Cell suspensions

Cell Differ, 1988 Aug, 24(3), 179 - 90
A monoclonal antibody recognizing a common antigen on neurons and fibroblasts in chicken and quail; Graniou M et al.; A monoclonal antibody, FiN1, obtained by immunization of a mouse with homogenates of embryonic quail nodose ganglia, was found to react with a surface antigenic determinant, both in quail and chick, present on practically all neurons of the spinal cord and of the peripheral nervous system and on a subpopulation of fibroblasts . An ontogenetic study performed on tissue sections, cell suspensions and cultures showed that FiN1 defines a differentiation marker which appears relatively late in development, during the second half of embryonic life, and persists after hatching . The onset and evolution of its expression during development varies in a tissue-specific manner.

Biophys J, 1988 Aug, 54(2), 241 - 7
Hypophosphite ion as a 31P nuclear magnetic resonance probe of membrane potential in erythrocyte suspensions; Kirk K et al.; Hypophosphorus acid has a single pKa of 1.1 and at physiological pH values it is therefore present almost entirely as the univalent hypophosphite ion . When added to a red cell suspension the ion crosses the cell membrane rapidly, via the anion exchange protein, and the intra- and extracellular populations of the ion give rise to separate 31P NMR resonances . From a single 31P NMR spectrum it was possible to determine the relative amounts of hypophosphite in the intra- and extracellular compartments and thereby estimate the corresponding concentrations . The ratio of intracellular to extracellular hypophosphite concentration was independent of the total hypophosphite concentration for cells suspended in NaCl solutions and was independent of hematocrit . The hypophosphite distribution ratio increased as extracellular NaCl was replaced iso-osmotically with citrate or sucrose, through it remained very similar to the corresponding hydrogen ion distribution ratio . Incorporation of the hypophosphite distribution ratio into the Nernst equation yielded an estimate of the membrane potential . For cells suspended in NaCl solutions the estimated potential was consistently around -10 mV.

Cell Biol Int Rep, 1988 Aug, 12(8), 637 - 46
Preliminary studies on the cultivation and characterization of mini-pig prostate epithelial cells; Costello LC et al.; Mini-pig prostate epithelial cells exhibited the unique metabolic characteristics associated with the specialized function of production and secretion of high levels of citric acid . Epithelial cell suspensions from mini-pig prostate were successfully grown in primary and secondary cultures . The cultured epithelial cells exhibited rapid proliferation reaching confluency in approximately 6 days . Growth and proliferation of fibroblasts were markedly restricted by the dominance of epithelial cell growth . Confluent cultures could be maintained for approximately 6 weeks . The epithelial cells retained their polymorphic appearance in primary and secondary cultures and exhibited the characteristic formalin-resistant acid phosphatase reaction . Testosterone stimulated mitochondrial aspartate aminotransferase (mAAT) activity and citrate production by confluent epithelial cell cultures . These initial results indicate that cultured epithelial cells derived from mini-pig prostate might be an excellent model related to human for studies of prostate biology and hormonal regulation.

Neuroscience, 1988 Aug, 26(2), 417 - 31
Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures; Hsiang J et al.; We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum . In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells . We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system . Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin . Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days . The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase-positive acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations . Representative reaggregate profiles were then re-embedded for electron microscopic examination . In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied . In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration . Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact . In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact . By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates . In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells . In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Arch Biochem Biophys, 1988 Aug 1, 264(2), 400 - 9
Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells; Fedde KN et al.; Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane . ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5'-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility . Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity . The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (microM) of PEA and PLP . TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) greater than 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C . Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH.

Cancer Res, 1988 Aug 1, 48(15), 4427 - 33
Activity of the human blood group ABO, Se, H, Le, and X gene-encoded glycosyltransferases in normal and malignant bladder urothelium; Orntoft TF et al.; Immunohistochemistry has led to the finding of an expression of ABO-related blood group antigens in normal and malignant bladder urothelium which is different from that found on erythrocytes from the same individual . This includes a loss of blood group ABO expression in malignant urothelium, and the expression of Leb antigens in normal and malignant cells from individuals with Le(a+b-) and Le(a-b-) erythrocytes . To elucidate the mechanism of this blood group antigen expression in urothelium we have analyzed the activity of the specific glycosyltransferases encoded by the ABO, Se, H, Le, and X blood group genes in normal and malignant urothelium . Biopsies of normal urothelium were obtained from 22 individuals and biopsies of urothelial tumors from 20 individuals . The tissue donors were typed for ABO, Lewis, and secretor status on erythrocytes and saliva . The biopsies were disaggregated to single cell suspensions, and the activity of the individual glycosyltransferases was determined as pmol of labeled sugar incorporated by oligosaccharide acceptors per 100,000 cells . The A (alpha-3-N-acetyl-D-galactosaminyl) and B (alpha-3-D-galactosyl) gene-specified transferases showed no activity in malignant cells, whereas all other enzymes examined were expressed in both normal and malignant cells . Secretors and nonsecretors showed the same alpha-2-L-fucosyltransferase activity in both normal and malignant cells, whereas the alpha-3-L-fucosyltransferase was reduced (P less than 0.02) in malignant cells from Lewis positive individuals . The Lewis gene-encoded alpha-4-L-fucosyltransferase showed a similar activity in Lewis positive and negative individuals . These results indicate that the disappearance of A and B blood group antigens in bladder tumors and the expression of Leb antigens in normal and malignant cells from individuals with Le(a+b-) and Le(a-b-) erythrocytes are due to corresponding differences in glycosyltransferases . The results indicate that the ABO, H, Se, and Le genes are subjected to a tissue-dependent differential expression.

Am Rev Respir Dis, 1988 Aug, 138(2), 451 - 7
Bronchoalveolar lavage cell differential on microscope glass cover . A simple and accurate technique; Laviolette M et al.; We describe a quick and easy technique to perform cell differentials on bronchoalveolar lavage: the microscope glass cover . Lavage fluids of 72 subjects were analyzed by 3 techniques: glass cover, filter, and cytocentrifuge preparations . Seventy-seven other lavages were analyzed by glass cover and cytocentrifuge preparations alone . Data for the 72 subjects studied by all 3 techniques showed that the cell counts on glass cover and filter preparations were similar, e.g., lymphocytes, 19.2% (range, 0.5 to 94%) and 20.9% (range, 3 to 95%), respectively (Spearman's correlation coefficient, 0.98) . However, on cytocentrifuge preparations, lymphocyte counts were lower (8.3%; range, zero to 87%) and macrophage counts were higher (p less than 0.005) . Comparison of glass cover and cytocentrifuge preparation mixtures with varying amounts (20 to 80%) of purified blood leukocytes labeled with 51Cr (greater than or equal to 72% lymphocytes) showed that a significant amount of radioactive cells was lost during the cytocentrifuge technique in contrast to the glass cover technique . Because neutrophils represented a low proportion of lavage cells, we also evaluated cell suspensions with known neutrophil contents (10 to 70%); we found no difference in neutrophil counts obtained with the 3 techniques . Lavage data analysis of 40 young nonsmoking volunteers showed that glass cover lymphocyte count was also higher than counts on cytocentrifuge preparations: 16.5% (range, 3 to 45%) and 8.2% (range, 2.5 to 35%), respectively . In this group, the distribution of glass cover lymphocyte percentages was normal (p = 0.21, chi 2 test), and the one-tailed 95% confidence interval was 18.6 to 34.7% (mean plus 1.65 standard deviation).(ABSTRACT TRUNCATED AT 250 WORDS)

Zh Mikrobiol Epidemiol Immunobiol, 1988 Aug, (8), 90 - 4
{Solid-phase immunoenzyme analysis of Coccidioides immitis antigens}; Khrapova NP et al.; A highly effective and specific solid-phase enzyme immunoassay system has been developed . The enzyme immunoassay is a highly sensitive technique for the detection and identification of C . immitis cellular and metabolic antigens . This technique is suitable for the study of strain differences in the antigenic composition of C . immitis, rendered harmless by different methods . The expediency of the preliminary sonification of cell suspensions of C . immitis, the causative agent of coccidioidomycosis, has been experimentally confirmed.

Allergy, 1988 Aug, 43(6), 464 - 8
IgE on Langerhans cells in the skin of patients with atopic dermatitis and birch allergy; Tigalonowa M et al.; Epidermal cell suspensions from the skin of seven patients with atopic dermatitis (AD) and seven healthy non-atopic controls were investigated for the presence of surface HLA-DR and CD1 antigen, and IgE using indirect and double-staining immunofluorescence techniques . Fifty-seven percent of all CD1+ and 68% of all HLA-DR+ cells from the patients demonstrated IgE on their surface, indicating that Langerhans cells (Lc) in AD may be a heterogeneous population with regard to surface characteristics . No IgE+ cells were found in the epidermal cell suspensions from the healthy non-atopic controls . An attempt to demonstrate birch pollen antigen on the surface of Lc from the same patients all strongly allergic to birch pollen, using indirect immunofluorescence techniques, was unsuccessful, also after in vitro incubation of the Lc with high concentration of the birch antigen.

Appl Environ Microbiol, 1988 Aug, 54(8), 2107 - 11
Influence of unsaturated fatty acid membrane component on sensitivity of an Escherichia coli fatty acid auxotroph to conditions of nutrient depletion; Massa EM et al.; The unsaturated fatty acid auxotroph Escherichia coli AK7 was provided with either oleic acid (cis 18:1) or linolenic acid (cis 18:3) to vary the degree of unsaturation of cell membrane lipids . The susceptibility of oleic acid- and linolenic acid-grown cells to starvation at 37 degrees C in 154 mM NaCl was compared following the decline in the number of CFU by plating the cells on agar medium . The decline in CFU was faster for linolenic acid-than for oleic acid-grown cells, but it was not indicative of cell death, since culturable CFU was recovered after respirable substrate was added to the starved cell suspension . Cell envelope microviscosity (determined by fluorescence polarization) of oleic acid- and linolenic acid-grown cells was equal in the presence of a respirable substrate, but in its absence the microviscosity of linolenic acid-grown cells was lower than that of oleic acid-grown cells . The results suggest that cell envelope microviscosity is an important factor in determining the sensitivity of E . coli to conditions of nutrient depletion.

Exp Eye Res, 1988 Aug, 47(2), 269 - 82
Selective failure of long-term survival of isolated photoreceptors from both homozygous and heterozygous rd (retinal degeneration) mice; Politi L et al.; Retinas from homozygous rdle/rdle and heterozygous rdle/++ C57BL/6J mice were dissected and dissociated on postnatal day 2, when they are still essentially indistinguishable . The resulting cell suspensions were seeded on highly adhesive substrata, to which the cells attach as individual units, and grown in vitro for 2 weeks in serum-free, chemically defined media . The behavior of neurons and photoreceptors in vitro was investigated with several techniques; essentially no differences were found between rdle/rdle and rdle/++ cells . Three distinctive cell types could be recognized in cultures of both genotypes towards the end of the first week in vitro: process-free cells, multipolar neurons and rod photoreceptors . There were similarities between rdle/rdle and rdle/++ cultures in the number and morphology of photoreceptor cells, to include the presence of a cilium and a short neurite terminating in a spherule-like body . Moreover, in cultures of both genotypes, only photoreceptors showed opsin immunoreactivity and the antigen recognized by the rod-specific monoclonal antibody RET-P1 . Biochemical and autoradiographic studies demonstrated that rdle/rdle and rdle/++ cells also showed similar uptakes of the putative amino acid neurotransmitters glutamate and aspartate (associated with most of the photoreceptors and only some neurons), and gamma-aminobutyric acid (associated with neurons but absent in photoreceptors) . Thus, according to several parameters, the properties shown by photoreceptor cells were similar in rdle/rdle and rdle/++ cultures during the first week in vitro . Massive photoreceptor cell death was observed in both genotypes during the second week in vitro, coinciding with the time when photoreceptor degeneration occurs in vivo in rd/rd, but not in rd/+ retinas . Photoreceptor death in culture appeared to be specific, since approx . 80% of the non-photoreceptor neurons survived normally during the period when photoreceptor degeneration took place . Several reports from the literature suggest that the period around postnatal days 8-10 represents a critical stage for rd/rd photoreceptors, since they survive until this time but degenerate thereafter . Genetically normal photoreceptors apparently undergo a comparable crisis during maintenance in primary culture, suggesting the involvement of cell-cell contacts and/or retina-derived environmental signals in the survival or rod visual cells.

Toxicol Appl Pharmacol, 1988 Aug, 95(1), 61 - 71
Allylamine-induced vascular toxicity in vitro: prevention by semicarbazide-sensitive amine oxidase inhibitors; Ramos K et al.; The present studies were designed to evaluate the role that metabolic activation plays in allylamine (AAM)-induced vascular toxicity . The effects of AAM were evaluated in primary cultures of rat vascular endothelial (VEC) and smooth muscle cells (SMC) . Semicarbazide (SC) and diethyldithiocarbamate (DDC) were used as inhibitors of semicarbazide-sensitive amine oxidase (SSAO) . Clorgyline and pargyline were used as inhibitors of monoamine oxidase (MAO) A and B, respectively . The effect of catalase, a hydrogen peroxide scavenger, on AAM-induced cytotoxicity was also evaluated . Lactate dehydrogenase (LDH) release and morphological alterations were chosen as indicators of cytotoxicity . Confluent cultures of VEC and SMC were exposed to various concentrations of AAM (2-200 microM) in the absence and presence of serum for 4, 12, or 24 hr . High concentrations of AAM (200 microM) alone produced a time-dependent increase in LDH release and morphologic alterations in cultures of both cell types . Lower concentrations of AAM did not compromise the structural integrity of the cells . Semicarbazide (200 microM) or DDC (2 mM), but not clorgyline (10 microM) or pargyline (10 microM), prevented the toxicity of AAM (200 microM) . Allylamine-induced cytotoxicity was partially prevented by catalase (2500 U/ml) . The presence of fetal bovine serum in the medium was not essential for the manifestation of cytotoxicity . Single cell suspensions of VEC or SMC formed acrolein (ACR) when incubated in the presence of AAM . The formation of ACR mediated by SMC was inhibited by SC (20 microM), but not clorgyline (10 microM) . These results support the concept that AAM is oxidatively deaminated by an SSAO present in vascular cells to generate toxic metabolic by-products capable of causing extensive cellular injury.

In Vitro Cell Dev Biol, 1988 Aug, 24(8), 771 - 7
Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations; Welder AA et al.; Primary myocardial cell cultures and freshly isolated cardiac cells in suspension represent two isolated, whole cell models for investigating cellular transsarcolemmal 45Ca++ exchange in response to a receptor-coupled stimulus . Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3- to 5-d-old Sprague-Dawley rats . In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations . All three preparations had saturable beta-adrenergic binding sites with the antagonist {125I}iodopindolol {( 125I}IPIN) . The suspensions had a significantly lower Bmax (42 +/- 6 fmol/mg protein) than the membranes and cultures (77 +/- 8 and 95 +/- 10 fmol/mg protein, respectively) . The KD of the cultures (218 +/- 2.0 pM) was significantly higher than that for the suspensions (107 +/- 1.3 pM) and membranes (93 +/- 1.3 pM) . Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer . Incubation of the cultures with 5.0 X 10(-7) M isoproterenol resulted in a significant increase in 45Ca++ exchange as early as 15 s . In contrast, 45Ca++ exchange into the suspensions was not increased . Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated 45Ca++ exchange . Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.

Mol Immunol, 1988 Aug, 25(8), 713 - 8
Cross-reaction of a monoclonal antibody to human MHC class II molecules with rabbit B cells; Dubiski S et al.; Monoclonal antibodies, 21w4 and 44H10, against human MHC class II determinants, were analysed for their reactivity with rabbit lymphoid cells . Both MAb bind to all human B lymphoblastoid lines, irrespective of their HLA-DR phenotype . HLA-DR antigens, purified by affinity to 21W4 IgG Sepharose, can be precipitated with 44H10 MAb, indicating that both antibodies react with the same molecules . Competitive inhibition studies with purified MAb IgG show that 44H10 and 21w4 recognize different epitopes of HLA-DR molecules . The 21w4 MAb, previously shown to cross-react with cells of pig, mouse and sheep, does not react with rabbit lymphoid cells . The 44H10 MAb binds to lymphoid cell suspensions prepared from rabbit appendix, spleen and mesenteric lymph nodes . Its reactivity correlates with that of RABELA, a polyclonal antibody specific for rabbit B cells . Mesenteric lymph node and spleen cell suspensions, depleted of sIg+ cells, are devoid of 44H10+ cells, while similarly-treated appendix B cells still contain a subset of B cells which are sIg-, RABELA+ and 44H10+ . These studies thus report on the presence of MHC class II determinants on rabbit B cells, cross-reacting with human HLA-DR.

Cell Biophys, 1988 Aug, 13(1), 55 - 63
Light-stimulated ultraweak photon reemission of human amnion cells and Wish cells; Scholz W et al.; Photon reemission in the ultraweak intensity range that is observed after irradiation of cell suspensions with light, reveals characteristic differences between normal human amnion cells and transformed Wish cells from the same parental tissue . The reemission kinetics, approximated best by a hyperbolical process, were studied as a function of cell density, showing that: malignant Wish cells have a photon storage capacity that is not improved by increasing the cell density; and that normal amnion cells exhibit a photon storage capacity that strongly increases with increasing cell density . The interpretation of this effect and the nature of the emitter are discussed.

FEBS Lett, 1988 Jul 18, 234(2), 349 - 52
NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions; Sullivan SG et al.; NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions . Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms . The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water . Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time . Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population . Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.

Cancer Res, 1988 Jul 15, 48(14), 4065 - 72
Morphological study of the interaction of intravascular tumor cells with endothelial cells and subendothelial matrix; Crissman JD et al.; Multiple steps or events have been described as essential in the metastatic cascade . Tail vein injection of single cell suspensions was used to study the ultrastructural details of the events involved in the initial arrest and attachment of circulating tumor cells . Lewis Lung Carcinoma (3LL) and a mammary adenocarcinoma (16c) were compared to a previous ultrastructural study of B16 amelanotic melanoma (B16a) detailing morphological events in the initial arrest and attachment of tumor cells in lung . The three murine tumors followed similar steps and varied only slightly in the time sequence of the steps . We observed the following steps: (a) initial arrest of tumor cells was characterized by an intimate tumor endothelial cell contact; (b) platelet activation and aggregation was noted by two minutes . Platelet aggregation continued for 1-4 h until a thrombus formed; (c) after approximately 4 h endothelial cell separation with extension of the tumor cell to the subendothelial matrix was noted; (d) at approximately 24 h the tumor cell associated thrombus dissipated and the attached tumor cells were exposed to a reestablished circulation . (e) mitoses were observed after 24 h with cell division and the development of intravascular tumor nodules; (f) the final step in the extravasation sequence was dissolution of the basement membrane by the attached tumor cells.

Eur J Biochem, 1988 Jul 15, 175(1), 187 - 91
Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver; Puschel GP et al.; In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow . These changes could be inhibited completely by alpha-receptor blockers . The possible involvement of inositol phosphates in the intracellular signal transmission was studied . 1 . In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate . 2 . In the perfused rat liver 1 microM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions . 3 . In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate . These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.

Biochim Biophys Acta, 1988 Jul 7, 942(1), 96 - 106
The influence of tetraphenylborates (hydrophobic anions) on yeast cell electro-rotation; Arnold WM et al.; The action of a series of tetraphenylborate ion (TPB) derivatives on yeast cells was studied by electro-rotation of the pre-treated cells . TPB derivatives in which all four phenyl groups were substituted with fluorine, chlorine or trifluoromethyl were much more toxic than the unsubstituted compound, the effect increasing dramatically with increasing size of substituents . These observations suggest that the toxicity of these hydrophobic ions is determined mainly by their size and possibly also by the chemical inductivity of their substituent groups . The order of the toxicities of these ions was in fair agreement with literature values for their translocation rates across artificial bilayers . Incubation times of 3 h were used as standard, longer incubations (up to 48 h) showed that the number of cells affected by low doses of TPB increased with the logarithm of time after the first hour of incubation . Although measurements of the percentage of cells showing co-field rotation showed that controls were not adversely affected by incubations as long as 9 h, rotation spectra showed that some cells suffer loss of internal conductivity during extended incubations . Decrease of the pH of the incubation medium, or inclusion of high concentrations of NaCl or KCl, potentiated the effects of these hydrophobic ions . The toxicity developed slowly, and the sensitivity of the assay was only very weakly dependent on the cell suspension density.

J Reprod Fertil, 1988 Jul, 83(2), 647 - 53
Effect of A-nor steroids and oestradiol on progesterone production by human luteal cells; Wang HZ et al.; Cell suspensions were prepared from human corpora lutea obtained during the mid-luteal phase . Progesterone production was assessed after short-term incubation of luteal cell suspensions . Luteal cells were very sensitive to hCG, the concentration required for 50% maximum response being 0.01 i.u./ml, and the response was 5 times higher than the basal production . Oestradiol (1-100 microM) induced a significant dose-related decrease in both basal and hCG-stimulated progesterone production . The A-nor steroidal compounds anordrin and AF-45 reduced hCG-stimulated progesterone production only at the high concentration of 100 microM . The ED50 values were approximately 3 microM, 75 microM and 100 microM for oestradiol, AF-45 and anordrin respectively . Anordrin showed no significant effects on basal progesterone production . In addition, oestradiol markedly inhibited the activity of 3 beta-hydroxysteroid dehydrogenase in luteal cells, expressed by the conversion of pregnenolone to progesterone, but the inhibitory effects of anordrin and AF-45 were negligible or relatively low . The effects of anordrin and AF-45 were different from those of oestradiol on progesterone production by human luteal cells in vitro, indicating that neither substance is likely to be a useful luteolytic agent in women.

Brain Res, 1988 Jul 1, 470(1), 146 - 50
Early appearance of type II astrocytes in developing human fetal brain; Elder GA et al.; To compare rat and human glial development, we examined the cellular composition of human fetal brain in short-term cultures and fresh cell suspensions from fetal ages ranging from 7 to 16 weeks, utilizing the cell type-specific markers which define glial subsets in rats . Here we report that unlike the early rat central nervous system (CNS), 7-10 week human fetal brain contains mostly astrocytes that can be characterized as type II rather than type I . Type I astrocytes become more prevalent in 16-week gestational age human brain . Although cells morphologically and immunocytochemically similar to the rat 02-A cell are found in human fetal brain and spinal cord, these cells were not induced to express galactocerebroside in serum-free media and did not have vimentin-containing intermediate filaments as do rat 02-A cells . These results suggest that functional differences may exist between rat type I and type II astrocytes and phenotypically similar cells found in humans.

Hematol Oncol, 1988 Jul-Sep, 6(3), 205 - 11
Rearranging antigen-receptor genes in enriched Reed-Sternberg cell fractions of Hodgkin's disease; Cossman J et al.; Molecular genetic analysis of rearranging antigen-receptor genes in non-Hodgkin's lymphomas has revealed the clonality and lineage in the majority of cases . In an analogous approach, we sought to apply gene rearrangement analysis to Hodgkin's disease to understand better the clonality and origin of this disorder . However, the putative neoplastic cell of Hodgkin's disease, the Reed-Sternberg cell and its variants, is extraordinarily rare in most cases of Hodgkin's disease . On the average, Reed-Sternberg cells and variants represent 0.1 per cent of total cell suspensions of nodular sclerosing Hodgkin's disease . As this frequency is below the minimum threshold of sensitivity of the Southern blot assay, we attempted to enrich for Reed-Sternberg cells before DNA extraction and analysis . Using either elutrition or Percoll density gradient centrifugation, we were able to enrich the percentage of Reed-Sternberg cells and variants to above 1 per cent in five cases of nodular sclerosing Hodgkin's disease . In three of these cases, immunoglobulin gene rearrangements were identified, but no T cell receptor gene rearrangements were seen . No rearrangements were detected in unseparated cells or in the Reed-Sternberg cell-depleted fractions . In addition, the L428 Hodgkin's disease cell line was found to have one rearranged and one deleted heavy-chain gene, a rearranged kappa gene, a rearranged lambda gene, and a single rearranged beta allele . No rearrangements of the T gamma gene were found in L428 . Taken together, these findings indicate that clonal cell populations are present in Hodgkin's disease and suggest the possibility of a clonally expanded lymphoid cell in this disorder.

J Neurosurg, 1988 Jul, 69(1), 121 - 6
Brain xenografts: the effect of cyclosporin A on graft survival; Howard MA 3rd et al.; Animal models of Parkinson's disease and Alzheimer's disease have shown dramatic functional improvement after transplantation of embryonic neurons into denervated regions of the adult brain . Because of the ethical and logistic problems associated with the use of human embryonic brain tissue, cross-species transplants are an attractive alternative . An experimental model of cross-species brain transplantation was developed to evaluate cell survival in untreated and cyclosporin A (CyA)-treated animals . Cholinergic ventral neurons from embryonic mice were transplanted into the frontal lobes of 18 adult Sprague-Dawley rats using a cell suspension technique . Nine animals were treated for 13 days with CyA (10 mg/kg/day) and nine were not treated . Twelve weeks after transplantation, frozen sections through the transplant volume were obtained . Alternate sections were prepared with hematoxylin and eosin and acetylcholine esterase stains . Cell counts through a 2-cu mm volume incorporating the transplant were compared to a contralateral control volume . Eight of the nine untreated transplants were successful (mean transplant cells +/- standard error of the mean: 90.7 +/- 19.4/2 cu mm) . All of the nine CyA-treated transplants survived, with mean transplant count 28.7 cells/2 cu mm greater than untreated transplants (mean increase 28.7: p less than or equal to 0.05, Wilcoxon matched-pairs signed ranks test) . It is concluded that: 1) this model is useful for quantitating transplant cell survival; 2) untreated xenografts survive well; and 3) a 13-day course of CyA improved long-term graft survival.

J Immunol, 1988 Jul 1, 141(1), 315 - 23
Age-related variation in the proportion and activity of murine liver natural killer cells and their cytotoxicity against regenerating hepatocytes; Itoh H et al.; We investigated the distribution of liver NK cells in mice of various ages and their cytotoxicity against regenerating hepatocytes . Liver NK cells were identified by asialo GM1 antibody in mononuclear cell suspension from the liver, whereas NK activity was assayed against YAC-1 target cells . Mononuclear cells in the liver consisted of more than 25% NK cells with potent NK activity in C3H/He mice, 8 wk of age . The strain-specific distribution (C3H/He greater than C57BL/6 greater than DBA/2) of liver NK cells was the same as those in the spleen and blood . The proportion of liver NK cells and the level of NK activity in C3H/He mice were further demonstrated to vary depending on age, in that both the proportion and the function were generated at 4 wk of age, reached a maximum between the 6th and 8th wk, and then rapidly decreased around the 9th wk . The appearance of an increased number of NK cells in the liver seemed to coincide with the slowing of the rapid increase of murine liver weight . We then investigated whether liver NK cells mediated their cytotoxicity against regenerating hepatocytes . Both specific 51Cr-release assay and single cell cytotoxicity assay demonstrated that liver NK cells were significantly cytotoxic against regenerating hepatocytes in partially hepatectomized liver, but to a lesser extent against normal hepatocytes in resting liver . Morphologic study revealed that normal liver predominantly consisted of hepatocytes with binuclei (greater than 60%) but that regenerating liver mainly consisted of hepatocytes with a single nucleus (greater than 70%) . One-nucleus hepatocytes were more susceptible to the cytotoxicity of liver NK cells . A comparative study of restoration kinetics of the liver weight and the number of liver NK cells after partial hepatectomy also showed a unique relationship . These results raise the possibility that liver NK cells might be responsible for regulating hepatocyte growth.

Endocrinology, 1988 Jul, 123(1), 113 - 9
Altered differentiated cell surface properties of transformed (RINm5F) compared with native adult rat pancreatic B cells; Halban PA et al.; RINm5F cells, a line derived from a rat insulinoma, are frequently used as a model for studying pancreatic B cell structure and function . These transformed cells are known, however, to be different from native B cells in a number of biochemical respects . We have now compared the surface features of RIN cells and native B cells in two different ways: 1) Dispersed cells from islet obtained from adult rats can reassociate spontaneously in culture to form aggregates (pseudoislets) with cellular organization similar to that of intact native islets (a central B cell core surrounded by a discontinuous mantle of non-B cells) . Native islet cells and RIN cells were mixed together and allowed to reaggregate . Examination by immunocytochemistry and transmission electron microscopy showed that the aggregates contained all cell types present in the original mixed cell suspension (native B- and non-B cells, and RIN cells) . The native B cells were centrally located, surrounded by zones of non-B cells, as in islets . The RIN cells, however, were restricted to the periphery and as such not recognized as native B cells . 2) R2D6, a monoclonal antibody, binds selectively to a ganglioside on the surface of islet B, but not non-B, cells . The role of this ganglioside is not known . RIN cells were incubated with R2D6 followed by a fluorescently labeled second antibody . Analysis by flow cytofluorometry indicated that the monoclonal antibody had bound (stained) only 3-15% of the RIN cells . These R2D6 positive cells were sorted from R2D6 negative cells and subsequently shown to have a lower DNA content . Expression of the R2D6 target ganglioside on RIN cells thus appears to be cell cycle dependent . Based on two different criteria, RINm5F cells do not therefore share surface features in common with native B cells . The cell cycle dependent expression of a B cell surface antigen by the RIN cells might, however, be a useful model for studying the regulation of ganglioside turnover.

Rev Argent Microbiol, 1988 Jul-Sep, 20(3), 107 - 18
{Effect of quinones and nitrofurans on Trypanosoma mega and Crithidia fasciculata}; de Pahn EM et al.; Demonstration of trypanocidal effects in vitro is a first step for the development of new antichagasic drugs . In order to obtain an experimental model allowing the pre-screening of potential trypanocides for Trypanosoma cruzi in a short time and under safe conditions, the trypanosomatids T . mega and C . fasciculata were assayed for their response to a) compounds known for their action on T . cruzi, and b) compounds not tested before on the latter . The drugs were assayed on the organisms growth in a liquid culture medium, cell multiplication being measured by the medium turbidity increase, using a photoelectric colorimeter previously calibrated with cell suspensions of known concentration . A series of quinones (Lapachones and related compounds), naftoquinone-imines, benzoquinones (perezone and dihydroperezone), a quinol (miconidine) and several nitrofurans, including nifurtimox and (5-nitro-2-furfurylidene)-amino (NF-group) derivatives, inhibited the flagellates growth, specially T . mega, with half-maximal inhibitory concentrations lesser than 5.0 microM, for the most active compounds . T . mega response to nifurtimox, NF-derivatives and beta-lapachone was in close agreement with that of T . cruzi . Cultures of T . mega in the presence of NF-pyrazole, NF-indazoles and NF-imidazole but not nifurtimox, showed irreversible damage since, after re-incubation in fresh medium without inhibitor, these cells grew significantly less than their corresponding controls . Similar effects were observed in C . fasciculata, with beta-lapachone and one naftoquinone-imine . Our results qualify T . mega as an adequate experimental model for the assay of antichagasic agents, as C . fasciculata and T . brucei brucei do for the african trypanosomes.

No To Shinkei, 1988 Jul, 40(7), 623 - 8
{Transplant-induced recovery from 6-OHDA lesions of the nigro-striatal dopamine neurons in mice}; Shimizu K et al.; Attempts to reconstruct the damaged nigrostriatal pathway in experimental models of Parkinson disease have thus far been carried out in animals with neurotoxically induced dopamine deficiency . Our study established that unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal-dopamine (DA) neurons produced a well-characterized functional asymmetry in the behavior of C57BL/6 (H-2b) mice . The intraperitoneal administration of methamphetamine induced ipsilateral rotation at 7-20 turns/min . 11 x 10(6) syngenic DA-rich cells of embryonic ventral mesencephalon were stereotaxically transplanted in the caudate-putamen . A complete recovery of methamphetamine-induced rotational response was produced around the 60-th day after the syngenic cell suspension graft . And a complete compensation of the rotational response was also brought about with the DA-rich cells from embryonic ventral mesencephalon (crown-rump length; 10-13 mm) of allogenic C 3 H/HeN (H-2k) mice . The FACS IV analysis revealed no H-2 (Kk and Iak) antigens before transplantation of these embryonic cells . Immunohistochemistry showed that the dopaminergic fibers had grown predominantly into the ipsilateral caudate-putamen . These results provide evidence of integration of syngenic and allogenic grafts and host tissue . And the immunological response in the transplanted brain are under investigation.

