas peritoneal macrophages were not . D19 astrocytes express macrophage colony-stimulating factor (CSF-1) activity at a high level, and their conditioned media induced the proliferation of brain and bone marrow Mac-1-positive cells . A specific anti-CSF-1 antiserum completely blocked bone marrow macrophage progenitor proliferation and significantly reduced the multiplication of microglial precursors induced by the D19-conditioned medium . These data indicate that the embryonic and adult mouse brain parenchyma contains potential progenitors for microglial cells.

J Immunol, 1991 Feb 15, 146(4), 1169 - 74
Accumulation of Th-2-like helper T cells in the conjunctiva of patients with vernal conjunctivitis; Maggi E et al.; A total number of 132 T cell clones (TCC) were obtained by PHA-stimulation of single T cells from mononuclear cell suspensions of conjunctival flogistic infiltrates of three patients with vernal conjunctivitis (VC) . The phenotype and functional properties of these TCC were compared with those of 122 TCC contemporarily established from PB mononuclear cell suspensions of the same patients, 120 TCC established from lymph nodes of three patients with nonspecific hyperplastic lymphoadenitis and 159 TCC established from thyroid lymphocyte infiltrates of three patients with Graves' disease . The great majority of conjunctival TCC displayed the CD4+ CD8- phenotype (CD4/CD8 ratios ranging from 6.1 to 7.0), whereas the mean CD4/CD8 ratios for control TCC ranged from 0.9 to 2.4 . After stimulation with either PHA or PMA plus anti-CD3 mAb, conjunctival TCC differed from control TCC for their ability to produce cytokines . In particular, a large number of conjunctival TCC produced IL-4, but no, or limited amounts of, IFN-gamma, whereas no difference was observed between conjunctival and control TCC with regard to the production of IL-2 . The failure of IFN-gamma production by conjunctival TCC was apparently not caused by delay or block in cytokine production, but actually reflected the lack of IFN-gamma transcription . Virtually all conjunctival TCC able to produce IL-4, but not IFN-gamma, as well as most of those producing both cytokines, provided helper function for IgE synthesis in allogeneic normal B cells . The accumulation in the conjunctiva of patients with vernal conjunctivitis of CD4+ T cells that, apart from the production of IL-2, resembles murine Th2 cells for their profile of cytokine production and helper function suggests a possible role for these cells in the pathogenesis of the disease.

Biochem Pharmacol, 1991 Feb 15, 41(4), 533 - 41
Fluorescent markers for hypoxic cells . A study of novel heterocyclic compounds that undergo bio-reductive binding; Hodgkiss RJ et al.; The bioreductive metabolism and binding of nitroaromatic compounds has been suggested as a method for the identification of hypoxic tumour cells . Bound metabolites of suitable nitroaryl compounds (and some other reducible aromatic compounds) may fluoresce, offering an alternative to radiolabelling or NMR etc . as a diagnostic method . In this paper, the synthesis of some heteroaromatic nitro-compounds is given together with the results obtained from testing of these and other mainly nitroaromatic compounds in vitro as potential bioreductive fluorescent probes for hypoxic cells in tumours . Compounds were incubated with oxygenated or hypoxic mammalian cell suspensions for various times before evaluation of the cellular fluorescence from bioreductive metabolites by fluorescence microscopy and flow cytometry . Among those compounds yielding fluorescent metabolites in cells, considerable variation in hypoxic:oxic differential fluorescence was observed . The in vitro mammalian cell test system showed several of the compounds to be sufficiently promising to merit further investigation in vivo.

Orv Hetil, 1991 Feb 3, 132(5), 245 - 7
{Dermabrasion for tattoo removal complemented by cell suspension for wound healing}; Sebok B et al.; The authors completed the surgical removal of tattoos with epithelization using epidermal cell suspension . The cells were separated from the removed tattood skin . The advantages of this method that there is no need for separate donor skin, the procedure is cheap and good epithelization can be achieved.

Vet Immunol Immunopathol, 1991 Feb, 27(4), 277 - 92
Characterization of bovine haemopoietic progenitor cells using monoclonal antibodies and fluorocytometry; Fritsch G et al.; Monoclonal antibodies against bovine leucocyte cell surface differentiation antigens were used in combination with a fluorescence activated cell sorter to enrich bovine haemopoietic progenitor cells present in bone marrow cell populations prior to in vitro culture . After two sequential centrifugations of the bone marrow cell suspension through Ficoll-Paque, the interface fraction was stained with a cocktail of monoclonal antibodies directed against mature monocytes/macrophages, granulocytes and lymphocytes . Using appropriate electronic window settings on a FACStar Plus, cells with a high 90 degrees light scattering property (granular cells), a low forward light scattering property (erythrocytes and reticulocytes) and cells positive for monoclonal antibodies specific for lineage-restricted leucocyte markers were removed and the negative cell fraction collected . These negatively-selected cells were stained with monoclonal antibodies specific for a pan-leucocyte or a MHC class II marker and the positive cell population was collected in a second sort and subsequently submitted to culture . All erythroid and granulocyte/macrophage colony forming cells expressed MHC class II antigens, as well as the pan-leucocyte antigen . These same progenitors did not bind any of a variety of monoclonal antibodies directed against lineage-specific antigens on lymphocytes, granulocytes or monocytes/macrophages, although they did bind monoclonal antibodies recognizing MHC class I antigens . Between 85% and 91% of the isolated cells seeded were capable of forming erythroid or granulocyte/macrophage colonies within 5 to 10 days, thus increasing the plating efficiency of these cell types in bone marrow populations by at least 60 fold.

J Steroid Biochem Mol Biol, 1991 Feb, 38(2), 119 - 26
Influence of luteinizing hormone-releasing hormone agonists on human mammary carcinoma cell lines and their xenografts; Vincze B et al.; The specific binding of luteinizing hormone-releasing hormone (LH-RH) agonist in estradiol-dependent MCF-7 and estradiol-independent MDA-MB-231 human breast cancer cells has been studied using {3H}Ovurelin {(D-3H-Phe6),des-Gly10-LH-RH- ethylamide} . The results of Scatchard analyses suggest the presence of a single class of receptor sites, both in cell suspensions and membrane fractions . Evaluation of these peptide receptors appears to reflect additional characteristics of biological behaviour of these human breast cancer cells . The synthetic LH-RH agonist Ovurelin {(D-Phe6),des-Gly10-LH-RH-ethylamide} can directly interfere (25-30%) with the proliferation of MDA-MB-231 human breast cancer cells in culture . The inhibitory effect of Ovurelin in vitro was negligible in the MCF-7 cell line . In the in vivo experiments the treated immunosuppressed mice bearing either MCF-7 or MDA-MB-231 xenografts responded to the high-dose LH-RH analogue Zoladex depot and Decapeptyl depot therapy . Since the MDA-MB-231 tumour was found to be ER-negative it seems possible that the regression of this xenograft results from the direct antitumor action of the LH-RH agonist.

Exp Hematol, 1991 Feb, 19(2), 122 - 7
Effect of lipopolysaccharide on the production of colony-stimulating factors by the stromal cells in long-term bone marrow culture; Wang SY et al.; The production of colony-stimulating factors (CSFs) by murine bone marrow stromal cells was studied with Dexter long-term bone marrow culture (LTBMC) . For induction of CSF release, various concentrations (0.5-40.0 microgram/ml) of bacterial lipopolysaccharide (LPS) were added to nonrecharged 3-week-old LTBMCs consisting of an intact or macrophage-depleted adherent cell layer . The depletion of monocytes/macrophages from freshly prepared bone marrow cell suspension was performed by carbonyl-iron incorporation before establishment of LTBMC . The supernatants (Sup) of normal LTBMCs contained a low level of macrophage colony-stimulating factor (M-CSF) that was produced by the adherent cells but not by the nonadherent cell elements . No colony inhibitor was found in the Sup of LTBMCs, whereas a colony-promoting activity (CPA) was detected in medium conditioned by the adherent marrow cells (AC-CM) . CPA could enhance the colony formation of myeloid progenitor cells when used in combination with recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) . The production of CSFs peaked at about 24 h after refeeding, but it then declined to only half the optimal activity at the end of the week . Addition of LPS to the intact LTBMC invariably increased the production of a GM-CSF-like cytokine . The release of this cytokine was dose dependent and peaked at a dosage of 20 micrograms/ml of LPS at 24 h after treatment . In contrast, macrophage-depleted marrow-adherent cells failed to respond to LPS for CSF secretion . These results suggest that LPS can stimulate marrow macrophages to directly release CSF or to potentiate the production of CSF by other stromal cells.

Ann Thorac Surg, 1991 Feb, 51(2), 204 - 7
Endothelial cell viability in the rat aortic wall; Christy JP et al.; Aortic allografts may offer advantages over prosthetic materials for aortic valve replacement or reconstruction with a valved aortic conduit . Cellular viability may partly determine long-term allograft performance . To evaluate endothelial cell viability in a rat model, valved aortic conduits were subjected to collagenase digestion . The resulting endothelial cell suspension was labeled with Griffonia simplicifolia agglutinin-fluorescein isothiocyanate (GSA-FITC), a marker specific for vascular endothelial cells of the rat . The cells were then incubated with propidium iodide, which is excluded by viable cells . Flow cytometry evaluated endothelial cell viability by determination of percentage of GSA-FITC-positive cells that were negative for propidium iodide . Aortas were studied immediately after harvest or after storage at 4 degrees C in a nutrient medium for 3 to 21 days . Percentage of viable endothelial cells showed a progressive decline with increasing duration of storage . These results demonstrate flow cytometric measurement of endothelial cell viability, a factor of possible importance in assessing allograft storage methods, and show that endothelial viability declines with prolonged storage at 4 degrees C in a nutrient medium.

J Neuroimmunol, 1991 Feb, 31(2), 165 - 77
T cell immunity and interferon-gamma secretion during experimental allergic encephalomyelitis in Lewis rats; Mustafa MI et al.; An immunospot assay that detects single secretory cells was used to enumerate interferon-gamma secreting cells (IFN-gamma-sc) in mononuclear cell suspensions from the central nervous system (CNS) and peripheral lymphoid organs after actively induced experimental allergic encephalomyelitis (EAE) in Lewis rats . In the CNS compartment there was a significant increase in the number of IFN-gamma-sc preceding the onset of the clinical signs of EAE . Both in rats with EAE and rats immunized with Freund's complete adjuvant (FCA) the number of IFN-gamma-sc increased in peripheral lymphoid organs, as compared to non-immunized controls . In view of the potent immunoregulatory effects of IFN-gamma, its intra-CNS secretion may play a crucial role for clinicopathological events in EAE . To study the numbers of primed T cells that in response to myelin antigens produced IFN-gamma, mononuclear cell suspensions from peripheral lymphoid organs were precultured to allow for antigen uptake, presentation and T cell triggering, followed by enumeration of IFN-gamma-sc . T cells responding to a peptide of myelin basic protein (MBP) that previously have been shown encephalitogenic in Lewis rats, appeared initially and were quantitatively dominant over the course of EAE . Later, T cell reactivities to multiple regions of MBP appeared, showing that the concept of immunodominance in EAE is non-absolute and time dependent . Splenocyte cultures from EAE rats exposed to the different antigens showed a reduced number of IFN-gamma-sc compared to cultures not exposed to antigen, suggesting an antigen-induced suppression of T cell effector molecules.

Blood, 1991 Feb 1, 77(3), 569 - 78
IgE-mediated anaphylactic degranulation of isolated human skin mast cells; Dvorak AM et al.; Isolated human skin mast cells (HSMC) were prepared and cultured overnight before functional and electron microscopic studies . Mast cell suspensions were examined after stimulation with anti-IgE to produce anaphylactic degranulation or examined in buffer-incubated controls . Histamine release was measured in replicate samples . Control, isolated HSMC studied by electron microscopy were well preserved and fully granulated . Although all granule patterns reported for human mast cells were found, crystal granules were the most prevalent, as is true for HSMC in situ . Individual mast cells containing both crystal and scroll granules occurred . Lipid bodies were rare, as in HSMC in situ . Control, isolated mast cells did not express granule changes associated with either piecemeal degranulation or recovery during wound healing in situ; nor were morphologic changes of anaphylactic degranulation present . Spontaneous histamine release was 0% in control samples . Anaphylactic degranulation of isolated HSMC was accompanied by 24% maximum histamine release and characteristically showed extrusion of altered, membrane-free granules through multiple pores in the plasma membrane to the exterior of the cell . Other morphologic aspects of anaphylactic degranulation, as expressed in isolated human lung mast cells, were also present . These events included granule swelling, fusion, alteration of matrix contents, degranulation channel formation, pore formation, and shedding of granules, membranes, and surface processes . The ultrastructural morphology of isolated HSMC and their IgE-mediated degranulation shows some differences from similar studies of isolated human lung mast cells and of human lung and gut mast cells in biopsy samples . These differences include crystal granules as the predominant granule pattern, minor numbers of lipid bodies, and extrusion of granules during anaphylactic degranulation as characteristic for HSMC . By contrast, isolated human lung and gut mast cells have more scroll granules and particle granules, respectively, and more lipid bodies . In isolated human lung mast cells, anaphylactic degranulation is almost exclusively an intracellular fusion event characterized by the formation of complex degranulation channels within which altered granule matrix materials solubilize . In addition to morphologic differences between mast cells of skin, lung, or gut origin, functional differences have also been reported among mast cells of these organs . The ultrastructural morphology of isolated HSMC is identical to that of skin mast cells in biopsy samples, thereby validating the usefulness of this new source of HSMC for correlative functional and morphologic studies.

Exp Neurol, 1991 Feb, 111(2), 166 - 74
Blood-brain barrier permeability is not altered by allograft or xenograft fetal neural cell suspension grafts; Geist MJ et al.; Alterations in blood-brain barrier (BBB) function after brain grafting seem dependent on the donor phenotype and possibly on the grafting technique . Intracerebral blood grafts of nonneural tissue permanently disrupt the host BBB, while fetal neural block grafts probably do not . Cell suspensions, an alternative technique in brain grafting, disrupt the extracellular matrix of the graft . Fetal cell suspension allografts appear to form a functional BBB . We confirm and extend this finding to include fetal neural xenografts . Allograft and xenograft fetal neural cell suspensions were intracerebrally injected, and the BBB was examined using intravenous horseradish peroxidase (HRP) . Neither graft type showed disruption of the BBB at the graft site from 2 weeks to more than 6 months after grafting . Vascular supply was prominent at all time points . Xenograft survival was improved with cyclosporine, yet cyclosporine did not affect BBB permeability . Cyclosporine did not interfere with repair of the BBB after simple brain trauma was induced by a control injection of saline . We conclude that fetal allograft and xenograft neural cell suspensions rapidly form and maintain a BBB impermeable to intravenous HRP.

