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-beari