Z Naturforsch {C}, 1988 Jul-Aug, 43(7-8), 479 - 84
Novel glucoalkaloids from Rauwolfia cell cultures--acetylrauglucine and related glucosides; Ruyter CM et al.; From cell suspension cultures of Rauwolfia serpentina grown in an optimized production medium for the glucoalkaloid raucaffricine, a novel glucoalkaloid was isolated and identified as 17-O-acetyl-21-O-beta-D-glucopyranosyl-ajmaline (acetylrauglucine) . This alkaloid is formed in very small amounts (less than 5 x 10(-4)%) . The biogenetically related N alpha-demethylated base (acetyl-nor-rauglucine) and the deacetyl product rauglucine have also been detected in culture extracts . In addition 21(R)-(beta-D-glucopyranosyl)-hydroxy-sarpagan-17-al has been isolated and identified as an artifact which originates from raucaffricine.

ASAIO Trans, 1988 Jul-Sep, 34(3), 773 - 7
In vitro evaluation of a pyridoxalated hemoglobin polyoxyethylene conjugate in reversing cell sickling; Yabuki A et al.; In sickle cell disease (SCD) microcirculatory blockage by red blood cells (RBC) occurs because of their low oxygen concentration, which results in both sickling and painful crises . Pyridoxalated hemoglobin polyoxyethylene (PHP), developed from human RBC hemoglobin (Hb) by chemical modification as an oxygen carrier, was evaluated in vitro for its ability to reverse cell sickling . PHP solutions of 6 or 8 g % Hb and a P50 of 20 mmHg were evaluated . RBC were obtained from SCD patients treated by exchange transfusion . The in vitro positive pressure filtration method (47 mm 5 microns Nuclepore membrane) was used . Comparisons of PHP with low oxygen carrier solutions (Hespan, saline) were made at flows of 0.43 to 6.0 ml/min . PO2 increases in a 20% mixture of air saturated solutions with deoxygenated sickle cell suspensions (SCS), at a hematocrit of 1%, were significantly higher in PHP as compared with saline and Hespan . The filtration resistance of deoxygenated SCS mixed with PHP was significantly lower than that of deoxygenated SCS mixed with saline and Hespan, and was comparable to that of air oxygenated SCS . By the 20% (v/v) addition of air saturated PHP to deoxygenated SCS, 89 +/- 2% of the sickled cells were unsickled . A novel artificial capillary system (ACS) modeling the dynamics of the microcirculation of the body was used . With the ACS plugged with deoxygenated cells, perfusion with oxygenated cell-PHP solutions was significantly more efficient in reversing the blockage than oxygenated saline and Hespan solutions . PHP reverses cell sickling by its effective delivery of oxygen.

ASAIO Trans, 1988 Jul-Sep, 34(3), 578 - 80
An in vitro model for human endothelial cell seeding of a small diameter vascular graft; Kent KC et al.; A precise system was devised to measure the kinetics of attachment of human venous endothelium to a variety of materials and substrates . Cells were labelled in a postconfluent state with tritiated thymidine, harvested, and a cell suspension seeded into a 4 mm PTFE graft . After a 90 minute incubation period, one half of the graft segment was sacrificed and the remaining portion placed in a perfusion system (225 cc/min) for 1 hour . Graft segments, effluents, and seeding suspension were assayed in a beta scintillation counter . The percentage of cells that attached pre- and postperfusion were determined, as well as the retrieval of tritium from the system . Initially, 71% of seeded cells attached to grafts coated with fibronectin, with significantly less (60%) remaining attached after perfusion . Only 10% of cells initially attached to uncoated grafts, with 4% retained postperfusion . Retrieval of tritium averaged 102 +/- 10% for all experiments . This system determines both pre- and postperfusion attachment of human endothelial cells to vascular grafts following manipulation of numerous variables, including graft material, substrate, incubation time, and seeding density . An optimal seeding protocol for human trials can thus be determined.

ASAIO Trans, 1988 Jul-Sep, 34(3), 375 - 85
Couette membrane filtration with constant shear stress; Fischel RJ et al.; Recent developments in the field of blood component separation have revealed the usefulness of membrane filtration using couette type configurations and Taylor vortices as an efficient and effective method . The authors have analyzed in detail the physical and chemical effects on whole blood separated into protein rich plasma, and concentrated red blood cell suspensions, using this technique . The authors also have calculated and demonstrated the technical specifications required to provide laminar flow with Taylor Vortex formation throughout the device, as well as those required to retain constant shear stress on the blood components as viscosity changes . By maintaining constant shear stress below a critical level, it is possible to avoid shear induced hemolysis and to maintain maximal separation efficiency throughout the procedure . The device has further been designed to alter the filtration velocity along the membrane so that the critical filtration velocity is nowhere exceeded, i.e., concentration polarization effects are prevented.

Agents Actions, 1988 Jul, 24(3-4), 261 - 5
Enhancement by cimetidine of chemotactic peptide-stimulated ATP release and chemiluminescence in human neutrophils; Tarnok I et al.; As determined by luciferase-luciferin, we recently found that the H2-blocker CIM considerably increased the ATP release from fMLP-stimulated PMN . This observation correlates well with our previous {1} regarding the enhancement of superoxide output (chemiluminescence) in in human neutrophils . by CIM plus fMLP . In order to compare the ATP release from PMN of different donors, a standard procedure has been developed consisting of the determination of the ATP present initially in the cell suspension (without stimulation), ATP release after stimulation with fMLP, and ATP release in the presence of CIM plus fMLP . The whole ATP content per neutrophil was determined after ultrasonication of the cells as well . The mean value of the initially present ATP was 0.45 x 10(-17) mol/cell in the suspension . Stimulation with fMLP plus CIM yielded within 5-10 minutes considerably higher ATP amounts than fMLP alone . The corresponding and statistically significantly different mean values were 2.46 x 10(-17) mol/PMN (s.d . = 1.047) and 1.38 x 10(-17) mol/PMN (s.d . = 0.55), respectively . The whole ATP per neutrophil was found to be 1.22 x 10(-15) mol (mean; s.d . = 0.60) and thus, the stimulation with CIM plus fMLP released about 2.0 per cent, with fMLP alone about 1.0 per cent of the whole ATP . CIM without fMLP did not enhanced the ATP release during the reaction time applied . On the other hand, fMLP-stimulated, lucigenin-amplified chemiluminescence determinations were carried out in the presence of CIM as well; contrarily to our previous method, CIM was dissolved in PBS without DMSO, because DMSO inhibited the chemiluminescence slightly.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunobiology, 1988 Jul, 177(3), 293 - 304
Lack of correlation between in vivo peroxidatic activity patterns and tumoricidal activity in vitro of peritoneal macrophages after intraperitoneal immunization with tumor cells; Dullens HF et al.; Immunization of C57BL mice with one inoculum of 10(7) DBA/2-derived SL2 lymphosarcoma cells resulted in a +/- 20-fold increase in the total number of peritoneal cells . The number of macrophages showed a 10-fold increase from 3 x 10(6) (control mice) to 3.4 x 10(7) cells at day 8 after immunization . Within this macrophage population, four different cell types, based on the ultrastructural peroxidatic activity patterns, could be distinguished: exudate macrophages, resident macrophages, resident-exudate macrophages and peroxidatic-activity-negative macrophages . The number of exudate macrophages significantly increased in the peritoneal cavity after immunization: at day 8 after immunization, a peak value of 10(7) cells was observed . At the same time, there were 2.2 x 10(7) peroxidase-activity-negative macrophages present (representing the control value x 50) . Significant in vitro tumoricidal activity of the isolated macrophages could not be measured until 8 days after immunization . At that time, a cytotoxicity index of 68 was reached . After immunization of the C57BL mice with 3 injections with allogeneic SL2 cells, there were no dramatic changes in the number of peritoneal cells after the last immunization . Only immediately after the last immunization was a minor increase in peroxidatic-activity-negative macrophages seen . But already at 5 days after the last immunization, the composition of the peritoneal suspension was similar to that of non-immunized mice with predominantly resident macrophages . The cytotoxicity of the peritoneal macrophages from hyperimmunized mice was constantly high during 1-15 days after the last immunization (cytotoxicity index ranged from 66-72) . In order to study which type(s) of macrophage(s) (resident, exudate, resident-exudate or peroxidatic-activity-negative) is/are responsible for the cytotoxicity measured in vitro, peritoneal cell suspensions (obtained after immunization) were fractionated according to their affinity to wheat germ agglutinin (WGA) coupled to Sepharose columns . Comparison of the values of cytotoxicity measured before and after separation into "subtypes" of the macrophages revealed that the expression of cytotoxicity is not correlated with any of the "sub-types", especially when the peroxidatic activity pattern is is taken as a criterion.

Ann Inst Pasteur Immunol, 1988 Jul-Aug, 139(4), 361 - 81
Mechanism of K562-induced human natural killer cell inactivation using highly enriched effector cells isolated via a new single-step sheep erythrocyte rosette assay; Abrams SI et al.; In this study, we used preparations highly enriched in human natural killer (NK) cells to further characterize the mechanism of target-cell-induced NK inactivation . Highly enriched populations of NK cells were obtained by a newly developed, single-step sheep red blood cell rosette assay . This method, which did not require any incubation steps to facilitate cell contact, permitted a rapid and efficient isolation of NK cells from adherent-cell-depleted peripheral blood lymphocytes . The non-rosetted cells had high NK activity, possessed large granular lymphocyte (LGL) morphology and expressed the NK-associated antigens Leu-11a, Leu-7, OKM1 and NKH-1 . In contrast, the rosetted cells had significantly lower NK activity, possessed typical lymphocyte morphology and expressed the T-cell-associated marker OKT3 . Next, we examined the ability of these NK-enriched effector cells (ECc) to become inactivated by K562 . Functional studies revealed that ECc lost greater than or equal to 95% of their lytic capacity following incubation with K562 at a ratio of 2/1 for 6 h . However, to achieve this level of inactivation, it was essential that the cell suspension be gently mixed every 90-120 min . Inactivation was not due to cell death and did not reflect changes in the percentages of cells bearing the Leu-11a, Leu-7, OKM1 and NKH-1 antigens, but was associated with an increase in cell surface concentration of OKM1 . As judged by gross morphology, the percentages of LGL in ECc before and after treatment with K562 were essentially the same . Finally, K562-treated ECc also lost their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), suggesting that both NK-cell-mediated cytotoxicity and ADCC may involve a common lytic pathway.

Blood, 1988 Jul, 72(1), 282 - 6
Bivariate flow karyotyping in human Philadelphia-positive chronic myelocytic leukemia; Arkesteijn GJ et al.; Chromosome analysis on clinical leukemia material was done by means of flow cytometry (flow karyotyping) to investigate the applicability of this technique in the detection of leukemia-associated abnormalities . Flow karyotyping was performed on blood or bone marrow samples from eight patients with chronic myelocytic leukemia (CML) after a culture period of four days and arresting the cells in metaphase during the last 16 hours . Discontinuous density gradient centrifugation proved to be essential in removing debris and dead cells from the cell suspensions . By this procedure the mitotic index increase ranged from 2 to 80 times initial values . Chromosomes were isolated and stained with two base pair-specific fluorochromes, ie, chromomycin A3 and Hoechst 33258, and run through a specially designed dual-laser beam flow cytometer . Generally, 20,000 chromosomes or more were measured . The data were computer stored in list mode . Besides the clear detection of the specific Philadelphia chromosome, trisomies and other additional chromosomal aberrations {like an i(17q)} were visualized . Quantitative analysis revealed the percentage of subclones containing a certain chromosomal anomaly . Conventional cytogenetic analysis confirmed these findings . In seven of eight cases, CML could be diagnosed on the basis of the presence of a Philadelphia chromosome in the flow karyogram . In one of these seven, the conventional cytogenetic analysis was unknown at that time . The remaining six all matched the standard cytogenetics . The one failure out of eight could be attributed to the specific stimulating conditions in the culture . Although it is impossible by this technique to determine the position of the breakpoint, the involved chromosomes in the translocation event could be identified . In some cases, low percentages of aberrations could not be detected . This study shows that CML can be diagnosed on the basis of flow karyotypic results . Additional chromosomal aberrations can be detected provided that changes in the amount of DNA per chromosome have occurred . Exact quantification of the composition of subclones in the case of mosaicism appear difficult.

J Comp Neurol, 1988 Jul 1, 273(1), 26 - 41
Development of intracerebral dopaminergic grafts: a combined immunohistochemical and autoradiographic study of its time course and environmental influences; Abrous N et al.; The aim of the study was to obtain a description of some aspects of the development of intracerebral dopaminergic grafts, namely, the time course of the glial reaction and its relation to cell division on one hand, and the development of graft-originated innervation and its dependence on adequate matching of the implanted neurons and target site on the other hand . Cell suspensions obtained from the mesencephalon or hypothalamus of embryonic day (ED) 14 rat embryos were implanted into the striatum or lateral hypothalamus of adult rats following the destruction of the nigrostriatal system of the hosts . Animals were sacrificed at different postimplantation times, and the development of the graft was followed by immunohistochemistry by using antisera directed against tyrosine hydroxylase (TH) or glial fibrillary acidic protein (GFA) . Furthermore, the existence of cell division at various times following implantation was examined by performing autoradiography on immunostained sections after prior intraventricular administration of 3H-thymidine to the host . The first stage of the development of intracerebral grafts was characterized by the existence of intense cell division within the grafted tissue, lasting about 2 weeks, and also in the host tissue surrounding the graft, lasting only about 6 days . The cell division in the host tissue was paralleled by the existence of a strong glial reaction which, however, did not extend into the graft itself . Glial reaction in the host tissue gradually decreased at later times and disappeared by 4 weeks postimplantation without leaving behind a noticeable glial scar . The graft itself was, however, transiently filled with a population of reactive astroglial cells between 3 and 6 weeks postimplantation . Within grafts of mesencephalic tissue located in the striatum TH-positive neurons were distributed evenly at short times postimplantation (2-6 days) . At later time a compartmentation could be observed, with TH-positive neurons being aligned along the graft-host interface or clustered within the graft itself . Innervation of the host tissue by TH-positive fibers increased between 1 and 6 weeks postimplantation . On the other hand, no compartmentation and reinnervation of surrounding host tissue was observed for intrahypothalamic grafts of mesencephalic tissue or intrastriatal grafts of hypothalamic tissue . This last observation indicates that adequate matching of implanted neurons and target tissue plays an important role in the development of intracerebral dopaminergic grafts.

Am J Physiol, 1988 Jul, 255(1 Pt 2), F22 - 32
Asymmetry of plasma membrane lipid order in Madin-Darby Canine Kidney cells; Le Grimellec C et al.; Fluorescence anisotropy experiments have been done to estimate, in situ, the lipid order of the plasma membrane of polarized Madin-Darby Canine Kidney cells (MDCK) grown on glass cover slips and labeled by 1-{4-(trimethylamino)phenyl}-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific marker of the plasma membrane of living cells . Fluorescence microscopy, back-exchange, and quenching experiments indicated that TMA-DPH labeled the highly ordered (r greater than or equal to 0.32, 37 degrees C) apical domain of the plasma membrane of confluent monolayers . Opening of tight junctions or addition of the probe to cell suspensions resulted in a homogeneous distribution of TMA-DPH over the cell surface and in a marked decrease in anisotropy (0.27 less than or equal to r less than or equal to 0.29) that was due neither to a direct effect of Ca2+ on the probe nor to a change in fluorescence lifetime . Our data indicate that the apical domain, likely the external leaflet, of the plasma membrane of polarized MDCK cells is much more ordered than its basolateral counterpart.

Tsitologiia, 1988 Jul, 30(7), 903 - 7
{The possible use of PKZh-type devices (liquid purity control devices) in the analysis of cell suspensions}; Ivanov NR et al.; PKZh means "device for liquid purity control" . A possibility is considered to use the native PKZh type device for carrying out quantitative analyses of cellular suspension components, for routine bacterial suspension, agglutinated bacterial suspension and erythrocyte suspension . The flowing photometric principle of particle recording, used in the device, allows to analyse biological suspensions with small amounts of components . The device provides a differential count of some cells and their conglomerates in six dimensional ranges, within the frames of 1-25 micron or higher . The time consumption for one sample analysis is 10-15 seconds.

Cytometry, 1988 Jul, 9(4), 303 - 8
Identification of cell subpopulations by dual-color surface immunofluorescence using biotinylated and unlabeled monoclonal antibodies; Cohen JH et al.; A new method of dual-color immunofluorescence is presented for analysis of surface antigen distribution among heterogeneous cell suspensions . It involves flow cytometric analysis of cells stained with a biotinylated first monoclonal antibody and/or with an unlabeled second monoclonal antibody . After addition of streptavidin-phycoerythrin and/or fluoresceinated goat antimouse immunoglobulin antibody, single-cell fluorescence intensities are measured and biparametric graphic representations are obtained, allowing one to determine the percentage of cells stained by each of the monoclonal antibodies or both . The validity of the method was assessed on human peripheral blood mononuclear cells by using three sets of two monoclonal antibodies: CD8 and CD5, CD3 and CD4, CD11 and HLA-DR . The results showed that dual staining did not induce significant quenching or competition between pairs of antibodies . The procedure is simple and sensitive . It requires only minute amounts of monoclonal antibodies . It is readily applicable to the screening of hybridoma supernatants and to the characterization of new antibodies to cell surface antigens with respect to well-defined markers.

Clin Exp Metastasis, 1988 Jul-Aug, 6(4), 325 - 32
Correlation between tumour antigens and malignancy in BKV-transformed hamster cells; Rosciani C et al.; Hamster kidney cells transformed by BK virus (HKBK cells) were studied in order to correlate the membrane tumour antigens to the metastatic capability . The presence of the tumour associated surface antigen (TASA) on the surface of HKBK cells was detected by the immunofluorescence test on live cell suspensions . The metastatic ability was investigated by inoculating HKBK cells subcutaneously (s.c.) into newborn hamsters, s.c., intraperitoneally (i.p.) and in the foot pads into adult hamsters, and s.c . into adult hamsters previously immunized with surface antigens extracted from HKBK cells . The results indicate that there is a correlation between the appearance of tumour antigens on the cell surface and the metastatic ability: HKBK cells at low passage (about 30 subcultures after transformation) showed the capping of TASA in the cell membrane and low metastatic ability, whereas HKBK cells at high passage (about 130 subcultures after transformation) exhibited a diffuse appearance of TASA in the cell surface and were highly metastatic.

Immunol Lett, 1988 Jul, 18(3), 219 - 23
Preparation of both DNA and RNA for hybridization analysis from limiting quantities of lymphoid cells; Pearse MJ et al.; This report describes a method for preparing both DNA and RNA simultaneously from as few as 5 X 10(5) lymphoid cells . The method is suitable for cultured cells or any tissue from which a cell suspension can be prepared and for the small samples of purified cells obtained by fluorescence activated cell sorting . Cells are lysed with Nonidet P-40 and the nuclear and cytoplasmic fractions separated by centrifugation . Nuclei are embedded in low-gelling-temperature agarose and the proteinase K and restriction enzyme digestions performed whilst the DNA is immobilized in this form . Total RNA is prepared from the cytoplasmic fraction . This method is simple but reliable and is therefore particularly useful for preparing and analyzing the DNA and RNA from multiple samples when material is limited.

J Cell Biol, 1988 Jul, 107(1), 163 - 75
Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures; Meyer DJ et al.; Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies . Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes . One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point . This component was conspicuously labeled by {3H}proline in vivo . The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan . Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin . Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin . We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin . Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane . This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion . The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues . However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated . This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

Blood, 1988 Jul, 72(1), 150 - 8
Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils; Dvorak AM et al.; The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils . This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein . The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome . Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods . Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques . Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum . CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules . These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules . The crystalloid-containing, large, specific granules did not stain for CLC protein . Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein . The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections . The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct "primary" granule population that serves as a major intracytoplasmic repository for this enzyme.

J Biol Chem, 1988 Jun 25, 263(18), 8803 - 9
Purine accumulation in human fat cell suspensions . Evidence that human adipocytes release inosine and hypoxanthine rather than adenosine; Kather H; Human adipocytes are of limited viability (7 +/- 2% release of lactate dehydrogenase/h) and contain active ectophosphatases which are capable of sequentially degrading ATP to adenosine . At densities of 30,000-40,000 cells/ml, human fat cell suspensions accumulated adenosine, inosine, and hypoxanthine, and their concentrations were 38 +/- 8, 120 +/- 10, and 31 +/- 7 nmol/liter after 3 h of incubation . Dipyridamole (10 mumol/liter), an inhibitor of nucleoside transport, caused a 5-7-fold increase in adenosine accumulation which was reduced by 85% on inhibition of ectophosphatases by beta-glycerophosphate and antibodies against ecto-5'-nucleotidase or alpha, beta-methylene 5'-adenosine diphosphate (10 mumol/liter), respectively, indicating that most of the adenosine is produced in the extracellular compartment . Accordingly, the spontaneous accumulation of adenosine was reduced beyond 5 nmol/liter on inhibition of ectophosphatase activities or removal of extracellular AMP by AMP deaminase (4 units/ml) . Added adenosine (30 nmol/liter) disappeared until its concentration approached 5 nmol/liter . Isoproterenol (1 mumol/liter) had no effect on adenosine accumulation regardless whether purine production from extracellular sources was minimized or not . In contrast to adenosine, the concentrations of inosine and hypoxanthine displayed only a modest decrease (30-50%) on inhibition of ectophosphatase activities . In addition, isoproterenol caused a 2-3-fold increase in inosine and hypoxanthine production which was concentration-dependent and could be inhibited by propranolol . It is concluded that the adenosine that accumulates in human adipocyte suspensions is almost exclusively derived from adenine nucleotides which are released by leaking cells . By contrast, inosine and hypoxanthine are produced inside the cells, and the release of these latter purines appears to be linked to ATP turnover via adenylate cyclase.

Neurosci Lett, 1988 Jun 17, 89(1), 108 - 13
Fetal serotoninergic transplants in the fourth ventricle of adult rodents reverse sleep disturbances induced by neonatal administration of 5,7-dihydroxytryptamine; McRae-Degueurce A et al.; Rats received a neonatal (at 4 days of age) intracisternal injection of 5,7-dihydroxytryptamine which eliminates serotonin (5-HT) throughout adulthood . Sleep recordings, performed on these rats at adult age, demonstrated significant decreases in paradoxical sleep . Dissociated fetal 5-HT cell suspensions transplanted in the IVth ventricle of these rats restored paradoxical sleep . However, fetal neuronal noradrenergic or cholinergic transplants did not restore paradoxical sleep . These results suggest that paradoxical sleep is directly or indirectly mediated through serotoninergic mechanisms.

In Vitro Cell Dev Biol, 1988 Jun, 24(6), 530 - 6
Clonal growth characteristics of adult human prostatic epithelial cells; Peehl DM et al.; The ability of human epithelial cells derived from adult prostatic tissues to undergo clonal growth in culture was examined . In a previously described serum-free culture system, such cells exhibited a density-dependent growth requirement . It was found that raising the level of one of the constituents of the culture medium, bovine pituitary extract, to 100 micrograms/ml permitted excellent clonal growth when as few as 100 cells were inoculated/60-mm2 dish . Raising the levels of supplements other than pituitary extract (cholera toxin, epidermal growth factor, hydrocortisone, or insulin) did not produce this result . The average colony-forming efficiency of cells derived from primary or early passage cultures was approximately 25% . When single cell suspensions were prepared from tissue isolates and directly analyzed for clonal growth, colony-forming efficiencies were approximately 5%, perhaps indicating the proportion of stem cells with proliferative potential in the original isolates . The colony-forming efficiency of a cell population derived from cancer tissue was not significantly different from those of populations derived from normal tissues.

Clin Biochem, 1988 Jun, 21(3), 163 - 5
A simpler procedure to study sodium-22 uptake (ouabain insensitive) in human erythrocytes; Gambhir KK et al.; We report a modification of the technique of Mahoney et al . (Blood 1982; 59: 439) for the determination of sodium-22 (22Na+) uptake in human erythrocytes . This modification facilitates the separation of 22Na+ taken up by erythrocytes from the free 22Na+ in the buffer by the addition of dibutyl phthalate, which forms an immiscible layer between the two . To further improve the sensitivity of 22Na+ uptake, we incubated a range of known numbers of erythrocytes with 22Na+ as opposed to the single cell suspension of known hematocrit used in Mahoney's et al . procedure (1) . Erythrocytes are incubated in KCI buffer containing 2627 Bq (0.071 microCi) 22Na+ in a total volume of 0.5 mL for 0.5 h at 37 degrees C . Incubation is terminated by placing the tubes in ice for 10 min and the amount of 22Na+ taken up by the erythrocytes determined . We observe a linear relationship between erythrocyte concentrations (0.5 to 2.5 X 10(9) cells/mL) and percent uptake of 22Na+ (0.37 +/- 0.06 (1 SD) to 1.85 +/- 0.27 (1 SD) of the total 22Na+, respectively) . The procedure is simple and sensitive, and can be used in clinical laboratories for the routine evaluation of 22Na+ uptake in erythrocytes.

Biull Eksp Biol Med, 1988 Jun, 105(6), 720 - 3
{Formation of bone tissue by mouse bone marrow cell suspensions in organ culture}; Luriia EA et al.; Adult mouse bone marrow cell suspensions prepared by trypsinization were cultivated in gelatin sponges on millipore filters . When HAWP filters were used, multilayer bone structure was formed . It contained mineralized ground substance, incorporated bone cells and osteoblast layer . With the use of AUFS filters, bone tissue developed not only on the top surface, but also inside the filter.

Lab Invest, 1988 Jun, 58(6), 706 - 17
The differentiation potential of tracheal basal cells; Inayama Y et al.; The objective of this study was to examine the differentiation potential of basal cells purified from rabbit tracheas by means of centrifugal elutriation . Cell suspensions containing between 83 and 94% basal cells were inoculated into tracheal grafts denuded of their own epithelium and transplanted to the backs of nude mice . Within 1 week after inoculation, the basal cells reestablished a continuous poorly differentiated epithelial sheet which at 2 to 4 weeks showed typical mucociliary differentiation . Basal cells, ciliated cells, goblet cells, and secretory cells with small granules were present . Some of the latter contained abundant rough endoplasmic reticulum and others contained predominantly smooth endoplasmic reticulum . These data indicate that basal cells can give rise to all major tracheal cell types suggesting that they are the main stem cell in the large airways of rabbits.

Radiology, 1988 Jun, 167(3), 825 - 9
Meningeal carcinomatosis in the VX2 rabbit tumor model: detection with Gd-DTPA-enhanced MR imaging; Frank JA et al.; Meningeal carcinomatosis developed in 14 of 14 New Zealand White rabbits after infusion of a VX2 tumor cell suspension into the cisterna magna . All died or were killed 7-15 days after inoculation . Within days of the tumor infusion, magnetic resonance (MR) imaging with gadolinium-diethylenetriaminepentaacetic acid (DTPA) at 0.5 or 1.5 T demonstrated enhancement of the cerebrospinal fluid (CSF) secondary to disruption of the blood-CSF barrier by plaquelike lesions along the meninges . Eventually, meningeal enhancement was observed along the base of the brain and cervical spine . Quantitative assessment of the contrast enhancement on T1-weighted images revealed an increase in mean signal intensity of 213% +/- 130% . Contrast enhancement was not observed in four control animals who received an infusion of cell culture medium . These results demonstrate in an animal model that contrast material-enhanced MR imaging can be used to detect meningeal carcinomatosis by revealing breakdown of the blood-CSF barrier.

J Bacteriol, 1988 Jun, 170(6), 2676 - 82
Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion; Ohsumi Y et al.; A specific effect of Cu2+ eliciting selective changes in the permeability of intact Saccharomyces cerevisiae cells is described . When 100 microM CuCl2 was added to a cell suspension in a buffer of low ionic strength, the permeability barrier of the plasma membranes of the cells was lost within 2 min at 25 degrees C . The release of amino acids was partial, and the composition of the amino acids released was different from that of those retained in the cells . Mostly glutamate was released, but arginine was mainly retained in the cells . Cellular K+ was released rapidly after CuCl2 addition, but 30% of the total K+ was retained in the cells . These and other observations suggested that Cu2+ caused selective lesions of the permeability barrier of the plasma membrane but did not affect the permeability of the vacuolar membrane . These selective changes were not induced by the other divalent cations tested . A novel and simple method for differential extraction of vacuolar and cytosolic amino acid pools by Cu2+ treatment was established . When Ca2+ was added to Cu2+-treated cells, a large amount of Ca2+ was sequestered into vacuoles, with formation of an inclusion of a Ca2+-polyphosphate complex in the vacuoles . Cu2+-treated cells also showed enhanced uptake of basic amino acids and S-adenosylmethionine . The transport of these substrates showed saturable kinetics with low affinities, reflecting the vacuolar transport process in situ . With Cu2+ treatment, selective leakage of K+ from the cytosolic compartment appears to create a large concentration gradient of K+ across the vacuolar membrane and generates an inside-negative membrane potential, which may provide a driving force of uptake of positively charged substances into vacuoles . Cu2+ treatment provides a useful in situ method for investigating the mechanisms of differential solute pool formation and specific transport phenomena across the vacuolar membrane.

J Neuroimmunol, 1988 Jun, 18(3), 217 - 22
Failure to detect the presence of pluripotential haemopoietic stem cells in the mouse brain; Stedra J et al.; Single cell suspensions prepared from adult mouse brains were tested for the presence of pluripotential haemopoietic stem cells (colony-forming units, CFU) by transfer into an irradiated recipient and enumeration of the CFU in the recipient's spleen . In contrast to the findings of others (Bartlett, 1982), we did not detect CFU after injection of brain cell suspensions, although they were detectable after inoculation with bone marrow cells . The number of CFU in recipients after transfer of increasing numbers of brain cells was the same as that detected in the irradiated controls which had not received any transferred cells . Finally, cells from the brain, in contrast to bone marrow cells, were not able to protect recipient animals from the effects of lethal irradiation.

Cancer Res, 1988 Jun 1, 48(11), 3040 - 4
Tissue uptake, distribution, and potency of the photoactivatable dye chloroaluminum sulfonated phthalocyanine in mice bearing transplantable tumors; Chan WS et al.; The potency of chloroaluminum sulfonated phthalocyanine (ClAlSPc) as a photosensitizing agent for photodynamic therapy of cancer was evaluated in vivo by its ability to be taken up and retained by murine tumors of diverse histological origin . Antitumor effects following laser irradiation were evaluated by measurement of the tumor weights of dissected-out tumor masses . Three tumors (Colo 26, a colorectal carcinoma; M5076, a reticulum cell sarcoma; and UV-2237, a fibrosarcoma) growing s.c . in the flank region retained substantially greater quantities of ClAlSPc than did adjacent skin and muscle achieving peak values 24-48 h after the i.v . administration of ClAlSPc (10 mg/kg) . The relative magnitude of ClAlSPc retention by these tumors was Colo 26 greater than M5076 greater than UV-2237 . However, normal liver and spleen were organs which retained the greatest amounts of ClAlSPc even compared to the s.c . grown tumors and other normal tissues examined . Flow cytometric analysis of tumor cell suspensions obtained from collagenase-digested tumors showed that individual neoplastic cells were capable of taking up and retaining ClAlSPc . Photodynamic therapy, undertaken by i.v . administration of dye (5 mg/kg) followed 24 h later by local laser light irradiation (675 nm, 100 J), brought about significant (Colo 26, M5076, and 3LL tumors) and obvious but nonsignificant (UV-2237 tumor) reductions in tumor weights, as assessed 5 days later . Thus, selective tumor retention of ClAlSPc coupled with a significant response to red light produced dramatic alterations in cancer growth.

Int J Radiat Biol Relat Stud Phys Chem Med, 1988 Jun, 53(6), 921 - 33
Variations in the spectrum of lesions produced in the DNA of cells from mouse tissues after exposure to gamma-rays in air-breathing or in artificially anoxic animals; Murray D et al.; Gamma-ray-induced DNA-protein crosslinks (dpc) are preferentially induced in cultured cells irradiated at very low oxygen tensions (Meyn et al . 1987) . Since some cells within mouse tumors may be radiobiologically hypoxic, dpc may also be induced in such cells after irradiation in vivo . To examine this possibility, mice bearing either an FSa or NFSa fibrosarcoma in their hind legs were whole-body irradiated either while breathing atmospheric oxygen or 15 min after cervical dislocation, which induces uniform anoxia . DNA single-strand breaks (ssb) and dpc were then assayed both in tumors and normal tissues by alkaline elution . The level of dpc was inferred from the observed increase in ssb yield after digestion of the cell lysates with proteinase K . In addition, cell suspensions were irradiated in vitro, on ice, exposed to atmospheric oxygen tensions . Few dpc were detected in the DNA from tumor cells irradiated in vitro; however, in cells from both FSa and NFSa tumors irradiated in situ there was a significant level of protein-concealed ssb, and thus of dpc . These data are most likely the result of the relative hypoxia of a proportion of cells from both the FSa and NFSa tumor in the air-breathing animals . Induction of dpc was further enhanced in the DNA from tumor cells irradiated under anoxic conditions . A significant level of dpc was also observed in jejunal and spleen cells irradiated in vivo; however, since a significant level of protein-concealed breaks was also observed in cells irradiated in vitro, oxygenation appears not to be the only parameter capable of modifying the proportion of protein-concealed ssb, and the effects of proteinase K on the DNA elution rate for normal mouse tissues may be complex.