J Immunol Methods, 1991 Jan 24, 136(1), 125 - 31
An improved method for the detection of DNA fragmentation; Facchinetti A et al.; An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis . DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe . The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation . In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold . This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.

Biochim Biophys Acta, 1991 Jan 23, 1073(1), 213 - 20
New substrates of the multispecific bile acid transporter in liver cells: interference of some linear renin inhibiting peptides with transport protein(s) for bile acids; Bertrams AA et al.; Interactions between some stable linear peptides with renin inhibitory activity and a multispecific transport system in the basolateral plasma membrane of liver cells was studied on cell suspensions . The peptides used in our experiments were taken up by liver cells and subsequently eliminated without any biotransformation (e.g., proteolysis) . No degradation products could be detected in the extracellular medium by thin-layer chromatography . All peptides tested inhibited the uptake of physiological and of some foreign substrates of the multispecific bile acid transporter (MT) . The phalloidin response of liver cells was also inhibited to a similar degree in a concentration-dependent manner . The potency of inhibition did not correlate with the lipophilic properties of the peptides . On the other hand a tight correlation could be documented between the inhibition of cholate transport and that of the phalloidin response . Transport inhibition of typical substrates of the MT by the above renin inhibitors was competitive . In contrast, the transport of a typical substrate of the bilirubin carrier (rifampicin), of amino acids (alpha-aminoisobutyric acid), long chain fatty acids (oleic acid) and cationic compounds (thiamin hydrochloride) was not inhibited by the same renin inhibitors . These results indicate that linear renin inhibiting peptides are taken up into liver cells by carrier proteins related to the MT.

Int J Dev Neurosci, 1991, 9(4), 427 - 37
Individual C6 glioma cells migrate in adult rat brain after neural homografting; Goldberg WJ et al.; Cultured C6 glioma cells were prelabeled with the plant lectin Phaseolus vulgaris leuco-agglutinin (PHAL) and grafted as a cell suspension (10(6) cells in 5.0 microliters) into freshly made cortical implantation pockets in adult host rats . Animals were killed 1-21 days post-implantation (DPI) . The brains were removed, dehydrated, embedded in paraffin and sectioned at 8 microns . Paraffin sections were processed for light level immunofluorescent double labeling for PHAL, a marker for graft derived cells, and glial fibrillary acidic protein (GFAP), a specific marker for C6 glioma cells and astrocytes . Cells positive for both PHAL and GFAP were graft-derived C6 cells . By 7 DPI a large mass developed which extended above the surface of the brain and invaded (displacement of host tissue by a cell mass) the host parenchyma . This mass increased in size over the next 14 days . The invading tumor mass contained double labeled cells at all time periods examined . In addition to the invasion process, grafted C6 cells spread through the host parenchyma by migration (movement of single cells) . Individual graft-derived C6 (GFAP/PHAL positive) cells migrated into host cortex surrounding the implantation pocket, corpus callosum ventral to the implantation pocket, ipsilateral internal capsule and bilaterally in the habenula.

Neuroscience, 1991, 42(2), 407 - 26
Development of intrastriatal striatal grafts and their afferent innervation from the host; Labandeira-Garcia JL et al.; The morphological maturation of cell suspension grafts of fetal striatal tissue (obtained from 14-15-day-old rat fetuses) was followed from two days to eight weeks after implantation into intact and ibotenic acid-lesioned striata of adult rats . The development of host afferent innervation of the grafts from the substantia nigra (tyrosine hydroxylase immunoreactive), mesencephalic raphe (serotonin immunoreactive), and the frontal cortex (anterogradely labelled with Phaseolus vulgaris leucoagglutinin) were revealed by immunohistochemistry . During the first weeks post-grafting, the striatal implants consisted of a mixture of mature- and immature-looking cell clusters . Grafts implanted into ibotenic acid-lesioned striatum grew rapidly (about five-fold) in volume over the first week . The areas of immature (probably proliferating) cells gradually disappeared, and by six to eight weeks the grafts had a fully mature appearance with patches of neurons which stained densely for DARPP-32 (i.e . were striatum-like) embedded within areas of essentially DARPP-32-negative (i.e . non-striatum-like) tissue . Peripheral clusters of grafted cells gradually intermingled with nearby areas of the surrounding lesioned host, and already by two to four days after implantation, coarse and densely immunoreactive host fibres from the substantia nigra, mesencephalic raphe and frontal cortex were present within the grafts . By four to five days the first DARPP-32-immunoreactive neurons appeared in patches within the mature portions of the grafts, and one to two days later the tyrosine hydroxylase-positive fibres began to sprout thin axons selectively within the DARPP-32-positive patches . Similarly, the serotonergic and cortical fibres in the grafts increased in number over the next two weeks, but they showed no preference for the DARPP-32-positive regions . Rich terminal networks were established by two to three weeks post-grafting, and by six to eight weeks the nigral, raphe and cortical afferents had reached terminal densities similar to those seen previously in long-term surviving grafts . Grafts implanted into dopamine-denervated hosts showed a normal morphological maturation of both DARPP-32-positive and -negative areas, although no tyrosine hydroxylase-positive innervation appeared within the grafts . Grafts implanted into non-lesioned striata did not grow beyond their initial size . The implanted cells showed less intermingling with the surrounding host striatum, thus resulting in sharply delineated graft-host borders . DARPP-32-positive patches developed, but they were smaller in size and generally present only in the most peripheral graft portions.(ABSTRACT TRUNCATED AT 400 WORDS)

J Chromatogr, 1991 Jan 4, 536(1-2), 265 - 72
Quantitation of adenosine, inosine and hypoxanthine in biological samples by microbore-column isocratic high-performance liquid chromatography; Gayden RH et al.; This paper describes a simple and sensitive high-performance liquid chromatographic method for measuring adenosine, inosine and hypoxanthine in cell suspensions . The method involves direct injection of the filtered sample on a microbore C18 reversed-phase column with UV detection at 259 nm . The mobile phase consisted of 125 mM potassium dihydrogenphosphate, 1.0 mM tetrabutylammonium hydrogen-sulfate, 1.5% acetonitrile and 20 mM triethylamine, pH 6.5 . The minimum detectable amounts (signal-to-noise ratio of 3:1) were 2.0 pmol of adenosine, 2.5 pmol inosine and 3.5 pmol of hypoxanthine . The limits of quantitation were 2.9 +/- 0.2 pmol for adenosine, 4.2 +/- 0.3 pmol for inosine and 4.9 +/- 0.4 pmol for hypoxanthine . This method was used to quantitate adenosine release by dispersed rat renal outer medullary cells (tubules) under conditions of normoxia and hypoxia.

Brain Res, 1991 Jan 4, 538(1), 1 - 8
A rapid method to quantify neurons in mixed cultures based on the specific binding of {3H}ouabain to neuronal Na+,K(+)-ATPase; Markwell MA et al.; The high-affinity binding of {3H}ouabain to Na+,K(+)-ATPase was characterized in primary cultures of hippocampal neurons grown on feeder layers of astrocytes . The specific binding of {3H}ouabain was found to be time-dependent, high-affinity (apparent Kd = 8.5 nM), saturable (Bmax = 20.6 pmol/mg protein), dependent upon the presence of ATP, inhibited by K+, and directly proportional to neuronal, but not glial, cell number . Similar results were obtained using either sonicated cell suspensions or intact whole cells in culture . At the concentration of neurons routinely used, the specific binding of {3H}ouabain to the astrocyte feeder layer constituted less than 10% of total specific binding . Agents that selectively kill neurons rather than glia, such as the excitotoxins N-methyl-D-aspartate (NMDA) and kainate, reduced the amount of {3H}ouabain specifically bound in mixed cultures in a time- and concentration-dependent manner . Measurement of high-affinity {3H}ouabain binding to the neuronal form of Na+,K(+)-ATPase provides a simple, rapid, and reproducible method to quantify neurons in mixed culture.

Eur J Obstet Gynecol Reprod Biol, 1991 Jan 4, 38(1), 59 - 62
DNA ploidy and the expression of tissue-carcinoembryonic antigen in grade III carcinoma of the breast; van Dam PA et al.; A series of 50 Stage I and II, Grade III ductal carcinomas of the breast was characterized by DNA flow cytometry and further analyzed with a monoclonal antibody for carcinoembryonic antigen (CEA) . The antigen was detected with the indirect immunoperoxidase technique on paraffin sections and on cytologic smears of the cell suspensions that were used for flow cytometric DNA analysis . A significant higher incidence of CEA-positivity in cytologic smears and tissue sections was found in DNA-diploid tumors (13/18 and 12/18, respectively) than in DNA-aneuploid tumors (13/22 and 12/32, respectively) (P less than 0.05 and P less than 0.025, respectively).

J Urol (Paris), 1991, 97(2), 63 - 71
{A new therapeutic concept in the treatment of metastatic cancers of the kidney: the TIL (tumor infiltrating lymphocytes)}; Peyret C; The metastases of cancers of the kidneys raise difficult problems for treatment . In fact, the classical treatments combining surgery, radiation therapy, chemotherapy and hormono therapy are hardly effective to treat this condition . This explains the interest arising from the new therapeutic approach of immunotherapy, which is just beginning and offers appealing prospects . Experimental studies have shown that the cells with the highest cytolytic activity are tumor-infiltrating lymphocytes (TIL) . The TIL are obtained by cultivating a mixed cell suspension--tumor cells/lymphocytes-infiltrating the tumor . After a three-weeks culture with Interleukin 2, the tumoral cells have been completely destroyed, and only a pure population of activated lymphocytes remains, which can be injected back to the patient . The first clinical trials with these activated lymphocytes have yielded encouraging results . Efforts are currently made to improve the therapeutic efficacy of this approach.

Folia Biol (Praha), 1991, 37(1), 52 - 4
Cultivation of psoriatic keratinocytes and an attempt to isolate and detect a specific psoriatic antigen; Novotny J et al.; Keratinocytes from psoriatic lesion and healthy skin region obtained from the skin of a psoriatic patient were cultivated on lethally irradiated 3T3 cells . They multiplied twice as quickly as normal keratinocytes from healthy skin . Ten antigen fractions were prepared from cell suspensions of psoriatic keratinocytes and from the medium left after their cultivation . Later, they were evaluated in ELISA tests performed with the sera of psoriatic patients and healthy persons . The presence of specific antigens was not demonstrated.

Acta Otolaryngol, 1991, 111(2), 410 - 3
A new concept for reconstruction of atresias of larynx and trachea: lining of wound surfaces with autologous isolated respiratory epithelial cells; Gerhardt HJ et al.; A new method, first applied two years ago in our clinic, has proved to be reliable for achieving a rapid reepithelialisation of epithelial defects after scar removal inside the trachea . The defect is seeded with isolated respiratory epithelial cells harvested the day before from the ethmoid . Isolation of epithelial cells was achieved by keeping the mucosa in 0.25% trypsin buffer solution under room temperature for about 16 h . Afterwards, the epithelial layer was separated from the submucosa using small forceps and knife . Cells were then isolated by pipetting . For seeding the wound the surface was covered with silastic sheeting and the cell suspension then injected into the cleft between both of them . Cell distribution occurred by capillary attraction . The tracheal lumen was maintained by inserting a silastic stent for about three weeks . So far, 10 patients between 6 and 45 years have been treated in this way . In 4 patients the tracheal wall additionally had to be stabilized using rip cartilage . Only in one case above the tracheostoma, considerable scar formation occurred again requiring a second operation some months later . In 8 patients decannulation was meanwhile possible.

Acta Neuropathol (Berl), 1991, 81(3), 303 - 11
Implantation of neuronal suspensions into contusive injury sites in the adult rat spinal cord; Hoovler DW et al.; Implants of various types of neuronal and nonneuronal tissue have shown promise for the amelioration of certain disorders of the adult mammalian brain . Implants may also have therapeutic potential for some lesions of the spinal cord . To examine the feasibility of implantation for clinically relevant spinal cord injuries, we have implanted cells into injury sites produced by a well-characterized and standardized rat model of contusive injury . To reduce the possibility of the implantation procedure itself causing damage to the spinal cord, the tissue was dissociated and a suspension of cells introduced into the cord via a small bore needle . To test the implantation procedure, dissociated adult rat dorsal root ganglia were used because of the ease with which these neurons could be distinguished after implantation . The extent to which functional deficits were produced or exacerbated by the implantation procedure was assessed by behavioral tests of groups of rats that had been implanted (implant controls), contused (injury only) or contused and implanted (injury-implant) . Survival of the implanted neurons was assessed by quantitative morphological analysis of histological sections taken through the injury/implant sites at different times following injury . In addition, the histopathology of the contusive injury sites was compared for rats that had or had not received immediate or delayed implants . Results indicated that cell suspensions could be implanted into the spinal cord without causing a functional deficit in an otherwise uninjured animal or exacerbating a standardized incomplete contusive injury . Implanted neurons survived for at least 4 weeks in all contusion sites whether implantation was performed immediately following injury or after a delay of 1 week.(ABSTRACT TRUNCATED AT 250 WORDS)

Patol Fiziol Eksp Ter, 1991 Jan-Feb, (1), 36 - 7
{Changes of blood flow structure in precapillary microvessels during significant slow-down of flow}; Lominadze DG et al.; The experiments were carried out with a special "biological model" in which the rabbits' red blood cell suspension possessing low hematocrit circulated in frogs' mesenterial microvessels . Red blood cell behaviour was investigated in microvessels of 19-45 microns in diameter under conditions of arbitrarily changed flow velocity in mesenterial microvessels . Automatic frame-to-frame analysis of cinematographic films with the texture analysis system (Ernst Leitz, FRG) showed that the velocity fluctuations of individual red blood cells and their radial displacements increased significantly, while their velocity profile became blunt, during slowing-down of flow from 0.7 to 0.2 mm/s . Thus the normal blood flow structure in microvessels becomes disordered under ischemic conditions entailing disturbance of blood rheological properties and creating additionally increased resistance in the vessels.

Int J Hyperthermia, 1991 Jan-Feb, 7(1), 125 - 30
Whole-body hyperthermia maintains the secondary immune response of specific antitumour immune T cells; Ozawa H et al.; The effect of hyperthermia on the spleen cells of Meth A-hyperimmunized BALB/c mice (Meth A-Im-SPL) was examined . Three kinds of heating were employed: (a) a cell suspension of Meth A-Im-SPL was directly heated for 1 h at 41 degrees C; (b) the whole body of Meth A-hyperimmunized mice was heated in the same manner--the antitumour activity of Meth A-Im-SPL was examined by Winn assay after heating; (c) the whole body of BALB/c mice intradermally inoculated with a mixture of Meth A-Im-SPL and Meth A cells (Winn assay) was heated in the same manner . The results showed that the antitumour activity of Meth A-Im-SPL was not affected by heating in vivo (Experiments B and C), although it was affected in vitro (Experiment A) . Furthermore, slight augmentation of the antitumour activity of Meth A-Im-SPL was observed on heating in vivo . This shows the possibility of combination therapy involving adoptive transfer and whole-body hyperthermia.

Cytometry, 1991, 12(2), 188 - 92
Retinoblastoma cell cycling; Kaleta-Michaels S et al.; Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter . The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors . Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material . Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd . BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy . Approximately 14% of viable cells were in the synthesis-phase of the cell cycle . The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48) . DNA analysis of these tumors was also performed using flow cytometry . Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak) . There was no correlation between abnormal DNA content and the percent of cells in synthesis.

Cytometry, 1991, 12(2), 157 - 66
Progesterone receptor detection and quantification in breast tumors by bivariate immunofluorescence/DNA flow cytometry; Remvikos Y et al.; A method was developed for the detection of progesterone receptors (PgR) by flow cytometry (FCM) in cell suspensions obtained from mechanically dispersed fragments of operated breast cancers . Two monoclonal antibodies were tested for sensitivity and specificity on four breast cancer cell lines of known PgR expression and a calibration curve thus established . A simple procedure was used to calculate the level of PgR expression, taking into account the relative displacement of total cellular fluorescence compared to nonspecific fluorescence for each sample and the average DNA content of the cells derived from the corresponding histograms . The PgR-specific immunofluorescence of the tumor specimens measured in arbitrary units (channels) was then transformed to fmoles/mg DNA by comparison with the calibration curve . The FCM-derived results were compared with those of a conventional immunoenzymatic PgR assay on 30 surgical samples . PgR content ranged from 10 to 22,000 fmoles/mg DNA and linear regression analysis yielded a good correlation (r = 0.86) . With a threshold of positivity of 300 fmoles/mg DNA, the two methods concurred for 28 of 30 tumors (93%) . Nine specimens were analyzed repeatedly, showing good reproducibility . This method could prove to be more useful than the biochemical assays on homogenates, since it allows the simultaneous analysis of receptor expression in individual cells and of DNA index (ploidy).

Acta Histochem, 1991, 90(1), 43 - 50
Macrophage subsets in the rat gut: an immunohistochemical and enzyme-histochemical study; Sminia T et al.; Macrophages' subsets including dendritic cells were characterized in situ and in cell suspensions of Peyer's patches, intestinal villi, and mesenteric lymph nodes using monoclonal antibodies, lectins, and enzyme-histochemistry . By combined labelling procedures, it was found that Peyer's patches comprise dendritic cells in addition to 2 types of macrophages; dendritic cells were not observed among the various macrophage-like cells in the lamina propria of the villi . The lectins Dolichos biflorus and Griffonia simplicifolia I agglutinin appeared to be useful for discriminating between several subtypes of macrophages.

Ter Arkh, 1991, 63(2), 84 - 8
{Cell-membrane aspects of the pathogenesis of hypoxia in vibration disease induced by local vibration}; Sukharevskaia TM et al.; A study was made of the blood and tissue oxygen regime in patients with vibratory disease (VD) induced by local vibration and of the importance of lipid peroxidation (LPO) in oxygenation disorders . Venous hyperoxia, a decrease of the arteriovenous difference according to oxygen, the percentage of oxygen utilization by tissues, shift of the acid-base balance towards metabolic acidosis were established, attesting to tissue hypoxia that increased with the gravity of VD . The importance of a steady activation of LPO and depression of the antioxidant system in the pathogenesis of hypoxia associated with VD was supported by the correlation analysis data on oxygen balance and LPO, the functional and metabolic characteristics of red blood cells (according to the viscosity of red blood cell suspension and the content in the cells of SH-groups, lipoproteins and histidine) and platelets (according to aggregation in response to ADP and thrombin) as well as by the level of blood serum fluorescence . The authors provide evidence for the use of antioxidants (a complex of alpha-tocopherol with ascorbic acid and methionine and calcium antagonists of the nifedipine group), giving a membranostabilizing effect, in multimodality treatment of patients afflicted with VD.

Gastroenterol Clin Biol, 1991, 15(3), 194 - 8
Phenotypes and spontaneous immunoglobulin production in mononuclear cells suspensions isolated from colonic biopsies of patients with mild active and quiescent ulcerative colitis; Squarcia O et al.; Lamina propria mononuclear cells can be isolated from mucosal specimens of human colon . In the present study, we have explored whether both the phenotypes and functional properties can be studied in lamina propria mononuclear cell suspensions isolated from the same set of endoscopic biopsies in patients with ulcerative colitis . The counts of CD11b+ lamina propria mononuclear cells in mild active ulcerative colitis were significantly higher than those of both quiescent ulcerative colitis and controls . Similarly, the CD16+ and the CD19+ lamina propria mononuclear cells were significantly increased in mild ulcerative colitis patients in comparison to both quiescent ulcerative colitis and control lamina propria mononuclear cells . Lamina propria mononuclear cells from all the biopsy samples appeared to produce detectable amounts of immunoglobulins of the three classes . The production of IgG in mild ulcerative colitis cultures was significantly higher than that observed in quiescent ulcerative colitis and controls . In contrast, the production of IgA in active ulcerative colitis lamina propria mononuclear cell cultures appeared to be significantly lower than that of both quiescent ulcerative colitis and controls . This study shows that morphology, phenotypes, and functional properties can be assessed in lamina propria mononuclear cell suspensions obtained from the same set of endoscopic biopsy samples . We have also shown that changes in phenotypes and functional status of lamina propria mononuclear cells occurred in mild active ulcerative colitis while no significant abnormality of these parameters was found in quiescent ulcerative colitis . This indicates that a normalization of mucosal immune functions occurs in ulcerative colitis patients when complete clinical and histological remission is achieved.

Mutagenesis, 1991 Jan, 6(1), 3 - 9
Partial pronase digestion of rat gastric mucosa isolates cells undergoing replicative DNA synthesis; Larsson H et al.; A pronase digestion procedure for the isolation of gastric mucosal cells was evaluated for its usefulness in measuring unscheduled DNA synthesis (UDS) . The method has been claimed to be suited for assessing the genotoxicity potential of compounds . Compounds were given orally to rats . After 13 h {3H}thymidine was injected and after another hour the animals were killed . The dissected stomachs were treated with pronase for 45 min and the incorporation of radioactivity into DNA was determined . Autoradiography was also performed both on the mucosa and on the isolated cell suspension . The cell suspensions were found to include cells undergoing normal replicative (S phase) DNA synthesis . Of all cells isolated, 4.8 +/- 0.6% consisted of S phase cells . The total amount of DNA recovered (corresponding to the number of cells isolated) was variable and ranged from 20 to 671 micrograms DNA, i.e . approximately 30-fold variation . Neither omeprazole (10, 30 and 80 mg/kg) nor ranitidine (215 mg/kg) had any effect on {3H}thymidine incorporation . The known carcinogen 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) increased incorporation . Refeeding of fasted animals increased incorporation of {3H}thymidine almost 4-fold, showing that animal feeding status influences incorporation . Hydroxyurea, the selective inhibitor of S phase DNA synthesis, inhibited {3H}thymidine incorporation in control and omeprazole-treated animals as well as that induced by MNNG by 92-98% . The results clearly show that the pronase digestion procedure used releases cells undergoing normal, replicative DNA synthesis and can therefore neither be used for measurements of UDS nor for the assessment of the genotoxic potential of drugs.

Cytometry, 1991, 12(3), 268 - 74
Sorting minor subpopulations of cells: use of fluorescence as the triggering signal; McCoy JP Jr et al.; Flow cytometric cell sorting is commonly used to obtain purified subpopulations of cells for use in in vitro and in vivo assays . This can be time-consuming if the subpopulations of interest represent very low percentages of the cell suspension under study . Often the desired subpopulations are identified by two-color immunofluorescence staining . Generally, cell sorting is performed with a flow cytometer configured to trigger on light scatter signals, then sort windows are set based upon the signals from both fluorescent markers . We demonstrate that triggering the cytometer with the fluorescence signal from antibody staining common to both of the desired subpopulations, then sorting the subpopulations based upon staining of a second marker, substantially increases the speed of cell sorting vis-a-vis traditional methods . This is because undesired events are not analysed, allowing an increase in the throughput rate . While desired subpopulations of cells can be obtained by this method, undesired (i.e., nonstaining) cell "contaminants" increase and may require a second sort . The combined time for the initial enrichment sort and a second sort can be less than sorting once using standard methodology . Alternatively, the degree of contamination may be controlled by adjusting the concentration of the cell suspension and by the sample flow rate.

Cancer Immunol Immunother, 1991, 33(1), 65 - 9
Application of isoelectric focusing for studies of major histocompatibility complex class I antigen expression on human carcinomas and sarcomas; Wang P et al.; By one-dimension isoelectric focusing we analysed the major histocompatibility complex class I antigen expression on human tumours . Blood lymphocytes of the patients, processed in parallel, served as a basis for comparison . The prerequisite for the analysis is the preparation of metabolically active tumour cell suspensions devoid of significant leucocyte contamination . The method was found to be suitable for study of the expression of HLA alleles on ex vivo tumour cells and allowed the detection of changes imposed by in vitro treatment with interferon gamma and tumour necrosis factor alpha.

Blood Cells, 1991, 17(1), 105 - 21; discussion 121-5
The role of the blood island during normal and 5-fluorouracil-perturbed hemopoiesis; Vogt C et al.; The blood island is found in all hemopoietic organs . It consists of a central macrophage surrounded by other hemopoietic cells, notably developing erythroblasts and cells of the myeloid lineage . In this report, several lines of investigation are combined to discuss the role of the erythroblastic island in erythropoietic differentiation and to question the source and role of circulating erythropoietin (epo) . In both cultured and fresh normal bone marrow macrophages, we demonstrate epo gene expression and, simultaneously, localize epo intracellularly . The following observations have also been made: a) red cell production initiated in vitro in mouse fetal liver and normal bone marrow cell suspensions by the addition of horse serum usually occurs in the presence of macrophages that form the blood island functional unit; however, epo can only be detected in the fetal liver supernatants and not in the supernatants derived from bone marrow cultures; b) in 5-fluorouracil-treated mice, circulating epo levels, measured immunologically by ELISA, RIA, and biologically by the in vitro stimulation of mouse bone marrow CFU-E, converged between days 10 and 12 to give epo concentrations that did not correspond with the degree of anemia; c) when bone marrow cells from day 5, 5-fluorouracil-treated mice are placed in culture for 24 hours, an active erythropoiesis is observed in which developing and mature erythrocytes surround macrophages expressing the epo gene (blood islands); at this time immunologically active epo (but little biologically active epo) is present . After 14 days in culture, no erythropoiesis is observed and the culture consists of macrophages, some of which are expressing the epo gene, and biologically, rather than immunologically, active epo is present . These results show that the blood island is a necessary functional unit for active erythropoiesis.

Anticancer Res, 1991 Jan-Feb, 11(1), 343 - 6
Flameless atomic absorption spectrophotometry analysis of cell-linked platinum after wet ashing; Tabary T et al.; Wet ashing of K562 cell suspensions by means of a sulphonitroperchloric acid digestion for electrothermal atomic absorption spectrometry of platinum has been achieved . The limit of detection was about 1 ng platinum per 10(6) cells . Platinum concentrations in K562 cells were measured after exposure to platinum coordination complexes such as cis-dichlorodiamminoplatinum (DDP) and Trans-1 diamminocyclohexanooxalatoplatinum (1-OHP) . Cell linked platinum was measured after a 24 hours exposure to a concentration of 6.8 nM ml-1 of both forms 1-OHP and DDP (i.e . 1.3 micrograms platinum per ml) . Platinum concentrations were found to be respectively (mean +/- S.D.) 14.8 +/- 2.7 and 10.0 +/- 4.0 ng platinum per 10(6) cells . These 1-OHP and DDP concentrations were cytotoxic and about twenty times the 50 percent cell growth inhibitory concentrations (0.45 nM ml-1 and 0.33 nM ml-1 respectively).

In Vitro Cell Dev Biol, 1991 Jan, 27(1), 86 - 8
Gene transfer into mammalian cells by rapid freezing; Sasaki K et al.; In this paper, we describe a simple technique to introduce DNA into cells through cracks and/or pores in cell membranes caused by intracellular ice crystal formation induced by liquid nitrogen . We mixed mouse BALB 3T3 cells and pSV2-neo DNA and froze the cell suspension under various conditions to determine those optimum for the introduction of DNA into mammalian cells . We found that brief treatment with liquid nitrogen, which showed only moderate cell killing, resulted in the induction of G-418 resistant colonies . These results suggest that this new technique is useful for transfection of genes into mammalian cells.

Cancer Chemother Pharmacol, 1991, 27(6), 417 - 22
The influence of tumor cell density on cellular accumulation of doxorubicin or cisplatin in vitro; Takemura Y et al.; The effect of tumor cell density on the cellular pharmacokinetics of doxorubicin (DXR) and cisplatin (CDDP) was studied using MOLT-3 human acute lymphoblastic leukemia cells . As determined by the MTT assay, the growth-inhibitory effect of DXR was approx . 40 times lower when cell density was increased from 10(6) to 10(8) cells/ml (positive inoculum effect), whereas little or no influence of cell density was observed in CDDP-induced cell-growth inhibition . As measured by high-performance liquid chromatography using a fluorescence detector, the cellular accumulation of DXR showed 6- and 18-fold decreases after 1 h incubation when the cells were concentrated from 10(6) to 10(7) and 10(8) cells/ml, respectively . Only at low cell density (10(6) cells/ml) did the amount of DXR in the cells increase with increasing exposure times of up to 6 h . The DXR concentration in the supernatant that was separated from a cell suspension showing a density of 10(8) cells/ml fell to 20% of that obtained at 10(6) cells/ml . The metabolites of DXR, including Adriamycinol and Adriamycinone, were not detectable in the cell extracts or supernatants at any cell density examined . In contrast, the cellular accumulation of CDDP calculated from the platinum concentration, which was measured with a flameless atomic absorption spectrophotometer, was essentially identical at all cell densities examined; moreover, extension of the exposure period resulted in a linear increase in the amount of CDDP in the cells . CDDP concentrations in the supernatants were equally retained, irrespective of cell densities . These observations indicate that the positive inoculum effect shown in DXR-induced cell-growth inhibition results from the decreased cellular accumulation of the drug at high cell densities . We found no influence for cell density on the cellular accumulation of CDDP that might be relevant to the therapeutic potentiation of this drug at high tumor-cell density.