J Histochem Cytochem, 1988 Jun, 36(6), 679 - 83
Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy; De Waele M et al.; We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS) . Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy . In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background . The sensitivity of detection was much higher than that of brightfield microscopy . Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension . However, non-specific staining was also better visualized . The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions . A 25% reduction of the silver enhancement time was necessary for this purpose . However, these weaker labeling conditions also reduced the intensity of the specific staining . Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure . Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.

Cell Biol Toxicol, 1988 Jun, 4(2), 211 - 23
A novel class of unstable 6-thioguanine-resistant cells from dog and human kidneys; Turker MS et al.; Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion . In medium containing a relatively high concentration (10 micrograms/ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10(-4) to 10(-5) . A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity . When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell-free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity . In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell-free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity . Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones . These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanthine phosphoribosyltransferase expression may occur in dog and human kidney cells.

J Neurocytol, 1988 Jun, 17(3), 351 - 60
Transplantation of oligodendrocytes and Schwann cells into the spinal cord of the myelin-deficient rat; Duncan ID et al.; Transplantation of oligodendrocytes or Schwann cells into the spinal cord of the newborn myelin-deficient (md) rat, an X-linked myelin mutant, was carried out and the extent of myelination of CNS axons studied . Dissociated glial cell suspensions, prepared from the spinal cords of female litter-mates, were injected into the lumbar spinal cord of 15 md rats and 5 normal litter-mates . In eight of the md rats examined 12 to 21 days post-transplantation patches of myelin produced by the transplanted oligodendrocytes were found in the dorsal or ventral columns . In two rats, small patches of myelination were found in more than one site . The myelin in these patches was positive on immunocytochemical staining for proteolipid protein . These observations were interpreted as evidence of the origin of this myelin from donor oligodendrocytes, as the md rat has an abnormality in synthesis of this protein . In addition, this myelin differed in its ultrastructure from host myelin, having a normal intraperiod line . Injection of cultured Schwann cells also resulted in extensive myelination of axons in the dorsal columns by these cells.

Appl Environ Microbiol, 1988 Jun, 54(6), 1330 - 3
Continuous-sterilization system that uses photosemiconductor powders; Matsunaga T et al.; We report a novel photochemical sterilization system in which Escherichia coli cells were sterilized with photosemiconductor powders (titanium oxide) . For sterilization that could be used in practice, it was necessary to separate the TiO2 powders from the cell suspension . Therefore, semiconductor powders were immobilized on acetylcellulose membranes . We constructed a continuous-sterilization system consisting of a TiO2-immobilized acetylcellulose membrane reactor, a mercury lamp, and a masterflex pump . As a result, under the various sterilization conditions examined, E . coli (10(2) cells per ml) was sterilized to less than 1% survival when the cell suspension flowed in this system at a mean residence time of 16.0 min under irradiation (1,800 microeinsteins/m2 per s) . We found that this system was reusable.

Immunol Invest, 1988 Jun, 17(4), 309 - 19
Flow cytometric analysis of lymphocyte activation in the mixed lymphocyte response; Prince HE et al.; Interleukin 2 receptor (IL2R) expression may be a useful parameter for assessing lymphocyte activation in the mixed lymphocyte response (MLR) . However, the contribution of irradiated stimulator (S*) cells to the levels of IL2R+ cells recovered must first be defined . We have used a flow cytometric parameter termed the sip count to assess this potential contribution of S* cells . This parameter, which is the number of cells within a defined cell gate, is a reflection of the viable cell number per culture well, since (a) a constant number of cells were plated per well on day 0, (b) cells recovered from a well were resuspended in a constant volume, (c) the flow cytometer aspirated (sipped) a constant volume of cell suspension, and (d) nonviable cells were not included in the gate . Sip count assessment showed that only 5% of S* cells were recoverable by day 4 of culture; in contrast, 70% or more of unstimulated responder cells were recoverable . Sip counts of MLR cultures identified an increase in cell number beginning on day 5, reflecting DNA synthesis and cell division . We then used the sip count to assess changes in the levels of IL2R+ cells in MLR cultures . The number of IL2R+ cells continued to increase up to day 7, even though maximal DNA synthesis occurred on day 5 . Further, dual color analysis revealed that the proportion of CD4 cells expressing IL2R was maximal on day 5, whereas the proportion of CD8 cells expressing IL2R continued to increase until day 9 . These findings show that flow cytometry can be used to study lymphocyte activation by alloantigens.

J Neurol Sci, 1988 Jun, 85(2), 197 - 207
Myogenicity in vitro and in vivo of mouse muscle cells separated on discontinuous Percoll gradients; Morgan JE; Mouse muscle cells, obtained by enzymatically disaggregating newborn mouse muscle, were separated on a discontinuous Percoll gradient . The myogenicity in vitro of the resultant cell fractions was examined by counting the percentage of nuclei in myotubes . Myogenicity in vivo was assessed by implanting a cell suspension of one of the allotypes of glucose-6-phosphate isomerase (GPI) into a regenerating skeletal muscle graft of a second GPI allotype: the finding of hybrid GPI indicated that the implanted cells were myogenic . Separation of mouse muscle cells on a discontinuous Percoll gradient gave rise to two myogenic fractions, one of which was more myogenic in vitro than were the unseparated cells and one of which was less myogenic . Both of these fractions were myogenic in vivo . A cell fraction was also produced which was non-myogenic in vitro as well as in vivo . In vitro and in vivo measurements of myogenicity were therefore in broad agreement.

Biochem Biophys Res Commun, 1988 May 31, 153(1), 203 - 8
Human B and T lymphocytes convert leukotriene A4 into leukotriene B4; Odlander B et al.; Incubation of human tonsillar B lymphocytes and peripheral blood T lymphocytes with leukotriene A4 led to the formation of leukotriene B4 . The purity of these cell suspensions was more than 99%, containing less than 0.5% monocytes . Incubation of purified B or T lymphocytes with the calcium ionophore A23187 did not lead to the formation of any detectable amounts of leukotrienes . Several established cell lines of B and T lymphocytic origin were also found to convert leukotriene A4 into leukotriene B4, showing that monoclonal lymphocytic cells possess leukotriene A4 hydrolase activity.

Eur J Biochem, 1988 May 16, 174(1), 189 - 97
Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials . An explanation for the apparent uncoupler insensitivity of ATP synthesis; Kaesler B et al.; The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein) . In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative) . When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the {K+}in/{K+}out ratio . Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ({K+}in/{K+}out = 1000) to 100 mV ({K+}in/{K+}out = 40) had no effect on the rate of methane formation and on the intracellular ATP content . At delta mu H+ values below 100 mV ({K+}in/{K+}out less than 40) both the rate of methanogenesis and the ATP content decreased . Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ({K+}in/{K+}out less than 7) . The data show that methanogenesis from H2 and CO2 and ATP synthesis in M . thermoautotrophicum are possible at relatively low electrochemical proton potentials . Similar results were obtained with Methanosarcina barkeri . Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M . thermoautotrophicum even when used at high concentrations (400 nmol/mg protein) . This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.

Arch Biochem Biophys, 1988 May 15, 263(1), 191 - 8
Induction of phytoalexin synthesis in soybean: enzymatic cyclization of prenylated pterocarpans to glyceollin isomers; Welle R et al.; A microsome preparation from elicitor-challenged soybean cell suspension cultures catalyzed an NADPH-dependent and oxygen-dependent cyclization of a mixture of 2- and 4-dimethylallylglycinols to the glyceollin isomers I-III . This is the last committed step in glyceollin biosynthesis . The cyclase was inhibited in a light-reversible manner by carbon monoxide in the presence of oxygen . Cyclase was also inhibited by cytochrome c, NADP+, and a number of inhibitors of cytochrome P-450 enzymes . NADH in the presence of low concentrations of NADPH had a synergistic effect . On a Percoll gradient, the position of cyclase coincided with marker enzymes for the endoplasmic reticulum . These properties identify the cyclase as a cytochrome P-450-dependent monooxygenase . Unstimulated soybean cell culture did not contain detectable cyclase activity . Challenge with either a glucan elicitor from Phytophthora megasperma f.sp . glycinea or with yeast extract caused strong stimulation of cyclase activity with a maximum at about 24 h after elicitor addition.

Cancer Res, 1988 May 15, 48(10), 2779 - 83
Effects of hyperoxia on growth characteristics of metastatic murine tumors in the lung; Margaretten NC et al.; Suspensions of an oxygen-sensitive (MT-7) and of an oxygen-insensitive(M109) tumor cell line were injected i.v . into BALB/c mice . Exposure to 100% O2 after injection of the cells did not modify the initial arrest of either cell line in the lung . Exposure of animals given injections of MT-7 cells for 60 h to 100% oxygen decreased the number of lung colonies formed even when onset of oxygen exposure was delayed up to 10 days after injection of the cell suspension . Cell cycle time and growth fraction in lung colonies growing in vivo were estimated from an analysis of the percentage of mitoses labeled . In lung colonies formed by MT-7 cells, hyperoxia produced a mitotic delay and a 30 to 40% reduction in the growth fraction . In M109-derived colonies, oxygen did not change cell cycle times or reduce growth fraction . In earlier experiments done in vitro and reported by others it had been found that, in tumor cell lines other than the ones used in the present study, a prolongation of the early prophase was the most oxygen-sensitive event . The present data show that in vivo oxygen inhibits lung colony formation in MT-7 cells by a similar mechanism.

J Immunol, 1988 May 15, 140(10), 3541 - 6
Granulocyte-macrophage colony-stimulating factor modulates the excitation-response coupling sequence in human neutrophils; Naccache PH et al.; We have investigated the effects of granulocyte-macrophage (GM) CSF on two biochemical responses thought to play internal signaling roles in the neutrophils, namely the increase in the concentration of free calcium and the changes in the internal pH . The changes in the right-angle light scatter of the cell suspensions were also examined . GM-CSF was found not to affect the resting levels of calcium or the internal pH of the cells . However, pre-incubation of the neutrophils with the growth factor resulted in an increase in the magnitude of the calcium transients that follow the stimulation of the cells with chemotactic factors as well as a profound depression of the cell alkalinization that is induced by fMet-Leu-Phe, leukotriene B4, and platelet-activating factor, as well as by phorbol esters . The rapid cytoplasmic acidification elicited by these agents was apparently magnified . GM-CSF did not directly affect the Na+/H+ antiport as GM-CSF-treated cells were as capable as untreated cells of recovering from an acid load . The right-angle light scatter responses of the cells to the chemotactic factors were found not to be affected by GM-CSF . These results provide an initial description of the effects of GM-CSF on the cellular physiology of the neutrophils and insights into the mechanism of action of this factor as well as into the excitation-response coupling sequence activated by chemotactic factors.

J Natl Cancer Inst, 1988 May 4, 80(5), 351 - 60
Mixed phenotype lymphomas of B10.H-2a-H-4bp/Wts mice: characterization of component cell populations; Willoughby PB et al.; Three murine lymphomas of H-2a origin were investigated . Each of these lymphomas was derived in B10.H-2aH-4bp/Wts (2a4b) mice after a hyperimmunization protocol involving sheep red blood cells . Analysis of cell suspensions derived from the splenic tumors revealed a mixed lymphoid phenotype of the total cell population . Both the T-cell and B-cell populations remained after multiple passages of the tumors in syngeneic mice . A multiparameter approach combining immunologic and molecular genetic techniques was taken to determine the malignant cell population within these lymphomas . This analysis included depletion experiments utilizing histocompatible (B10.A X 21M)F1 mice to distinguish normal host-derived cells from the malignant tumor cells, fluorescence-activated cell sorting of the two cell populations followed by in vivo growth of the sorted cells, in vitro and in vivo cloning attempts, and molecular analysis of genomic DNA derived from the splenic tumors for clonal rearrangements of the immunoglobulin heavy-chain and T-cell receptor loci . Each of the three lymphomas was shown to be a malignant B-cell tumor that has an associated nonneoplastic T-cell component . The nature and functional significance of the numerous T-cells present in the B-cell lymphomas remain unknown and are being investigated.

J Surg Res, 1988 May, 44(5), 473 - 8
A possible mechanism for the release of serotonin from the gut caused by pentagastrin; Gronstad KO et al.; Pentagastrin (PG) is a potent agent causing release of serotonin (5-HT) from patients with carcinoid tumors . The physiological release of 5-HT from gut enterochromaffin cells is controlled by beta-adrenoceptors . Studies on carcinoid tumor cell suspensions, acute or in culture, have shown that catecholamines (CA), but not PG, release 5-HT, thus indicating an indirect mode of action by the peptide . In this study the mechanism for release of 5-HT from the gut induced by PG was investigated in animal models . The test protocol for patients was used in anesthetized cats . Portal blood samples were drawn after PG injection (0.6 microgram/kg iv), which resulted in significantly increased levels of 5-HT at 3 and 5 min postinjection . The PG-induced release was totally inhibited after blockade of beta-adrenoceptors (propranolol) or of slow calcium channels (verapamil) as well as after adrenalectomy . Blockade of beta-adrenoceptors or slow calcium channels decreased the basal levels of 5-HT, while adrenalectomy caused no change . In separate experiments CA were measured after PG injection in caval blood, drawn at the level of the adrenal veins . There was a significant increase in the levels of dopamine and epinephrine postinjection, while the levels of norepinephrine were stable . The changes of CA levels after PG injection could be prevented by adrenalectomy . These results further suggest an indirect action of PG in the release of 5-HT from the feline gut via activation of beta-adrenoceptors by epinephrine released from the adrenals.

J Cell Physiol, 1988 May, 135(2), 262 - 8
Growth characteristics of human epidermal melanocytes in pure culture with special reference to genetic differences; Hirobe T et al.; Human melanocyte cultures were established using disaggregated epidermal cell suspensions derived from foreskins and plated onto culture dishes in medium containing 2% fetal bovine serum, growth factors, hormones, and melanocyte growth factor (MGF) extracted from bovine hypothalamus (Wilkins et al., J.Cell . Physiol., 122:350, 1985) . After 2 days in culture the cells were transferred to serum-free medium to eliminate keratinocyte and fibroblast growth . Melanocytes grew preferentially and pure melanocyte populations could be harvested after 12-16 days in vitro . Melanocytes were later subcultured in the presence of 1% FBS . Pure melanocyte cultures were characterized by light and electron microscopic criteria, as well as by cytochemical demonstration of the melanocyte-specific enzyme, tyrosinase . At the ultrastructural level, cultured melanocytes derived from black (negroid) neonatal skin (B-M) had numerous mature rod-shaped stage IV melanosomes, while white (caucasoid) skin-derived melanocytes (W-M) in culture contained no mature melanosomes . Growth rate, cell yield, and in vitro lifespan for B-M were more than twice that for W-M in pure melanocyte cultures in the presence of MGF . Our results suggest that MGF-dependent growth of B-M differs from that of W-M.

Blut, 1988 May, 56(5), 221 - 7
Sulfide silver amplification of ferritin iron cores in blood and bone marrow cells . Methods, adaptations to microphysical analyses, and the impact of advanced iron overload; Hausmann K et al.; A short exposure of cell suspensions to gaseous hydrogen sulfide, appropriate fixations, and subsequent physical development of silver shells around sulfidated insoluble metals were used to amplify ferritin iron cores in blood and bone marrow cells . The methods described are suitable for both light microscopy and transmission electron microscopy . These techniques made it possible to visualize Prussian Blue stainable ferritin and haemosiderin, as well as a large variety of isoferritin iron and other smaller particles beyond the sensitivity of Prussian Blue staining . Admixtures of sulfidatible zinc and traces of other heavy metals had to be taken into consideration . For further research, adaptations of sulfide silver staining to microphysical analyses were developed . However, conventional energy dispersive X-ray analysis was not sensitive enough to signalize the presence of Fe in sulfide silver amplified iron cores of a single or a few ferritin molecule(s) . Proton-induced X-ray emission was used to measure Fe and Zn down to 1 fg/single cell in unstained or sulfide silver stained smears on thin foils . However, multielement analysis of homogeneous cell concentrates was much easier to perform and far more sensitive . In advanced iron overload, highly increased sulfide silver staining was found in peripheral blood cells including lymphocytes, monocytes, eosinophils, basophils, and--in extreme cases--also in neutrophils and platelets.

Ultrasonics, 1988 May, 26(3), 168 - 70
Ultrasonic attenuation in red blood cell suspensions; Dai HP et al.; A theoretical analysis of ultrasonic attenuation in aqueous suspensions of red blood cells was carried out . This verified the relative importance of several mechanisms: absorption accounts for 60% of the attenuation, viscous relative motion loss accounts for less than 30%, and the sound scattering contribution is negligible.

Arch Biochem Biophys, 1988 May 1, 262(2), 445 - 54
Purification and properties of S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase from cell suspension cultures of Glycine max L; Upmeier B et al.; A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the nitrogen atom of pyridine-3-carboxylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean (Glycine max L.) . Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties . S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 degrees C . The apparent KM values were determined to be 78 microM for nicotinic acid and 55 microM for the cosubstrate . S-Adenosyl-L-homocysteine was a competitive inhibitor of the methyltransferase with a KI value of 95 microM . The native enzyme had a molecular mass of about 90 kDa . The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence of the enzyme reaction . The purified methyltransferase revealed a remarkable specificity for nicotinic acid . No other pyridine derivative was a suitable methyl group acceptor . To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation . This was achieved by affinity chromatography on S-adenosyl-L-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.

Immunology, 1988 May, 64(1), 147 - 53
A role for mature B cells in bone marrow transplantation; Yoshio T et al.; The proliferative potential of membrane Ig (mIg)-bearing B lymphocytes was assessed in an adoptive transfer system based on the use of non-inbred rabbits matched for major histocompatibility (MHC) antigens and mismatched for immunoglobulin (Ig) allotypes . Cell suspensions made from spleens (SP), mesenteric lymph nodes (LN), or bone marrow (BM) of allotype b4b5 rabbits were deprived of B cells with mIg of the b4 type by adherence to plastic dishes coated with affinity-purified anti-b4 . When such b4-depleted cell populations were injected into newborn hosts of allotype b6b6, stable and lasting chimerism promptly resulted, in which donor-derived products were almost entirely of the b5 allotype . Chimeras formed by transfer of unfractionated cells from b4b5 donors, on the other hand, exhibited a predominance of the b4 allotype, as seen in the living donors . BM but not SP or LN contained precursors capable of differentiating into mIg+ lymphocytes in culture, but no evidence was obtained for engraftment and differentiation by such B-cell precursors or more primitive stem cells in vivo . These studies suggest a potentially significant role for mature B cells in reconstituting the immune system of human transplant recipients.

J Chem Neuroanat, 1988 May-Jun, 1(3), 133 - 46
Laminin facilitates and guides fiber growth of transplanted neurons in adult brain; Zhou FC et al.; Laminin has been shown in vitro to act as a surface adhesive molecule for neuronal process elongation . To test whether laminin has a similar role in the brain, we sequentially injected laminin and transplanted fetal neurons into various brain regions to determine if the fetal neurons would preferentially grow along a laminin injection tract . In the fetal brain, the raphe area of the rostral rhombencephalon is rich in serotonergic (5-HT) neurons; the rostral ventral mesencephalon is rich in dopamine (DA) neurons, while the lateral rhombencephalon is rich in norepinephrinergic (NE) neurons . These three areas were transplanted to the motor cortex, neostriatum or hippocampus of adult animals . The tract used for microinjection of cell suspension was then immediately filled with laminin in a suspension media or a laminin-collagen (type IV) mixture . In other animals, laminin or control solution was injected in a separate needle tract displaced 0.3-1 mm from the transplant injection tract . Straight and thick 5-HT, DA or NE immunoreactive (IR) fibers (stained with anti-5-HT or anti-tyrosine hydroxylase antiserum) were predominant within the laminin-treated tracts, or were directed toward the laminin-treated parallel tracts when it was positioned less than 0.5 mm from the transplant site . The density of 5-HT-, DA- and NE-IR fibers in the injection tracts in all three brain areas was much higher for laminin and laminin-collagen mixture than control media . Thin axonal fibers of fetal 5-HT and NE neurons were observed surrounding the laminin-treated tracts, but not around vehicle-injected tracts . In addition, a number of transplanted 5-HT, DA and NE neuronal cell bodies were seen within the laminin-treated tracts, but not in vehicle-treated tracts . Finally, laminin injection to the hippocampus, motor cortex or neostriatum of the adult brain did not stimulate sprouting of undamaged adult 5-HT or NE fibers . These results suggest that purified laminin can facilitate and guide process outgrowth of 5-HT, DA and NE neurons during early developmental stage, but does not induce sprouting on these same fiber types in the adult brain.

Biull Eksp Biol Med, 1988 May, 105(5), 621 - 3
{Structural and immunologic indicators of Peyer's patches in the human fetus}; Khlystova ZS et al.; Histological and immunological methods were used for the investigation of the development of Peyer's patches in the ileum of 110 human fetuses of 8-29 weeks of gestation . The formation of Peyer's patches and the increased number of lymphatic follicles in them are described . Beginning from 8th-9th week of gestation T- and B-lymphocytes and their subpopulations (auto-RFC, EAC-RFC, IgM- and IgG-positive cells) can be identified in mononuclear cell suspension obtained from Peyer's patches . An increase in their number during embryogenesis and the pathways of lymphocyte migration are shown . By the moment of birth "O" cells dominant in Peyer's patches and later on E-RFC and IGM-positive cells prevail . Auto-RFC are the most scanty . The authors regard Peyer's patches as peripheral organs of immunogenesis involved in local defensive reactions and in the fetal immunogenesis system on the whole.

Cell Tissue Kinet, 1988 May, 21(3), 143 - 7
The spleen colony technique . II . Errors in estimation of the fraction of CFU-s synthesizing DNA; Znojil V et al.; A refinement of the method for determining the fraction of CFU-s in the S phase is presented . The new procedures were tested by means of a model experiment in which the fraction of CFU-s 'killed' was simulated by diluting the cell suspension . Another test was to determine the fraction of the CFU-s in the S phase using duplicate samples.

J Immunol, 1988 May 1, 140(9), 2956 - 63
Induction and functional characterization of class II MHC (Ia) antigens on murine keratinocytes; Gaspari AA et al.; To induce Ia molecules on the surface of murine keratinocytes (KC), healthy mice were treated daily with i.p . injections of rIFN-gamma at a dose of 50,000 U/day for 6 days . This resulted in strong Ia expression by KC as determined by immunofluorescence of epidermal sheets or cell suspensions with anti-class II mAb . To obtain a population of Ia-bearing KC devoid of Langerhans cells, a method of depleting Langerhans cells from such suspensions was developed . Although Ia+ KC were unable to stimulate allogeneic T cells in a primary epidermal cell-lymphocyte reaction (less than 5% control), they did induce a proliferative response in an allospecific T cell line . Ia+ KC were unable to present native peptide molecules to class II restricted, Ag-specific T cell hybridomas . However, Ia+ KC were able to present a peptide fragment of pigeon cytochrome c to a hybridoma, suggesting that although these cells cannot process native protein Ag, they can present antigenic peptides . Ia+ (but not Ia-) KC also served as targets for class II restricted cytolytic T cell clones . These data indicate that the Ia expressed by KC is a functional molecule, and that Ia+ KC can participate in some immunologic reactions.

Cell Biol Int Rep, 1988 May, 12(5), 337 - 46
Flow cytometric analysis of cell-surface binding elements for fibronectin on mouse lung cell isolates; Rosenkrans WA Jr et al.; Fresh lung cell isolates from LAF1 mice were examined for the presence of fibronectin-binding elements using flow cytometric analysis . Thoroughly perfused lungs from adult male mice were dissociated using an elastase-trypsin digestion, gentle pipetting and filtering . The resulting heterogeneous cell suspension was incubated with fibronectin coated 0.5 micron fluorescent beads . Subsequent flow cytometric analysis indicated the presence of two species of specific fibronectin-binding populations; one of higher binding affinity which can be blocked with exogenous plasma fibronectin and one of lower binding affinity . We tentatively identify the lower affinity binding element with the fibronectin adhesion receptor and the higher affinity element with the putative matrix assembly receptor.

J Pharmacol Methods, 1988 May, 19(3), 193 - 203
Determination of beta adrenoceptor density on rabbit mononuclear leukocytes; Earley KJ et al.; The purpose of the present study was to devise a technique for the isolation of a relatively homogeneous mononuclear leukocyte (MNL) preparation from rabbit whole blood and determine the density and affinity of beta-adrenoceptors on MNL . A modified method based upon that by Boyum was developed to maximize isolation of MNL from red blood cells (RBC), granulocytes, and platelets . The method involved an initial centrifugation to remove platelets and two centrifugations with a Ficoll solution to eliminate RBC and granulocytes . beta-Adrenoceptor density as determined with {125I}cyanopindolol and MNL membrane or whole cell preparations ranged between 317 and 360 sites per cell . Affinity for the binding sites was dependent upon whether membrane or whole cell preparations were studied, being 56.3 +/- 9.9 and 11.4 +/- 1.4 pM, respectively . Binding sites were found to be saturable and noncooperative . In addition, the binding sites demonstrated selectivity and stereospecificity for beta-adrenoceptor ligands . It is concluded that the modified method of harvesting MNL from rabbit whole blood provides a relatively homogeneous cell suspension that can be used to study the beta-adrenoceptor system.

Transplantation, 1988 May, 45(5), 953 - 7
Absence of allogeneic T cell response to human insulin-secreting cells following long-term culture; Demidem A et al.; Lymphocyte proliferative responses (3H-Thymidine uptake) were studied in mixed culture combinations of peripheral blood lymphocytes as responder cells and human insulinoma cells as stimulator cells (MILR) . Influence of culture time on the ability of insulinoma cells to stimulate allogeneic T cell proliferation was examined . Crude cell suspensions initiated a strong lymphoproliferative response with a stimulation index (SI) that ranged between 3 and 7, whereas insulinoma cells did not stimulate allogeneic T cells after one month of culture . Expression of cell surface determinants coded by the major histocompatibility complex (MHC) was evaluated simultaneously by indirect immunofluorescence using monoclonal antibodies to class I shared-determinant or class II molecules . Human insulinoma cells expressed class I but not class II molecules . Crude insulinoma cell suspensions were found to be contaminated by 2% of DR+ cells from nonislet components . It is postulated that loss of these DR+ lymphoreticular cells with culture time resulted in absence of immune recognition and lymphoproliferative response . These results emphasize the need of culturing human islet cells prior to transplantation in order to reduce cell immunogenicity.

J Neurosci Res, 1988 May, 20(1), 46 - 53
Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes; Gard AL et al.; Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA) . A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture . At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures . The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures . As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar . Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA . As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain . The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.

Int J Radiat Oncol Biol Phys, 1988 May, 14(5), 983 - 8
The use of dansyl lysine to assess heat damage and thermotolerance of normal tissues; Sekiguchi R et al.; The fraction of cells excluding the fluorescent dye dansyl lysine has previously been shown to correlate well with heat-induced cell killing in a variety of mammalian cell lines and in murine tumors in vivo . Here we evaluate the usefulness of dansyl lysine as a probe for assessment of thermal damage and for measuring the kinetics of thermotolerance development and decay in murine normal tissues . Skin cells were heated in vivo with an initial treatment of 44 degrees C for 20 min by local radiofrequency . Bone marrow cells were heated at 42.5 degrees C for 20 min by whole body water bath immersion . Cell suspensions were prepared, heated in vitro for various lengths of time at 44 degrees C (skin) or 43 degrees C (bone marrow), and scored for the fraction of dansyl lysine-excluding cells . Skin and bone marrow cells expressed maximum thermotolerance by 8 and 6 hr, respectively and returned to normal heat sensitivity by 48 and 146 hr, respectively . The assay was not useful with skeletal muscle and liver, as we were not successful in obtaining viable, dansyl lysine-excluding cells from these tissues . Also, in our hands red blood cells, normal human leukocytes, mouse spleen and thymus cells all failed to stain dansyl lysine even after extreme heating . Dansyl lysine staining, particularly when combined with flow cytometry analysis, has been shown to be a useful method for assessing thermal damage and thermotolerance relatively rapidly in all tumor systems tested to date, and, as shown here, may possess utility in measuring similar endpoints for certain nonclonogenic normal tissues.

Biochim Biophys Acta, 1988 Apr 25, 969(2), 194 - 7
Characterization of the renal epithelial cell line, PKE 5, using 31P- and 13C-NMR spectroscopy; Jans AW et al.; In order to characterize intracellular pH regulation and cellular metabolism in PKE 5 cells, a mutant of the renal epithelial cell line LLC-PK1 supposed to lack Na+-H+ exchanger activity, 31P and 13C-NMR studies were conducted . The 31P studies on intact cell suspensions revealed that these cells have an ATP content and an ATP/ADP ratio similar to the parent cell line . Their intracellular pH, in the presence of 5 mM HCO3-, was 7.17 +/- 0.04 (n = 5) - identical to that of LLC-PK1 cells . After acid loading the cells with 15% CO2, the initial rate of realkalinization was 0.027 pH units/min (n = 6), 50% lower than in the parent cells . The recovery rate was not affected by the removal of extracellular sodium or by the addition of 1 mM amiloride . These results indicate that PKE 5 cells are devoid of Na+-H+ exchange activity, but are able to regulate their intracellular pH by amiloride-insensitive, sodium-independent mechanisms . Extracts prepared from PKE 5 cells incubated with {13C}lactate showed 13C spectra identical to those of the parent cell line . In particular, no synthesis of 13C-labeled D-glucose was observed.

Cancer, 1988 Apr 15, 61(8), 1587 - 93
Circulating cerebriform lymphoid cells (Sezary-type cells) in a B-cell malignant lymphoma; O'Briain DS et al.; Circulating cerebriform lymphoid cells (Sezary cells) are considered to be highly predictive of cutaneous T-cell Lymphoma (CTCL) . A leukemic peripheral blood (leukocyte count 24.5 x 10(9)/l) composed predominantly of cerebriform cells was found in a 75-year-old man presenting with weight loss and generalized lymphadenopathy but without skin lesions . Cell suspensions studies and immunohistochemistry of peripheral blood revealed that the cerebriform cells were B-cells (IgM+ Kappa+, HLA DR+, Leu 1+, CALLA-, B1+, and OKT 10+) . A variety of T-cell markers (other than Leu1) was negative . Computer-assisted morphometry confirmed a nuclear profile typical of CTCL (mean nuclear contour index, 7.47) . A lymph node that underwent subsequent biopsy revealed a follicular malignant lymphoma of small to intermediate cells with similar morphologic and immunologic characteristics to the circulating cerebriform cells . The findings of a leukemic presentation of a cerebriform B-cell lymphoma extends the recent observation of nodal B-cell lymphomas composed of cerebriform cells and indicates that circulating cerebriform cells should not be considered to be exclusively of T-cell origin.

Cancer, 1988 Apr 15, 61(8), 1571 - 8
Differences between subcutaneous and intraperitoneal forms of three human testicular teratocarcinomas in nude mice; Niederberger M et al.; Three human testicular teratocarcinomas were serially passaged following subcutaneous transplantation into nude mice . Tumor cell suspensions from selected passages were injected intraperitoneally . The subcutaneous transplants of each tumor conserved the morphological characteristics of one component of the primary tumor, namely an embryonal carcinoma in one case and a yolk sac tumor in two . The latter maintained the capacity to synthesize alpha-fetoprotein (AFP) . After intraperitoneal injection of cell suspensions, tumors, either attached to or even invading abdominal organs or in the form of free-floating tumor spheroids, were observed . AFP could be localized within the attached growths but not in the spheroids . A critical tumor volume and/or vascularization seemed to be necessary for AFP production in tumor cells . In spheroids from one tumor, cytogenetic analysis revealed both human and murine cells . Thus, these spheroids, apparently composed of tumor cells in the center and murine cells at the periphery, can not be considered to be embryoid bodies.

Science, 1988 Apr 8, 240(4849), 204 - 7
Genetically transformed maize plants from protoplasts; Rhodes CA et al.; Genetically transformed maize plants were obtained from protoplasts treated with recombinant DNA . Protoplasts that were digested from embryogenic cell suspension cultures of maize inbred A188 were combined with plasmid DNA containing a gene coding for neomycin phosphotransferase (NPT II) next to the 35S promoter region of cauliflower mosaic virus . A high voltage electrical pulse was applied to the protoplasts, which were then grown on filters placed over feeder layers of maize suspension cells (Black Mexican Sweet) and selected for growth in the presence of kanamycin . Selected cell lines showed NPT II activity . Plants were regenerated from transformed cell lines and grown to maturity . Southern analysis of DNA extracted from callus and plants indicated the presence of the NPT II gene.