Arch Oral Biol, 1991, 36(1), 77 - 83
Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax; Bozner P et al.; Proteolytic activities in crude extracts and culture filtrates from Trichomonas tenax were determined using hide powder azure as substrate and the proteinase profiles in both samples were analysed in SDS-polyacrylamide gels containing copolymerized gelatin . The enzyme activity in the crude extract was detected over a broad pH range and was strongly activated by dithiothreitol, mainly in the pH range 5-8, and inhibited by cysteine proteinase inhibitors . Extracellular enzyme activity in culture filtrates was SH-dependent and increased continuously during incubation of the cell suspension, suggesting proteinase release . A total of seven distinct proteolytic bands could be detected in crude preparations . Three of these, with apparent Mr values 35,000, 45,000 and 56,000 and a pH optimum of 4-7, were SH-dependent and their inhibitory sensitivities were characteristic for cysteine proteinases . The 45,000 and 56,000 proteinases probably corresponded to those found in the culture filtrates . Proteolytic bands with apparent Mr 76,000, 87,000, 102,000 and 270,000 and pH optima in the alkaline region, pH 8-9, were independent of SH groups and were inhibited by a chelating agent EDTA, suggesting that they belong to the metalloproteinase family.

Int J Cell Cloning, 1991 Jan, 9(1), 24 - 42
Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C3H/He mice: cytological properties and kinetics of its leukemic stem cells; Hirabayashi Y et al.; Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C3H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation . Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia . MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells . There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice . A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057 . Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture . Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase . Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used . The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining . Cells with up to 32N-DNA content were detected . After separating MK-8057 by counter-flow CE, the larger cell fraction (mode at 540 microns3) produced more leukemic colonies when injected into irradiated mice than did the small cell fraction (mode at 240 microns3) . A higher percent of the larger cell fraction (61.9%) was killed by the addition of tritiated thymidine cytocide than in the smaller cell fraction (14.9%) . Thus, the smaller cell fraction is considered to have more leukemic spleen colony-forming units (L-CFU-s) in the resting state.

Clin Endocrinol (Oxf), 1991 Jan, 34(1), 5 - 11
The relationship between growth hormone (GH) messenger ribonucleic acid levels and hormone release from individual cells derived from human GH-secreting pituitary adenomas; Hofland LJ et al.; GH mRNA expression and GH release by individual cells derived from four GH-secreting pituitary adenomas were studied by in-situ hybridization and the reverse haemolytic plaque assay, respectively . In addition the percentage of PRL mRNA-containing cells was determined in these cell suspensions . The percentages of GH mRNA-containing cells varied between 52 and 89 while the percentages of GH plaque forming cells varied between 25 and 77 . Frequency distributions of GH mRNA levels in individual cells and of individual GH plaque areas showed a majority of the cells having low GH mRNA levels and secreting low amounts of GH respectively, while there is a low proportion of cells expressing high GH mRNA levels and forming large GH plaques . There was a significant correlation between the GH mRNA levels and the GH plaque areas of individual cells from the four adenomas (P less than 0.001) . The percentages of PRL mRNA-containing cells in the four different adenomas amounted to less than 1, 5, 2 and 18 . Cultured cells from the adenomas consisting of 5 and 18% PRL mRNA-containing cells also contained and released measurable amounts of PRL . Our data show that individual cells from GH-secreting pituitary adenomas are heterogeneous with respect to GH mRNA expression, a small proportion of the cells expressing a high amount of GH mRNA . The heterogeneity in GH mRNA expression is correlated with the heterogeneity in GH release . These observations suggest that a considerable part of GH secreted from a GH-secreting pituitary adenoma is produced by a minority of the GH-secreting tumour cell population.(ABSTRACT TRUNCATED AT 250 WORDS)

J Am Acad Dermatol, 1991 Jan, 24(1), 70 - 3
Immune sensitization against epidermal antigens in polymorphous light eruption; Gonzalez-Amaro R et al.; To get further insight into the pathogenesis of polymorphous light eruption, we studied nine patients with polymorphous light eruption and six healthy persons . Two skin biopsy specimens were obtained from each person, one from previously ultraviolet light-irradiated skin and another one from unirradiated skin . An epidermal cell suspension, skin homogenate, or both were prepared from each specimen . Autologous cultures were made with peripheral blood mononuclear cells combined with irradiated or unirradiated skin homogenate and peripheral blood mononuclear cells combined with irradiated or unirradiated epidermal cell suspension . Cell proliferation was assessed by 3H-thymidine incorporation assay . The response of peripheral blood mononuclear cells to unirradiated epidermal cells or unirradiated skin homogenate was similar in both patients and controls . However, peripheral blood mononuclear cells from patients with polymorphous light eruption showed a significantly increased proliferative response to both irradiated epidermal cells and irradiated skin homogenate . Our results indicate that ultraviolet light increases the stimulatory capability of polymorphous light eruption epidermal cells in a unidirectional mixed culture with autologous peripheral blood mononuclear cells . This suggests that an immune sensitization against autologous ultraviolet light-modified skin antigens occurs in polymorphous light eruption.

Oncogene, 1991 Jan, 6(1), 113 - 8
Cell type-specific tumor induction in neural transplants by retrovirus-mediated oncogene transfer; Aguzzi A et al.; Using a neural transplantation model which mimics structural and functional properties of the normal rat brain to a high extent, we have taken a novel approach to study the transforming potential of activated oncogenes in the developing brain . Single cell suspensions prepared from fetal rat brains were infected with replication-defective retroviral vectors encoding oncogenes and stereotaxically injected into the caudoputamen of adult F344 rats . Rats carrying transplants expressing the polyoma middle T antigen developed endothelial hemangiomas in the graft which in 70% of the recipient animals led to fatal cerebral hemorrhage within 13-50 days after transplantation . Expression of the v-src gene caused astrocytic and mesenchymal tumors with a 70% incidence after latency periods of 2-6 months, but no endothelial lesions . It was found by in situ hybridization that these oncogenes are expressed in all cell types present in the graft . This indicates that cell-type specific transformation is due to differential susceptibility of the respective target cell to the oncogenes, rather than selective integration or expression of the retroviral construct . The highly efficient gene transfer by retroviral vectors into fetal brain transplants provides a challenging experimental strategy to study differentiation and oncogenesis in the CNS.

Life Sci, 1991, 48(7), 613 - 5
Biotransformation of 4-androstene-3,17-dione by green cell suspension of Marchantia polymorpha: stereoselective reduction at carbon 17; Hamada H et al.; The biotransformation of 4-androstene-3,17-dione by a green cell suspension of Marchantia polymorpha was studied . It was found that the cultured cells stereoselectively reduce the carbonyl group of 4-androstene-3,17-dione from the re-face at C-17 to form testosterone as the primary metabolite.

J Surg Res, 1991 Jan, 50(1), 15 - 23
The specificity of lymphokine-activated killer (LAK) cells in vitro: fresh normal murine tissues are resistant to LAK-mediated lysis; Lefor AT et al.; We have previously reported that murine splenocytes incubated in the lymphokine interleukin-2 acquire the ability to mediate the lysis of a variety of fresh tumor cells in short-term chromium-51 release assays . This study was designed to evaluate the effect of lymphokine-activated killer (LAK) cells on fresh normal murine tissues . The susceptibility to lysis by LAK cells of single cell suspensions from a variety of murine tissues including lung, kidney, bone marrow, peripheral blood mononuclear cells, intestinal mucosa, liver, and fetus was studied . While kidney, intestinal mucosa, and peripheral blood mononuclear cells were clearly not lysed, there was a very low level of lysis of lung, bone marrow, liver, and fetus in repeated experiments . Separation of the cell suspensions of lung and bone marrow demonstrated much higher lysis of the adherent cell population, corresponding to an increased number of macrophages in the target cell suspension . Macrophages represent a population of cells particularly sensitive to lysis by LAK cells . All of the normal tissues were highly lysable by LAK cells in antibody-dependent cellular cytotoxicity assays in the presence of the appropriate anti-H-2 antibody . In a series of cold target inhibition studies, fresh normal murine kidney, lung, and bone marrow did not inhibit the lysis of the LAK-sensitive tumor target MCA-102, further demonstrating that fresh normal tissues share little if any of the determinant recognized by LAK cells on tumor targets . However, the MCA-105 and MCA-106 tumors, and the YAC cell line, all of which are sensitive to LAK cell lysis, did inhibit the lysis of MCA-102 tumor . These studies suggest that a common determinant is present on LAK-sensitive tissues that is absent or present in very low amounts on fresh normal tissues.

Clin Orthop, 1991 Jan, (262), 298 - 311
The osteogenic potential of culture-expanded rat marrow mesenchymal cells assayed in vivo in calcium phosphate ceramic blocks; Goshima J et al.; When whole marrow is introduced into porous calcium phosphate ceramic, bone forms on the walls of the pores . As an extension of earlier studies, bone marrow cells derived from the femora of inbred rats were introduced into tissue culture, and the adherent cells were cultivated, mitotically expanded, subcultured, harvested, placed in small cubes of porous calcium phosphate ceramic, and grafted into subcutaneous sites of syngeneic rats . Primary marrow-derived, cultured mesenchymal cells introduced into ceramic showed strong osteogenic potential, with bone forming in the pore regions of ceramic as early as two weeks after in vivo implantation; cartilage was observed infrequently in pores that appeared to be avascular . Osteogenesis could be observed after the 18th subculture (over 36 population doublings) when the cells were tested in ceramic at subcutaneous sites, whereas chondrogenesis was observed with only the first and second subcultured cells in the ceramic delivery vehicle . With increasing numbers of subcultures, the initiation of osteogenesis and the apparent rate of bone formation declined, and the course of osteogenesis was delayed . Cultured, marrow-derived mesenchymal cells, even after the 21st subculture (over 40 population doublings), exhibited a positive histochemical reaction for alkaline phosphatase . However, the in vivo osteogenic potential of these cells was not correlated with their alkaline phosphatase activity . The implantation of cell pellets or the injection of cell suspensions of fresh or cultured, adherent marrow cells never produced bone or cartilage in heterotopic sites . These data indicate that porous ceramic provides an excellent delivery vehicle for cells that are capable of osteogenic expression and suggest that the composite graft of marrow-derived mesenchymal cells and porous ceramic may be useful for repair of massive bone defects . It may be possible to culture marrow mesenchymal cells as a source for reparative cells for implantation back into autogeneic sites.

J Urol, 1991 Jan, 145(1), 171 - 5
Cytotoxic effects of high energy shock waves in different in vitro models: influence of the experimental set-up; Smits GA et al.; High energy shock waves (electromagnetically generated, Siemens Lithostar) were studied for their effects in vitro on different (tumor) cell types . Cells were exposed to the shock waves as a single cell suspension or as a cell pellet on the bottom of a test tube . In both cases, a dose dependent direct cytotoxicity, established by trypan blue dye exclusion, was observed after treatment with 1000 or 2000 shock waves . Also, the antiproliferative capacity as determined by clonogenic potential (double layer soft agar) and growth rate (plastic) were affected in this way . However, comparing the results after treatment in suspension or pellet, a discrepancy was evident . The cell lines showed a different susceptibility in pellet vs . suspension . Also the differential sensitivity of the cell types varied in these two treatment models . Furthermore the outcome depended on the cell concentration; direct cytotoxicity in a cell suspension was more pronounced at higher cell concentrations, while in a pellet this was increased by decreasing the number of cells . Finally, no shock wave induced cytotoxicity could be seen after fixation of cells in gelatine or by placing the pellet on a bottom layer of gelatine . Pressure measurements revealed no adequate explanation for this phenomenon . These results indicate that in vitro effects depend on the way cells are exposed to the shock waves and can be greatly influenced by changing the conditions of the microenvironment . Therefore, precise descriptions of the experimental set-up and careful interpretations of their outcome are obligatory.

Ultrasound Med Biol, 1991, 17(4), 401 - 6
Single strand breaks in CHO cell DNA induced by ultrasonic cavitation in vitro; Miller DL et al.; Ultrasonic cavitation induces a multiplicity of bioeffects in cell suspensions exposed in a 72 RPM rotating-tube exposure system . Single strand DNA breaks (SSBs) were found in cultured Chinese hamster ovary (CHO) cells exposed directly to 1.61 MHz ultrasound at a continuous 8 W/cm2 spatial peak temporal average (SPTA) intensity with cavitation for 10 min at 2 degrees C . Viability assessed by the trypan blue test was less than 1%, which indicates that these SSBs were in dead cells . Burst mode exposure with 10.5 microseconds bursts repeated each 21 microseconds not only caused SSBs at 5.6 W/cm2 and 8 W/cm2 (SPTA) for 10 min, but also allowed 20% and 7% viability, respectively . In order to determine if any of these breaks resided in the viable fraction of cells, the exposures were repeated with a 30 min postexposure incubation period at 37 degrees C to allow breaks in viable cells to repair . No significant repair occurred, relative to the samples which remained at 2 degrees C to prevent repair . A similar result was obtained with 10.5 microseconds bursts repeated each 42 microseconds at 4 W/cm2 (SPTA) with 46% viability . Thus, the observed ultrasonically induced SSBs reside primarily in the nonviable fraction of cells.

Immunopharmacol Immunotoxicol, 1991, 13(3), 221 - 35
Amphotericin B selectively stimulates macrophages from high responder mouse strains; Wolf JE et al.; Lymphoid cells from most inbred mouse strains respond to amphotericin B (AmB)-induced immunostimulation . However, C57BL/6 mice and related strains display low or absent lymphoid cell stimulation by AmB and enhanced susceptibility to AmB toxicity . Experiments reported here show that in vitro incubation with AmB can stimulate AKR (AmB-high responder strain) macrophage proliferation . Intraperitoneal injection of AKR mice with AmB also elicits a population of macrophages primed for enhanced oxidative burst activity after triggering by zymosan particles . Under the same experimental conditions, AmB elicits a population of very weakly responsive macrophages from C57BL/6 mice . The low responsiveness of C57BL/6 macrophages correlates with previous observations that AmB is a potent immunoadjuvant and B cell mitogen in most inbred strains, but it selectively lacks immunoadjuvant effects in C57BL/6 mice and it also fails to induce polyclonal B cell stimulation in their spleen cell suspensions . Similarly, in measurements of protein synthesis in vitro, high concentrations of AmB produce a greater inhibition of protein synthesis in C57BL/6 peritoneal macrophages than in parallel cultures of AKR macrophages . These findings support the hypothesis that the macrophage is an important target cell in the mediation of AmB-induced immunomodulation.

Toxicon, 1991, 29(6), 589 - 601
Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver; Hooser SB et al.; Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol . wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to {3H}-dihydro-microcystin-LR ( {3H}-2HMC-LR), and purified to greater than 99% purity by C-18 reverse-phase high-performance liquid chromatography . The uptake and subcellular distribution of {3H}-2HMC-LR were determined in suspensions of hepatocytes at 0 degrees C and 37 degrees C, or following rifampicin pretreatment, and in perfused rat liver . The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation . Suspensions of 7.5 x 10(6) hepatocytes also were incubated with 10 micrograms/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin) . Isolated rat livers were perfused with media containing {3H}-2HMC-LR and the uptake of radiolabel was determined . Sequential biopsy samples were collected for histologic examination . The remaining liver was homogenized and subcellular fractions prepared . Uptake of radiolabel was rapid in both cell suspension at 37 degrees C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0 degrees C and by rifampicin (50 micrograms/ml) pretreatment . Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with {3H}-2HMC-LR . In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction . In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions . Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein . Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein . These data suggest that the uptake of {3H}-2HMC-LR occurs primarily by an energy-dependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).

Eur Biophys J, 1991, 19(6), 327 - 34
Difluorophosphate as a 19F NMR probe of erythrocyte membrane potential; Xu AS et al.; Erythrocyte membrane potential can be estimated by measuring the transmembrane concentration (activity) distribution of a membrane-permeable ion . We present here the study of difluorophosphate (DFP) as a 19F NMR probe of membrane potential . This bicarbonate and phosphate analogue has a pKa of 3.7 +/- 0.2 (SD, n = 4) and therefore exists almost entirely as a monovalent anion at physiological pH . When it is incorporated into red cell suspensions, it gives two well resolved resonances that arise from the intra- and extracellular populations; the intracellular resonance is shifted approximately 130 Hz to higher frequency from that of the extracellular resonance . Hence the transmembrane distribution of DFP is readily assessed from a single 19F NMR spectrum and the membrane potential can be calculated using the Nernst equation . The membrane potential was independent of, DFP concentration in the range 4 to 59 mM, and haematocrit of the cell suspensions of 31.0 to 61.4% . The membrane potential determined by using DFP was 0.94 +/- 0.26 of that estimated from the transmembrane pH difference . The distribution ratios of intracellular/extracellular DFP were similar to those of the membrane potential probes, hypophosphite and trifluoroacetate . DFP was found to be transported across the membranes predominantly via the electrically-silent pathway mediated by capnophorin . Using magnetization transfer techniques, the membrane influx permeability-coefficient of cells suspended in physiological medium was determined to be 7.2 +/- 2.5 x 10(-6) cm s-1 (SD, n = 4).

Biotech Histochem, 1991, 66(4), 200 - 2
A new technique facilitating studies of scant cell specimens; Duarte L; A simple method is described by which multiple cytological and cytochemical studies can be done on a clinical sample that contains relatively few cells . The cells are concentrated by centrifugation . The cell pellet is fixed, frozen and embedded in plastic . Thin (2-microns) sections are cut from the plastic . Thus, each cell may appear in several sections and many slides can be made from a single specimen . The advantages of this method over cytospins and Millipore filter preparations of cell suspensions are optimal utilization of all cells, excellent morphological and immunological preservation and ease and reproducibility of this technique.

Plant Mol Biol, 1991 Jan, 16(1), 81 - 94
cDNA cloning and characterization of a putative 1,3-beta-D-glucanase transcript induced by fungal elicitor in bean cell suspension cultures; Edington BV et al.; Synthetic oligonucleotides based on similarity between tobacco 1,3-beta-D-glucanase and barley 1,3-1,4-beta-D-glucanase were used to prime the synthesis and amplification of a 162 bp bean (Phaseolus vulgaris L.) beta-glucanase cDNA by the polymerase chain reaction (PCR) . The PCR product was used to isolate a near full-length beta-glucanase cDNA corresponding to an approximately 1400 bp full-length transcript, from a library containing cDNA sequences complementary to mRNA from fungal elicitor-treated bean cells . At the amino acid level, the bean beta-glucanase cDNA was 59% similar to tobacco 1,3-beta-D-glucanase, 46% similar to barley 1,3-beta-D-glucanase and 46% similar to barley 1,3-1,4-beta-D-glucanase . At the nucleotide level, the similarities were 65, 50 and 53% respectively . The beta-glucanase appeared to be encoded by a single gene with similar genomic organization in bean cultivars Canadian Wonder, Imuna and Saxa . On the basis of predicted Mr, isoelectric point, sequence similarity, and comparisons of rate of transcript appearance with induced enzyme activity, it was concluded that the cDNA encodes the basic bean endo-1,3-beta-D-glucanase . Glucanase transcripts were induced, from very low basal levels, with similar kinetics to chitinase transcripts in elicitor-treated bean cell suspension cultures.

Izv Akad Nauk SSSR Biol, 1991 Jan-Feb, (1), 31 - 42
{The effect of a cationic surface-active substance (catamine AB) on the physiological-morphological properties of B . cereus and E . coli}; Pronin SV et al.; Catamine AB (0.05-0.5%) promotes transfer of B . cereus st 96, and E . coli st . 906 cell cultures into metabolic rest . Detergent-treated cells have no energetic metabolism and autolytic processes, have high light-scattering coefficient, and peculiar ultrastructural organization . Viable cells can be observed in the detergent-treated cell suspension after 1 year of incubation . Difference in action of different catamine AB concentrations on stationary and exponential B . cereus cells has been revealed.

Neuroscience, 1991, 40(1), 123 - 31
Correlation of functional recovery after a 6-hydroxydopamine lesion with survival of grafted fetal neurons and release of dopamine in the striatum of the rat; Rioux L et al.; Female rats were lesioned with 6-hydroxydopamine in the left substantia nigra . At least two weeks later they were tested with amphetamine (5 mg/kg, s.c.) and apomorphine (0.25 mg/kg, s.c.) . A cell suspension from the ventral mesencephalon of rat embryos was distributed in three sites in a triangular fashion in the center of the denervated striatum . The amphetamine test was then repeated every month for six months . The pattern of circling to amphetamine before the graft was strictly ipsiversive in all animals . From the first month we observed a progressive change and three patterns of rotation could be observed . In 21% of animals, the total number of ipsiversive turns in 90 min actually increased but during the first 20 min the animals turned contralaterally to the lesion (and to the graft) . In 38% of animals, the total number of turns switched from ipsiversive to contraversive with the animals turning initially toward the intact side and during the second half of the test toward the lesion . Finally 41% of rats progressively switched to turning only toward the intact side . In all cases, maximal contraversive turning occurred during the initial 20 min . In these rats, tyrosine hydroxylase-positive cells were detected mainly in the dorsal striatum with a few in the central portion . Moreover there was a strong correlation between the number of surviving grafted neurons and the growth of their fiber into the host striatum and the extent of recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroscience, 1991, 42(2), 427 - 39
Increase of striatal methionin enkephalin content following lesion of the nigrostriatal dopaminergic pathway in adult rats and reversal following the implantation of embryonic dopaminergic neurons: a quantitative immunohistochemical analysis; Manier M et al.; The aim of the present study was to test whether intrastriatal implants of embryonic dopaminergic neurons are able to normalize the lesion-induced dysfunction of striatal enkephalinergic neurons, one of the major output systems of the striatum . The ascending dopaminergic pathway of adult rats was unilaterally lesioned . Three weeks later a cell suspension obtained from the mesencephali of ED14 rat embryos was implanted into the denervated striatum and striatal methionin enkephalin immunostaining was quantified six months later by the use of an image analyser . Methionin enkephalin immunostaining was unevenly distributed in the striatum of control animals . Besides the classical patch/matrix pattern, a mediolateral gradient was also present and, moreover, immunostaining decreased towards caudal levels . Seven months after the lesion of the nigrostriatal dopaminergic pathway, methionin enkephalin immunostaining was found to be increased in the denervated striatum by about 50% . However, relative increases were more sustained in the areas where basal methionin enkephalin immunostaining were lowest, i.e . the lateral striatum and posterior striatal areas . This resulted in an attenuation of the global gradients seen in the normal striatum . Increased immunostaining was also found in the ipsilateral globus pallidus . The implantation, into the denervated striatum, of embryonic dopaminergic neurons led to a reversal of the lesion-induced increase of striatal and pallidal methionin enkephalin immunostaining six months later . Moreover, this reversal resulted in an overshoot, as the level of immunostaining in the graft-bearing striatum was found to be lower than the levels found in the normal striatum . It is concluded that grafts of embryonic dopaminergic neurons can normalize the function of one of the major output systems of the striatum and, through it, influence more distant targets of this structure . This suggests a physiological basis for the behavioral effects observed previously with such grafts.

Acta Pharm Hung, 1991 Jan, 61(1), 32 - 47
{Use of isolated myocytes and the patch clamp technic in pharmacological studies}; Meszaros J; Effects of palmitylcarnitine, a toxic metabolite of ischaemia, on the electrophysiological properties of single ventricular myocytes of guinea pig were studied by means of patch clamp technique . Cells were enzymatically isolated by perfusing the heart with Ca-free Tyrode solution containing collagenase enzyme . Before experimentation, cells were allowed to recover in a regenerating solution for at least one hour at room temperature . An aliquot of the cell suspension was placed in a small (0.5 ml volume) chamber mounted on the stage of an inverted microscope . After a settling period of 10 minutes, the cells were superfused with Tyrode solution at a rate of 2 to 4 ml/min . Bath temperature was maintained at 35 degrees C . Patch electrodes were fabricated from glass capillary tubes . The electrodes having resistances of 2 to 4 M were connected to a LIST EPC-7 voltage clamp amplifier . The passive parameters (membrane resistance, time constant, capacitance), and resting and action potentials of the cells were recorded in current clamp mode, while the ionic currents (inward calcium, outward potassium) were recorded in voltage clamp mode . Palmitylcarnitine at 10(-6) M concentration increased the membrane resistance, time constant, and depolarized the cell membrane which was accompanied by a marked prolongation of the action potential duration . Under the effect of the drug, the outward potassium current responsible for the repolarization was decreased, while the inward calcium current responsible for the plateau phase was significantly increased mainly at positive membrane potentials . The steady-state activation and inactivation curves were shifted to less negative potentials . The results indicate that the changes in the membrane electrical properties play an important role in the development of the well-known arrhythmogenic effect of palmitylcarnitine.

Neuroscience, 1991, 41(2-3), 703 - 11
Normal cerebrovascular regulatory mechanisms are present in intracerebral neuronal transplants; Sharkey J et al.; Local cerebral blood flow and local cerebral glucose utilization were measured using quantitative autoradiography in parallel groups of rats (n = 5-7) which 12-15 weeks previously had undergone limited unilateral ibotenate-induced lesion of the nucleus basalis magnocellularis, followed by implantation into ipsilateral neocortex of primordial basal forebrain cell suspensions . Surviving transplants were visualized by acetylcholinesterase histochemistry . Neither lesion alone nor the presence of a transplant produced significant side-to-side differences in either blood flow or glucose use in any of the 20 brain areas measured . Glucose use within the transplant was independent of the site of implantation . When sited in neocortex, glucose use in the transplant (66 +/- 4 mumol/100 g per min) was significantly lower than in the corresponding contralateral site (113 +/- 3 mumol/100 g per min), whereas when sited in subcortical white matter, glucose use (53 +/- 3 mumol/100 g per min) was significantly higher than in the contralateral side (29 +/- 4 mumol/100 g per min) . In the host brain as a whole, the ratio of blood flow to glucose use ipsilateral to the transplant (m = 1.27, r = 0.88) was not significantly different from that of the contralateral side (m = 1.30, r = 0.94) . This relationship was also observed within the transplanted tissue itself despite the fact that alkaline phosphatase histochemistry revealed a relative hypervascularization associated with the implantation site.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunology, 1991 Jan, 72(1), 34 - 9
Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to herpes simplex virus in vitro; Williams NA et al.; The role of epidermal Langerhans' cells in infection with herpes simplex virus (HSV) was investigated using a culture system that supports antigen-specific primary and secondary T-cell proliferative responses . Epidermal cell suspensions were capable of restimulating the response of in vivo primed T cells to UV-inactivated HSV . This capability was also present in cell suspensions enriched for Langerhans' cells, but was abrogated by the depletion of I-A-bearing cells . The magnitude, kinetics and phenotype of the responding cells were similar to those elicited when HSV was presented to primed T cells by antigen-presenting cells from the spleen . In marked contrast, whereas splenic antigen-presenting cells induced strong antigen-specific proliferation of unprimed T cells (primarily of the helper phenotype), Langerhans' cells failed to invoke any detectable reaction of such cells.

Immunopharmacol Immunotoxicol, 1991, 13(1-2), 73 - 86
Induction of helper T cells by pertussis toxin during in vivo priming to insulin; Grenier-Brosette N et al.; We have studied the effect of pertussis toxin (PT) on in vivo priming of T lymphocytes to insulin . Mice were immunized with bovine insulin in complete Freund's adjuvant and antigen-specific DNA synthesis was measured in lymphoid cell suspensions from lymph nodes and spleens . Insulin-specific response was greatly enhanced both in spleen and lymph nodes if mice were given PT at the time of immunization . Mice given PT presented 3 times more cells in spleen and 4 times less in lymph nodes . However, the major antigen-specific response was still observed in lymph nodes . PT had a strong mitogenic effect in vitro on lymph node cells but a weak effect on spleen cells indicating that the adjuvant activity of PT involves other effects besides the mitogenic activity.

Eur J Cancer, 1991, 27(6), 703 - 10
In vitro and clinical characterisation of a Newcastle disease virus-modified autologous tumour cell vaccine for treatment of colorectal cancer patients; Liebrich W et al.; A virus-modified autologous tumour cell vaccine prepared from human colorectal cancer cells is described . After dissociation an average of 5 x 10(7) cells/g tissue were obtained from primary tumours and 9 x 10(7)/g tissue from metastases with an average viability of 72% and 51%, respectively . Following irradiation (200 Gy), inactivation of the proliferative activity of the cells was demonstrated by their degeneration in tissue culture and the absence of incorporation of 3H-labelled thymidine . One third of the cells were still metabolically active, as shown by the incorporation of 3H-uridine and a mixture of 3H-aminoacids . The dissociated cells expressed MHC class I and II antigens in a qualitatively similar way to tissue sections . Epithelium-specific antigens (detected by MAb HEA125) were expressed on an average of more than 75% cells of the suspension, while leucocyte-specific antigens (detected by MAb CD53) were expressed on an average of less than 25% cells . The vaccine was prepared by admixing the nonlytic strain Ulster of Newcastle disease virus (NDV) with the tumour cell suspension . The NDV adsorption at tumour cells was shown by electron microscopy . Clinically, the treatment with the vaccine was associated with an increased sensibilisation against autologous tumour cells, measured by DTH skin reactivity . First results in 23 patients with colorectal liver metastases who underwent "curative" liver resection followed by vaccination show a clear correlation between the induced increase of DTH skin reaction against autologous tumour cells and the recurrence-free interval . No correlation was found for DTH reaction caused by standard antigens (Merieux test), NDV alone or autologous normal liver tissue . The results demonstrate the possibility of preparing immunogenic virus-modified autologous tumour cell vaccine from colorectal cancer tissue, which could be used for cancer therapy.