Brain Res, 1988 Apr 5, 445(2), 325 - 37
Adrenal medullary cells transmute into dopaminergic neurons in dopamine-depleted rat caudate and ameliorate motor disturbances; Nishino H et al.; Adrenal medullary cell suspensions, derived from newborn rats (postnatal day 1-6), were implanted into the head of the caudate nucleus in 35 rats with unilateral 6-hydroxydopamine (6-OHDA) lesions in the nigrostriatal dopamine (DA) pathway . Behavioral recovery from Met-amphetamine induced circling, cell growth and morphological features (tyrosine hydroxylase positive cells), and release of adrenaline (Ad), noradrenaline (NA), DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were investigated for 40 weeks after transplantation . Met-amphetamine induced circling decreased significantly in 43% (15/35) of the rats . The decrease was concurrent with transmutation of the tyrosine hydroxylase-like immunopositive (THLI) cells into mature neurons that had abundant elongated neurites with varicosities and synapses on neuronal elements in the host caudate . In the absence of behavioral recovery (57%, 20/35) THLI cells were very scant . DA, DOPAC and HVA were reduced more than 90% in perfusates collected by in vivo dialysis from the striata of the animals that were not improved by transplant . These levels recovered to 20-50% of controls in animals whose behavior recovered . Ad and NA were not detected in the perfusates of either recovered or non-recovered animals . The results suggest that some grafted adrenal medullary cells transform into dopaminergic neurons and the release of DA from these grafted cells functionally affects behavior improvement for at least 40 weeks.

J Chromatogr, 1988 Apr 1, 438(1), 61 - 72
High-performance liquid chromatography of carthamin, safflor yellow A and a precursor of carthamin . Application to the investigation of an unknown red pigment produced in cultured cells of safflower; Nakano K et al.; A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed to analyse and purify carthamin, safflor yellow A (safflomin-A) and the yellow precursor of carthamin . A red pigment similar to carthamin was extracted from cell suspension cultures of safflower (Carthamus tinctorius L.) as an application of biotechnology . The RP-HPLC method was used to characterize the unknown red pigment . Various spectroscopic methods were used to characterize purified carthamin and the purified unknown pigment . Based on these spectral observations, it was concluded that the unknown red pigment produced in cultured cells of safflower differed from carthamin, although the unknown pigment might be a glycoside . From other observations, the possibility of anthocyanins and carotenoids were also discounted . It was assumed that the cultured cells lacked the biosynthetic pathway of the yellow precursor of carthamin, although they possessed the activity of the enzyme mediating the synthesis of carthamin from the yellow precursor.

Brain Res, 1988 Apr 1, 467(2), 233 - 43
Survival and function of dissociated rat dopamine neurones grafted at different developmental stages or after being cultured in vitro; Brundin P et al.; The in vitro culture approach was combined with the cell suspension grafting technique to examine whether the maturation of dopamine (DA) neurones in vitro imposed similar limitations on their ability to survive grafting as when they are allowed to develop in situ in the fetus . The functional capacity, survival and growth of DA neurones from 2.5- and 7-day-old cultures, grafted to rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway, was compared with similar grafts freshly prepared from fetal donors of embryonic days 14, 16 and 20 . Grafts of freshly dissociated mesencephalic DA neurones, taken from embryonic day 14-16 donors and 2.5-day-old cultures, generally survived well and markedly reduced amphetamine-induced rotational asymmetry in the recipient rats . However, when cultured for 7 days prior to grafting, or when taken from 20-day-old fetuses, the mesencephalic DA neurones survived very poorly and the grafts did not have any functional effects . Plating of aliquots of cell suspension used for grafting indicated that the survival rate of dissociated DA neurones is in the same order of magnitude when grown in vitro (about 2 DA neurones per 1000 cells) as when grafted in vivo to the rat striatum (about 1-5 DA neurones per 1000 cells) . When the number of surviving grafted DA neurones was plotted against the behavioural effects of the grafts, a threshold number of around 100-200 DA neurones was found necessary to obtain a marked reduction (greater than 50%) in amphetamine-induced rotational asymmetry . Moreover, the survival of 300-500 DA neurones seemed to produce a 'ceiling effect' beyond which additional surviving DA neurones gave rise to little or no further effect on the amphetamine-induced rotational behaviour.

J Trauma, 1988 Apr, 28(4), 453 - 7
Effects of total hip replacement and bed rest on blood rheology and red cell metabolism; Kaperonis AA et al.; In order to better understand the pathophysiologic changes in the immediate postoperative period after total hip replacement surgery and to distinguish alterations due to the surgical operation from those due to bed rest, we examined rheologic parameters and red cell metabolism of patients before, 1 day after, and 5 to 6 days after total hip replacement and compared the results to those obtained from normal volunteers placed at bed rest for 5 days . Bed rest in the control group led to increases in hematocrit, mean corpuscular hemoglobin concentration, red cell DPG and ATP levels, and plasma concentrations of total proteins, globulins, and fibrinogen, with attendant increases in whole blood viscosity, plasma viscosity, blood viscosity, relative blood viscosity with hematocrit adjusted to 45%, and viscometric aggregation index, and the viscosity of red cell suspensions in Ringer's solution at 45% hematocrit decreased at low shear rate . The patient group, despite the postoperative lowering of their hematocrit, mean corpuscular hemoglobin concentration, and total plasma proteins and a consequent decrease of whole blood viscosity, revealed disproportionate increases in blood viscosity, relative blood viscosity, and viscometric aggregation index . These rheologic changes, which reflect an enhanced red cell aggregability, may contribute to complications of thrombophlebitis . Enforced inactivity, when superimposed on the effects of trauma, blood loss, transfusion with bank blood, and the low-flow state, would exaggerate these rheologic problems . The results suggest that management of total hip replacement patients should include hemorrheologic considerations (e.g., preoperative intentional hemodilution) and early postoperative activity.

J Bacteriol, 1988 Apr, 170(4), 1962 - 4
Lack of carbon substrate repression of uptake hydrogenase activity in Bradyrhizobium japonicum SR; van Berkum P et al.; The expression of ex planta uptake hydrogenase (Hup) activity in Bradyrhizobium japonicum SR induced in the absence or presence of carbon substrates was compared . Hup activity was influenced by pH, indicating that acidification of induction medium with low buffering capacity resulting from carbon substrate metabolism inhibited Hup activity . Cell suspensions in medium with adequate buffering capacity and carbon substrate were limited in O2; increasing O2 availability to cells during induction stimulated Hup expression . The data showed a lack of carbon substrate repression of Hup activity in cell suspensions provided with adequate O2 and buffering capacity.

Cancer Res, 1988 Apr 1, 48(7), 1864 - 73
UM-EC-1, a new hypodiploid human cell line derived from a poorly differentiated endometrial cancer; Grenman SE et al.; The University of Michigan endometrial carcinoma cell line UM-EC-1 was derived from a poorly differentiated endometrial adenocarcinoma of a 66-yr-old white female . Cell cultures were started using both tumor explants and a cell suspension obtained from collagenase-treated tumor tissue . The collagenase-derived cell suspension gave rise to monolayer cultures which grew rapidly from the outset . This subline of UM-EC-1 has now been subcultured more than 50 times . Cells derived from the tumor explants grew more slowly initially, but after a lag phase of 5 to 6 wk, this subline also exhibited rapid logarithmic growth and reached the same growth rate as that of the collagenase-treated cells . The explant subline has been subcultured more than 37 times . The doubling time of both sublines is 24 h under optimal growth conditions . The karyotype of both cell cultures is 43, XX, inv(1)(p32q42), -4, +der(8) t(8;12)(p23.1;q22), del(9)(q11), -13, -13, +t(13;13) (p13;p13), del(18)(q), -19, -22, -22, +t(22;22)(p11;p11) . The net result of the chromosome losses and rearrangements was monosomy 4, duplication 8p23.1----qter, deletion 9q11----9qter, duplication 12q22----qter, deletion 18q, and monosomy 19 . The t(13;13) and the t(22;22) were dicentric by C-banding . Virtually all of the chromosome changes were stable in multiple passages except that there was mosaicism for chromosome 13 . Some cells contained a single copy of 13 and others had t(13;13) . The available evidence indicates the t(13;13) is an isochromosome . UM-EC-1 cells produced tumors histologically similar to the original tumor in male, female, and ovariectomized female athymic mice . UM-EC-1 cells express human class I histocompatibility antigens as assessed by binding of antibodies to nonpolymorphic HLA and beta-2-microglobulin antigens . Blood group antigens A and H were absent although the patient is blood type A and these antigens are normally expressed in endometrial glands . A rearrangement involving the region of chromosome nine that carries the ABH locus may be related to the absence of blood group antigen expression by these cells . The E7 membrane antigen, the locus for which resides on the short arm of chromosome 11, was expressed strongly which is consistent with the presence of two intact copies of chromosome 11 in these cells.

Hum Pathol, 1988 Apr, 19(4), 394 - 402
Immunophenotyping of hematopoietic malignancies in paraffin sections; Andrade RE et al.; Immunophenotyping of hematopoietic malignancies is usually accomplished in frozen sections or cell suspensions . To determine whether this procedure was also feasible in paraffin sections, we performed a double-blind immunoperoxidase study of 65 hematopoietic tumors whose phenotypes had been determined previously in fresh tissue . A selected antibody panel was used, including anti-LN2, UCHL-1, anti-cathepsin B, anti-Leu M1, anti-MB2, and anti-MT1 . A correct phenotype was obtained on paraffin sections in 95% of cases . All 31 B-cell malignancies were properly classified, showing reactivity for LN2 and MB2 . In 14 of 15 T-cell hematopoietic malignancies, all cells reacted with anti-MT1 and/or UCHL-1; the 1 case negative for these antigens was misdiagnosed as a B-cell tumor because of misinterpreted LN2 reactivity in benign histiocytes . Four of 5 true histiocytic neoplasms were positive for cathepsin B and LN2 but lacked other antigens; the fifth case was wrongly considered a B-cell proliferation because only bland histiocytes displayed cathepsin B . Only 1 of 7 Hodgkin's lymphomas was misdiagnosed (as a T-cell tumor); in the other 6 cases, Reed-Sternberg cells were reactive for LN2 and LEU M1 . Five of 6 extramedullary myeloid leukemias also stained for LN2, MT1, and LEU M1 . One showed LN2, MB2, and MT1; this case was classified as a B-cell neoplasm and indeed represented a pre-B-cell transformation of chronic myelogenous leukemia . These results show that the specified panel of antibodies may be useful for immunophenotyping of hematopoietic neoplasms when only paraffin sections are available for analysis . However, it cannot supplant traditional cell-marker studies of hematopoietic tumors because of its lesser accuracy.

Invest Ophthalmol Vis Sci, 1988 Apr, 29(4), 534 - 43
Development of neonatal mouse retinal neurons and photoreceptors in low density cell culture; Politi LE et al.; We describe here a culture method which allows the growth of dissociated mouse retinal neurons and photoreceptors in chemically defined medium . Neural retinas from 2-day-old C57/BL mice were dissected from other ocular tissues, including the pigment epithelium, and dissociated into a cell suspension after brief trypsination . Most cells attached as single, unaggregated units to substrata pretreated with polyornithine and the neurite-promoting factor (PNPF) . The cells were cultured in serum-free, high pyruvate Dulbecco's modified Eagle's medium containing chemically defined supplements . Under these conditions, onset of cell process development was rapid, giving rise to extensive neurite networks . Three morphologically distinct cell types were apparent during the first week in vitro . Some cells retained a circular outline and failed to produce processes, while 50-60% of the cells developed as multipolar neurons showing a large cell body and several neurites . Approximately 90% of these cells reacted with an amacrine cell-specific monoclonal antibody . Some 30% of the cultured cells expressed phenotypic properties characteristic of rod photoreceptors, including a small cell body, an apical cilium, a short neurite with a spherule-like terminal body, and immunoreactivity with antibodies against opsin as well as a rod cell-specific monoclonal antibody . No further signs of outer segment differentiation were observed in these cells . Non-neuronal "flat" cells, which represented less than 0.5% of the total cell number, reacted with an antibody against the glial fibrillary acidic protein . The number of neurons and photoreceptors remained relatively stable during the first 4-7 days in vitro . During the second week in culture, however, there was specific degeneration of greater than 90% of the photoreceptor cells, while less than 20% of the multipolar neurons were similarly affected . Consequently, in addition to providing a system for studying the differentiation of retinal neurons and photoreceptors, the specific degeneration of photoreceptors in these mouse retinal cell cultures makes this system ideal for investigating factors influencing photoreceptor survival.

Eur J Immunol, 1988 Apr, 18(4), 545 - 50
Class-specific suppression of human B cell maturation by IgA-binding factors; Millet I et al.; IgA-binding factors (IgA-BFs) were prepared by chromatography on Sepharose 4B beads covalently linked to dimeric and polymeric monoclonal IgA1 from supernatants of peripheral blood mononuclear lymphocytes (PBMC) and human B cell lines incubated in serum-free medium . Receptors for IgA, as revealed by the binding of biotinylated monoclonal IgA1, were expressed on monocytes, T-enriched and T-depleted lymphocytes . IgA-BFs or control eluates were added to pokeweed mitogen (PWM)-stimulated PBMC cultures, and their effects on the terminal differentiation of polyclonally activated human B cells were assessed by enumeration of intracytoplasmic IgM-, IgG- or IgA-containing cells . A selective decrease of IgA-containing cells was observed in the presence of IgA-BFs whereas IgM- and IgG-containing cells remained unchanged . Differential counts of B blasts and plasma cells revealed that only the former were decreased following addition of IgA-BFs . Kinetic studies indicated that maximum inhibition of IgA-containing cell generation was achieved when IgA-BFs were added during the first 5 days of PWM-stimulated PBMC cultures, whereas no inhibition could be demonstrated when IgA-BFs were added 24 h before harvesting . IgA-BFs did not decrease {3H}thymidine incorporation in PWM-stimulated PBMC cultures . They diminished the proliferation of the surface IgA+ monoclonal human B cell line DAKIKI, but not that of the surface IgA- IM-9 cell line . Several control eluates obtained from the same cell supernatants absorbed on Sepharose 4B, Sepharose 4B-IgG or Sepharose 4B-beta 2-microglobulin had no effect . Finally, IgA-BFs prepared from supernatants of two human B cell lines bearing receptors for IgA selectively depressed the generation of intracytoplasmic IgA+ cells in PBMC cultures stimulated by PWM . Altogether the data indicate that IgA-BFs obtained by spontaneous release from heterogeneous mononuclear cell suspensions or from IgA receptor-positive human monoclonal B cell lines selectively depress the maturation of B cells into IgA plasma cells and the proliferation of a surface IgA+ B cell line.

Am J Reprod Immunol Microbiol, 1988 Apr, 16(4), 151 - 8
Antigen presenting cells in human decidual tissue: III . Role of accessory cells in the activation of suppressor cells; Oksenberg JR et al.; Human decidual antigen presenting cells (DAPCs) exposed to fetal cells in vitro induce generation of suppressor T cells among a population of peripheral blood lymphocytes . Human lymphocyte antigen (HLA)-class II positive antigen presenting cells were isolated from early normal human decidual tissue and from peripheral blood (PAPCs) by adhering Ficoll-Paque separated cell suspensions to fibronectin . In contrast to PAPCs, DAPCs pulsed with fetal antigens induced a radio-sensitive, Leu 1,2-positive T suppressor cell population . A nylon wool adherent B cell population is required during the in vitro induction of the suppressor cells . These suppressor cells impair primary mitogen and mixed lymphocyte culture (MLC) responses, generation of anti-trinitrophenyl (TNP) cytotoxic T lymphocytes, and antibody response of autologous and allogeneic lymphocytes . Only intact viable embryonic cells can effectively confer upon DAPCs the ability to induce T suppressor cells . The T suppressor cell induction by DAPCs primed with fetal antigens is restricted by the major histocompatibility complex . Our results show that the HLA-DR molecules are the most prominent restriction elements.

Biull Eksp Biol Med, 1988 Apr, 105(4), 464 - 6
{Controlling effect of T lymphocyte suppressors on the proliferation of cells in various tissues}; Kraskina NA et al.; The changes in mitotic index of mouse bone marrow, spleen, liver, kidneys, small intestine, cornea were studied during alterations in the number of T-suppressors . It was found that mitotic activity in all tissues investigated enhanced significantly after a decrease in the number of T-suppressors caused by single injection of an antiserum against T-suppressors . On the contrary, the mitotic index diminished significantly after the transfer of tolerant spleen lymphoid cell suspension enriched by T-suppressors to normal syngeneic mice . These data indicate that T-suppressors are responsible for the control over cell proliferation in nonlymphoid tissues.

J Exp Med, 1988 Apr 1, 167(4), 1479 - 85
Thymus and autoimmunity . Transplantation of the thymus from cyclosporin A-treated mice causes organ-specific autoimmune disease in athymic nude mice; Sakaguchi S et al.; Organ-specific autoimmune diseases such as gastritis, oophoritis, thyroiditis, or insulitis developed in athymic nu/nu mice after engraftment of the thymus from euthymic nu/+ mice treated with cyclosporin A (CsA), a potent immuno-suppressant . The development of autoimmune disease in the nu/nu mice was prevented by inoculation of thymocyte suspensions prepared from normal nu/+ mice, but not by thymocyte suspensions from CsA-treated nu/+ mice . Cotransplantation of normal nu/+ mouse thymus with CsA-treated thymus also suppressed the development of autoimmune disease . Inoculation of spleen cell suspensions prepared from normal adult nu/+ mice prevented autoimmune disease, but inoculation of those from newborn nu/+ mice did not . Thus, CsA appears to interfere selectively with the thymic production of certain suppressor T cells controlling self-reactive (autoimmune) T cells, allowing the latter to expand and cause autoimmune disease.

Mol Gen Genet, 1988 Apr, 212(1), 93 - 8
Two alleles of the single-copy chalcone synthase gene in parsley differ by a transposon-like element; Herrmann A et al.; Two types of genomic DNA hybridizing with a chalcone synthase cDNA were isolated from cell suspension cultures of parsley (Petroselinum crispum cv . Mooskrause) and cloned in lambda EMBL4 . Their fragmentation patterns with several common restriction enzymes were identical, except for the occurrence of a 927 base pair insertion in one type relative to the other . This insertion is located 538 base pairs upstream of the first of two transcription start sites and has characteristic features of a transposable element . The two types of cloned DNA most likely represent two alleles of a chalcone synthase gene occurring in one copy per haploid parsley genome . The nucleotide sequence and exon-intron structure of the larger allele were determined . Analysis of plants either heterozygous or homozygous with respect to the chalcone synthase gene revealed that both allelic forms were expressed and activated by UV light.

Scand J Immunol, 1988 Apr, 27(4), 451 - 9
Generation and metabolism of leukotrienes and release of histamine from human dispersed tonsillar cells; Schluter B et al.; We studied the generation and metabolism of leukotrienes (LT) and the release of histamine by human tonsillar cell suspensions . Human tonsils were dissected and mechanically dispersed . This procedure yielded a single cell suspension with 1.6 +/- 0.5 X 10(8) cells/g tissue consisting of 97.3 +/- 0.4% lymphocytes, 1.4 +/- 0.3% granulocytes, 1.3 +/- 0.3% macrophages/monocytes, and 0.03 +/- 0.02% mast cells/basophils . The cells were stimulated either with Ca-ionophore A 23187, melittin, or anti-human IgE . Determination of the 5-lipoxygenase products LTB4 and LTC4 was performed with specific radioimmunoassays (RIA), and histamine release was measured by the fluorophotometric technique . A time- and dose-dependent release of the mediators was monitored . LTB4 exceeded the amount of LTC4 in the supernatants . The concentration of leukotrienes ranged between 0.8 and 5.4 ng LTB4/1 X 10(8) cells or 0.5 and 1.5 ng LTC4/1 X 10(8) cells, depending on the stimulus . Histamine release after stimulation ranged between 25 and 35% of the total histamine content, whereas buffer controls amounted to 17% . The incubation of the cells (1 X 10(8) with exogenously added LTB4 resulted in the formation of omega-oxidated products (20-OH and 20-COOH-LTB4) and a novel unpolar metabolite, as identified by thin layer chromatography . This metabolite was not immunoreactive in the LTB4-RIA used . LTC4 and LTD4 were converted into LTE4 when added either to sonicated cells or to the cell-free supernatants of prestimulated tonsillar cells, indicating the release of gamma-glutamyltranspeptidase and dipeptidase, respectively . Our data clearly demonstrate the generation and metabolism of the 5-lipoxygenase products LTB4 and LTC4 as well as the release of histamine from human dispersed tonsillar cells, suggesting that they have a modulatory function with respect to the inflammatory potential at local sites.

Nature, 1988 Mar 31, 332(6163), 448 - 50
Cholinergic-rich brain transplants reverse alcohol-induced memory deficits; Arendt T et al.; Alcohol-induced memory impairment in man has been attributed to deficiencies in subcortical noradrenergic and cholinergic systems, as well as to damage in midbrain structures . Korsakoff's psychosis, a disease in which alcohol poisoning causes apparently irreversible memory defects, is characterized by lesions in cholinergic and noradrenergic nuclei and by a decrease in the activity of choline acetyltransferase (ChAT) and the content of noradrenaline (NA) in forebrain areas such as cerebral cortex and hippocampus, innervated by these nuclei . Prolonged intake of ethanol in rodents similarly produces signs of noradrenergic and cholinergic deafferentation in the cortex and hippocampus, as well as persistent memory deficits . To test whether alcohol-induced memory impairments depend on cholinergic deafferentation, we transplanted cholinergic-rich fetal basal forebrain cell suspensions into the cortex and hippocampus of alcohol-treated rats . The substantial and persistent memory losses produced in our rats by ethanol intake were associated with an impairment of cholinergic function, and were reversed by cholinergic-rich transplants into cortex and hippocampus.

FEBS Lett, 1988 Mar 28, 230(1-2), 35 - 7
Muscarinic receptor-mediated intracellular Ca2+ mobilization in embryonic chick heart cells; Schmidt H et al.; Activation of muscarinic receptors of heart cells elevates the intracellular Ca2+ concentration . The increase is considered to be due to influx of extracellular Ca2+ . We show that intracellular Ca2+ mobilization is involved . Cell suspensions prepared from hearts of 6-day-old chick embryos were loaded with the fluorescent Ca2+ chelator chlortetracycline . Muscarinic stimulation induces a dose-dependent fluorescence decrease (ED50 = 2.6 x 10(-6) M) indicating intracellular Ca2+ mobilization.

Biochim Biophys Acta, 1988 Mar 22, 939(1), 29 - 39
Measurement of rapid membrane permeation in cell suspensions by application of a generalized capillary method; Klosgen B et al.; An improved version of the capillary technique for the determination of diffusion coefficients has been developed as a simple method of measuring membrane permeabilities of single cells suspended at relative densities between 0.70 and 0.97 . A new, generalized theoretical formulation to describe the diffusion process of a solute in a composite system was derived using a series-parallel-pathway model with explicit consideration of the diffusion pathways inside and between the cells . This renders the technique insensitive to unstirred layer effects . Any single cell population of known size distribution may be investigated . High permeabilities (above 5.10(-3) cm/s) can be measured with the greatest precision, but lower permeabilities, down to a limit of about 5.10(-4) cm/s, may also be determined by the method . Measurements in erythrocyte suspensions have been made using non-electrolytes such as hexanol, water and ethylene glycol as test solutes . The permeabilities obtained agree with the values obtained by much more sophisticated equipment . Cell shape was shown to be without significant influence on the permeability data obtained . The procedure may become of particular interest for measurement of suspensions of membrane vesicles.

Biochim Biophys Acta, 1988 Mar 3, 938(3), 334 - 44
Hepatocellular uptake of aflatoxin B1 by non-ionic diffusion . Inhibition of bile acid transport by interference with membrane lipids; Muller N et al.; Aflatoxin B1 permeates isolated rat hepatocytes by non-ionic diffusion . Its uptake is neither saturable nor influenced by metabolic energy and not inhibited by treatment of cells with proteases . The initial rate of aflatoxin B1 uptake measured at 7 degrees C is between 40 and 50% compared to that at 37 degrees C . However, after an incubation period of 7 minutes identical equilibrium uptake is reached at both temperatures . The apparent activation energies, calculated for aflatoxin B1 uptake by Arrhenius diagrams ranged between 1.69 and 4.5 kcal/mol . A Q10 value of 1.34 was calculated for a temperature interval of 7-17 degrees C but decreased to 1.05 for the interval of 27-37 degrees C . Liposomes or lipoproteins added to the cell suspension inhibited the aflatoxin B1 uptake into hepatocytes . Liposomes mainly composed of unsaturated fatty acids bind twice as much aflatoxin B1 as those composed of saturated ones, indicating that the lipophilicity of the mycotoxin is crucial in the determination of its uptake into liver cells . At concentrations above 5 micrograms/ml, aflatoxin B1 inhibited the carrier-mediated uptake of cholic acid and of phalloidin into hepatocytes . This effect was reversible and abolished by washing the cells after preincubation with aflatoxin . In concentrations below 5 micrograms/ml the uptake of phallotoxin and cholic acid was however stimulated by 15-25% . These results indicate, that a carrier-mediated uptake into hepatocytes via the multispecific bile salt transporter is not responsible for the organoselective clearance of aflatoxins by the liver . On the other hand, the cholestatic effect of aflatoxin B1 results at least partially from the inhibition of the multispecific bile acid transport system . This inhibition may arise from affinity of aflatoxins to lipid domains of the cell membrane.

Poult Sci, 1988 Mar, 67(3), 487 - 92
Posthatch ontogeny and sex-related differences in somatotroph numbers in the chicken adenohypophysis; Vasilatos-Younken R; Adenohypophyseal total cell and somatotroph numbers were determined in male and female chicks over the early (12 wk) growth period . In a first experiment, adenohypophyses from male and female broiler-strain (Hubbard x Hubbard) chicks were harvested at 2 wk intervals from 2 to 12 wk of age . Glands were enzymatically dispersed to yield uniform cell suspensions that were fixed and stained (Herlant's Tetrachrome system) to distinguish somatotrophs . Percentages of somatotrophs in the total adenohypophyseal cell population were similar for both sexes (P greater than .05) and declined with age (P less than .01) from a high of 16.8 +/- .79% (least squares mean +/- SEM) at 2 wk to 6.3 +/- .88% at 12 wk . From 2 to 6 wk of age, total adenohypophyseal cell numbers increased over two-fold (P less than .01) but sexes did not differ significantly . From 8 to 12 wk of age, however, total cell numbers reached a plateau and were higher in males than females (P less than .01) . Similarly, the absolute numbers of somatotrophs per adenohypophysis increased from 2 to 6 wk of age, where it reached a plateau . Somatotroph numbers in males were twice those in females over the 8 to 12-wk growth period . In a second experiment, the percentages of somatotrophs in the total adenohypophyseal cell population from males of a slower growing, layer-type breed (White Leghorn) were compared to those of Hubbard males at 4, 8, and 12 wk of age . Leghorn males were higher than Hubbard males over all ages in percentages of somatotrophs (14.9 +/- .87% vs . 10.8 +/- .71%; P less than .01), however, the absolute numbers of somatotrophs per pituitary of the two breeds did not differ.

J Androl, 1988 Mar-Apr, 9(2), 102 - 8
A microperfusion chamber for study of mammalian spermatozoa; Burkman LJ; The design of a microperfusion chamber is presented for use with spermatozoa or other cell suspensions . This chamber allows perfusion of a small number of spermatozoa during simultaneous observation of cell behavior at the microscope . The chamber is made from a flat glass capillary tube that is fitted at both ends with a filter unit containing Millipore filter discs . The entire assembly is designed to fit the stage of an inverted microscope . A population containing as few as several hundred sperm cells may be observed in the chamber during successive changes of the suspending medium as controlled by a perfusion pump . Several experiments are presented demonstrating sperm survival in the sealed chamber and the response of rabbit and human sperm motility after the washing process . For these manipulations, the percentage of motile cells, linear swimming speed and incidence of hyperactivated motility are reported . Simple incubation in the chamber for 1 hour was not deleterious to the motility of rabbit spermatozoa . Human seminal spermatozoa showed no decline in vigorous motility after the washing procedure . Compared with in vitro capacitated spermatozoa, however, washing of rabbit seminal spermatozoa showed a variable response . Finally, partially capacitated human spermatozoa were examined for any alteration of motility during chamber incubation with a subsequent wash . When small numbers of spermatozoa or other cell types must be manipulated, the methodology can be effectively substituted for the standard washing procedure that uses repeated centrifugation and resuspension.

Cell Differ, 1988 Mar, 23(1-2), 77 - 86
Expression of the Ca2+ mobilizing muscarinic system in the chick embryo correlates with morphogenesis; Oettling G et al.; We describe muscarinic receptors and intracellular Ca2+ mobilization after cholinergic stimulation in cell suspensions prepared from chick embryos between day 2 (stage 12/13) and day 13 (stage 40) of development . Cell suspensions are prepared from whole chick embryos and from embryonic hearts, heads or brains, limb buds, and trunks . Muscarinic receptors are measured using {3H}quinuclidinylbenzilate as specific ligand . Intracellular Ca2+ mobilization is determined by changes of chlorotetracycline fluorescence . (1) Considerable amounts of muscarinic receptors are found in all parts of the embryo and at all stages tested . (2) The intracellular Ca2+ response after stimulation by muscarinic agonist shows a peak at day 3-4 (stage 23) . (3) The pharmacological profile of the Ca2+ response remains constant during embryonic development and differs from the profiles of most adult systems . (4) The 'embryonic muscarinic system' is uniformly expressed in cells from neural and non-neural tissues . It appears and disappears independently of innervation.

Cytometry, 1988 Mar, 9(2), 121 - 5
Discrimination of myogenic and nonmyogenic cells from embryonic skeletal muscle by 90 degrees light scattering; Yablonka-Reuveni Z; A myogenic cell suspension was isolated from the breast muscles of 10-day-old chicken embryos by trypsin digestion . The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblast-like cells present . The Percoll-isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90 degrees light scattering as the parameter for sorting . Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones . Cells exhibiting higher scatter (120-200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones . Cells with intermediate light scatter were also detected . The latter produced both myogenic and nonmyogenic clones . The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells . Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.

Toxicol Lett, 1988 Mar, 40(3), 247 - 55
Effects of culture conditions on susceptibility of alveolar epithelial cell monolayers to NO2; Cheek JM et al.; The effects of nitrogen dioxide (NO2) exposure on primary cultured monolayers of rat type II pneumocytes were investigated as a function of the isolation and culture conditions . Monolayers were cultured in Eagle's minimum essential medium (MEM) and in MEM supplemented with Ham's F-12; in some experiments, the initial cell suspension was also replated after 3 h . Both supplementation of the basal medium and replating increased the sensitivity of the monolayers to NO2, as measured by reduction in dome formation of plastic dishes 24 h post-exposure . These findings suggest that comparisons of in vitro toxicologic observations may be complicated by the effects of specific experimental conditions.

Immunology, 1988 Mar, 63(3), 477 - 82
Increased production of Kurloff cells and accompanying lymphocyte subset changes in immunized guinea-pigs treated with cyclophosphamide and cyclosporin A; Thomson AW et al.; We have used a panel of novel monoclonal antibodies to investigate the influence of cyclophosphamide (Cy) and cyclosporin A (CsA) on blood and spleen mononuclear cell populations in guinea-pigs immunized with ovalbumin (OVA) in complete Freund's adjuvant (CFA) . Female animals received, on Day 0, 100 micrograms OVA/CFA in each hind footpad and were treated with either Cy (300 mg/kg i.p., Day -3) or CsA (25 mg/kg orally, daily from Day 0) or with both drugs . Two weeks later, mononuclear cell suspensions were prepared from blood and spleen . Immunocytochemical (alkaline phosphatase anti-alkaline phosphatase) analyses were performed using monoclonal antibodies directed against pan T cells, T-suppressor/cytotoxic (Ts/c) cells, a putative T-helper (Th) cell marker, B lymphocytes and to Ia antigen . Cy and CsA, particularly the former, caused depletion of T cells, although no striking differential effect of either drug was observed on the T-suppressor cell population . Cy caused a more severe depletion of B-cell numbers, whilst CsA selectively spared these cells . The number of Ia-positive mononuclear cells also decreased markedly in the blood of animals given either drug and in the spleens of those guinea-pigs given both Cy and CsA . In contrast, absolute numbers of Kurloff cells (mononuclear leucocytes unique to the guinea-pig and possessing a proteoglycan-containing inclusion body) were markedly increased in the blood and spleen of animals given Cy and CsA compared with animals given Cy or CsA alone . Phenotypic analysis revealed that the Kurloff cells bore the pan T-cell marker, (but neither the Ts/c nor Th subset markers) and were Ia positive . The model described provides an opportunity for further characterization of these cells, their function and of factors regulating their production.