Glia, 1991, 4(2), 225 - 32
The origin of remyelinating cells in the adult central nervous system: the role of the mature oligodendrocyte; Wood PM et al.; The sequence of events by which new oligodendrocytes are generated in the adult mammalian central nervous system has not been clearly defined . Here we review old evidence that remyelinating cells can arise from the division of mature oligodendrocytes . In addition, we report the results of a tissue culture study comparing oligodendrocytes and immature progenitors with regard to their capacity for proliferation, for the generation of new oligodendrocytes and for myelination . Monoclonal antibodies 04 and 01 were used to distinguish oligodendrocytes (04+01+) from progenitors (04+01-) . Dissociated cell suspensions from adult rat spinal cord were separated by flow cytometry into 01+ and 01- cell fractions, at greater than 93% purity . The 01+ fraction contained approximately 0.7% 04+01- cells while the 01- fraction contained approximately 4.4% 04+01- cells . Cells from these fractions were plated onto cultures of purified dissociated dorsal root ganglion neurons . The cultures that received 01+ cells developed numerous expanding colonies of cells expressing both 01 and 04, or 04 only, by 8 days and were essentially covered by oligodendrocytes by 16 days . In marked contrast, oligodendrocyte colonies were rare in cultures receiving 01- cells . By 24 days, myelination was extensive in cultures receiving 01+ cells; in contrast, only a few myelin segments were observed in cultures receiving the 01- fraction . Thus, oligodendrocytes (01+ cells) appear more capable than progenitors (04+01-) of generating new myelinating oligodendrocytes in this culture system.

Cytometry, 1991, 12(1), 33 - 41
Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine; Fogt F et al.; Flow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker-256 carcinoma cells . After both in vivo and in vitro pulse labelling with BrdUrd, Walker-256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase . BrdUrd-labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle . To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro . The results indicated exponential growth characteristics for this tumor between days 5 and 8 after implantation . Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar . The mean time for DNA synthesis was estimated from the relative movement of BrdUrd-labelled cells towards G2 . The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration . This study demonstrates that BrdUrd labelling of rat Walker-256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor-bearing rat.

J Periodontal Res, 1991 Jan, 26(1), 17 - 23
Biochemical comparison of proteolytic enzymes present in rough- and smooth-surfaced capnocytophagas isolated from the subgingival plaque of periodontitis patients; Soderling E et al.; Four rough-surfaced (R) and three smooth-surfaced (S) clinical isolates of Capnocytophaga obtained from the subgingival plaque of periodontitis patients were studied for their peptidase and protease profiles . The results were compared with those obtained with C . gingivalis (which has a smooth morphology) . All cell extracts obtained by ultrasonic treatment displayed high peptidase activity toward N-aminoacyl-2-naphthylamines, the best substrates being the arginyl, aspartyl, and leucyl derivatives . The R and S isolates did not differ in these enzyme activities . Also the protease profiles studies with 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-proly l-D-arginine (PZ-PLPGA) and casein were similar . All extracts also hydrolyzed furylacryloyl-L-leucylglycyl-L-prolyl-L-alanine (FALGPA), reconstituted type I {3H}-collagen, and gelatin . N alpha-Benzoyl-DL-rginyl-2-naphthylamine was hydrolyzed faster by the R than the S strains . Comparison between cell suspensions and cell extracts of C . gingivalis showed the suspensions to be enzymatically more active than the extracts . In general, peptidase substrates and PZ-PLGPA were hydrolyzed at a higher rate by suspensions than by extracts, while protease substrates (such as casein) were hydrolyzed faster by the extracts . Gelatin and FALGPA were hydrolyzed by cell extracts only . Fast protein liquid chromatography of peptidases on a gel column was found to be a suitable method to differentiate between R and S isolates in diagnostics, while the chromatographic profiles of proteases were not suitable for this purpose.

Neuromuscul Disord, 1991, 1(5), 345 - 55
Allografts of muscle precursor cells persist in the non-tolerized host; Watt DJ et al.; Implantation of normal muscle precursor cells into myopathic fibres to alleviate recessively inherited diseases of skeletal muscle has received much attention since the discovery of a defective or deficient gene coding for the protein dystrophin in the Duchenne and Becker forms of muscular dystrophy . Therapeutic allografting of cells would require some means of preventing their immune rejection . Here we have allografted muscle into the non-tolerant and non-immunosuppressed murine host . Precursor cells introduced in the form of a single cell suspension survive for prolonged periods post-implantation . Allografts of minced muscle often failed to survive, even though host and donor were compatible at the major histocompatibility locus . Differences at minor loci may well have contributed to such rejection . Where allografted tissue was rejected, there was a decrease in the amount of surviving host muscle at the graft site, an important observation in terms of the therapeutic implantation of cells.

Tsitologiia, 1991, 33(8), 110 - 2
{The dispersion analysis of cell suspensions by a conductometric method}; Aleshin AM et al.; A conductometric device-analyser ADS-05 designed by the authors is proposed for dispersive analysis of cell suspensions . The device makes it possible to do a wide range express analysis of suspensions (0.1-600 microns) according to any required number of classes (from 2 to 2(10)) . The results of dispersion analysis of the yeast cells of Saccharomyces aragilis crop are given (according to the 16 classes).

Chin J Physiol, 1991, 34(4), 355 - 69
The purine adenosine amplifies the response to gonadotropins and inhibits prostaglandin F2 alpha (PGF2 alpha) suppression of gonadotropin stimulation of bovine luteal cells in vitro; Weems CW et al.; Dispersed bovine luteal cells collected on day 7 or 13 postestrus were incubated in vitro with human chorionic gonadotropin (hCG) or prostaglandins E1 or E2 (PGE1; PGE2) in the presence or absence of adenosine, dipyridamole and/or prostaglandin F2 alpha (PGF2 alpha) . Secretion of progesterone by day 7 or 13 bovine luteal cells was increased by hCG, PGE1 or PGE2 which was increased further when co-incubated with adenosine (p less than 0.05) . Adenosine alone increased secretion of progesterone by day 7 luteal cells (p less than 0.05) but not by day 13 luteal cells (p greater than 0.05) . PGF2 alpha inhibited the gonadotropic response to hCG, PGE1 or PGE2 (p less than 0.05) which was reduced by adenosine (p less than 0.05) by day 7 or 13 bovine luteal cell suspensions . Dipyridamole reduced the adenosine-facilitated response to hCG, PGE1 or PGE2 by day 7 or 13 luteal cell suspensions (p less than 0.05) . It is concluded that adenosine enhances the steroidogenic response by functional bovine luteal cells to hCG, PGE1 or PGE2 and that adenosine reduces the luteolytic response to PGF2 alpha by gonadotropin-stimulated bovine luteal cells in vitro.

Tsitologiia, 1991, 33(11), 166 - 71
{The role of changes in the cytoplasmic concentration of Ca2+ in dexamethasone-induced thymocyte death}; Kotelevskaia SM et al.; The putative role of changes in cytosolic Ca2+ concentration ({Ca2+}i) in the dexamethasone (DM) induced thymocyte death was investigated . Incubation of rat thymocytes with 10(-7) M DM for different time intervals from 0.1 to 8 h did not change the basal {Ca2+}i level ca 100 nM as measured with Ca(2+)-fluorescent probe Quin-2 . Ca2+ influx measured by the rate of 45Ca2+ uptake was also just the same in DM treated and control cells . At the same time a 6-8 h incubation of cell suspension with 10(-7) M DM results in significant increase in DNA fragmentation and pyknosis, and a 24 h incubation is associated with the decrease in the percentage of cells not staining with Trypan blue . Thus, the results obtained indicate that 10(-7) M DM induces thymocyte death without any significant and constant {Ca2+}i rise during the first 8 h after hormone application.

Diabetes Res, 1991 Jan, 16(1), 41 - 5
CELISA for rapid screening of monoclonal islet cell surface antibodies using living rat insulinoma cells as target; Ziegler B et al.; An improved rapid cell enzyme-linked immunosorbent assay (CELISA) is described which is suitable for the large scale screening of monoclonal antibodies to islet cell surface antigens . 5 x 10(4) insulin-producing rat insulinoma (RIN) cells were seeded per well in a 96-well flat-bottomed polystyrene plate coated one day before a 0.01% poly-D-lysine solution in PBS . After culture for 4 days in 200 microliters/well RPMI 1640 supplemented with 7.5% heat-inactivated fetal calf serum, the cell number per well was up to 2.1 x 10(5) . These monolayer RIN cell cultures were used as a target for the detection of islet cell surface antibodies (ICSA) in the supernatants of hybridomas . The cells were used without fixation to avoid modification of sensitive surface antigens . Poly-D-lysine did not cause non-specific binding of immunoglobulins to the plastic wells as tested with irrelevant monoclonals . The specificity and sensitivity of the method is comparable to indirect immunofluorescence . All mc-ICSA primary screened by indirect immunofluorescence using viable RIN cell suspensions were positive in this CELISA . There was a correlation (r = 0.7; n = 44) between the antibody binding measured by CELISA and the indirect immunofluorescence technique . The advantage of this CELISA is that cell surface structures are well preserved in a viable cell monolayer used as target without chemical fixation . This assay procedure should be generally suitable for the initial screening of monoclonal antibodies to cell surface antigens of cells growing under culture conditions.

Ter Arkh, 1991, 63(12), 113 - 6
{Glycosylated proteins and the rheological properties of the erythrocytes in carbohydrate metabolic disorders}; Galenok VA et al.; The level of glycosylated hemoglobin (Hb AIc), the concentration of glycosylated proteins in red blood cell membranes (GPCM), and fructosamine were measured in patients with different carbohydrate metabolism abnormalities (glucose tolerance test disorders, insulin-dependent diabetes mellitus/IDDM/) . The "pyrene" probe was used to examine rheological disorders in different shift rates and microviscosity of red blood cell membranes . Close correlations were established between glycosylated proteins and microviscosity as well as between GPCM and viscosity of red blood cell suspension . In addition to an increase of the content of glycosylated proteins, deterioration of the rheological properties, and a rise of microviscosity associated with hypoxic phenomena, a group of patients suffering from IDDM with low microviscosity and graver clinical manifestations (microangiopathies, coronary heart disease, cerebral atherosclerosis) were distinguished . Low microviscosity can also be seen in normoglycemia . The cumulative action of the hemorheological shifts, a decrease of red blood cell deformity accompanied by tissue hypoxia favour the development of diabetic angiopathies . A possibility is revealed of the use of microviscosity of red blood cell membranes together with Hb AIc as markers of early carbohydrate metabolism abnormalities and early microcirculatory disorders.

Cytometry, 1991, 12(8), 707 - 16
Attachment of A172 human glioblastoma cells affects calcium signalling: a comparison of image cytometry, flow cytometry, and spectrofluorometry; Szollosi J et al.; The intracellular free calcium concentration ({Ca2+}i) of indo-1 loaded A172 human glioblastoma cells stimulated by platelet-derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry . With all three techniques, the percentage of responsive cells, peak {Ca2+}i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose . The maximum response occurred at a PDGF concentration of about 20 ng/ml . Basal and peak {Ca2+}i did not differ significantly from method to method even though different calibration procedures were used . Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells . This did not appear to be a direct effect of trypsinization . Spectral analysis of indo-1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency . Calibration of {Ca2+}i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.

Int J Rad Appl Instrum B, 1991, 18(7), 727 - 33
Evaluation of two 111In-oxinate formulations for labelling of white blood cells; Leners N et al.; This study compares the cell labelling characteristics of two 111In-oxinate formulations . The two preparations differ by the solubilizing agent of the chelate and the total amount of oxine . White blood cell suspensions were obtained by standard separation techniques and were labelled with either of these formulations . The labelling efficiency was higher for 111In-oxinate in aqueous solution (compound B) compared to the preparation where an organic solubilizer was added (compound A) (79.2 +/- 7.7 vs 68.6 +/- 17.6%, respectively, P = 0.03) . Red blood cells contaminating the cell suspensions incorporated a higher fraction of 111In if the cells were incubated with the aqueous 111In-oxinate preparation (22.6 +/- 4.6 vs 4.8 +/- 4.6%, respectively, P less than 0.0001) . The uptake of activity by polymorphonuclear cells was reduced with compound B (46.1 +/- 12.8 vs 63.8 +/- 15.8%, respectively, P = 0.0002) whereas the fraction retained by mononuclear cells and platelets was similar (31.3 +/- 13.9 vs 31.4 +/- 15.0%, respectively) . The recovery from the vial was higher for 111In-oxinate in an organic solution (86.6 +/- 1.82 vs 60.3 +/- 14.3%, respectively, P less than 0.0001) . Twenty four hours after administration of the labelled cells, the vascular compartment was less frequently visualized if cells were labelled with compound A (8% of the scintigrams vs 62.5% respectively, P less than 0.0001) . High quality images were more often recorded after the administration of cells labelled with compound A (60.0% of the images vs 23.5%, respectively, P less than 0.02) . The image quality of scintigrams was not related to any of the other cell labelling parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Microbiol, 1991, 156(6), 507 - 12
Aluminium toxicity and binding to Escherichia coli; Guida L et al.; The toxicity and binding of aluminium to Escherichia coli has been studied . Inhibition of growth by aluminium nitrate was markedly dependent on pH; growth in medium buffered to pH 5.4 was more sensitive to 0.9 mM or 2.25 mM aluminium than was growth at pH 6.6-6.8 . In medium buffered with 2-(N-morpholino)ethanesulphonic acid (MES), aluminium toxicity was enhanced by omission of iron from the medium or by use of exponential phase starter cultures . Analysis of bound aluminium by atomic absorption spectroscopy showed that aluminium was bound intracellularly at one type of site with a Km of 0.4 mM and a capacity of 0.13 mol (g dry wt)-1 . In contrast, binding of aluminium at the cell surface occurred at two or more sites with evidence of cooperativity . Addition of aluminium nitrate to a weakly buffered cell suspension caused acidification of the medium attributable to displacement of protons from cell surfaces by metal cations . It is concluded that aluminium toxicity is related to pH-dependent speciation {with Al(H2O)6(3+) probably being the active species} and chelation of aluminium in the medium . Aluminium transport to intracellular binding sites may involve Fe(III) transport pathways.

Ann N Y Acad Sci, 1991, 637, 258 - 76
Endocytosis of human sex steroid-binding protein in monkey germ cells; Gerard A et al.; We investigate in this study the hypothesis of human sex steroid-binding protein hSBP internalization into germ cells in a primate model . Human SBP was purified from late-pregnancy serum and labeled either with colloidal gold particles (18 nm) or with {3H}delta 6-testosterone by photoaffinity treatment . The germ cells were isolated from sexually mature monkey testis or caput epididymis (Macaca fascicularis) by mechanical means and cell suspensions (4 x 10(6) per 100 microliters culture medium) were incubated in presence of hSBP-gold complex (60 ng/100 microliters) or hSBP-{3H}delta 6-testosterone complex (66 ng/100 microliters, 20,000 cpm) for 2, 5, 15, 45, and 60 min . The samples were processed for electron microscopy followed by autoradiographic treatment for the radiolabeled samples . Localization of the label occurred over the whole germ cell lineage whichever tracer was used . Spermatogonia, spermatocytes, spermatids, testicular and epididymal spermatozoa exhibited specific binding sites over the plasma membrane associated with clathrin-like coated pits and vesicles . At 34 degrees C, intracellular localization of the labeled ligand was found within coated vesicles, in early and late endosomes . In addition, in early spermatogenic cells, labeled ligand was detected in the nuclei and/or associated with the nuclear envelope whereas in late spermatids and residual bodies, the labeling was accumulated in multivesicular, prelysosomal structures . Quantitative analysis of the "labeled cells/total cells" ratio exhibited a negative correlation to the maturation steps, epididymal spermatozoa being the least labeled . The cellular distribution is similar with one or the other protein in the same spermatogenic cells . Unlabeled hSBP treatment prior to labeled hSBP reduced significantly the internalization . Lowering the temperature to 4 degrees C prevented endocytosis and enhanced membrane binding . EDTA pretreatment strongly decreased hSBP internalization and modified the early endocytic steps, namely, the pinching off of the coated vesicles . It is concluded that monkey germ cells are able to internalize the human sex steroid-binding protein through specific endocytic organelles . This endocytosis leads to the labeling of the nuclei in the early spermatogenic cells and of the multivesicular bodies in the late germ cells . This strongly suggests that steroid-binding proteins may be required for spermatogenesis in acting at the germ cell lineage level either by themselves or by serving as steroid transmembrane carriers.

Wien Med Wochenschr, 1991, 141(21), 485 - 92
{Current methods in blood preservation: recovery of blood components using a multi-bag system}; Bergmann H et al.; A new method of blood preparation by use of a multiple plastic bag system is prescribed . Buffy coat free red cell suspensions in additive solution (SAG-M), fresh frozen (recovered) plasma and random platelet concentrates can thus be produced . Apart from the so far clinically not instantaneously available stored platelets the storage time of the red cells is remarkably increased up to 42 to 49 days and the cellular contaminations (red cells and leucocytes in platelet concentrates, thrombocytes and leucocytes in FFP) are markedly diminished leading to a higher quality of these components . The Red Cross Blood Transfusion Service of Upper Austria presents its data obtained by the use of a quadruple plastic bag (RC Transfusion Production Center Eugendorf/Salzburg) with cellular contamination parameters far below the generally accepted limits . Apart from the increased quality of the components a very high level of clinical acceptance concerning especially the stored random platelet concentrates has been experienced . In conclusion the necessity of changing to a multiple bag system is strongly emphasized thus not only better fulfilling the clinical needs for the different blood components but also delivering higher qualities . Red cell preparations containing the buffy coat should therefore not be used any longer.

Autoimmunity, 1991, 9(3), 217 - 23
Experimental autoimmune oophoritis . II . Both lymphoid cells and antibodies are successful in adoptive transfer; Damjanovic M; Experimental autoimmune oophoritis can be readily induced by passive transfer of peripheral blood lymphocytes, lymph node cells, spleen cells, T- and B-enriched cell suspensions, immune serum and gamma globulins, from ovary antigen immunized rats to naive recipients . Adoptive transfer was markedly enhanced when recipient rats were injected simultaneously with sensitized lymphoid cells and anti-ovary antibodies . Histologically, this passively induced disease was much the same as the actively induced disease . By syngeneic lymph node assay it was shown that regional lymph nodes of neonatally thymectomized rats did not enlarge upon injection of EAOO lymphocytes which otherwise produced a marked effect in lymph nodes of normal recipient rats . Therefore, it appears that enlargement of the draining lymph node was dependent on the participation of host T cells . The possibility that development of EAOO may involve cooperation between antigen-reactive and effector classes of lymphocytes was discussed.

Neuroscience, 1991, 45(3), 587 - 607
Effects of cholinergic-rich neural grafts on radial maze performance of rats after excitotoxic lesions of the forebrain cholinergic projection system--I . Amelioration of cognitive deficits by transplants into cortex and hippocampus but not into basal forebrain; Hodges H et al.; After ibotenate (10.0 mg/ml) lesions to the nucleus basalis and medial septal regions, at the source of the cortical and hippocampal branches of the forebrain cholinergic projection system, rats displayed long-lasting stable impairment in reference and working memory in both spatial (place) and associative (cue) radial maze tasks . Cell suspension transplants of cholinergic-rich fetal basal forebrain tissue dissected at embryonic day 15 substantially improved all aspects of radial maze performance to a comparable degree whether sited in cortex, hippocampus, or both regions of the host brain . No additive effects were obtained with grafts in both terminal regions, but total graft volume, assessed stereologically, showed a significant negative correlation with error scores . Rats with behaviourally effective grafts, like controls, were disrupted in the place task when tested in dim light which obscured extra-maze spatial cues . Lesioned rats were not affected by change in lighting . Grafts of cholinergic-poor fetal hippocampal tissue did not improve radial maze performance; neither did grafts of cholinergic-rich tissue placed within the host basal forebrain lesion sites . In rats with cholinergic-rich terminal grafts, cortical and hippocampal choline acetyltransferase activity was restored to control level, commensurate with site of transplant, whereas it was significantly reduced in lesioned animals and those with functionally ineffective grafts . The indiscriminate error pattern and insensitivity to changes in lighting shown by lesioned rats suggested that lesioning primarily disrupted attention rather than short- or long-term spatial or associative memory processes . Since rats with cholinergic-rich grafts showed both reduced errors and recovery of stimulus control, the data indicated that grafts affected information processing, rather than changes in motor or motivational processes . Changes in choline acetyltransferase activity and the behavioural efficacy of cholinergic-rich grafts are consistent with the involvement of acetylcholine in the behavioural deficits and recovery displayed by lesioned and grafted groups, but do not rule out contributions from other factors . The equipotency of grafts within each terminal region suggests also that there may be a considerable degree of functional cooperation between the two branches of the forebrain cholinergic projection system . Functional recovery may involve local, nonspecific synaptic or paracrine mechanisms within the target regions, since grafts were efficacious only when placed in the terminal areas, but not when sited homotopically in the basal forebrain, indicating that they did not achieve any functionally significant structural repair to the host brain at that site.

Invasion Metastasis, 1991, 11(4), 216 - 26
Parathymic lymph nodes during growth and rejection of intraperitoneally inoculated tumor cells; Dullens HF et al.; The omental lymphoid organ (OLO) is a part of the greater omentum composed of vascularized milky spots situated between fat cells and containing lymphocytes, plasma cells and macrophages . We analysed the disappearance of intraperitoneally injected tumor cells from the peritoneal cavity and their infiltration into and disappearance from the OLO and the parathymic lymph nodes (PTLN) that drain the peritoneal cavity . After intraperitoneal inoculation of irradiated syngeneic tumor cells, they were visible in the OLO within 24 h . After 3 days, no tumor cells were seen anymore, but there were many macrophages that had phagocytosed tumor cells . After intraperitoneal inoculation of nonirradiated syngeneic tumor cells, a solid growing tumor mass developed in the OLO . The PTLN were also invaded by these nonirradiated tumor cells within 24 h as transfer of cell suspensions of these lymph nodes into naive mice leads to the death of the recipient mice due to tumor growth . However, from day 6 onwards, after tumor inoculation, these lymph nodes contained no tumor cells, despite progressive tumor growth intraperitoneally and in the liver and lungs . In allogeneic mice, tumor cells were rejected in the OLO after 7-10 days . Simultaneously, the number of lymphocytes and macrophages increased and a plasma cell reaction developed . The PTLN did not have tumor-inducing potency at any stage after intraperitoneal tumor injection . This study suggests, that the PTLN are more effective in the (local) eradication of tumor cells than the OLO . Still, considering its immunological potential, the OLO might be important as 'inducer' of immunological reactions in the peritoneal cavity.

Cytometry, 1991, 12(5), 445 - 54
Cell kinetic analysis of mixed populations using three-color fluorescence flow cytometry; Begg AC et al.; The development of antibodies to DNA-incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters . More recently, these techniques have been applied to clinical tumor material . One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material . In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro . An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative) . The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours . Selecting out cells from the mixture required the application of three-color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd-DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA) . This allowed single laser excitation . The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC-conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE-conjugated second antibody) and lastly by the DNA stain containing RNase . Two analysis methods of the IdUrd/DNA cytograms were applied: a mid-S window analysis and a relative movement (RM) analysis . Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately . A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms . By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis . Little difference was seen, with both the mid-S and RM analyses, between cells gated from mixtures or stained separately . It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors.

Neuroscience, 1991, 43(1), 135 - 50
Cryopreservation of postnatal rat retinal ganglion cells: persistence of voltage- and ligand-gated ionic currents; Sucher NJ et al.; Established methods for cryopreservation of living cells were modified for freeze-storage of postnatal retinal ganglion cells from rat . Retinal cell suspensions containing fluorescently labeled ganglion cells were frozen after addition of 8% dimethyl sulfoxide and stored at -80 degrees C for up to 66 days . Viability of identified retinal ganglion cells was assessed by their ability to take up and cleave fluorescein diacetate to fluorescein . No significant difference was found in the number of living retinal ganglion cells when cells obtained from the same dissociation were counted before and after freezing (6.65 +/- 2.37 x 10(4) vs 7.05 +/- 3.67 x 10(4) retinal ganglion cells per ml, respectively; mean +/- S.D., n = 4) . In culture following cryopreservation, the cells appeared morphologically normal, and developed neurites and growth cones similar to their freshly dissociated counterparts . Since very little is known about the electrophysiology and membrane properties of neurons after cryopreservation, we used the whole-cell configuration of the patch-clamp technique to study voltage- and ligand-gated conductances in cryopreserved retinal ganglion cells . The cryopreserved retinal ganglion cells studied under current-clamp maintained resting potentials of -60.9 +/- 6.6 mV (n = 10) and upon depolarization fired action potentials . During voltage-clamp in the whole-cell mode, depolarizing voltage steps activated Na(+)-(INa), Ca(2+)-(ICa), and K(+)-currents in all cells tested (n = 122) . INa could be reversibly blocked by 1 microM tetrodotoxin added to the external solution . ICa was blocked by external 250 microM Cd2+ or 3 mM Co2+ . In some cells, ICa consisted of both a transient and prolonged component . The outward K(+)-current consisted of Ca(2+)-dependent and -independent components . The Ca(2+)-insensitive portion of the K+ outward current was separated into four distinct components based upon pharmacological sensitivity and biophysical properties . In many cells, a rapidly inactivating current similar to the A-type K(+)-current (IA) observed in freshly cultured retinal ganglion cells was isolated by its greater sensitivity to 4-aminopyridine (5 mM) than to tetraethylammonium (20 mM) . A tetraethylammonium-sensitive current with a more prolonged time course reminiscent of IK, the delayed rectifier, was also found . When the 4-aminopyridine- and tetraethylammonium-insensitive portions of the outward current were further analysed with voltage protocols, an additional slowly decaying potassium current became apparent . The inhibitory amino acids, GABA (20 microM) and glycine (100 microM), activated chloride-selective currents that were selectively blocked by bicuculline methiodide (10 microM) and strychnine (5 microM), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Lab Delo, 1991, (6), 32 - 3
{A method of determining erythrocyte deformability}; Shiliaev RR et al.; Red cell suspension filtration through synthetic filters is the most prevalent method for estimation of red cell deformability . A modified technique with the use of cellulose acetate filters manufactured by the Polimersintez Research and Production Amalgamation in the town of Vladimir is suggested . Employment of these filters will help introduce this test in clinical practice.

Neuroscience, 1991, 42(2), 365 - 77
Morphological, neurochemical, and behavioral studies on serotonergic denervation and graft-induced reinnervation of the rat hippocampus; Van Luijtelaar MG et al.; A procedure was developed to conduct simultaneously immunocytochemical and neurochemical studies on the serotonergic system in adjacent 300-micron-thick slices of rat hippocampus . This procedure was applied to correlate morphological (innervation pattern and density), neurochemical (5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and {3H}5-hydroxytryptamine uptake and release) and behavioral (spatial learning) effects of neurotoxin-induced denervation and reinnervation by grafting fetal mesencephalic raphe cells . Intracerebroventricular injections of a low dose of 5,7-dihydroxytryptamine caused a discrete serotonergic denervation of the hippocampus . Eleven months after lesioning, 5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and {3H}5-hydroxytryptamine uptake capacity were decreased by 50-60% . By this time, the residual fibers displayed an enhanced vulnerability towards K(+)-induced depolarization . Grafting of a fetal raphe cell suspension resulted in a reinnervation of the host hippocampus . The pattern of reinnervation was comparable to control innervation and the density was supranormal at the level of the graft . As observed semiquantitatively, the innervation density decreased with distance from the core of the graft . Neurochemical studies showed that the fibers were capable of synthesizing, metabolizing and releasing 5-hydroxytryptamine . The turnover of 5-hydroxytryptamine in both the denervated and the reinnervated hippocampus was comparable to that in control tissue . Previous behavioral testing of the denervated and of the denervated and implanted animals did not reveal any effect on spatial learning, either in an individual or in a social test paradigm . The latter data substantiate the notion that interference with the hippocampal serotonergic innervation does not hamper adequate spatial learning.