J Cell Biol, 1988 Mar, 106(3), 657 - 66
Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2+ levels; Di Virgilio F et al.; Cytosolic free Ca2+ ({Ca2+}i) homeostasis was investigated in mouse peritoneal macrophages and in the macrophage-like cell line J774 . {Ca2+}i measurements were performed in both cells in suspension and cells in monolayers loaded with either quin2 or fura-2 . Resting {Ca2+}i was 110-140 and 85-120 nM for cell suspensions and monolayers, respectively . There were no significant differences in {Ca2+}i between the two macrophage populations whether quin2 or fura-2 were used as Ca2+ indicators . Addition of heat-aggregated IgG, IgG-coated erythrocyte ghosts, or a rat monoclonal antibody (2.4G2) directed against mouse Fc receptor II induced a rise in {Ca2+}i . This {Ca2+}i increase was consistently observed in J774 and peritoneal macrophage suspensions and in J774 macrophage monolayers; in contrast it was observed inconsistently in peritoneal macrophages in monolayer cultures . The increase in {Ca2+}i induced by ligation of Fc receptors was inhibited totally in macrophages in suspension and by 80% in macrophages in monolayers by a short preincubation of macrophages with PMA; however, phagocytosis itself was unaffected . The effect of reducing cytosolic Ca2+ to very low concentrations on Fc receptor-mediated phagocytosis was also investigated . By incubating macrophages with high concentrations of quin2/AM in the absence of extracellular Ca2+, or by loading EGTA into the cytoplasm, the {Ca2+}i was buffered and clamped to 1-10 nM . Despite this, the phagocytosis of IgG-coated erythrocytes proceeded normally . These observations confirm the report of Young et al . (Young, J . D., S . S . Ko, and Z . A . Cohn . 1984 . Proc . Natl . Acad . Sci . USA . 81:5430-5434) that ligation of Fc receptors causes Ca2+ mobilization in macrophages . However, these results confirm and extend the findings of McNeil et al . (McNeil, P . L., J . A . Swanson, S . D . Wright, S . C . Silverstein, and D . L . Taylor . 1986 . J . Cell Biol . 102:1586-1592) that a rise in {Ca2+}i is not required for Fc receptor-mediated phagocytosis; and they provide direct evidence that Fc receptor-mediated phagocytosis occurs normally even at exceedingly low {Ca2+}i.

J Urol, 1988 Mar, 139(3), 650 - 2
Bone resorption induced by transplanted bladder tumor (MBT-2) in mice; Nemoto R et al.; The high incidence of metastatic bone resorption in urological cancer makes it necessary for clinicians to look for a valid experimental model to investigate basic interaction between cancer cells and bone in order to improve the treatment . A new model of bone resorption has been defined, namely the subcutaneous injection of tumor cell suspensions of invasive transitional cell carcinoma of the bladder (MBT-2) into mice after the periosteum of the calvaria has been disrupted . The tumor induced osteolysis associated with osteoclast proliferation with reactive bone formation . Moreover, we were able to show hypercalcemia accompanied with local bone resorption in this model.

J Bacteriol, 1988 Mar, 170(3), 1369 - 72
Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri; Peinemann S et al.; Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein . Correspondingly, the proton motive force increased to a steady-state level of -120 mV . The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV . The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation . The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased . The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP . The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M . barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established . Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed . Na+ is not involved in the CO dehydrogenase reaction.

Gastroenterology, 1988 Mar, 94(3), 576 - 81
Selective recognition of mucosal lymphoid high endothelium by gut intraepithelial leukocytes; Schmitz M et al.; Circulating precursors of mucosal immunoglobulin A plasma cells and T-cell immunoblasts migrate selectively into mucosal sites from the blood, but the mechanisms controlling this selective trafficking have not been determined . One possibility is that the site-specific extravasation of circulating effector cell populations is determined by organ-specific endothelial cell recognition mechanisms . Here we have assessed the ability of isolated mouse gut intraepithelial lymphocytes to recognize and bind to mucosal versus nonmucosal lymphoid organ high endothelial venules, vessels that support high levels of lymphocyte traffic in vivo . In an in vitro assay of lymphocyte interaction with high endothelial venules in frozen sections, intraepithelial leukocytes bind well to high endothelial venules in Peyer's patches but, unlike most circulating B and T lymphocytes, are unable to interact with peripheral lymph node high endothelial venules . Furthermore, we show by in situ immunohistology and in cell suspension immunofluorescence studies that intraepithelial leukocytes fail to stain with a monoclonal antibody, MEL-14, against putative lymphocyte receptors for lymph node high endothelial venules . Thus, they lack a cell surface glycoprotein required for homing to peripheral nodes . The demonstration of organ-specific recognition of endothelial cells by a normal mucosal effector lymphocyte population suggests that selective interactions with endothelium may play an important role in controlling the distribution of effector cells in vivo . The utilization of organ-specific endothelial cell recognition mechanisms by circulating precursors of mucosal effector cells could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from nonmucosal immune mechanisms.

Biol Reprod, 1988 Mar, 38(2), 413 - 21
Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum; Farin CE et al.; Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells . In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group . Blood samples were drawn daily from the jugular vein for quantification of progesterone . On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions . Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined . Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea . Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01) . The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05) . In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Tissue Kinet, 1988 Mar, 21(2), 123 - 31
Comparative analysis of different approaches to investigate cell kinetics; Silvestrini R et al.; The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas . Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by {3H}-thymidine autoradiography {( 3H}TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67 . A good correlation was observed between the values of {3H}TdR LI and BrdU LI (rs = 0.90; P less than 0.01), {3H}TdR LI and S phase (rs = 0.62; P less than 0.01) and {3H}TdR LI and Ki67 (rs = 0.64; P less than 0.01) in individual lymphomas . Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between {3H}TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between {3H}TdR LI and S phase (73%) or Ki67 (76%) . In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.

Can J Physiol Pharmacol, 1988 Mar, 66(3), 270 - 5
Bioactive atrial natriuretic factor-like peptides in rat anterior pituitary; Gutkowska J et al.; The presence of biologically active atrial natriuretic factor (ANF)-like peptides was demonstrated in rat anterior pituitary . ANF-like immunoreactivity was detected in rat anterior pituitary by specific radioimmunoassay and was extracted from rat anterior pituitary homogenates by heat-activated Vycor glass beads; extracts were purified by reverse-phase high performance liquid chromatography . Two peaks containing ANF immunoreactive material were obtained . The first peak was eluted from the C18 mu Bondapak column at a position similar to the 28-amino acid carboxy terminal peptide (Ser99-Tyr126)-ANF of prohormone . The second peak had the same pattern of elution as the 126-amino acid prohormone, (Asn1-Tyr126)-ANF . The biological activity of the smaller molecular weight peptide (28 amino acid) was assessed by its inhibitory effect on 10(-8) M ACTH-stimulated aldosterone secretion in rat zona glomerulosa cell suspension . This ANF-like material also displaced I125-labelled ANF from rat glomerular receptors with a potency similar to synthetic (Arg101-Tyr126)-ANF . Immunocytochemical localization revealed a distribution of ANF-stained cells similar in pattern and location to that of gonadotrophs . These results suggest the existence of biologically active ANF-like peptides and ANF prohormone within the anterior pituitary . However, their role remains to be elucidated.

Transplantation, 1988 Mar, 45(3), 591 - 600
Effects of in vitro and in vivo anticanine T lymphocyte and anticanine class II-specific monoclonal antibodies in the beagle; Fuller L et al.; Two murine anticanine lymphocyte monoclonal antibodies, designated T83 and B1F6, were assessed for (1) in vitro antiepitope activity to circulating lymphocyte subsets, their functional effects on lymphoproliferative assays and binding specificities to diverse cell suspensions and tissue sections; (2) in vivo effects after administration on cells expressing the target epitopes, lymphoproliferation, and allograft survival; and (3) the host immune response to the injected murine immunoglobulins . The monoclonal antibody T83 (IgG3) appeared to be specific for a T cell subset with an up-regulating function on lymphoproliferation . It caused a profound depletion of cells with the appropriate epitope after intravenous administration but it bound to lymphocyte membrane epitopes on some cells in peripheral blood that did not become depleted and putatively caused other modulating effects on lymphocyte function . The monoclonal antibody B1F6 (IgG2a, previously described) was immunologically specific in vitro for cells expressing class II major histocompatibility complex antigens . In the dog, this also consisted of 50% of T lymphocytes . After intravenous administration, there were functional effects similar to those of T83 . A modest prolongation of survival of renal allografts was observed when both mAbs were used as the sole immunosuppressive agent . These studies also demonstrated the occurrence of natural canine antimurine IgG antibodies . Administration of either monoclonal antibody was followed by a rapid increase in the concentration of the antimouse antibody(s) . We postulate that the presence of canine natural antimouse IgG markedly influenced the biologic effects of in vivo administered monoclonals.

J Leukoc Biol, 1988 Mar, 43(3), 224 - 31
Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process; Akiyama Y et al.; Elutriator-purified human monocytes were cultured in a serum-free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages . Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered . When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture . In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability . Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium . When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate . We attempted to characterize certain of the serum protein(s) found in HuAB which promoted the monocyte maturation process . Human immunoglobulin G (IgG) was found to be the most potent serum protein in increasing 5'-N activity and decreasing peroxidase activity of suspension cultured monocytes . Immunoglobulin M (IgM) and albumin (Alb) were shown not to have significant monocyte maturation activity . Heat-treated human gamma globulin and IgG purified by high-performance liquid chromatography (HPLC) were shown to have patterns identical with that of untreated HGG and IgG with regard to promoting monocyte maturation; F(ab')2 was not an active maturation promoter, indicating the need for an intact Fc portion of the IgG molecule . Fibrinogen and fibronectin also had maturation promoting activity . Finally, addition of the potent monocyte functional activators, muramyl dipeptide (MDP), polyriboinosinic:polyribocytidilic acid (Poly I:C), and lipopolysaccharide (LPS) had no effect on the monocyte maturation process . Thus, neither cell adherence or activation appear to be critical for the monocyte to macrophage maturation process . Instead, we hypothesize that in addition to proper nutritional support, a group of serum proteins (unified mainly by their ability to interact with monocyte membrane receptors) appear to be the principal promoters of this process.

J Virol, 1988 Mar, 62(3), 987 - 97
Pathogenesis of murine cytomegalovirus infection: identification of infected cells in the spleen during acute and latent infections; Mercer JA et al.; Spleen cells which replicate murine cytomegalovirus (MCMV) during acute infection in vivo were identified by electron microscopy and combined immunocytochemical staining and in situ cytohybridization . Most infected cells, as defined by in situ hybridization for viral RNA with MCMV-specific probes, were shown to be positive for factor VIII-related antigen and negative for Ia, Thy-1, and F4/80 antigens . Electron microscopic ultrastructural observations indicated that the infected cells in the spleen are predominantly sinusoidal-lining cells . We also studied reactivation of MCMV from latently infected mice by cocultivation of spleen cells with mouse embryo fibroblasts . Virus was only recovered from cells in preparations of stromal (or reticular) fragments, and not from spleen cell suspensions . Neither removal of immunoglobulin-bearing cells from the stromal fragments by panning nor depletion of Thy-1- and Ia-bearing stromal cells by treatment with monoclonal antibodies and complement reduced the frequency of reactivation of MCMV . These data suggest that T lymphocytes, mature B lymphocytes, and other Ia-bearing cells are not predominant reservoirs of latent MCMV.

Neurosci Lett, 1988 Feb 15, 85(1), 158 - 62
Independent developmental regulation of migratory granule neurons and their cerebellar ligand in the mouse; Bolin LM et al.; Interactions between migratory granule neurons and the developing molecular layer of the mouse cerebellum were examined using an in situ binding assay . Single cell suspensions of postnatal granule neurons specifically adhere to unfixed frozen cerebellar tissue sections . We investigated the influence of postnatal age of granule neurons and of tissue on this interaction . Granule neurons from P10 (the time of peak migratory activity) bind preferentially to the molecular layer . Premigratory granule neurons, P5, do not bind age-matched cerebellar tissue . Postmigratory granule neurons, P14 and older, adhere to the molecular and internal granular layers of age-matched and older cerebellar tissue but not to younger tissue . These binding patterns are most simply explained as a single receptor-ligand system in which both elements exhibit independent developmental regulation . Although granule neurons lose the ability to bind with increasing age, the molecular layer ligand retains its capacity for this interaction into adulthood, long after normal migration has ceased.

J Comp Neurol, 1988 Feb 8, 268(2), 204 - 22
Comparison of growth and reinnervation properties of cholinergic neurons from different brain regions grafted to the hippocampus; Nilsson OG et al.; Grafts of five different types of central cholinergic neurons, from the septal-diagonal band region, the nucleus basalis magnocellularis region (NBM), the striatum, the pontomesencephalic tegmentum of the brainstem, and the spinal cord, were compared with respect to their ability to grow and to reinnervate the cholinergically denervated hippocampal formation of adult rats . The areas were dissected from 14 to 15-day-old rat fetuses, and the same number of viable cells (35 X 10(4) from each of the different regions were stereotaxically injected as cell suspensions into the hippocampus of rats subjected to a transection of the intrinsic septo-hippocampal cholinergic pathways . At 17-19 weeks after transplantation, the various graft types differed considerably in their volume, the total amount of acetylcholinesterase (AChE)-positive fiber outgrowth, and the innervation pattern and morphology of the AChE-positive fibers growing into the host hippocampus . On average the NBM and spinal cord grafts had grown to become three to four times larger than the septal and the brainstem grafts, and 15-20 times larger than the striatal grafts . By contrast, the total ingrowth score of AChE-positive fibers in the host hippocampus from the septal grafts was about twice that of the NBM and brainstem grafts, about five times greater than the striatal grafts, and about six times greater than that of the spinal cord grafts . The large NBM grafts thus exhibited similar fiber outgrowth to the much smaller brainstem grafts, and the AChE-positive neurons of the grafted spinal cord grew very poorly into the hippocampus despite the fact that they survived very well . The innervation pattern and morphological features of the ingrowing AChE-positive fibers in the host hippocampus proper and in the dentate gyrus resembled those of normal rats in animals with grafts from any of the three forebrain regions (i.e., septum, NBM, or striatum), whereas ingrowth from the brainstem and spinal cord grafts were markedly abnormal with respect to both innervation pattern and fiber morphology . These results provide further evidence that the overall survival, growth, and fiber outgrowth of intracerebral neural grafts depend on interactions with the surrounding host tissue . Since the ability to reinnervate the previously denervated host target was greatest for the neuron type normally innervating that area, i.e., the septal-diagonal band neurons, we conclude that neuronal properties beyond the transmitter type are essential for the optimal performance of implanted neurons in intracerebral grafting experiments.

Science, 1988 Feb 5, 239(4840), 635 - 7
Labeling of neural cells by gold-filled Sendai virus envelopes before intracerebral transplantation; Ardizzoni SC et al.; Gold-filled Sendai virus envelopes were fused with cell suspensions from the basal forebrain of fetal rat donors, and the resulting gold-labeled cells were transplanted into the neocortex of adult rat recipients . Not only did large numbers of labeled cells remain intact through 3 months in the neocortex, but sizable numbers migrated subcortically to the recipient's lesioned nucleus basalis region (a distance of 4 to 5 millimeters) . Since this technique is capable of labeling most transplanted cells for long periods of time, it may be useful in determining the survival, migration, and connectivity of intracerebrally transplanted tissues.

Proc Natl Acad Sci U S A, 1988 Feb, 85(3), 812 - 6
Enzyme stimulation upon fertilization is revealed in electrically permeabilized sea urchin eggs; Swezey RR et al.; Sea urchin eggs and embryos subjected to high-voltage electric discharge in a medium mimicking the intracellular milieu retain their structural integrity and remain permeable, permitting substrates to enter the cytoplasm and thus assay of enzyme activity . At saturating concentrations of substrates, five of six enzymes assayed for more active (three to fifteen times) in permeabilized embryos than in permeabilized eggs, but no fertilization-related differences are seen in homogenates prepared from these same permeabilized cells . Furthermore, enzyme activity in homogenates always exceeds that in the permeabilized cell suspensions . This difference in enzyme reaction rates between unfertilized eggs and fertilized eggs is not due to differences in the diffusibility of substrates into the permeabilized cells . The activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in permeabilized cells was studied in greater detail and has the following characteristics . (i) Regulation of activity persists during early development . (ii) This regulation is not mediated by diffusible allosteric agents . (iii) Stimulation at fertilization is initiated by a rise in intracellular calcium and is further promoted by cytoplasmic alkalinization . (iv) The microenvironment experienced by this enzyme intracellularly differs from that of the enzyme in homogenates as evidenced by markedly different pH vs . activity profiles . These results indicate that the regulatory status of enzymes is preserved in electrically permeabilized cells and suggest that this regulation depends on some cell structural feature(s) that is (are) destroyed upon homogenization.

No To Shinkei, 1988 Feb, 40(2), 127 - 31
{Growth activity of meningeal carcinomatosis--immunohistochemical study using anti-BrdU monoclonal antibody}; Izumoto S et al.; We have developed an experimental model of leptomeningeal tumor by inoculating Walker 256 carcinosarcoma cells into the cisterna magna of rats . This model was considered to be useful in studying pathophysiology and treatment of malignant brain tumors . In this study, the growth kinetics of this experimental tumor was investigated by using the immunohistochemical technique with an anti-BrdU monoclonal antibody . Walker 256 carcinosarcoma was subcutaneously passaged in female Wistar rats . Seven days after subcutaneous inoculation, the tumor was aseptically removed and minced in Hank's medium by scissors to make single cell suspension of the tumor . The cell suspension was adjusted to 1 x 10(5) cells/ml . And 0.1 ml was inoculated percutaneously into the cisterna magna of female Wistar rats weighing 150 gr . Every day after tumor inoculation, the animal (5 on each day) was sacrificed 30 minutes after intravenous BrdU (200 mg/kg) and perfused by saline . Then, the brain was removed, fixed in ethanol and embedded in paraffin . Coronal sections of the brain 6 mu in thickness were cut and stained by the indirect immunoperoxidase (ABC) method . The anti-BrdU monoclonal antibody (Becton-Dickinson) was diluted in 1:100 . The sections were counterstained by hematoxylin . Labelling index (L.I.) of the tumor was obtained by counting immunoreactive cells under the microscope . L.I . of the subcutaneous tumor 7 days after inoculation was 52.4% . In the tumor 1 to 3 days after inoculation, L.I . was still low and between 11.9 and 15.1% . Four or 5 days after inoculation, the tumor cells grew in several layers in the subarachnoid space . L.I . at this stage of the tumor growth was 26.6 to 34.8%.(ABSTRACT TRUNCATED AT 250 WORDS)

Microcirc Endothelium Lymphatics, 1988 Feb, 4(1), 21 - 44
Effect of EDRF release from freshly harvested endothelial cells on the coronary circulation of the isolated working rabbit heart; Hartmann A et al.; Vascular relaxation in rabbit aortic preparations by acetylcholine is endothelium-dependent . Because of the short half-life of the endothelium-derived relaxing factor(s) (EDRF), a constant source of this material is necessary to study its effect in perfused hearts or hearts in situ (reported half-life 6-50 sec) . To investigate the effect of EDRF(s) on the large coronary arteries and the resistance vessels of the isolated working rabbit heart, freshly harvested porcine endothelial cells were used . The cells were stimulated with acetylcholine to produce EDRF(s) . To extend the half-life of EDRF, superoxide dismutase was added to the cell suspension . Left atrial infusion of these cells increased coronary flow and reduced total coronary resistance . No significant effect on the diameter of the endothelium-deprived obtuse marginal coronary artery was noted . The vasoconstrictor effect on resistance vessels of topically applied histamine was significantly reduced during the infusion of activated endothelial cells . It can be concluded that EDRF(s) released from freshly harvested endothelial cells increases coronary flow and diminishes total coronary vascular resistance in the working heart . In this preparation coronary arterioles are more sensitive to EDRF(s) than large coronary arteries . EDRF may contribute to the regulation of regional myocardial flow.

In Vitro Cell Dev Biol, 1988 Feb, 24(2), 91 - 9
In vitro cytodifferentiation of perinatal rat islet cells within a tridimensional matrix of collagen; Amory B et al.; Cell suspensions prepared by collagenase digestion of pancreases obtained from rat fetuses (21.5 d old) and newborns (2.5 d old) were mixed with a collagen solution and inoculated on a collagen base layer . At the onset of the culture, most acinar cells became necrotic, whereas other epithelial cells proliferated . Most of the cell clusters arranged themselves into simple polarized structures composed of epithelial cells forming hollow spheres, and from these budded neoformed endocrine islets . Scarce fibroblasts were located close to these structures . Immunocytochemical localization of insulin and glucagon, as well as ultrastructural characteristics of the cell types revealed an intrainsular distribution similar to the in vivo localization . Tridimensional matrix of collagen offers, to perinatal pancreatic cells in culture, an environment close to the in vivo conditions: cells reorganize themselves in tissuelike structures and cell interactions concerned in the cytodifferentiation of pancreatic islets occur . This system allows for the study of undifferentiated epithelial cells--the presumed stem cells--differentiating and differentiated endocrine cells in the same preparation.

J Pathol, 1988 Feb, 154(2), 133 - 40
Expression of Leu-8 surface antigen in B-cell lymphomas . Correlation with other B-cell markers; Carbone A et al.; Using in situ immunohistological analysis, expression of Leu-8 and its correlation with other B-cell markers were investigated in 21 selected lymphomas of different categories, each one expressing its own typical immunophenotype . These categories included eight follicular centroblastic/centrocytic (CB/CC) lymphomas, eight intermediately differentiated lymphocytic lymphomas (ILL)/mantle zone lymphomas (MZL), and five lymphocytic lymphomas (LL) associated with chronic lymphocytic leukaemia (CLL) . Four reactive lymph nodes and three tonsils were also studied using double immunolabelling procedures . Cell suspensions were also performed in three CB/CC and four ILL/MZL cases . Leu-8 was consistently expressed in ILL/MZL and LL but it was absent in most (7/8) CB/CC lymphomas . In reactive tissues, the Leu-8-positive B cells were strictly confined to the mantle zones . A close association emerged between Leu-1 (CD5) and Leu-8, both being present in ILL/MZL and LL but absent in CB/CC . A consistent lack of association was found between Leu-8 or CD5 antigens and common acute lymphoblastic leukaemia antigen (CD10) and BA-2 (CD9) antigen, whereas Leu-8 and CD5 were strictly associated with surface IgD . Reactivity with Leu-8 provides a means of distinguishing between CB/CC and ILL/MZL . Furthermore, shared immunoreactivity for Leu-8 in ILL/MZL and LL may represent a potential clue to the still uncertain cellular derivation of LL/B-CLL.

J Invest Dermatol, 1988 Feb, 90(2), 201 - 6
Immunostimulatory capabilities of highly enriched Langerhans cells in vitro; Picut CA et al.; Langerhans cells (LC) are epidermal antigen-presenting cells capable of inducing allogenic, antigen-specific, and cytotoxic T cell proliferation . Previous studies have examined the dynamics of LC maintained in vitro in crude epidermal cell (EC) suspensions in which the major cell type is the keratinocyte (KC) . To avoid the confounding effects of KC and other immunoregulatory cells on LC dynamics in vitro, highly enriched murine LC (85%) were studied, through 72 h of incubation in vitro, for their ability to present alloantigen (in a primary allogenic proliferation assay) and foreign antigen (in a secondary autologous proliferation assay) . The results were compared to similar studies using crude EC suspensions . Freshly prepared LC are very poor stimulators of a primary allogenic proliferation response, with a 12- to 16-fold increase in stimulatory capacity by 72 h using panned-enriched and crude EC suspensions, respectively . Similarly, freshly prepared LC are weak stimulators of a secondary autologous proliferation response, with a 2.5- to 6-fold increase in immunostimulatory capability by 72 h . The overall increased stimulatory effect observed with the crude EC suspensions compared to highly enriched LC is most likely attributed to the effect of KC on T cell proliferation, rather than to a maturation effect of KC on LC during the 72 h of in vitro incubation . Using back-scattered electron imaging, the surface density of MHC-class II molecules (Ia) increased three- to fourfold through culture, which parallels the increase in functional ability . This study demonstrates that LC in either a crude or highly enriched cell suspension mature into potent immunostimulatory cells after incubation in vitro with an increased surface expression of Ia molecules . Keratinocytes are not necessary for LC maturation in vitro, but seem to exert some stimulatory effect by enhancing lymphocyte proliferation in the functional assay system.

Nippon Geka Gakkai Zasshi, 1988 Feb, 89(2), 245 - 50
{Allotransplantation of islet endocrine aggregates}; Kakizaki K et al.; Dissociated pancreatic islets form aggregates from single cell suspension by rotation culture . Islet cell aggregates, or neoislets, newly formed from Lewis islet cells were transplanted into diabetic ACI rats intraportally and prolongation of neoislet grafts was observed (mean survival time greater than 45.8 +/- 6.4 days) . Five out of seven recipients were still normoglycemic at 60 days after transplantation . Transplantation of neoislets combined with 3 day cyclosporine therapy for recipients resulted in 100% survival at 60 days after transplantation . These findings indicate that neoislets can provide endocrine constitution for streptozotocin diabetic rats and enjoy prolonged survival . As predicted by cell-cell recognition in rotation mediated aggregation, neoislets can exclude mesenchymal cells which bear Ia antigen . Therefore reduced immunogenicity is accomplished.

Kidney Int, 1988 Feb, 33(2), 543 - 54
Isolation by monoclonal antibody of intercalated cells of rabbit kidney; Koseki C et al.; We produced a monoclonal antibody, gamma G6, that reacts only with one cell type in the connecting (CNT) and collecting tubules (CT) of rabbit kidney . The gamma G6 antibody-reactive cells revealed carbonic anhydrase activity, showing one of the characteristics of intercalated (IC) cells . Using immunoelectron microscopy, we demonstrated that IC cells in cortical CT consist of the gamma G6 antibody-reactive and non-reactive cells, whereas all IC cells in medullary CT were reactive with the gamma G6 antibody . We used a cell sorter to enrich this cell type from the isolated kidney cell suspension . When we measured hormone-sensitive adenylate cyclase (ACase) activities of the sorted cells, the presence of parathyroid hormone (PTH) and isoproterenol (ISO) almost doubled ACase activities when compared with the basal values; however, no additive effect of PTH and ISO was observed . They showed no calcitonin-sensitive ACase and negligible arginine vasopressin (AVP)-sensitive ACase . We suggest that the IC cells recognized by the gamma G6 monoclonal antibody possess a receptor(s) for PTH and/or ISO but not for AVP in the CNT and CT, although it remains to be clarified whether the reactivities to PTH and ISO in these cells originate from single or dual cells.

J Mol Recognit, 1988 Feb, 1(1), 9 - 18
Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro; Lea T et al.; By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns . Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes . Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies . So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood . These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas . High speed and specificity are the most conspicuous features of immunomagnetic cell separation . These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens . Both higher sensitivity and specificity have been obtained . The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions . The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision . The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail . Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process . Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.

J Exp Zool, 1988 Feb, 245(2), 187 - 93
Gonadotropin action on androgen synthesis in short-term incubation of explants and dispersed cells of the testis of Xenopus laevis; Lecouteux A; Testis cells of the toad Xenopus laevis were dissociated with collagenase and the cell suspension was enriched for steroidogenic cells by Percoll gradients . Results suggested that cells should be preincubated during a 6-h period before stimulation with gonadotropin . Our results indicate that a 2-h incubation period with gonadotropin was necessary to obtain a significant response . Furthermore, the cells can be maintained in a functional state longer than mammalian testis cells . Different gonadotropins were used to stimulate androgen production, and their effects were compared in both dissociated cells and testicular explants . Cells were more sensitive to luteinizing hormone (LH) and follicle stimulating hormone (FSH) than the explants (ED50LH = 0.041 +/- 0.003 micrograms for cells and 0.097 +/- 0.002 micrograms for explants: ED50FSH = 0.41 +/- 0.03 micrograms for cells and 0.63 +/- 0.03 micrograms for explants) . Moreover, human chorionic gonadotropin (hCG) which only stimulates testicular explants at high doses, failed to stimulate the androgen production of dissociated cells; this indicates a low sensitivity of amphibian testis to hCG and a possible damaging effect of collagenase on the receptors of isolated cells.

Mol Cell Endocrinol, 1988 Feb, 55(2-3), 183 - 92
Activation of adenylate cyclase in rat fat cells promotes an increase in GTP content which controls the enzyme activity; Lambert B et al.; In previous studies, we demonstrated that the treatment of adipocytes with cholera toxin or Bordetella pertussis toxin (IAP) promoted an increase in the total guanosine triphosphate (GTP) content of the cells concomitant with the increase in cyclic adenosine monophosphate (AMP) level and the resulting lipolysis . In the present studies, we show that the acute challenge of fat cells with 1 microM isoproterenol (IPNE) is associated with a transient increase in GTP level (3-fold in 6 min) . This increase may be attributed to an inhibition of the disposal of GTP or to a stimulation of its synthesis . To evaluate the actual role of GTP, we used virazole, an antitumor agent which inhibits inosinic acid dehydrogenase . After 2 h preincubation of the cells with 1 mM virazole, the effect of a 6 min challenge with 1 microM IPNE is decreased by 59% at the GTP level and by 42% in cyclic AMP production . One hour later, the resulting lipolytic efficiency is reduced by 57% . IAP treatment (10 micrograms/ml) produced its maximal effect on GTP and cyclic AMP levels and on lipolysis after 90 min incubation . The antilipolytic effect of 1 microM phenylisopropyladenosine (PIA) is almost abolished . When 1 mM virazole is added to the cell suspension to deplete the guanyl nucleotide pool, the resulting lipolysis due to IAP treatment is decreased by 85%, whereas GTP and cyclic AMP levels were decreased by 80 and 70%, respectively . We can conclude that the cyclic AMP synthesis in intact cells is accompanied by a parallel increase of their GTP content, whether the stimulation results from the activation of Gs or the inhibition of Gi . The reduction of the guanyl nucleotide pool under virazole results in a relatively less important inhibition of lipolysis when Gs is stimulated than when it is Gi.

Int J Radiat Oncol Biol Phys, 1988 Feb, 14(2), 361 - 5
A predictive assay for human tumor cellular response to hyperthermia using dansyl lysine staining and flow cytometry; Woo SY et al.; The heat response of five human tumor biopsies has been examined using the fluorescent probe dansyl lysine and multiparameter flow cytometry . Dansyl lysine has previously been shown to possess specificity for heat killed mammalian cells . The human tumors tested included a cervical squamous cell carcinoma, malignant melanoma, colon adenocarcinoma, ovarian carcinoma, and a mesothelioma . The samples were excised, mechanically disrupted into single cell suspensions and heated in vitro for various lengths of time at 45 degrees C . The cells were returned to 37 degrees C incubation for 12 to 15 hours prior to staining with dansyl lysine . The fraction of cells staining dansyl lysine was quantitated by flow cytometry after gating on high forward angle light scatter and 90 degrees C light scatter . This gate excluded much of the normal cell contamination within the tumor sample . The data show that the heat response of human tumor biopsies varied significantly, with cervical carcinoma and malignant melanoma being the most resistant and the mesothelioma and ovarian carcinoma the most heat sensitive . Finally, evidence is presented for the expression of thermotolerance in ovarian carcinoma and mesothelioma biopsies pre-heated in vitro . Dansyl lysine appears to be useful in measuring the intrinsic cellular heat sensitivity of human tumors and in determining the kinetics of decay of thermotolerance following an initial heat exposure.

J Biol Chem, 1988 Jan 15, 263(2), 1002 - 7
Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking; Zhang QY et al.; The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor . Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes . The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate} and a noncleavable (disuccinimidyl suberate) cross-linking reagent . The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis . Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000 . However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000 . The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins . The radiolabeled bands were not detectable in hCG down-regulated Leydig cells . Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species . The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces . Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.