Fiziol Zh SSSR Im I M Sechenova, 1991 Jan, 77(1), 62 - 7
{The mechanisms of cellular complementarity in the erythroblast islands of the bone marrow}; Rassokhin AG et al.; Phenylhydrazine was shown to reduce the content of erythroblast islands (EI) within 24 hrs in the rat bone marrow, the effect depending on the dose . Simultaneous strengthening of phagocytic activity, acidifying of lysosomes central macrophages EI and lowering of electrophoretic mobility, were observed . The proteinase inhibitor contrycal (50 and 500 i . u.) prevented the effect in the EI and intensified the regeneration of erythron . In vitro, trypsin and chymotrypsin decreased the EI content in the marrow cell suspension . Changing of functional condition of the central macrophages EI and, probably, products of its secretion, seems to affect the cell complementing in EI.

Lab Delo, 1991, (1), 63 - 6
{A rotational floating viscosimeter for hemorheological studies}; Bashkirov AB et al.; The authors describe a modified Zimm-Krothers viscosimeter fit for hemorheological studies . The device is easy to make; it permits work within the range of shear rates from 1 to 100 sec-1 using but 1.5 ml of blood, plasma, or red cell suspension . The error of a single measurement is under 5 percent.

Histochemistry, 1991, 95(5), 483 - 90
Light microscopical detection of leukocyte cell surface antigens with a one-nanometer gold probe; De Valck V et al.; The potential of ultrasmall gold particles for the light microscopical detection of leukocyte cell surface differentiation antigens was investigated . Suspensions and cytocentrifuge preparations of peripheral blood leukocytes were first incubated with monoclonal antibodies and then with goat antimouse antibodies coupled to colloidal gold particles of 1-nanometer diameter . Cytocentrifuge preparations were made from the cell suspensions . Silver enhancement was performed on all preparations . Then they were counterstained with May-Grunwald Giemsa and examined in light microscopy . The immunostaining appeared as fine dark granules on the surface membrane of the cells . Labeling conditions were determined which gave a dense specific immunostaining and a low background . High dilutions of the ultrasmall gold probe could be used to detect all antigen expressing cells in the samples . The labeling efficiency of the IGSS method with the 1 nanometer probe was comparable to that described earlier for 5 nanometer gold particles . Lymphocyte subsets enumerated with this method in normal peripheral blood were similar to those found with immunofluorescence microscopy . We concluded that one nanometer probes do not offer a major advantage in comparison with 5 nanometer probes for the study of cell surface antigens.

Cytometry, 1991, 12(4), 350 - 9
Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry; Segal GH et al.; Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM) . Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from true B-cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion . Autofluorescence was measured to establish a fluorescence threshold . A second control measured non-specific binding of isotypic control mouse Ig and non-immune rabbit IgG . Cell suspensions from 128 samples of various lymphoid proliferations were studied . In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis . Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated . SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively . Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas.

Exp Brain Res, 1991, 85(3), 501 - 6
Intrastriatal grafts derived from fetal striatal primordia . III . Induction of modular patterns of fos-like immunoreactivity by cocaine; Liu FC et al.; Cocaine, a catecholamine agonist, has been shown to produce a transient induction of the immediate-early gene c-fos and its protein product Fos in the striatum of normal rats . In the present study we report that the expression of Fos can be induced by cocaine challenge in intrastriatal grafts derived from cell suspensions of embryonic striatal primordia . Fos-like immunoreactivity in the nuclei of grafted neurons was detected 2 hr after the injection of 50 mg/kg cocaine into the host rats . Neurons with Fos-immunoreactive nuclei tended to form clusters in the striatal grafts . The Fos-rich clusters were aligned with acetylcholinesterase (AChE)-rich and tyrosine hydroxylase (TH)-rich patches demonstrated in adjoining sections . Previous studies have shown that presynaptic and postsynaptic cellular markers of the dopaminergic system in the striatum, including immunostaining for TH and dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein (DARPP-32), and binding for high affinity dopamine uptake sites and for dopamine D1 and D2 receptor sites, are all concentrated in the AChE-rich patch regions (P regions) of such embryonic striatal grafts . The preferential expression of Fos in neurons of the P regions of the grafts thus implies that the induction of Fos was cell-type specific in being concentrated in the parts of the grafts that express striatal phenotype and that are innervated by catecholamine-containing fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Exp Pathol, 1991, 43(3-4), 129 - 39
Metastatic potential of malignant fibrous histiocytoma of myxoid or giant-cell subtype in rats; Miyauchi Y et al.; We have previously reported on the induction of rat malignant fibrous histiocytomas by 4-hydroxiaminoquinoline 1-oxide . The present study describes cell number- and time-related formation of metastatic lung nodules after i.v . injection of cell suspensions containing various numbers (10(3) to 10(5)/ml) of myxoid or giant-cell subtype malignant fibrous histiocytoma cells . The metastatic potential of the myxoid subtype of rat malignant fibrous histiocytoma was significantly enhanced by i.v . injection of tumor cells selected from metastatic lung nodules and was further increased by repeating the selection procedure more than 7 times . In contrast, the growth activity of subcutaneous transplants of tumor fragments obtained from metastatic lung nodules derived from myxoid subtype did not differ from that of parent malignant fibrous histiocytomas.

Zh Nevropatol Psikhiatr Im S S Korsakova, 1991, 91(10), 14 - 7
{Functional activity of cell membranes in multiple sclerosis}; Predtechenskaia AV et al.; A study was made of the activity of the marker enzymes of plasma membranes Na+,K(+)-ATPase and AChE comparatively to the changes in red blood cell suspension viscosity . Interferometry and capillary viscosimetry were employed to examine red cell membranes of 100 patients suffering from multiple sclerosis . In the interval of physiological temperatures, the two temperatures areas -35 degrees and 40 degrees C characterized by specific behavior of the enzymes and viscosity changes were discovered . These temperature areas are viewed from the standpoints of temperature-induced structural transformations . Each of them has a definite clinical importance for the estimation of the activity of the underlying process . The fact of the existence of the complexly organized system of abnormal structural transformations attests to gross imbalance of red cell membranes . This phenomenon may also be observed as regards other membranes including the membrane of the oligodendrocyte . In addition to the evidence for the membrano-patho-chemical component in the pathogenesis of multiple sclerosis, the temperature-induced structural transformations play the role of diagnostic criteria for multiple sclerosis.

Dev Comp Immunol, 1991 Fall, 15(4), 369 - 81
The effect of infectious bursal disease virus on B lymphocytes and bursal stromal components in specific pathogen-free (SPF) White Leghorn chickens; Ramm HC et al.; The effect of infectious bursal disease virus (IBDV) was studied on adult specific pathogen-free (SPF) white Leghorn chickens through analysis of peripheral blood cell suspensions and histological staining patterns on various tissue types, with specific mAbs . A rapid, progressive loss of B lymphocytes was observed in the bursal cortex and medulla, peripheral blood and thymic medulla . There was, however, a resistant population of MUI-36+ cells at the bursal cortico-medullary junction and scattered around splenic periellipsoidal sheaths . These resistant cells were suggested to be a subpopulation of macrophages which expressed the MUI-36 marker; alternatively these may have phagocytosed virally infected B cells or their remnants . Throughout the period of infection, T lymphocytes appeared nonsusceptible . Further, while the distribution of stromal cell antigens within the bursal cortex remained unaltered, particular epitopes on the surface epithelium and in the medulla were lost as a consequence of viral infection . The data presented therefore suggests that immunodepression of chickens post-IBDV infection, may arise as a direct consequence of infection of B lymphocytes; additionally, it is possible that the elimination of certain crucial elements within the bursal microenvironment may contribute to this state.

Plant Mol Biol, 1991 Jan, 16(1), 21 - 37
Patterns of mitochondrial DNA instability in Brassica campestris cultured cells; Shirzadegan M et al.; We previously showed that the mitochondrial DNA (mtDNA) of a Brassica campestris callus culture had undergone extensive rearrangements (i.e . large inversions and a duplication) relative to DNA of the control plant {54} . In this study we observed that after continued growth, the mtDNA of this culture continues to change, with rearranged forms amplifying and diminishing to varying proportions . Strikingly similar changes were detected in the mtDNA profiles of a variety of other long- and short-term callus and cell suspension lines . However, the proportions of parental ('unrearranged') and novel ('rearranged') forms varied in different cultured cell mtDNAs . To address the source of this heterogeneity, we compared the mtDNA organization of 28 individual plants from the parental seed stock . With the exception of one plant containing high levels of a novel plasmid-like mtDNA molecule, no significant variation was detected among individual plants and therefore source plant variation is unlikely to have contributed to the diversity of mitochondrial genomes observed in cultured cells . The source of this culture-induced heterogeneity was also investigated in 16 clones derived from single protoplasts . A mixed population of unrearranged and rearranged mtDNA molecules was apparent in each protoclone, suggesting that the observed heterogeneity in various cultures might reflect the genomic composition of each individual cell; however, the induction of an intercellular heterogeneity subsequent to the protoplast isolation was not tested and therefore cannot be ruled out . The results of this study support our earlier model that the rapid structural alteration of B . campestris mtDNA in vitro results from preferential amplification and reassortment of minor pre-existing forms of the genome rather than de novo rearrangement . Infrequent recombination between short dispersed repeated elements is proposed as the underlying mechanism for the formation of these minor mtDNA molecules.

Biomed Biochim Acta, 1991, 50(2), 199 - 206
Effect of dextran sulfate on fusion of Sendai virus with human erythrocyte ghosts; Ohki S et al.; The effect of dextran sulfate on the fusion of Sendai virus and erythrocyte ghosts was studied as a function of dextran sulfate concentration and pH of cell suspension solutions . In order to examine the interaction of dextran sulfate with Sendai virion and erythrocyte ghost surfaces, the turbidity of cell suspensions was also measured with respect to the same parameters as above . It was found that dextran sulfate inhibited the fusion of Sendai virus with erythrocyte ghosts . The lower the pH of the suspension solution was, the more effective was dextran sulfate in suppressing virion erythrocyte ghost fusion . Dextran sulfate of a higher molecular weight was slightly more effective in suppressing fusion than that of a lower molecular weight . From turbidity measurements of Sendai virus and erythrocyte ghost suspensions, it is likely that dextran sulfate interacts preferentially with Sendai virion surfaces and inhibits interaction between Sendai virions and erythrocyte ghosts.

Int J Vitam Nutr Res, 1991, 61(1), 20 - 6
Superoxide generation in leukocytes and vitamin E; Okano T et al.; Superoxide generation in polymorphonuclear cells (PMNs) can be detected through determination of chemiluminescence by stimulation with opsonized zymosan in association with the Cypridina luciferin analogue, 2 methyl-6-phenyl-3,7-dihydroimidazol{1,2-a}pyradin-3-one . Following the addition of vitamin E to cell suspensions, chemiluminescence was suppressed by a vitamin E concentration of more than 80 micrograms/10(9) cells . Superoxide production in PMNs was suppressed by both very low and very high levels of vitamin E (less than 2 micrograms/10(9) cells and more than 50 micrograms/10(9) cells, respectively) . The high level of more than 50 micrograms/10(9) cells, which may be a dangerous level, was attained only by the intramuscular administration of 50 mg/kg/day for three days in rats . Young human adults (who received 600 mg/day for 3 months) or premature infants (who received 40 mg/kg/day for 8-14 days) developed PMN vitamin E concentrations not exceeding 30 micrograms/10(9) cells, and chemiluminescence was not affected . This indicates that oral administration of vitamin E does not impair PMN function, even in preterm infants . On the other hand, there was no enhancement of PMN function in premature infants and adults after vitamin E supplementation.

Free Radic Res Commun, 1991, 14(3), 195 - 208
Oestrogen-induced enhancement of myeloperoxidase activity in human polymorphonuclear leukocytes--a possible cause of oxidative stress in inflammatory cells; Jansson G; Micromolar concentrations of beta-estradiol, estrone, 16-alpha-hydroxyestrone and estriol enhance the oxidative metabolism of activated human PMNL's . The corresponding 2-hydroxylated estrogens 2-OH-estradiol, 2-OH-estrone and 2-OH-estriol act on the contrary as powerful inhibitors of cell activity . Equilenine, a naturally occurring steroid hormone structurally closely related to estrone, removes the estrogen-induced increase in oxidative metabolism of activated PMNL's without diminishing cell activity determined in the absence of enhancing hormone . A number of other female and male sexual hormones were without potentiating effect . The cell response to hormone treatment was assayed as increase (or decrease) in LU-dependent CL of activated PMNL's . When assaying LUC-dependent CL of the cells no stimulatory effects of the estrogens could be detected . This fact may imply that the myeloperoxidase enzyme system of the cells is the target for the hormonal action . Various inhibition experiments using activated PMNL's or purified MPO confirmed this conclusion . The efficicious hormones induced approximately a doubling of CL of activated cells and a tenfold increase of the activity of purified MPO . If cell activity was initiated by the additions of low concentrations of hydrogen peroxide, the presence of estrogens caused a remarkable enhancement of the luminol-dependent chemiluminescence . PMNL's activated with fMLP release MPO activity into the surrounding cell medium . It has been found here that the presence of estrogens in micromolar concentrations greatly increases such enzyme release . Release of MPO activity from the cells could be achieved by the mere addition of estrogenic hormones . Estrogen-induced release of enzyme activity was abrogated by the simultaneous presence of equilenine in the cell suspension . Released enzyme responded vigorously to estrogens in the presence of chloride ions and its substrate, hydrogen peroxide . About a tenfold increase in enzyme activity could be measured in the presence of 5 microM beta-estradiol or esteriol . The activity of the released enzyme (as well of purified MPO) was effectively inhibited by small amounts of anti-MPO antibodies . This observation together with other inhibition experiments was taken as evidence for the view that the released enzyme was identical with myeloperoxidase.

Eisei Shikenjo Hokoku, 1991, (109), 32 - 7
{Studies on the uptake of 8-anilino-1-naphthalene sulfonate by rat freshly isolated renal cells}; Ohno Y et al.; The addition of 8-anilino-1-naphthalene sulfonate (ANS) into rat renal cell suspension caused a rapid increase in fluorescence (Ex . 400 nm, Em . 470 nm) . The increase in fluorescence seemed to be composed of two phases . When the cells were disrupted by ultrasonic wave, the fast phase (0-ca 20 sec) increased and the slow phase (after ca 40 sec) disappeared . The rate of increase in the slow phase was dependent on the concentration of ANS and on the ambient temperature . A double reciprocal plot of the rate and the concentration exhibited straight line . It was decreased by bromophenol blue and rose bengal but not by other organic anions like p-aminohippuric acid and several metabolic inhibitors . It seemed that the uptake of ANS into the renal cell is mediated by a carrier which is different from that for p-aminohippuric acid.

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