Cancer Res, 1988 Jan 15, 48(2), 368 - 78
Promotion of growth and differentiation of rat ductular oval cells in primary culture; Germain L et al.; Oval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to alpha-fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45:673-681, 1985) . In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer . The use of monoclonal and polyclonal antibodies directed against cytokeratins of Mr 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS7) and normal hepatocytes (HES6) allowed us to establish the ductular phenotype of the oval cells . A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES6 antibody, and cell separation by isopyknic centrifugation in a Percoll gradient . The procedure yielded about 8 x 10(7) cells, of which 95% expressed CK39, CK52, and BDS7, 84% gamma-glutamyl transpeptidase, and 5% albumin and AFP . The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by {3H}thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase . Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled . Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells . When the cells were cultured in the presence of an elaborate combination of supplements {minimum essential medium containing 1 mM pyruvate, 0.2 mM aspartate, 0.2 mM serine, 1 mM tyrosine, 1 mM proline, 1 mM phenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 microM bilirubin, 10 ng/ml cholera toxin, 1 microM dexamethasone, 2.5 micrograms/ml insulin, 50 mM beta-mercaptoethanol, and 5 micrograms/ml transferrin (medium MX)}, the labeling index increased to around 30% and the level of cell fusion greatly decreased . The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1988 Jan 5, 263(1), 130 - 4
The contribution of magnetic susceptibility effects to transmembrane chemical shift differences in the 31P NMR spectra of oxygenated erythrocyte suspensions; Kirk K et al.; Triethyl phosphate, dimethyl methylphosphonate, and the hypophosphite ion all contain the phosphoryl functional group . When added to an oxygenated erythrocyte suspension, the former compound gives rise to a single 31P NMR resonance, whereas the latter compounds give rise to separate intra- and extracellular 31P NMR resonances . On the basis of experiments with intact oxygenated cell suspensions (in which the hematocrit was varied) and with oxygenated cell lysates (in which the lysate concentration was varied), it was concluded that the chemical shifts of the intra- and extracellular populations of triethyl phosphate differ as a consequence of the diamagnetic susceptibility of intracellular oxyhemoglobin but that this difference is averaged by the rapid exchange of the compound across the cell membrane . The difference in the magnetic susceptibility of the intra- and extracellular compartments contributes to the observed separation of the intra- and extracellular resonances of dimethyl methylphosphonate and hypophosphite . The magnitude of this contribution is, however, substantially less than that calculated using a simple two-compartment model and varies with the hematocrit of the suspension . Furthermore, it is insufficient to fully account for the transmembrane chemical shift differences observed for dimethyl methylphosphonate and hypophosphite . An additional effect is operating to move the intracellular resonances of these compounds to a lower chemical shift . The effect is mediated by an intracellular component, and the magnitude of the resultant chemical shift variations depends upon the chemical structure of the phosphoryl compound involved.

Tumour Biol, 1988, 9(4), 170 - 7
An antigen specific to hyperplastic liver nodules defined with monoclonal antibody: a new marker for preneoplastic cells in rat chemical hepatocarcinogenesis; Okita K et al.; Two monoclonal antibodies (MoAbs) were prepared against the hepatocytes of hyperplastic liver nodules from rats killed in the 13th week during hepatocarcinogenesis with 2-acetylaminofluorene . The specificity of these MoAbs was confirmed by cellular radioimmunoassay . By means of immunoperoxidase staining with 2 MoAbs (HAM 6 and HAM 7), HAM 6-defined antigen was detected on the plasma membrane and/or cytoplasm of the hepatocytes in hyperplastic nodules, while HAM 7 reacted to the hepatocytes surrounding hyperplastic nodules . Therefore, HAM 6 seemed to be specific to hyperplastic nodules, but HAM 7 to be a MoAb developed accidentally to the surrounding hepatocytes by contamination of the cell suspension of hyperplastic nodules.

Biol Struct Morphog, 1988, 1(2), 84 - 8
In vitro proliferation and differentiation of myogenic cells from adult Xenopus; Franquinet R et al.; A technique is described for isolating amphibian myogenic cells from the muscle of adult Xenopus laevis (Dauchin) . Muscles were dissociated with 0.2% collagenase and 0.1% trypsin . The resulting cell suspensions were separated from the remaining myofibres by filtration through nylon grids . Most of the cells remaining in the filtrate suspension were satellite cells or fibroblasts . When plated in Petri dishes, satellite cells adhered to the substrate, became spindle-shaped and proliferated activity in a culture medium supplemented with fetal calf serum . Mitotic waves lasted 4 days and consequently cell density markedly increased . Satellite cells came into contact and began to fuse into myotubes on day 8 of culture . Horse serum, which replaced fetal calf serum in the medium on day 12, accelerated cell fusions which were almost complete on day 18 . However, under these conditions, some mononucleated cells continued to undergo mitosis . Cell proliferation with a high rate of mitosis was prolonged by repeated trypsinization and replating in medium supplemented with fetal calf serum . When myofibres from dissociated muscles were cultured under the same conditions, they never fragmented or divided.

Bone, 1988, 9(2), 113 - 9
A morphologic study of osteoclasts isolated from osteopetrotic microphthalmic (mi/mi) mouse and human fetal long bones using an instrument permitting combination of light and scanning electron microscopy; Helfrich MH et al.; Cell surface structures of isolated osteopetrotic (mi/mi) and normal murine (+/+ and +/mi) and human osteoclasts were examined in a microscope combining light and scanning electron microscopy (LM/SEM) . Tartrate-resistant acid phosphatase (TrAP) was used as an histochemical osteoclast marker . In osteopetrotic bone, as in normal murine bone, TrAP activity was exclusively seen in osteoclasts and preosteoclasts and was therefore judged a suitable marker for identification of isolated osteoclasts . A method was developed for preparation of LM/SEM specimens from osteoclast-enriched cell suspensions . In the LM/SEM isolated osteoclasts were easily recognized in the LM mode by TrAP contents . In specimens prepared from murine cells, but not human cells, LM identification of osteoclasts by TrAP was essential . This was in particular true for small, mononuclear, mi/mi osteoclasts . All osteoclasts examined had a villous appearance and were well spread over the glass substrate . There were no differences in cell surface morphology and in adherence to glass between osteopetrotic and normal osteoclasts.

Physiol Chem Phys Med NMR, 1988, 20(1), 23 - 30
Dosimetry considerations in far field microwave exposure of mammalian cells; Meltz ML et al.; A circulating water bath exposure system has been designed for in vitro radiofrequency radiation (RFR) exposure studies in the 915 to 2450 MHz range . A Styrofoam float, in which 10 T-25 plastic tissue culture flasks are embedded, is rotated at approximately 20 rpm in a Plexiglas water bath at a distance beneath a rectangular horn . The continuous circular rotation of the flasks is designed to "average out" the heterogeneity present in stationary flask exposures . The rotation also serves to prevent the establishment of chemical gradients in the medium within the flasks . Several factors have been demonstrated to affect the specific absorption rate (SAR) measured in the medium in the exposed flasks . These factors include: 1) the position of the exposure flasks relative to the long axis of the antenna horn; 2) whether the flasks are exposed while stationary or in rotation; 3) the volume of the medium contained in the flask; and 4) the depth in the medium in the flask at which temperatures for SAR calculation are measured . The presence of cells in the exposure flask (as attached monolayer or cell suspension) did not result in an SAR different from that measured in the same volume of medium without cells present.

Exp Brain Res, 1988, 70(1), 192 - 208
Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: immunological aspects, spontaneous and drug-induced behaviour, and dopamine release; Brundin P et al.; We have used a rat model of Parkinson's disease (PD) to address issues of importance for a future clinical application of dopamine (DA) neuron grafting in patients with PD . Human mesencephalic DA neurons, obtained from 6.5-8 week old fetuses, were found to survive intracerebral cell suspension xenografting to the striatum of rats immunosuppressed with Cyclosporin A . The grafts produced an extensive new DA-containing terminal network in the previously denervated caudate-putamen, and they normalized amphetamine-induced, apomorphine-induced and spontaneous motor asymmetry in rats with unilateral lesions of the mesostriatal DA pathway . Grafts from an 11.5-week old donor exhibited a lower survival rate and smaller functional effects . As assessed with the intracerebral dialysis technique the grafted DA neurons were found to restore spontaneous DA release in the reinnervated host striatum to normal levels . The neurons responded with large increases in extracellular striatal DA levels after the intrastriatal administration of the DA-releasing agent d-amphetamine and the DA-reuptake blocker nomifensine, although not to the same extent as seen in striata with an intact mesostriatal DA system . DA fiber outgrowth from the grafts was dependent on the localization of the graft tissue . Thus, grafts located within the striatum gave rise to an extensive axonal network throughout the whole host striatum, whereas grafted DA neurons localized in the neocortex had their outgrowing fibers confined within the grafts themselves . In contrast to the good graft survival and behavioural effects obtained in immunosuppressed rats, there was no survival, or behavioural effects, of human DA neurons implanted in rats that did not receive immunosuppression . In addition, we found that all the graft recipients were immunized, having formed antibodies against antigens present on human T-cells . This supports the notion that the human neurons grafted to the non-immunosuppressed rats underwent immunological rejection . Based on an estimation of the survival rate and extent of fiber outgrowth from the grafted human fetal DA neurons, we suggest that DA neurons that can be obtained from one fetus may be sufficient to restore significant DA neurotransmission unilaterally, in one putamen, in an immunosuppressed PD patient.

Cancer Invest, 1988, 6(2), 161 - 5
Characterization of different cell subpopulations derived from an experimental tumor model; Gardner HA et al.; The R3230AC rat mammary adenocarcinoma is a transplantable tumor model, which can be subcultured in vitro and does not metastasize spontaneously . However, when cell suspensions of this tumor are injected intravenously, multiple lung foci develop . These foci were used as source for a cell subpopulation . Prolonged exposure of the original tumor cell line to increasing levels of lectins (concanavalin A and wheat germ agglutinin) resulted in the development of a lectin-resistant subpopulation . Through experimental in vitro exposure to the antiestrogenic compound tamoxifen citrate, a tolerant, karyotypically defined subpopulation was obtained . In the four cell lines studied, heterogeneity was observed in the following parameters: doubling time, steroid receptors, and median chromosome counts . Statistically discernible differences were found . This may represent a model for the effect of selective pressures in the tumor environment, such as endocrine manipulation, leading to the gradual development of resistant cell lines and treatment failure.

Arch Geschwulstforsch, 1988, 58(2), 99 - 104
Sensitivity of clonogenic cells of human ovarian ascitic cancer to antitumor drugs; Potapov SL et al.; The sensitivity of human primary ovarian tumors to cyclophosphamid, 5-fluorouracil and adriamycin was investigated using tumor cell cultivation in agar diffusion chamber . It was found that the growth of clonogenic cells of human ovarian cancer was most strongly inhibited by cyclophosphamid and 5-fluorouracil . Tumor sensitivity to cyclophosphamid and cis-platin was significantly higher when tumor cells were cultivated in the presence of macrophages, the effect being dependent on the macrophage concentration . Tumor sensitivity to 5-fluorouracil was higher if prior to treatment macrophages were removed from the tumor cell suspension.

Exp Brain Res, 1988, 69(3), 545 - 58
Behavioral deficits after intrahippocampal fetal septal grafts in rats with selective fimbria-fornix lesions; Dalrymple-Alford JC et al.; Fetal septal transplants have been shown to promote behavioral recovery in young adult rats with aspiration fimbria-fornix lesions, rats with septal lesions and in intact aged rats . The present study examined the behavioral impact of intrahippocampal septal cell suspension transplants (T) in young female rats that had received, 10 days earlier, either medial fimbria lesions (Group FI.T), dorsal (subcallosal) fornix lesions (Group FO.T) or these two lesions together (Group FIFO.T) . Relative to rats with lesions only (groups FI, FO and FIFO), grafted rats, irrespective of lesion locus, displayed unexpected impairments in (i) a serial alternation learning task, 5 weeks and 6 months after transplantation, and (ii) in a radial maze, 7 months after transplantation . In the first alternation test, Group FIFO showed impaired performance relative to Groups FI, FO and the sham-operated controls (Group S) . In the second alternation test, Groups FO.T and FO showed impaired performance relative to Groups FI.T and FI, and only the performance of Group FI did not differ from that of Group S . In the radial maze, Groups FI, FO and FIFO all showed impaired performance relative to Group S . By contrast, there were no deleterious effects of lesions or of grafts in the acquisition and retention of a step-through passive avoidance task, 10 weeks after transplantation . Our findings on the effects of selective fimbria-fornix lesions did not confirm the report that rats with FI lesions but not those with FO lesions are unable to learn a serial alternation task, nor the report that FO lesions impair passive avoidance retention . Acetylcholinesterase (AChE) histochemistry revealed that grafts were present but graft-derived innervation of the host hippocampus varied from extensive to almost non-existent in all transplant groups . AChE-positivity in the dorsal hippocampus (DH) was not related to behavioral performance . However, the grafts often grew to a considerable size within the host brain and in many rats, especially those in Group FI.T, produced moderate to extreme damage of the host DH . There was a significant positive correlation between errors in the radial maze and graft-induced DH damage but no relationship between errors and graft size . The results indicate that, after partial lesions of the fimbria-fornix, intrahippocampal septal grafts survive well but are likely to damage recipient structures and result in behavioral impairments.

Dev Neurosci, 1988, 10(1), 1 - 11
Extensive oligodendrocyte remyelination following injection of cultured central nervous system cells into demyelinating lesions in adult central nervous system; Blakemore WF et al.; Following the injection of central nervous system (CNS) cell cultures, prepared from 1-day-old rats and maintained in vitro for 7 days, into irradiated, demyelinating lesions in the spinal cord of adult isologous animals, extensive remyelination of axons by oligodendrocytes was observed . In addition, astrocytes, within the transplanted cell suspension, established normal relationships with oligodendrocytes, axons and other tissue elements, which led to the establishment of large CNS territories throughout the lesions . Outside these CNS domains, Schwann cells, which are present in the transplanted cell suspension, myelinated groups of axons . These observations indicate that the irradiated, ethidium bromide lesion provides an in vivo environment, devoid of the influences of host glia, in which to examine the interactions of transplanted glial cells with demyelinating axons.

Int J Biochem, 1988, 20(4), 393 - 5
Age-related norepinephrine induced lipolytic response of isolated rat adipocytes; Terblanche SE et al.; 1 . Groups of male Long Evans rats were sacrificed at the ages of 10, 16, 20, 24, 28, 32, 38 and 45 weeks . 2 . The two epididymal fat pads from each rat in each group (4-5 rats) were excised for the preparation of adipocytes . 3 . Cell suspensions were incubated in triplicate with each of seven norepinephrine concentrations ranging from 0.5694 to 569,400 nM . 4 . Lipolytic responses are expressed as nmol glycerol released/microgram DNA/90 min . 5 . The animals reached a peak response between the ages of 20 and 32 weeks . 6 . Aging resulted in a gradual increase in the apparent affinity (Km) of the response yielding system for norepinephrine . 7 . Initially an increase in the lipolytic capacity of the cells in response to norepinephrine, is observed, as reflected by the Vmax values up to an age of 20 weeks . 8 . Vmax then stays relatively constant at elevated levels up to an age of 32 weeks, followed by an abrupt decrease with further aging.

Anal Biochem, 1988 Jan, 168(1), 88 - 93
H2O2 as a DNA fragmenting agent in the alkaline elution interstrand crosslinking and DNA-protein crosslinking assays; Szmigiero L et al.; A method for DNA fragmentation by H2O2 in the DNA alkaline elution procedure is described . Treatment of cell suspensions for 1 h with 100 microM H2O2 or 5 mM H2O2 at 0-1 degree C resulted in DNA breakage equivalent to doses of 300 and 3000 rad of gamma-rays, respectively . The elution profiles were reproducible and H2O2 was used for measurements of interstrand crosslinks and DNA-protein crosslinks induced in HeLa cells by mitomycin C, cis-diamminedichloroplatinum(II), and trans-diamminedichloroplatinum(II) . The comparison of data obtained with the use of H2O2 and gamma-rays has shown that both methods have similar sensitivity and reproducibility.

Adv Exp Med Biol, 1988, 222, 299 - 307
Influences of carbon monoxide on the binding of oxygen, carbon dioxide, proton and 2,3-diphosphoglycerate to human hemoglobin; Yamaguchi K et al.; In an attempt to estimate the influences of CO on the CO2 Bohr effect and the 2,3-diphosphoglycerate (DPG) effect linked to the reversible binding of O2 to the hemoglobin molecule (Hb), O2 dissociation curves of human blood in the presence of CO were investigated at 37 degrees C over a DPG concentration ranging from 2.2 to 4.3 mmol/(1RBC) and a pH range of 7.2 to 7.6 . The sample with a low DPG concentration was made by incubating whole blood for 6 hrs, whereas the saturation of Hb with CO, SCO in the sample was adjusted by anaerobically mixing completely carboxygenated blood with that free of O2 and CO so as to give the final SCO at either 0, 10, 15, 20, 40 or 50% . The blood samples thus prepared were diluted at 1:100 in isotonic buffer solution and were equilibrated with gas mixtures containing O2 ranging from 1 to 9% and CO2 from 4 to 9% . The SO2 and SCO values of diluted red cell suspensions were examined by means of a dual-wavelength spectrophotometric method based on the isosbestic points for reduced Hb, HbO2 and HbCO . The extinction difference at 562.7 and 578.0 nm was monitored as a measure of SO2, while that at 570.1 and 584.6 nm was recorded for determining SCO . Fitting the experimental results by the Hill equation, the coefficients describing allosteric interaction induced by CO2 hydration and by DPG in the presence of both O2 and CO were calculated for the total saturation, ST defined as the sum of SO2 and SCO, ranging from 20 to 90%.(ABSTRACT TRUNCATED AT 250 WORDS)

Nauchnye Doki Vyss Shkoly Biol Nauki, 1988, (2), 105 - 9
{Effect of biologically active substances on lymphocyte energetics studied by using a simple luminometer-ATP meter}; Larionov VN et al.; A method of lymphocyte energetics investigation according to the ATP concentration in cell suspension has been described . A simple easily reproducible luminometer was applied for ATP measurement by luminescence of luciferin/luciferase system . The conditions of cell incubation were found when the changes in mitochondrial metabolic state reflected on ATP concentration . For all this rotenone (5 nM) decreases the ATP concentration heavily than inhibits the rate of oxygen consumption . Ecto-ATPases hydrolyze quickly the low concentrations of exogenous ATP . The examples given show the possibilities of this method for studying the effect of biologically active substances on cell energetics.

Biol Neonate, 1988, 53(1), 47 - 52
In vivo effects of glucocorticoid excess on the contents of the rat fetal liver in erythroid progenitors; Billat C et al.; Following a laparotomy of the pregnant rat at 12 days of gestation, erythroid cell suspensions prepared from the fetal livers at 14 days contained an increased proportion of progenitor cells forming colonies after 2 or 7 days of culture . When laparotomy was performed at 14 days and the fetal livers were sampled at 16 days, the opposite effects were observed . Injection of 0.25 mg/kg dexamethasone to the 12 days pregnant rat increased the proportion of erythroid progenitors in suspensions from 14 days fetal livers; injection of 10 mg/kg (a long-acting dose) produced the opposite effects . Both the composition of the erythroid cell line and its environment change between 12 and 14 days, and these modifications might explain the inversion of glucocorticoid effects between these two stages.

Arch Toxicol, 1988 Jan, 61(3), 224 - 8
Phosphatidylcholine synthesis in isolated type II pneumocytes from ozone-exposed rats; van Bree L et al.; Phosphatidylcholine (PC) synthesis by alveolar type II cells, as an indicator for the production of pulmonary surfactant, was studied after a 4-h exposure of rats to 4 mg ozone/m3 (2 ppm) . Lung ravage fluid analysis after exposure revealed significant increases in proteins, which is indicative for pulmonary injury . When type II cells were isolated immediately and thereafter cultured for 20 h, the rate of PC synthesis in cells derived from ozone-exposed rats was not significantly different from that in cells from unexposed controls . Yet, a decreased rate of PC synthesis was observed when these cells were subsequently exposed to ozone in vitro . The activity of the enzyme glycerolphosphate acyltransferase (GPAT) was slightly enhanced in cultured type II cells isolated from ozone-exposed rats, while the lysophosphatidylcholine acyltransferase (LPCAT) activity was unchanged . However, ozone exposure of rats did result in a significant decrease of PC synthesis when measured in freshly prepared type II cell suspensions, although both GPAT and LPCAT activities were not affected . It is concluded that a decrease in pulmonary surfactant related PC synthesis after ozone exposure of rats can be demonstrated in freshly isolated type II pneumocytes . Cultured type II cells from exposed rats lack this effect and are therefore less useful to study changes in phospholipid biosynthesis after in vivo ozone exposure . The data on in vitro ozone exposure of cultured type II cells, however, support the view that ozone may impair pulmonary surfactant production.

J Cancer Res Clin Oncol, 1988, 114(2), 170 - 6
A simple method of delayed processing of tumor tissue in a soft agar clonogenic assay; Krischke W et al.; Sarcoma 180 tumor tissue from C57 mice was processed in a soft agar clonogenic assay immediately after removal from the animal and after various methods of storage . The sensitivity to the antineoplastic drug cis-platinum was not affected by different storage methods . The highest yield of colony forming cells per 100 mg tumor tissue was 2.3 X 10(4) cells following immediate processing of fresh tumor material . A 24-h storage of solid tumor tissue at 4 degrees C as well as cryopreservation of solid tumor tissue or cryopreservation of a single cell suspension prepared from fresh tumor specimens were found to reduce this value to 24%-37% . Storage of a tumor single cell suspension for 24 h at 4 degrees C however, proved to be a useful and simple method of delayed processing of tumor in a soft agar clonogenic assay . The observed cell yield was 1.91 X 10(4) colony forming cells, 83% of the immediately processed value . If this procedure is suitable for use with human tumor material obtained from biopsies and surgery, the 24 h time interval would be useful for transport from hospital to an appropriate special laboratory . This would result in the broader application of the human soft agar clonogenic assay in predictive testing and drug screening.

J Cancer Res Clin Oncol, 1988, 114(1), 47 - 58
Mechanism of liver-specific metastatic tumor spread in a murine tumor model; Edel G; Malignant tumors frequently show an organ-specific metastatic spread, the causes of which are still largely unknown . Using an experimental tumor model, a methylcholanthrene-induced pleomorphic myofibrosarcoma ER 15-P of the C57Bl6J mouse, we wanted to find out whether this phenomenon is due to an adaptation or to a selection of tumor cells . After i.v . injection of tumor cells from the primary ER 15-P into the tail vein of male mice, metastases were regularly found in the lungs, mediastinal lymph nodes, and brain, as well as in the liver and kidneys, and occasionally in the adrenals . The following experimental procedures were used to isolate a tumor cell line with a possible liver preference: (1) Tumor cells from the primary ER 15-P were injected into a mesenteric vein of male mice . Tumor cells from the resulting liver colonies were again injected into the portal system of one group of mice . In a second group, part of the same cell suspension was injected into the tail vein . This procedure was performed four times . (2) Tumor cells from the primary ER 15-P were applied into the tail vein of male mice . Tumor cells from the resulting liver metastases were reinjected directly into the tail vein . This experiment was repeated three times . (3) Tumor cells from the primary ER 15-P were injected into the tail vein of male mice . Tumor cells from liver metastases were then injected, first, into the portal system of one group of male mice, and thereafter into the tail vein of another group of animals . This experiment was repeated twice . The following results were obtained: (1) By a repeated adaptation of tumor cells from the primary ER 15-P to liver tissue, no tumor cell line could be isolated that would show a preferential metastatic spread to this organ after tail-vein injection . (2) Repeated i.v . passages of tumor cells from liver metastases into the tail vein led to the selection of a tumor cell line with a tendency to liver metastasis . (3) Tumor cells selected from liver metastases induced via tail-vein injection showed, after a prolonged stay in the liver and a successive i.v . passage into the tail vein, a marked specificity for this organ . These results indicate that the liver-specific spread of tumor cells in our model is based on the selection of a tumor cell line from the primary ER 15-P influenced by the hepatic microenvironment.

J Neurosci Res, 1988, 19(1), 34 - 42
Migration of cultured fetal spinal cord astrocytes into adult host cervical cord and medulla following transplantation into thoracic spinal cord; Goldberg WJ et al.; Cell suspensions from 14-day-gestation rat spinal cord, which had previously been soaked for 1 hr in a 2 micrograms/ml solution of Phaseolus vulgaris leucoagglutinin (PHAL), were cultured on collagen gels containing laminin for 2 weeks . Pieces of the gel and attached cells were then transplanted into the dorsal column of adult host thoracic spinal cord . At 1, 2, and 3 months postimplantation (MPI), animals were sacrificed, and the spinal cords were removed, embedded in paraffin, and sectioned at 8 micron for immunohistochemistry at the light microscopic level . Sections were double labeled for PHAL and utilized as a marker for transplant-derived cells and glial fibrillary acidic protein (GFAP), a specific marker for astrocytes . Transplant-derived astrocytes (PHAL-GFAP positive cells) migrated from the transplantation site in both rostral and caudal directions and were observed within the host dorsal column ipsilateral to the transplantation site . At 2 months, lateral migration into the contralateral dorsal column and ipsilateral dorsal horn was observed . At 3 MPI transplant-derived astrocytes were observed in host medulla (nucleus gracilis) . Transplant-derived astrocytes were also observed on the glial limitans as far as nucleus gracilis . A migration rate of 0.72 mm/day was calculated, assuming a 14-day delay in the initiation of migration . The ramifications of such extensive migration are discussed with regard to return of function and amelioration of lesion-induced deficits.

J Surg Oncol, 1988 Jan, 37(1), 5 - 9
Inhibition of macrophage- and neutrophil-mediated cytotoxicity by verapamil; Cameron DJ et al.; Peripheral blood monocyte-derived macrophages and polymorphonuclear leukocytes (PMNs) obtained from normal donors kill tumor cells in vitro . However, if verapamil is added to the macrophages or neutrophil tumor cell suspensions in microgram concentrations (0.1 microgram to 0.1 mg), there is marked inhibition of tumor cell killing . The inhibitory effect for the macrophages resulted from an effect of verapamil on both the effector and target cells . When either the effector cells or target cells were preincubated with verapamil, they became resistant to the effects of the cytotoxic macrophages . Cytotoxicity was also inhibited when 0.1 mg of verapamil was added to the macrophages monolayers either at the time of addition of the tumor cells or 15-30 min after addition of the tumor cells, whereas no inhibition of cytotoxicity occurred when verapamil was added more than 30 min after the initiation of the cytotoxic reaction . For the neutrophils it was observed that the inhibitory activity resulted from an effect of verapamil on the effector cells rather than the target cells . When the effector cells were preincubated with verapamil they became incapable of killing the tumor cells, whereas preincubation of the target cells with verapamil had no effect on their ability to be killed by the neutrophils . Cytotoxicity was also inhibited when 0.1 mg of verapamil was added to the neutrophil monolayers either at the time of addition of the tumor cells or 15-60 min after addition of the tumor cells, whereas no inhibition of cytotoxicity occurred when verapamil was added more than 60 min after the initiation of the cytotoxic reaction.

J Clin Invest, 1988 Jan, 81(1), 119 - 25
Glomerular procoagulant activity in human proliferative glomerulonephritis; Tipping PG et al.; Mechanisms for initiation of glomerular fibrin deposition were studied using renal tissue obtained from two patients with rapidly progressive, crescentic glomerulonephritis . Histological examination showed extensive glomerular monocyte infiltration and fibrin deposition in both patients . Sonicated cell suspensions of isolated glomeruli from these patients contained markedly augmented levels of procoagulant activity (PCA) compared with the levels found in normal glomeruli . This PCA was characterized as tissue factor by its functional dependence on Factors VII and V, independence of Factors VIII and XII, inhibition by concanavalin A and phospholipase C, and association with cell membranes . Its coagulant activity was also inhibited by a specific monoclonal anti-human tissue factor antibody . Tissue factor could be identified in glomeruli from these two patients by indirect immunofluorescence using this antibody . These studies implicate extrinsic pathway activation via tissue factor in intraglomerular deposition of fibrin in these patients . Activated monocytes, known to be a potent source of procoagulant activity and seen in large numbers within glomeruli from these patients, are a likely source of this tissue factor.

Clin Exp Metastasis, 1988 Jan-Feb, 6(1), 27 - 37
Pulmonary tumor colony formation following i.v . inoculation of six human colorectal carcinoma xenografts in young gnotobiotic athymic mice; Zimmerman RJ et al.; The lung colonizing potential of 6 xenografted human colorectal adenocarcinomas following tail vein inoculation of tumor cell suspensions into gnotobiotic 3-4-week-old congenitally athymic mice was investigated . One of the lines, CRCo2, was of particular interest, as apparently distinctive lung colonizing phenotypes, large (greater than 2.5 mm diameter) and small (less than 1 mm diameter) colonies were identified, and variant lines with greater, equal, or lesser ability to grow in the lungs relative to the sc tumor of origin were observed . Another line, CRCo1, was also able to grow well in the lungs following tail vein inoculation, but subsequent cycles of lung tumor recovery and reinoculation i.v . did not result in an enhancement of the tumor's lung colonizing ability relative to the initial i.v . inoculation of the sc carried tumor . Scattered lung colonies were observed following i.v . inoculation of sc carried xenografts in three of the four other lines, but we could not consistently recover lung colonies with these tumors . The data are in accord with the clinical observation that pulmonary metastasis is not a high frequency event in human colorectal carcinoma, illustrating the selective nature and experimental utility of this model of metastasis . Further, there were indications of the inefficient and/or random nature of the metastatic process in some of the tumors, while in others, evidence for both effectively higher and lower metastatic variants were found, as might be predicted in heterogeneous tumor cell populations.

Cancer Res, 1988 Jan 1, 48(1), 14 - 8
Interactions of putative estrogens with the intracellular receptor complex in mouse Leydig cells: relationship to preneoplastic hyperplasia; Juriansz RL et al.; The interaction of 14 steroidal and nonsteroidal estrogen agonists and antagonists with the intracellular estrogen receptor system was examined in cell suspensions prepared from the testes of mice that develop malignant Leydig cell tumors after prolonged estrogen administration . The ability of these substances to stimulate DNA synthesis in short-term (3-day) studies and to provoke Leydig cell hyperplasia with prolonged (3-mo) administration was also measured . Our data were consistent with the proposal that, in Leydig cells, the carcinogenic effects of estrogens are mediated through the intracellular receptor complex that results in a localization of hormone bound to chromatin and nuclear matrix . All tested compounds displaced 17 beta-{3H}estradiol from the cytosolic estrogen receptor, but to varying degrees; and there was no discernible relationship between their ability to compete for this receptor and their efficacy in stimulating DNA synthesis . The effect of the test compounds on the levels of estrogen receptor in cytosol and in nuclei was measured by {3H}estradiol exchange . 17 beta-Estradiol, equilin, 17 alpha-ethinylestradiol, diethylstilbestrol, hexestrol, dienestrol, coumestrol, and nafoxidine provoked a complete estrogen receptor response: acutely a decrease in the level of cytosolic estrogen receptor and an increase in the nuclear estrogen receptor . All of these substances acutely stimulated DNA synthesis . Tamoxifen, clomiphene, and nitromifene provoked a decrease in cytosolic receptor but no increase in demonstrable nuclear estrogen receptor . 17 alpha-Estradiol, mestranol, and estriol did not significantly alter the levels of estrogen receptor in cytosol or nuclei . Only those substances that increased measurable nuclear estrogen receptor also acutely stimulated DNA synthesis . Chronic (3-mo) treatment of 2-mo-old male BALB/c mice with diethylstilbestrol, 17 beta-estradiol, ethinylestradiol, and nafoxidine led to Leydig cell hyperplasia . Chronic mestranol treatment also provoked Leydig cell hyperplasia; this is most probably due to induction of liver metabolism of mestranol to ethinylestradiol . Chronic treatment with 17 alpha-estradiol, tamoxifen, and clomiphene failed to produce significant histologic al changes in the testes . Only chronic administration of those substances that exhibited a complete estrogen receptor response and acutely stimulated DNA synthesis produced Leydig cell hyperplasia.

Bibl Anat, 1988, (31), 1 - 84
The human macrophage system: activity and functional morphology; Michna H; Macrophages of humans could be extracted in large numbers from the connective tissue using a newly developed, not particularly difficult method . These macrophages were compared with the peritoneal macrophages of mice using light-, scanning and transmission electron-microscopic methods . The sterility of the cell suspension and the high yield of macrophages has allowed the first in vitro study of histiocytes to take place, in contrast to the classic 'microexudate-coated surface method' . The activity of the human in comparison with peritoneal murine macrophages has been evaluated using numerous histochemical and immunological techniques . These methods prove a modulation of the macrophage activity of healthy humans and mice under exemplary conditions of extremely strenuous physical exercising, in accordance with earlier experimental findings on animals alone . The degenerative changes which occur under these experimental conditions in the skeletal muscular system show an invasion of cells of the immune system, which are integrated into an explanation of the increased activity of macrophages . These results find their place in a new theoretical concept supporting the general validity of the co-operation of macrophages and other cells of the immune system in pathological degeneration and regeneration processes in the skeletal muscular system . It has been shown that the increased activity of human and murine macrophages brought about by extreme strenuous physical exercising, insofar as one is able to order them into a progressive scheme of stress happenings, fit very well into the concepts of the 'alarm reaction' phase . The activity of macrophages proves to be sensitive to the mediators of tumours of mesenchymal origin, with respect to the initial stage of phagocytosis, to the further biochemical deterioration, to the cytotoxicity and to the amount of cells; this, however, is not able to halt the rapid growth of sarcoma in a long term experiment . The proof of a weakened migration of macrophages in sarcoma-bearing animals raises the interest in those substances which are able to positively modulate the migration activity . On the one hand the migratory performance of macrophages in sarcoma-bearing animals in a short-term experiment was increased by the introduction of an anabolic steroid hormone . On the other hand, however, a different degree of success was registered for the further parameters of macrophage activity during short- and long-term experimental investigations.(ABSTRACT TRUNCATED AT 400 WORDS)

Thymus, 1988, 11(1), 15 - 27
The entry of the prothymocyte into the thymus after lethal irradiation and bone marrow transplantation . I . Seeding of bone marrow cells into the thymus; Mulder AH et al.; Bone marrow (BM) cells arrive in the thymus of lethally irradiated mice as early as three hours after bone marrow transplantation (BMT) . They can be recognized by labeling of the injected cells with Hoechst 33342 (direct homing assay) . In order to relate the immigrated BM cells to thymocyte precursor cells, direct homing and thymus repopulation experiments were compared . It was shown that homing of BM cells depends on the time between lethal irradiation and BMT, while it was previously shown that thymus repopulation does not . In addition, thymic immigrants were smaller than precursor cells committed to the T cell limeage (prothymocytes) and their progenitors . A cell population obtained from normal BM cells and enriched in stem cells (purified stem cells) was previously shown to repopulate the thymus similarly as BM cells from mice pretreated in vivo with 5-fluorouracil (FUBM) . Both cell suspension showed a delayed thymus repopulation when compared to normal BM . This is indicative for a depletion of prothymocytes in these cell suspensions . In the direct homing assay, however, it was found that relatively many cells from FUBM seeded into the thymus, while purified stem cells did not . These results indicate that most if not all donor cells that are present in the thymus at three hours after BMT are not thymocyte precursor cells.

Biomed Biochim Acta, 1988, 47(12), 1007 - 11
Superoxide dismutase activity in thymus cell suspension: effects of methionine-enkephalin and morphine; Endroczi E et al.; Superoxide dismutase activity of rat thymus-derived cells was studied by the inhibition of L-adrenaline auto-oxidation oxidation in vitro . The incubation of cells in the presence of methionine-enkephalin (Met-Enk) or morphine (2-20.10(-7) M and 2-8.10(-6) M, respectively) was performed in Krebs-bicarbonate buffer at 37 degrees C for 180 min . After a lag period of 30 to 60 min of incubation, both Met-Enk and morphine decreased the inhibitory activity of cell suspension on the adrenaline autooxidation . Naloxone blocked the effects of opioids in near equimolar concentrations . The observations suggest the interaction of opioids on superoxide anion production of T cell lymphocytes.

Int Arch Allergy Appl Immunol, 1988, 87(4), 367 - 70
Suppression of human IgA-B-cell maturation by IgA-binding factors requires T cells; Millet I et al.; IgA-binding factors (IgA-BFs) prepared from human peripheral blood mononuclear cells selectively decrease the generation of cytoplasmic IgA-positive cells in peripheral blood mononuclear cell cultures stimulated by pokeweed mitogen or by Nocardia opaca delipidated cell mitogen . In N . opaca delipidated cell mitogen stimulated cultures, the suppressive effect of IgA-BFs was no longer demonstrable after removal of sheep erythrocyte rosetting T cells or after depletion of CD8+ cells, but it was not altered by depletion of CD4+ cells . In pokeweed mitogen stimulated cultures of T-depleted peripheral blood mononuclear cell suspensions, IgA-BFs were ineffective when irradiated (12 Gy) instead of control T cells were used for reconstitution . The data indicate that radiosensitive and CD8+ T cell subsets may be required for IgA-BFs to suppress the generation of IgA-containing B cells after polyclonal activation of human peripheral blood mononuclear cells.

Allerg Immunol (Leipz), 1988, 34(1), 27 - 34
{Functional capacity of T-cell subpopulations of the mouse . Effect of unstimulated and allogenic stimulated T-cell subpopulations on Con A stimulation of splenic lymphocytes}; Huckel C et al.; The distribution and the functional capacity of special subpopulations was examined by means of monoclonal anti-Lyt-1.2-, anti-Lyt-2.2-, anti-L3T4- and anti-Thy-1.2-antibodies . The proportion of Lyt-2+-cells increased after allogeneic stimulation of spleen cells compared with the Lyt-1+-cells . The proportion of L3T4+-cells did not change by allogeneic stimulation . Lyt-2- cells which were negatively selected by monoclonal antibody and complement effected the Con A stimulated 3H-thymidine incorporation of a spleen cell culture in a different way depending on their state of differentiation . While Lyt-2--cells selected from a fresh prepared spleen cell suspension increased the 3H thymidine incorporation, Lyt-2--cells selected from allogeneically stimulated cell suspension act as suppressor cells . The results demonstrate the dynamics in the functional capacity of the Lyt-2--T cell subpopulation depending on their state of differentiation.

J Urol, 1988 Jan, 139(1), 150 - 5
Anti-tumor reactivity of human lymphokine activated killer (LAK) cells against fresh and cultured preparations of renal cell cancer; Belldegrun A et al.; Immunotherapy utilizing the adoptive transfer of lymphokine activated killer (LAK) cells in conjunction with recombinant interleukin-2 (IL-2) is capable of mediating the regression of established cancer in a variety of animal tumor models as well as advanced metastatic cancers in humans . We have thus examined the variability of the anti-tumor lytic reactivity of LAK cells obtained from patients with metastatic renal cell cancer (RCC) . Tumor cell suspensions were prepared by enzymatic digestion from 37 consecutive renal cell tumors . The mean (+/- SEM) total number of cells recovered was 1.5 +/- 2.2 X 10(9) cells per tumor . The percentage of tumor cells in the suspension was 39.1 +/- 3.3% (range: 6 to 75%) . Thirteen of 13 different fresh renal tumor cell preparations tested in 57 experiments and tow of two renal tumor lines tested in 10 experiments were all lysed by LAK cells . RCC patients, like normal donors, generated good LAK effectors with broad antitumor activity against autologous as well as allogenic tumors . Both renal and nonrenal tumors were equally lysed by LAK cells . LAK killing of the erythroleukemic tumor lines K562 and Daudi was significantly better than the lysis of fresh autologous and allogeneic tumor targets or cultured RCC tumor lines . Short term tumor cultures derived from renal cancer preparations proved to be sensitive and reliable tumor targets for studying the in vitro killing by LAK cells . Clinical trials testing the therapeutic role of LAK cells and IL-2 in patients with advanced renal cell cancer are currently in progress.

Cancer Res, 1988 Jan 1, 48(1), 206 - 14
Interleukin 2 expanded tumor-infiltrating lymphocytes in human renal cell cancer: isolation, characterization, and antitumor activity; Belldegrun A et al.; We have described a method for the generation, from fresh human renal cell cancers, of lymphoid cells that are capable of exhibiting significant antitumor reactivity when tested in short term 51Cr release assays . Tumor cell suspensions obtained from 37 consecutive fresh human renal cell cancer specimens (35 patients) could be separated by using enzymatic techniques and culturing in medium containing recombinant interleukin 2 (IL-2) . The total cell recovery was 1.5 X 10(9) +/- 2.2 (SE) per tumor with a range of 1 X 10(8) to 5 X 10(9) cells . The percentage of tumor cells in the suspension ranged from 6 to 75% with a mean of 39.1 +/- 3.3% . The remaining cells were predominantly lymphocytes . Viability of mononuclear cells was greater than 90% . Activated tumor-infiltrating lymphocytes (TIL) within these tumors expand and by 10 to 14 days after initiation of culture a 5- to 15-fold increase in the number of lymphocytes could be achieved with elimination of all autologous tumor cells . Lymphocytes were recultured in fresh medium containing IL-2 and continued to expand between 2- and 10-fold every 4 to 6 days for an average of 33.7 +/- 4.5 days, resulting in greater than 50,000-fold increase in the total number of lymphocytes . The average number of splits was 4.9 +/- 0.8, with a range of 0 to 21 . In 11 of 11 cases tested, TIL exhibited a far better expansion capability in vitro compared to that of peripheral blood lymphocytes obtained from the same patient and grown under identical conditions . The majority of TIL were T cytotoxic/suppressor cells (Leu 2+ Leu 4+) . With continued in vitro expansion (up to 50 days) there was a concomitant increase in the helper T (Leu 3+) and pan T populations (Leu 4+) and decrease in Leu 2+ and HLA-DR+ cells . Compared with expanded peripheral blood lymphocytes, these cells demonstrated higher levels of IL-2+ receptors and HLA-DR+ antigens . Renal TIL effectors expanded in IL-2 could lyse almost all autologous tumor targets in 4-h chromium release assays . Allogeneic renal as well as nonrenal targets were equally lysed . TIL lysis of cultured tumor targets K562 and Daudi was significantly better than lysis of autologous, allogeneic-renal, and nonrenal targets . No statistically significant difference in the cytotoxic activity of renal TIL or peripheral blood lymphocyte effectors in killing autologous or allogeneic targets could be demonstrated.(ABSTRACT TRUNCATED AT 400 WORDS)

Adv Enzyme Regul, 1988, 27, 15 - 29
Photoaffinity analogues of methotrexate as folate antagonist binding probes; Freisheim JH et al.; A photoaffinity analogue of methotrexate, APA-{125I}ASA-Lys, specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation . An excess of methotrexate blocks incorporation of the photoprobe . Following cyanogen bromide digestion of the radiolabeled enzyme and high-pressure liquid chromatographic separation of the generated peptides, a majority of the label was centered around residues 63-65 (Lys-Asn-Arg), part of the inhibitor binding domain . This photoprobe is also transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Vmax similar to that for methotrexate . Ultraviolet irradiation at 4 degrees C of a cell suspension that had been incubated with the radiolabeled photoprobe resulted in the covalent modification of a 46-48 Kd protein . This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography . Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound . Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate . In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated . Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr = 38 Kd and 21 Kd) derived from the cell homogenate supernatant.

Eur J Nucl Med, 1988, 14(12), 621 - 3
Evaluation of a new leukocyte labeling procedure with 99mTc-HMPAO; Kelbaek H et al.; A technique for labeling leukocytes with 99mTc using hexamethylpropylene-amineoxime (HMPAO) was evaluated in vitro . The labeling procedure resulted in a cell bound fraction of radioactivity of 56% after 99mTc incubation and 96% after 1 washing of cells . In the final cell suspension 84% of the radioactivity was attached to the polymorphonuclear (PMN) leukocytes constituting 94% of white cells . Only 5% was bound to residual red blood cells . The stability evaluated in autologous plasma showed a decline of cell bound activity from 96% to 84% over 3 h . The chemotactic function of PMN leukocytes was unaffected by the labeling procedure . These findings demonstrate that HMPAO, albeit cell unspecific, is efficient for labeling PMN leukocytes with 99mTc . The stability of the labeling procedure is high and the technique does not affect cell function . No other current 99mTc leukocyte labeling technique possesses all these qualities.

Arch Immunol Ther Exp (Warsz), 1988, 36(2), 223 - 33
Evaluation of technical aspects of the preparation of haemopoietic cells from human fetal liver; Ratajczak MZ; Methods employing both mechanical and enzymatic treatment for obtaining single cell suspension from human fetal livers were compared . Subsequently, the effects of various media and sera on cell survival in fetal liver cell suspensions were studied with special attention to short term storage at 4 degrees C . The largest number of living cells was obtained after homogenization of fragments of fetal liver by teasing them through metal sieve . Cell survival was the best when HEPES buffered Dulbecco MEM supplemented with 20-30% of human AB group serum was used throughout . More than of 50% granulocytemonocyte progenitor cells (GM-CFU) were still present in this suspension after 96 hours of storage at 4 degrees C.

Cell Tissue Res, 1988, 254(3), 659 - 70
Formation of a new fibrous attachment to human dental roots . A new vitro model for studying periodontal regeneration; Bernstein AB et al.; This study was performed to improve currently employed in vitro models for the study of periodontal regeneration by using a porous filter upon which periodontal ligament cells were grown . Periodontal ligament cells were harvested and 0.3 mm root discs cut from three partially erupted and extracted third molar teeth of one patient . Experimental culturing was performed by seeding periodontal ligament cell suspensions on Puropor-200 filters supported by wire-mesh grids in Grobstein Petri dishes . The following day, an interdental space of 0.1 to 0.3 mm was created by gently placing two dental root discs upon the filter . Cultures were terminated after 42, 56, 112 and 124 days, and processed for light- and electron microscopy . Collagen fibril diameters were measured . Adjacent and often attached to large areas of cementum-lined root discs, a dense fiber fringe developed . This fiber fringe was not found on dentin-lined root discs . Although less organized, older cultures demonstrated a similar disc-culture interface, which depended upon the presence or absence of original root cementum . Collagen fibrils of early cultures had a mean diameter of about 42 nm, while in older cultures the diameters ranged from 47 to 68 nm . It is concluded that the fibrous matrix attached to cementum-lined root discs somewhat resembles the initial stages of the formation of dental root cementum in vivo.

Anat Embryol (Berl), 1988, 178(6), 529 - 36
An in vitro model of gonad differentiation in the chick embryo . Roller cultures in gas permeable biofoil bags; Drews U et al.; Embryonic gonads of 6 1/2 to 12 days old chick embryos were enzymatically dissociated . The cell suspensions were cultured in small gas permeable bags of foil (Biofolie Heraeus) in a roller culture apparatus . The cells formed multiple small aggregates, in which sex specific differences developed within two days . In cell suspensions of embryonic testes smooth spheric aggregates formed with well delineated testicular cords in the center and a tunica albuginea-like mesenchymal layer at the outside . Most of the male germ cells were incorporated in the central cords . A number of germ cells were barred from entering the cords by the tunica albuginea-like mesenchymal layer and populated the outer surface of the aggregates . The aggregates of left ovary were irregular in shape and characterized by clusters of germ cells residing in an outer cortical zone . The aggregates of the right ovary, which regresses in vivo, showed poor growth and did not differentiate, thus, indicating that the suppression of right ovary was not removed in culture . In the roller cultures of dissociated embryonic gonads male and female morphogenesis was mimicked in a reproducible manner, so that the system can be used for further experimental studies of gonadal development.

Biomater Artif Cells Artif Organs, 1988, 16(4), 747 - 69
Microencapsulation of mammalian cells in a hydroxyethyl methacrylate-methyl methacrylate copolymer: preliminary development; Stevenson WT et al.; Erythrocytes were microencapsulated in a thermoplastic copolymer of poly-2-hydroxyethyl methacrylate (79% mole%) - co-methyl methacrylate (21 mole %) with little apparent initial cell lysis . Droplets of cell suspension and polymer solution were blown from the tip of a coaxial needle assembly into a receiving bath of hexadecane over phosphate buffered saline (PBS) containing a low concentration of nonionic surfactant . Capsules were trapped at the hexadecane/PBS interface where they were cured by the removal of polymer solvent to precipitate a polymer coating around the cell suspension . Important principles which were considered in the development of the successful process, included the need to prevent intermixing of polymer solution and cell suspension, to fully surround the cells with polymer solution prior to precipitation, and to prevent direct mixing of the curing bath with the polymer solution.

Dev Comp Immunol, 1988 Fall, 12(4), 843 - 54
Dissociation of the RES and immune components in the transient splenic response of embryos and neonatal chicks to immunization; Seto F; Immunization with mouse erythrocytes (MRBC) elicited transient splenic enlargement as well as PFC formation in immunocompetent 7, 10, and 14 day chicks . An early enlargement within two days was followed by a decline and a second enlargement by day six . Only the early enlargement was observed when MRBC or Percoll was administered to neonatal chicks and late embryos . No significant splenomegaly was observed following cell transfer of bone marrow, spleen, leucocyte, thymocyte or bursal cell suspensions from B haplotype-compatible donors . During the adoptive immune response in embryo hosts, a minor early peak was observed that was followed by an elevated or progressive increase in spleen size . The data suggest that the early splenic response reflects primarily RES-vascular activity and the later enlargement is the consequence of immune proliferation.

Acta Physiol Hung, 1988, 71(4), 551 - 5
Immunobiological methods in the prenatal diagnosis and evaluation of foetal neural tube defects; Polgar K et al.; In cases of foetal neural tube defects (NTDs) macrophages are present in the amniotic fluid . These mononuclear cells were analysed with immunobiological methods: functional markers as Fc and C3b receptor-mediated phagocytosis and chemoluminescence have been studied . It was found that most of these pathognomic cells ingest haemolysin sensitized sheep red blood cells (sSRBCs) and zymosan (Mannozym) particles opsonized with fresh human serum . Amniotic fluid cell suspensions from pregnancies with and without foetal NTDs were stimulated by opsonized Mannozym; consistently higher chemoluminescence activities were found when open lesion was present . The evaluation of multiple functional markers is likely to provide a better basis for understanding the characteristics of amniotic fluid macrophages and may contribute to the prenatal diagnosis of NTDs.

Biorheology, 1988, 25(1-2), 349 - 54
Competitive role between fibrinogen and albumin on thixotropy of red cell suspensions; Lacombe C et al.; Plasmatic proteins, namely fibrinogen and globulins, play a major role in red blood cell (RBC) aggregation which is accountable for the three-dimensional structure of blood . Consequently, blood rheological properties linked to this structure must be modified when the protein plasma content changes . This paper gives results and related comments on thixotropic properties of RBC suspensions (0.45 hematocrit) in isotonic solutions containing various amount of fibrinogen to which albumin is added . Thixotropic behavior of these RBC suspensions is studied with a low inertia coaxial cylinders viscometer at a shear rate step of Y = 1 s-1 . Rheograms are interpreted in term of thixotropy coefficient . The main conclusion is that albumin improves RBC disaggregability of whole blood, resulting probably from a competitive effect between fibrinogen and albumin in the RBC aggregation process.

Biorheology, 1988, 25(1-2), 123 - 8
Effects of viscoelasticity of cytoplasm on the complex viscosity of red blood cell suspensions; Takano Y et al.; To consider the effects of the viscoelasticity of cytoplasm on the relaxation phenomenon of red blood cell suspensions, we calculate the complex intrinsic viscosity {eta*} = lim(eta* - eta)/eta c of the disperse system of spherical c----0 cells as a function of the frequency, where eta* is the complex viscosity in suspensions, eta the medium viscosity and c the volume concentration of the cells . The cell consists of a viscoelastic membrane and a viscoelastic cytoplasm . The viscoelasticity of the membrane is described by the Voigt model, while the viscoelasticity of the cytoplasmic region is described either by the Maxwell model or by the Voigt model . The interfacial tension is taken into account on both the interfaces of the membrane . The results of {eta*} are compared with the ones in the case in which the cytoplasmic region is purely viscous liquid.

Haematologia (Budap), 1988, 21(3), 163 - 7
Immunological markers in a coexisting chronic lymphocytic leukemia and Hodgkin's disease; Ojeda E et al.; A 57 year old patient in whom Hodgkin's disease (HD) and Chronic Lymphocytic Leukemia (CLL) was simultaneously diagnosed is described . The infiltration of peripheral blood and bone marrow by mature lymphocytes, with special immunological phenotype (SIg negative, mouse rosette positive and monoclonal antibodies B1+, B4+) consistent with B-CLL and histological findings of lymphnode and axillary mass biopsies were typical of HD . The immunological study of the cell suspension from the axillary mass displayed a phenotype similar to that of the peripheral blood lymphocytes . Whether HD and CLL are two processes of fortuitous association, or a single clinical entity remains to be elucidated . The immunological findings in our patient suggest a common origin for both disorders and that HD could sometimes be the result of a B cell proliferation.

Exp Cell Biol, 1988, 56(1-2), 67 - 73
Vesicle-mediated delivery of membrane to growth cones during neuritogenesis in embryonic rat primary neuronal cultures; Shea TB et al.; Single cell suspensions from 15-day embryonic rat hindbrain plated on collagen formed large clumps by day 1 in culture . Neurite outgrowth was visible within 2 days . By day 14, morphological synapses were observed in nearly all instances of contact of a neurite ending with another cell . At day 3 in culture, the Golgi apparatus consisted of relatively few, broad lamellae . By contrast, at day 7 in culture this organelle consisted of tightly packed lamellar stacks with a considerable increase in vesicles budding from lamellae . Electron-lucent vesicles, ranging in size from 60 to 180 nm, similar to those generated by the Golgi apparatus were noted in neurite shafts and growth cones, with fusion of these vesicles virtually exclusively at the growth cone leading edge . Monensin resulted in the loss of these vesicles in cell somata and neuritic profiles . The electron-dense marker horseradish peroxidase was not incorporated into these vesicles following its addition to the culture medium, indicating that the vesicles were exocytotic . The number of total vesicles increased during the first 7 days of neurite outgrowth with no further increase up to day 14 . This increase was due entirely to vesicles not labeled with the impermeable electron-dense stain ruthenium red, indicating that this increase represents actual vesicular elements and not increased surface convolutions . These data suggest that the 60- to 180-nm electron lucent vesicles are derived from the Golgi apparatus and, by fusion with the growth cone plasmalemma, provide new membrane required for neuritic outgrowth and maintenance.

Jpn J Physiol, 1988, 38(2), 145 - 58
Pressure-flow relationship of erythrocyte suspension in perfusion of nuclepore membrane and red cell deformability; Uyesaka N; The hemodynamic characteristics of Nuclepore (NP) membrane filtration were evaluated from a pressure (P)-flow rate (Q) relationship of erythrocyte suspension obtained by the vertical-tube method (Nichol et al., 1951) with a slight modification . It became evident that the vertical-tube method was a more quantitative and simple method than the conventional steady flow perfusion method using a variable speed pump . The P-Q relationship of erythrocyte suspensions in the perfusion of NP membrane consisted of a smooth curve convex to the P-axis at low P tending to a straight line at high P . The extrapolated linear segments of P-Q curves of erythrocyte suspension did not converge to the same point on the negative Q-axis, that is, the value of the negative intercept increased as flow decreased . This observation indicated clearly an obstruction or plugging of the pores by erythrocytes . It was made clear that flow rate measured with NP membrane filtration was influenced not only by intrinsic red cell deformability but also by several different factors, namely, the different distribution of pore size of NP membrane, microdust contaminated inevitably in red cell suspension, total volume of cell suspension through NP membrane, stagnation or retention of erythrocyte suspension and the hematocrit value of suspension . In conclusion, the present study shows that the precise definition of deformability depends on the method or rheological conditions, such as hematocrit value of suspension, used to measure it, even if confined within NP membrane filtration.

Acta Histochem Suppl, 1988, 36, 315 - 8
The fate of cerebellar allotransplants as determined with histochemical methods; Marani E; Allotransplants of cerebellar cell suspensions from neonate 1 day old rabbits into litter mates showed the survival of granule cells . No Purkinje cells survived this treatment . The transplanted granule cells kept their ability to express the Stage Specific Embryonic Antigen, indicating their recovery from the dissociation procedure and their positive selection of one cell type from the cell suspensions.

Exp Brain Res, 1988, 73(2), 236 - 48
Intrastriatal dopaminergic grafts restore inhibitory control over striatal cholinergic neurons; Herman JP et al.; The aim of the study was to examine the influence of intrastriatal dopaminergic grafts on the functioning of striatal cholinergic neurons using an in vitro superfusion method . Rats bearing unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic system received a cell suspension obtained from ED 14 rat embryonic mesencephali which was injected into the denervated striatum . Lesioned animals displayed an ipsilateral rotation in response to amphetamine (5 mg/kg i.p.) . This rotational response disappeared following grafting and there was even a significant contralateral rotation in response to the drug . Apomorphine (0.1 mg/kg s.c.) induced a contralateral rotation following the lesion . This latter response was attenuated in the grafted group . Three months after grafting 350 microns thick slices were prepared from striata from the control and experimental sides of lesioned and graft-bearing animals . The slices were preincubated either with 3H-dopamine (10(-7) M) or 3H-choline (10(-7) M) and then superfused with an oxygenated Krebs-Ringer solution . Stimulation with electrical pulses following preincubation with 3H-dopamine elicited a marked increase of tritium outflow from control slices . Stimulation-evoked overflow was of similar magnitude from slices from striata containing the graft, while it was much reduced in slices from lesioned striata . Amphetamine markedly potentiated the effect of electrical stimulation in slices obtained from control and graft-containing striata . Nomifensine (a dopamine uptake blocker) led to a significant decrease of the overflow of 3H-acetylcholine evoked by electrical stimulation from control striatal slices . This inhibition was antagonized by domperidone, a D2 dopamine receptor blocker, a finding which indicates that the action of nomifensine was indeed due to a potentiation of the action of endogenous dopamine released by the electrical stimulation . A similar, although somewhat attenuated, action of nomifensine and domperidone was observed for striatal slices containing the graft . Amphetamine inhibited the stimulation evoked overflow of 3H-acetylcholine in a dose-dependent manner from striatal slices obtained both from the intact and experimental sides of graft-bearing animals, while it had no action on slices from denervated striata . Finally, the dose-response curve for the inhibition of 3H-acetylcholine release by apomorphine was significantly shifted to the left for slices from the lesioned striata as compared with control slices . This leftward shift was totally abolished in the slices from the graft-containing striatum.(ABSTRACT TRUNCATED AT 400 WORDS)

Acta Neurochir Suppl (Wien), 1988, 43, 149 - 53
Transplant-induced recovery from 6-OHDA lesions of the nigrostriatal dopamineneurones in mice; Shimizu K et al.; Attempts to reconstruct the damaged nigrostriatal pathway in experimental models of Parkinson's disease have thus far been carried out in animals with neurotoxically induced dopamine deficiency . Our study established that unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopamine (DA) neurons produced a well-characterized functional asymmetry in the behaviour of the C57BL/6 (H-2b) mice . The intraperitoneal administration of methamphetamine induced ipsilateral rotation at 7-20 turns/min 1 x 10(6) syngenic DA-rich cells of embryonic ventral mesencephalon were stereotaxically transplanted in the caudate-putamen . A complete recovery of methamphetamine-induced rotational response was produced around the 60th day after the syngenic cell suspension graft . And a complete compensation of the rotational response was also brought about with the DA-rich cells from embryonic ventral mesencephalon (crown-rump length; 10-13 mm) of allogenic C3H/HeN (H-2k) mice . The FACS IV analysis revealed no H-2 (Kk and Iak) antigens before transplantation of these embryonic cells . Immunohistochemistry showed that the dopaminergic fibers had grown predominantly into the ipsilateral caudate-putamen . These results provide evidence of integration of syngenic and allogenic grafts and host tissue . And the immunological response in the transplanted brain are under investigation.

Dev Comp Immunol, 1988 Summer, 12(3), 657 - 68
Influence of interferon gamma on B cell replication; Vetvicka V et al.; Semisolid agar culture techniques, recombinant murine gamma interferon, (Mu IFN gamma) and monoclonal anti-Mu IFN gamma antibodies were used to evaluate the role of interferon in mitogen-dependent B cell proliferation . Subtle, but significant, effects of interferon addition were noted when unseparated spleen cells were cultured . Depending on culture conditions, the cloning efficiency of the B cells was slightly increased by optimal concentrations of interferon . When the spleen cells were cultured over unstimulated peritoneal macrophages separated by an agar spacer, and especially when no other potentiators were added to the cultures, substantial enhancement of B cell proliferation routinely resulted . The mechanism of this effect was then investigated with macrophage depleted spleen cells as well as highly enriched, positively selected, B cell suspensions . Results of these experiments indicated that B cell replication can be modulated by direct interaction with interferon and again, the conditions of culture determined the magnitude and direction of the effect . Addition of anti-interferon antibodies, indomethacin, or interleukin 1 (IL-1) to the cultures did not influence the cloning efficiency of normal splenic B cells . Therefore, we found no evidence that endogenously produced interferon, prostaglandins, and IL-1 influence B cell colony formation . Since interferon stimulated macrophages dramatically improved B cell proliferation without direct cell contact, some other monokine may be involved . Therefore, gamma interferon can participate in humoral immune responses by influencing the expansion of activated B cells directly as well as indirectly via stimulation of macrophages.

Eur J Immunol, 1988 Jan, 18(1), 167 - 72
Signals involved in T cell activation . T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies; Van Lier RA et al.; Monoclonal antibodies (mAb) directed against the T cell differentiation antigen CD28 (Tp44) induce proliferation of resting T lymphocytes in the presence of phorbol esters . Moreover, it has been reported that such antibodies augment and sustain T cell proliferation induced by soluble antigens, phytohemagglutinin and anti-CD3 mAb . Recently, we have shown that in monocyte-depleted T cell suspensions, anti-CD28 mAb 9.3 and Kolt-2 were able to circumvent the requirement for interleukin 2(IL2) in T cell proliferation induced by soluble anti-CD3 antibodies . Apart from the synergy of anti-CD28 antibodies with phorbol myristate acetate and anti-CD3 antibodies, we found that anti-CD28 mAb were able to induce T cell mitogenesis in combination with an E rosette-blocking anti-CD2 antibody . In this report, we show that antibodies directed against different epitopes on the CD2 antigen can synergize with anti-CD28 mAb . Furthermore, we demonstrate that proliferation induced through the synergistic action of anti-CD28 mAb with anti-CD2 antibodies can be induced in the absence of accessory cells and is accompanied by the production of IL2 and the expression of IL2 receptors . We were unable to induce detectable Ca2+ mobilization through the simultaneous binding of anti-CD28 and anti-CD2 mAb . Taken together, these data show that IL2-dependent proliferation can be induced through the simultaneous binding of anti-CD28 and anti-CD2 antibodies, possibly through phosphatidyl inositol-independent pathways . The observations may provide further insight into the activation mechanisms of human T cells.

J Cell Sci Suppl, 1988, 10, 29 - 44
Early haemopoietic stem cells in the avian embryo; Dieterlen-Lievre F et al.; Using 'yolk sac chimaeras', we have previously demonstrated that stem cells, destined to colonize haemopoietic organs other than the yolk sac, arise in the embryo proper . We have now investigated the emergence and potentialities of these cells in vivo and in vitro . The in vivo approach consisted of interspecies grafting between quail and chick embryos . The cell progeny from the grafts was detected by means of QH1, a monoclonal antibody specific for the quail haemangioblastic lineage . When grafted into the dorsal mesentery of the chick embryo, which is a haemopoietic microenvironment, the region of the aorta from E3-E4 quail embryos generated large haemopoietic foci . When associated with a chick attractive thymic rudiment, cells left the quail aorta, entered this rudiment and underwent lymphopoiesis . Cell suspensions prepared from 40-50 chick aortae, seeded in appropriate semi-solid media, yielded macrophage, granulocyte or erythrocyte clones . These colony forming cells were two to eight times more frequent than in cell preparations from hatchling bone marrow . By contrast, cells prepared from the whole embryonic body deprived of the aorta were not clonogenic . By interspecies grafting of somatopleural (ectoderm + mesoderm, e.g . limb bud) or splanchnopleural rudiments (endoderm + mesoderm, e.g . lung, pancreas, intestine), the endothelial lining of blood vessels was shown to arise by two entirely different processes according to the rudiment considered: angiogenesis, i.e . invasion by extrinsic endothelial cells, in the limb bud, and vasculogenesis, i.e . in situ emergence of endothelial cells, in internal organs . The spleen, which first develops as a continuum to the pancreatic mesoderm, acquires its endothelial network by vasculogenesis, and is colonized by extrinsic haemopoietic stem cells . Granulopoietic cells in the pancreas and accessory cells in the lung are also extrinsic . Thus, in the case of endomesodermal rudiments, interspecies grafting reveals separate origins of endothelial and haemopoietic cells.

Cell Tissue Res, 1988, 254(3), 487 - 97
Fate of intraocular chromaffin cell suspensions: role of initial nerve growth factor support; Stromberg I et al.; Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin . Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine-beta-hydroxylase (DBH) . After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if beta-nerve growth factor (NGF) was given at grafting . One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber networks and all cells were DBH-immunoreactive . Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris . Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes . The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls . A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections . In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathetically denervated iris . Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.

Acta Orthop Scand Suppl, 1988, 228, 1 - 39
DNA cytometry of osteosarcoma; Bauer HC; The relationship between cytochemical features and histomorphology in osteosarcoma, and the clinical significance of DNA content were investigated by microspectrophotometry (MSP) of tissue sections and flow cytophotometry (FCM) of cell suspensions . MSP of tissue sections entails the methodological error of determining the DNA content of sectioned cell nuclei . By analyzing 184 normal mesenchymal cell populations, an upper limit of diploidy (normal DNA content) was deduced . Applying this upper limit for 42 sarcomas, 6 were diploid and 36 hyperploid . Comparative analysis of the same lesions by MSP of imprint preparations and by FCM disclosed complete agreement in ploidy classification (diploid versus hyperploid) . Retrospective MSP analysis of bone tumors is often impeded by previous demineralization in acid, which destroys DNA . EDTA as an alternative was found to slightly reduce Feulgen DNA stainability of osteosarcomas, but did not affect tumor ploidy determination . Hence, EDTA offers a means of retaining DNA stainability of bone tumors requiring demineralization . MSP analysis of different histologic areas, and comparative FCM analysis of biopsy and surgical specimens, dislosed that individual osteosarcomas are cytochemically uniform despite morphologic heterogeneity . Hence, a single tumor sample for DNA analysis can be relied upon as representative for the tumor as a whole . In a consecutive series of 83 osteosarcoma patients treated by surgery and adjuvant Interferon, the 7-year survival rate was 0.44 . MSP DNA analysis gave no significant prognostic information . Multivariate analysis identified 3 risk factors for tumor related death, i.e., male sex, proximal tumor location, and histologic grade IV . In a prognostication model, the 7-year survival rates, for patients with 0, 1, 2, or 3 risk factors, were 0.80, 0.59, 0.42, and 0.13, respectively . Hence, it is possible to identify subgroups of high grade osteosarcoma patients with different prognosis . In a study of 166 primary bone tumors, the applicability of DNA analysis for differential diagnostic purposes was investigated . The series included high grade osteosarcomas, parosteal osteosarcomas and benign bone tumors, which may be mixed up histologically with osteosarcoma . Out of 166 tumors, 149 (90%) were histologically noncontroversial, whereas 17 (10%) posed diagnostic difficulties . In the diagnostically noncontroversial group, all benign tumors and parosteal osteosarcomas were diploid, whereas 97 of 102 osteosarcomas were hyperploid . Hence, hyperploidy seems to be a characteristic feature of high grade osteosarcoma.(ABSTRACT TRUNCATED AT 400 WORDS)

Dev Comp Immunol, 1988 Fall, 12(4), 855 - 64
The monoclonal antibody CVI-ChNL-68.1 recognizes cells of the monocyte-macrophage lineage in chickens; Jeurissen SH et al.; The characteristics of monoclonal antibody CVI-ChNL-68.1, which specifically reacts with a group of chicken non-lymphoid cells, are described . Both tissue distribution shown on cryostat sections using immuno-enzyme histochemistry, and quantitative data obtained on cell suspensions are presented . Functional characteristics of CVI-ChNL-68.1-positive cells, such as antigen uptake and glass adherence, are determined . Results show that CVI-ChNL-68.1 reacts with monocytes, macrophages, and interdigitating cells . Possible relationships between the various non-lymphoid cells are discussed.

Immunopharmacol Immunotoxicol, 1988, 10(3), 387 - 98
Ex vivo treatment of murine splenocyte-supplemented bone marrow inocula with mafosfamide prior to allogeneic transplantation in an attempt to prevent lethal graft-versus-host disease without compromising engraftment; Kohn FR et al.; Murine splenocyte-supplemented bone marrow cell suspensions were incubated with mafosfamide, an analog of "activated" cyclophosphamide, prior to transplantation across major histocompatibility barriers into lethally-irradiated recipient mice in an attempt to reduce the incidence of graft-versus-host disease (GvHD)-related mortality without compromising engraftment . Irradiated mice that received vehicle-treated splenocyte-supplemented bone marrow inocula developed symptoms of severe GvHD; the majority of such animals did not survive . Treatment of donor cells with 160 microM mafosfamide for 30 min resulted in a marked increase in animal survival without evidence of GvHD . Survival of bone marrow allografts was demonstrated by the persistence of donor-type mononuclear cells in the peripheral blood of surviving animals . Treatment of donor cells with a four-fold higher concentration of mafosfamide also resulted in a significant increase in survival without evidence of GvHD; however, host resistance to engraftment was indicated by a low percentage of donor mononuclear cells in the peripheral blood of the survivors . Treatment of donor cells with a four-fold lower concentration of mafosfamide resulted in a slight increase in survival; however, all animals developed symptoms of GvHD . These results indicate that, at appropriate concentrations, mafosfamide can effect the elimination of GvHD-causing T lymphocytes from donor bone marrow inocula without compromising its engraftment potential.

Bone Marrow Transplant, 1988 Jan, 3(1), 31 - 41
Immunomagnetic removal of B-lymphoma cells from human bone marrow: a procedure for clinical use; Kvalheim G et al.; B-lymphoma cells were purged from human bone marrow by incubating the cell suspension with a cocktail of three different pan-B cell mouse IgG1 monoclonal antibodies, and then with immunobeads charged with sheep anti-mouse antibody, followed by magnetic separation . The primary antibodies used, HD37 (CD19), HD6 (CD22), and HH1 (CD37), bind to a very high percentage of the cells in non-Hodgkin's lymphomas of poor prognosis . The secondary antibody is directed against the Fc portion of the IgG antibodies . In model experiments Burkitt's lymphoma cells (Rael) were admixed to mononuclear bone marrow cells in the ratio 1/9 . With a ratio of immunobeads/total antibody-binding B cells of 50/1 in a first treatment cycle and repeating the procedure with the same number of beads in a second cycle, a tumor cell depletion of more than 5 logs was achieved, as judged by a clonogenic assay . The concomitant reduction of CFU-GM and CFU-GEMM was about 20% . The purging procedure has been scaled up to clinical use . Equipment suitable for purging patients' marrow specimens, employing standard transfusion facilities, is described . With this equipment the efficacy of tumor cell removal was the same as in the model experiments, and the whole magnetic separation could be completed in 2 hours.

Chem Res Toxicol, 1988 Jan-Feb, 1(1), 41 - 6
Covalent binding of N-hydroxy-N-acetyl-2-aminofluorene and N-hydroxy-N-glycolyl-2-aminofluorene to rat hepatocyte DNA: in vitro and cell-suspension studies; Corbett MD et al.; Two 2-aminofluorene-derived hydroxamic acids that differ only in the nature of the N-acyl group were examined for their relative abilities to undergo covalent binding to nucleic acids . Studies of the bioactivation of N-hydroxy-N-acetyl-2-aminofluorene (N-OH-AAF) and N-hydroxy-N-glycolyl-2-aminofluorene (N-OH-GAF) were conducted with hepatocyte suspensions and subcellular fractions prepared from male Sprague-Dawley rats . Both hydroxamic acid substrates displayed equal binding to both DNA and RNA after incubations with hepatocyte suspensions . The extent of binding of each substrate was approximately the same for DNA and RNA . Investigations with subcellular fractions revealed some major differences between the probable mechanisms by which the two substrates were covalently bound to exogenous DNA . In agreement with the prior literature reports, N-OH-AAF was extensively bound to DNA through the action of cytosol enzymes, including both N,O-acyltransferase and sulfotransferase . The microsomal enzyme fraction also catalyzed binding to DNA, and this process was completely inhibited by paraoxon . The covalent binding of N-OH-GAF to DNA was catalyzed by cytosol enzymes to a significant extent only in the presence of 3'-phosphoadenosine-5'-phosphosulfate, which suggests the action of sulfotransferase . Covalent binding of N-OH-GAF to DNA was minimal through the action of cytosolic N,O-acyltransferase, which confirms our earlier observation that N-OH-GAF is a potent suicide inhibitor of this enzyme . The microsomal fraction catalyzed the binding of N-OH-GAF to DNA at a rate that was about twice that observed for N-OH-AAF.(ABSTRACT TRUNCATED AT 250 WORDS)

Biorheology, 1988, 25(4), 639 - 49
Theoretical and experimental study of the time dependent flow of red blood cell suspension through narrow pores; Bucherer C et al.; The Hemorheometer has been adapted to allow the recording of the flow rate during the filtration process . For newtonian fluids, the flow rate variation versus time through the pores is well approximated by Poiseuille's law . For dilute red blood cell suspensions, the same analysis can be applied by introducing the concept of "apparent filtration viscosity" which is higher than the usual viscosity measured by Couette viscometry . The apparent filtration viscosity parameter is related to the deformations undergone by red blood cells as they pass through the narrow pores . Apparent filtration viscosity can be used to obtain a precise determination of the erythrocyte deformability . Measurements performed, for a given blood sample, with pores of different diameters (5 microns, 8 microns and 12 microns) show that the error on the value of apparent filtration viscosity is less than 3% . As a result, the sensitivity of the filtration method allows to discriminate among normal blood samples . High concentrations of erythrocytes or leucocytes are found to modify the apparent filtration viscosity . These factors are apparent in the recorded filtration curves . Their effects on filtration measurements can be easily estimated.

Glia, 1988, 1(2), 156 - 64
Tetanus toxin binding to isolated and cultured rat retinal glial cells; Huba R et al.; The presence of immunocytochemically detectable membrane receptors for tetanus toxin, supposedly composed of higher gangliosides, is widely accepted as a marker of neuronal cells . We now demonstrate that Muller cells, a unique glial cell type of the vertebrate retina, possess specific tetanus toxin (TT)-binding sites . Single cell suspensions were prepared from adult rat retina by a gentle dissociation method, and the Muller cells, unequivocally identified by their morphology, could be immunocytochemically double-labeled by antisera to vimentin and to TT . The expression of complex gangliosides by identified Muller cells was also demonstrated by immunofluorescence labeling with the monoclonal antibody A2B5 . Using the double-immunolabeling method for the identification of Muller cells we show that specific tetanus toxin binding is acquired by these cells during postnatal maturation both in vivo and in vitro . In vivo the percentage of tetanus toxin-positive Muller cells increases from 0% in 4-day-old animals to 10% on postnatal day 8, reaching the adult level of about 95-100% around day 30 . In retinal monolayer cultures prepared from newborn rats, the majority (65%) of vimentin-positive non-neuronal cells became TT-positive during a 2-week culture period, indicating that this population of non-neuronal cells represents differentiating Muller cells . Again, comparable results were obtained with A2B5, supporting the conclusion that Mullerian glia expresses surface molecules, which are normally regarded as neuronal markers.

Hematol Pathol, 1988, 2(2), 73 - 8
Different expression of CD3 and CD22 in leukemic cells according to whether tested in suspension or fixed on slides; Rani S et al.; The expression of antigens detected by monoclonal antibodies of the CD22 and CD3 groups was studied, by immunofluorescence (IF) on cell suspension and by immunoperoxidase (IP) on cells fixed on cytospin slides, in a range of B- and T-cell malignancies . CD22 was demonstrated in the B-cell leukemias more frequently by IP than by IF; in B-lineage acute lymphoblastic leukemia (ALL) it was positive only by IP . In chronic lymphocytic leukemia and lymphoplasmacytic tumors approximately 40% of cases were CD22 positive by IP but few by IF . In other disorders of mature B cells, CD22 was positive by both methods in most cases . CD3 was demonstrated by IP in all 48 cases of T-cell leukemia and lymphoma regardless of the maturation stage (i.e., from early thymic to mature T cells) . This was not the case by IF, where only 16% of T-ALL and 73% of cases with postthymic T-cell leukemias were recorded as positive . The greater reactivity of CD3 and CD22 by IP reflect the early cytoplasmic expression of the antigens in immature T and B cells, respectively, as well as the possible greater sensitivity of the IP method . These differences should be taken into account when describing results in lymphoid malignancies . CD3 was not expressed in cells from any of the B-cell leukemias, and likewise, CD22 was not demonstrated in any of the T-cell disorders, confirming that both reagents are highly specific for their respective lineages.

J Leukoc Biol, 1988 Jan, 43(1), 80 - 90
Pulmonary interstitial macrophages: isolation and flow cytometric comparisons with alveolar macrophages and blood monocytes; Dethloff LA et al.; Pulmonary interstitial macrophages (IM) were isolated from rat lungs by an Fc gamma receptor-based affinity technique coupled with multiparameter flow cytometry . Single cell suspensions obtained by collagenase digestion of extensively perfused and lavaged lungs were applied to carpets of opsonized sheep red blood cells (SRBC-IgG) bound to plastic tissue culture flasks . At 0-4 degrees C, optimal binding of lung cells occurred within 60 min at plating densities of 1-2 X 10(6) lung cells/cm2 when the SRBC substrate was opsonized with 10 micrograms/ml anti-SRBC IgG . Nonadherent cells were removed by gently rinsing the plates and adherent cells were recovered by lysing the SRBC-IgG substrata . By light microscopy, the mixture of adherent cells was comprised of mononuclear cells (approximately 54%), many of which appeared to be macrophages, lymphocytes (approximately 20%), polymorphonuclear leukocytes (approximately 15%), plasma cells (approximately 8%), eosinophils (approximately 2%), and mast cells (approximately 0.5%) . The cells which adhered to the SRBC-IgG monolayers were further resolved into subpopulations by multiparameter flow cytometry and sorted according to their electro-optical characteristics . One subpopulation appeared morphologically to be macrophages, and greater than 90% of these cells readily phagocytized SRBC-IgG in vitro . Peroxidase staining of this population was minimal, indicating that these cells were not blood monocytes (BM) . Using a method by which alveolar macrophages (AM) were prelabeled with SRBC-IgG in situ, we demonstrated that alveolar macrophages constituted only approximately 5% of the total adherent cell population . We concluded from these observations that the macrophage population harvested in this manner were neither BM nor AM, but, rather, were harvested from the lung's interstitial compartment . Flow cytometric analyses indicated that the IM exhibited electro-optical characteristics intermediate between those of BM and AM, which is consistent with the concept of the lung's interstitium as a maturation compartment for the BM prior to migration into the alveolar compartment . However, the IM more closely resembled the BM than the AM, indicating that if the IM is in fact a precursor to the AM population, substantial maturation or differentiation must occur subsequent to its migration into the alveolar compartment . This isolation technique will be useful for harvesting highly purified IM for in vitro investigations.

Clin Exp Metastasis, 1988 Jan-Feb, 6(1), 17 - 25
Thymic factor-induced reduction of pulmonary metastases in mice with FSA-1 fibrosarcoma; Tomazic VJ et al.; The biological activities of two thymic factors, serum thymic factor thymulin normally present in serum and thymosin alpha-1 (Ta-1) extracted from the thymus gland, have been studied . The effects of the factors on the growth of pulmonary metastases and survival of mice were evaluated in pathogen-free C3H/fSed males . Mice were injected i.v . with the single cell suspension of the syngeneic methylcholanthrene-induced fibrosarcoma . The treatment with thymulin and Ta-1 started two days after injection of 5 x 10(4) to 2 x 10(5) tumor cells per mouse . Different doses of the thymic factors were administered S.C . in sets of 5 daily injections through a period of 2 or 3 weeks . Numbers of tumor colonies in the lung were determined two weeks after the cell injection . Treatment with 0.1 micrograms Ta-1 per injection through the period of two or three weeks, prolonged the survival of tumor-injected mice . Similar effects were observed in mice treated with 0.01 microgram thymulin per injection . Numbers of tumor colonies in lungs of these mice two weeks after the cell injection were also reduced in comparison with saline-treated controls . These findings correlated with prolonged survival time of identically treated mice . The effectiveness of thymic factors in reducing tumor growth was dependent on the tumour load . In addition, the effects induced by Ta-1 persisted longer than observed in thymulin-treated mice . Mice challenged 150 days after the primary tumor cell injection and treatment with Ta-1 demonstrated increased resistance to tumor, while mice treated with other factors behaved as saline-treated controls . The results indicate that both factors exert beneficial effects against tumor growth, although mode of action for each factor may be different.

Biol Cell, 1988, 64(1), 23 - 8
Cell cycle-related proteins: a flow cytofluorometric study in human tumors; Danova M et al.; We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67 . FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells . The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining . The investigated proteins have different relationships with the proliferative state of the cell . Trf-r is expressed mainly at the transition from G0/G1 to S-phase . PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M . The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases . Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation . Bivariate FCM is an easy method for obtaining these data from large numbers of cells.

J Ocul Pharmacol, 1988 Fall, 4(3), 203 - 14
Alpha 1-adrenoceptors in the albino rabbit ciliary process; Mallorga P et al.; 3H-Prazosin and 3H-rauwolscine binding sites were identified in a membrane suspension prepared from albino rabbit iris + ciliary body . Scatchard analysis of saturation binding experiments demonstrated that both 3H-prazosin and 3H-rauwolscine bind to a single population of binding sites with KD values of 0.87 nM and 5.33 nM, respectively . Bmax values of 65.7 and 198 fmol/mg protein were obtained for 3H-prazosin and 3H-rauwolscine, respectively . Displacement studies by several adrenergic agonists and antagonists indicated that 3H-prazosin and 3H-rauwolscine labelled alpha 1- and alpha 2-adrenoceptors, respectively, in the iris + ciliary body . Epinephrine, norepinephrine and phenylephrine were able to stimulate the synthesis of 3H-inositol phosphates in ciliary processes labelled with 3H-inositol, with EC50 values of 2.4, 12 and 10 microM, respectively . The corresponding maximum stimulations of basal activity were 433, 430 and 283%, respectively . Phenylephrine behaved like a partial agonist in this assay . The norepinephrine response could be potently antagonized by prazosin (Ki = 27 nM), with rauwolscine being 285-fold less potent . An epithelial cell suspension was prepared from the ciliary process . Stimulation of phosphatidylinositol turnover by norepinephrine (0.1 mM) was observed, and this could be blocked by prazosin (10 microM), thus, indicating the presence of alpha 1-adrenoceptors, coupled to phosphatidylinositol turnover, in epithelial cells of the rabbit ciliary process.

Acta Haematol, 1988, 80(2), 61 - 4
Early expression of MCS2 (CD13) in the cytoplasm of blast cells from acute myeloid leukaemia; Pombo de Oliveira MS et al.; The expression of two myeloid antigens identified by the monoclonal antibodies (McAb) MCS2 (CD13) and MY9 (CD33) was investigated in 136 cases of leukaemia . MCS2 was positive in blast cells of 78 of 88 (88.5%) and MY9 in 51 of 81 (64%) cases of acute myeloid leukaemia (AML) and chronic granulocytic leukaemia in myeloid blast crisis . One or other McAb, or both, were positive in all but 2 (2.3%) of these cases . MCS2 was more sensitive than MY9 to detect blasts of the myeloid lineage due to its most frequent reactivity in the cytoplasm of fixed cells by the immunoperoxidase (IP) technique compared with its membrane expression on cell suspensions by immunofluorescence (IF) . MY9 was not suitable for tests on fixed cells . MCS2 was positive by IP but not by IF in 24% of AML, but the reverse was not observed . This suggests that the antigen detected by MCS2 is expressed in myeloblasts first in the cytoplasm and later on the cell membrane, pattern which is similar to that of the early antigens CD3 and CD22 in T and B lineage lymphoblasts, respectively . MCS2 was always positive in FAB types of AML-involving myeloblasts (M1-M4), including cases of undifferentiated morphology (M0), whilst MY9 was more frequently positive in monocytic leukaemia (M5) . On the other hand, MCS2 was positive in 4 of 33 cases of acute lymphoblastic leukaemia and MY9 in 1 . We conclude that both McAb, particularly MCS2, contribute to the better characterisation of myeloid leukaemias but that other tests are required to clarify the nature of the blasts when unexpected reactivities are observed.

Comp Biochem Physiol A, 1988, 89(2), 113 - 7
Studies on the energy metabolism of opossum Didelphis virginiana erythrocytes--II . Comparative aspects of 2-deoxy-D-glucose catabolism in opossum and human red cells in vitro; Bethlenfalvay NC et al.; 1 . G-6-P, F-6-P, F-1, 6-P, DHAP and GA-3-P in opossum erythrocytes were found at levels above those reported in human red cells . 2 . About 1% of the radioactivity provided as {1-14C} DOG to red cells of both species was recovered as 14CO2 in 1 hr . 3 . Unlike {1-14C} DOG, radiochromatography of extracts of cells incubated DOG revealed two diffusible radiolabelled compounds in the supernatant of cell suspensions . 4 . The catabolism of DOG was quantitatively and qualitatively similar in opossum and human erythrocytes under the conditions of this study.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1988, 54(5), 273 - 7
DNA flow cytometry in metastases and a recurrency of malignant melanomas . A comparison of results from fresh and paraffin embedded material; Jacobsen AB et al.; Single cell suspensions from 16 biopsies from 15 patients with metastatic or recurrent malignant melanoma were prepared according to the method described by Vindelov (1977) and the nuclear DNA content was measured by a laboratory-built flow cytometer . The DNA histograms thus obtained were compared with those obtained from suspensions of single nuclei from the same biopsies after formalin fixation and paraffin embedding, according to the method of Hedley et al . (1983) . Linear regression analysis of ploidy values from fresh material compared with those from paraffin blocks showed a strong correlation (R2 = 0.85), while that of the S-phase fraction was somewhat weaker (R2 = 0.66) . It is concluded that archival wax preparations of malignant melanoma cell populations are suitable for FCM analysis of ploidy, and to a lesser extent for analysis of fraction of cells in various cell cycle phases.

J Pediatr Surg, 1988 Jan, 23(1 Pt 2), 3 - 9
Selective cell transplantation using bioabsorbable artificial polymers as matrices; Vacanti JP et al.; To date, selective cell transplantation has involved injecting cell suspensions into tissues or the vascular system . This study describes attaching cell preparations to bioerodable artificial polymers in cell culture and then implanting this polymer-cell scaffold into animals . Using standard techniques of cell harvest, single cells and clusters of fetal and adult rat and mouse hepatocytes, pancreatic islet cells, and small intestinal cells have been seeded onto biodegradable polymers of polyglactin 910, polyanhydrides, and polyorthoester . Sixty-five fetuses and 14 adult animals served as donors . One hundred fifteen polymer scaffolds were implanted into 70 recipient animals: 66 seeded with hepatocytes; 23 with intestinal cells and clusters; and 26 with pancreatic islet preparations . The cells remained viable in culture, and in the case of fetal intestine and fetal hepatocytes, appeared to proliferate while on the polymer . After four days in culture, the cell-polymer scaffolds were implanted into host animals, either in the omentum, the interscapular fat pad, or the mesentery . In three cases of fetal intestinal implantation coupled with partial hepatectomy, successful engraftment occurred in the omentum, one forming a visible 6.0 mm cyst . Three cases of hepatocyte implantation, one using adult cells and two using fetal cells, have also engrafted, showing viability of hepatocytes, mitotic figures, and vascularization of the cell mass . To date, no pancreatic islets have survived implantation . This method of cell transplantation, which we have termed "chimeric neomorphogenesis," is an alternative to current methods and requires further study.

Exp Hematol, 1988 Jan, 16(1), 21 - 6
Isolation of hemopoietic stem cell subsets from murine bone marrow: I . Radioprotective ability of purified cell suspensions differing in the proportion of day-7 and day-12 CFU-S; Ploemacher RE et al.; We have studied the ability of bone marrow cell suspensions greatly differing in the relative proportion of day-7 and day-12 spleen colony-forming units (CFU-S) to rescue mice from radiation-inflicted death, and to repopulate the irradiated bone marrow and spleen with nucleated cells . Counterflow centrifugal elutriation in combination with removal of adherent cells and fluorescence-activated cell sorting on differences in wheat germ agglutinin (WGA)-fluorescein isothiocyanate (FITC) affinity and light scatter properties were used consecutively to enrich large numbers of hemopoietic stem cells from mouse bone marrow . Enrichments of 50- to 200-fold have been achieved for day-12 CFU-S and radioprotective ability (RPA), permitting 50% of lethally irradiated mice to survive over a period of 30 days with as few as 50-80 donor cells . The ratio of day-7 and day-12 CFU-S in the various suspensions could be significantly modulated on the basis of their WGA binding and perpendicular light scatter characteristics . This finding enabled us to investigate the properties of day-7 and day-12 CFU-S with respect to their RPA . We found a highly significant log/log relationship between enrichment factors for (1) RPA, (2) the number of day-12 CFU-S, and (3) spleen cellularity as measured on day 13 . In addition, similar numbers of sorted and unfractionated day-12 CFU-S were required to obtain the same level of protection . Enrichment for RPA was significantly less related to either the number of day-7 CFU-S injected, or the bone marrow cellularity of the irradiated mice on day 13.

Toxicon, 1988, 26(12), 1187 - 91
Suppression of leukotriene synthesis in human leukocytes by a urea extract of Bordetella pertussis: evidence for mediation by adenylate cyclase toxin; Green FA et al.; Incubation of human leukocytes with a urea extract of Bordetella pertussis led to inhibition of zymosan-induced leukotriene generation . The proteins in this extract are known to include an adenylate cyclase which suppresses certain defense mechanisms of leukocytes against bacterial invasion . The formation of leukotriene B4 and leukotriene C4, induced by serum-coated zymosan, was almost completely inhibited in the presence of 75 micrograms of urea-extracted proteins/ml cell suspension . This suppression by the bacterial urea extract was rapid, with the maximum effect occurring in the first min of incubation . The reduction in leukotriene generation was accompanied by a dramatic increase in intracellular cyclic AMP levels . Since leukotrienes are potent pro-inflammatory compounds, the present study indicates that Bordetella pertussis-induced suppression of leukotriene formation might be an important factor in the increased susceptibility to secondary bacterial infection, which occurs as a result of this disease.

Psychoneuroendocrinology, 1988, 13(4), 317 - 23
Paradoxical effect of corticosteroids on pituitary ACTH/beta-endorphin release in stressed animals; Young EA et al.; We have previously demonstrated a number of changes in the anterior lobe proopiomelanocortin (POMC) system in chronically stressed rats . The purpose of the present experiments was to investigate whether chronically stressed rats demonstrate changes in pituitary sensitivity to glucocorticoid negative feedback . To study this question we compared the effects of glucocorticoids on ovine corticotropin releasing factor (oCRF)-stimulated ACTH and beta-endorphin release from cell suspensions prepared from naive unhandled rats versus chronically stressed rats . After dexamethasone, there was a 50% decrease in oCRF-stimulated hormone release in control rats but no inhibition of oCRF-stimulated hormone release in anterior lobe suspension from chronically stressed rats . Rather, the chronically stressed group exhibited a 50% hormone increase above the oCRF-stimulated baseline . The same pattern was observed after the addition of corticosterone to the medium . These findings suggest that there may be a positive feedback effect of glucocorticoids at the pituitary level under some conditions of chronic stress.

Bone, 1988, 9(1), 1 - 6
Isolation of osteoclasts from Pagetic bone tissue: morphometry and cytochemistry on isolated cells; Basle MF et al.; Giant osteoclasts and other cells were isolated from Pagetic bone tissue using 0.5 mM ethylene diamine tetraacetic acid on bone samples from 8 patients with Paget's disease . The cell suspension contained osteoclasts and osteoblasts as well as some mononuclear cells such as monocytes . The number of nuclei in isolated osteoclasts (33.85 +/- 20.92 nuclei/osteoclast) correlates fairly well (p less than 0.02) with the number of nuclei counted on histologic sections (15.88 +/- 11.80 nuclei/osteoclast) for samples from each patient . Enzyme histochemistry demonstrated acid phosphatase activity in isolated osteoclasts and in mononucleated cells, such as monocytes . Alkaline phosphatase was detected only in osteoblasts while succinate dehydrogenase was observed in osteoclasts, osteoblasts and monocytes . Esterases, such as nonspecific aliesterase and specific naphthol AS-D acetate esterase, were identified in osteoclasts and in macrophages . Inhibition of specific naphthol AS-D acetate esterase in osteoclasts by addition of sodium fluoride suggests that the enzyme could be of monocytic origin.

Gut, 1988 Jan, 29(1), 94 - 100
Gastric cell c-AMP stimulating autoantibodies in duodenal ulcer disease; De Lazzari F et al.; Gastric cell c-AMP stimulating antibodies (GCS-Ab) were studied in 30 patients with duodenal ulcer (DU) disease . Semipurified immunoglobulin (Ig) preparations from 13/30 patients stimulated c-AMP production in parietal cell enriched gastric cell suspensions obtained from male guinea pig stomachs . Maximum stimulation (varying between 260 and 547%) was reached after four hours incubation with 2 and 4 mg/ml Ig concentrations . The 13 patients with gastric cell stimulating antibodies (GCS-Ab), all male patients, developed the disease at a younger age (nine of 13 under the age of 30), had a longer duration of symptoms (mean 18.4 years), and had a higher incidence of DU in their families (61%) . Eight of 13 (61%) in the GCS-Ab+ group did not respond to anti-H2-R drugs, whereas in the negative patients only three of 17 (18%) were classified as 'non-responders' . Remarkably few conventional autoantibodies were detected in our series . Gastric cell stimulating antibodies are a new addition to the growing list of receptor antibodies in human diseases and the described in vitro test should provide an easier tool for screening large populations.

Exp Pathol, 1988, 34(2), 115 - 8
The effect of paraquat lung on mononuclear cells; Barabas K et al.; The mouse footpad swelling test was used to clarify the possibility of the induction of local cellular reaction with cell suspension of mouse lungs treated with paraquat in a syngeneous animal . Among the inbred strains used, the highest, statistically significant cellular reactivity was observed in C3H/He strain mice . These results suggest indirect evidence of macrophage activation in the lung toxicity of paraquat.

Ann Dermatol Venereol, 1988, 115(1), 11 - 8
{Use of the immunoblotting technic for the study of antibodies present in the serum of patients with bullous pemphigoid}; Reano A et al.; Bullous pemphigoid (BP) is an autoimmune disease characterized, at histology, by subepidermal bullous separations . Antibodies (Ab) and complement are present at the dermal-epidermal junction, and most patient with the disease have serum antibodies directed against a normal constituent of stratified epithelia (10, 25) . During the past few years, the immunotransfer technique has been used to study autoimmune bullous diseases of the skin . The antigen (Ag) of bullous pemphigoid has been described by Stanley et al . (16, 17) as the polypeptidic doublet 220-240 KD . However, different results in favour of an heterogeneous antigen have been obtained by Labib et al . (11) who reported the presence of 5 separate polypeptides: 240, 200, 180, 97 and 77 KD . The purpose of the present study was to confirm on infirm these findings . We recorded the antigens recognized at immunotransfer by the circulating antibodies of BP patients, then extended this study to sera from BP patients in whom no antibody had been detected by indirect immunofluorescence (IIF) . We report the results of our immunotransfer study in 9 seropositive and 9 seronegative BP patients and in 11 seronegative controls . The antigenic extract we used was prepared from suspensions of epidermal cells and represented the intracellular BP antigen . Epidermal cell suspensions were obtained by trypsinization of human abdominal skin . The cells were homogenized in a Tris-HCl 10 mM pH 7.8 buffer with SDS 2 p . 100 and B-mercaptoethanol 5 p . 100 . The soluble proteins were analyzed in a polyacrylamide gel in the presence of SDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Res, 1987 Dec 29, 437(2), 298 - 308
Histiotypic organization and cell differentiation in rat retinal reaggregate cultures; Akagawa K et al.; Reaggregate cultures have been formed from cell suspensions of neonatal rat retinas . Histological sections of the reaggregates showed evidence of lamination with central rosettes formed around a lumen, a clear neuropil layer and an outer cellular layer . Each of the major retinal cell types, except ganglion cells, could be positively identified using cell type-specific antibodies to label cryostat sections . Many of these were found to occupy positions within the reaggregates similar to those found in the intact retina . Electron microscopic observations showed abundant immature and mature synaptic endings within the neuropil layer, including a number of ribbon synapses . Examination of the rosettes showed an arrangement of Muller glia and photoreceptors that closely resembled that of the intact retina . Within the lumen of rosettes, photoreceptors were found to contain stacks of disc-like membranes bounded by a plasma membrane, analogous to immature outer segments . The photoreceptors within rosettes also underwent molecular differentiation and expressed an outer segment specific marker . The findings suggest that retinal cells have intrinsic properties that allow them to organize themselves into a correctly laminated structure and that particular cell interactions are necessary for continued differentiation of at least rod photoreceptors and Muller cells.

J Immunol Methods, 1987 Dec 24, 105(2), 165 - 9
Removal of Langerhans cells from human epidermal cell suspensions by immunomagnetic particles; Nilsson H et al.; The possibility of eliminating the class II antigen-expressing and antigen-presenting Langerhans cells from normal human epidermal cell suspensions was investigated . Cell suspensions containing 1.7-2.8% Langerhans cells were prepared by enzyme treatment of human skin obtained at plastic surgery . The cells were incubated with mouse monoclonal antibodies directed against the CD1 antigen present on Langerhans cells and were further incubated with magnetic particles, 4.5 micron in diameter, coated with sheep anti-mouse IgG . The optimal ratio between particles and target cells was found to be 40:1 . The rosetted Langerhans cells were removed by a cobalt-samarium magnet . In six experiments immunoperoxidase staining revealed 0-0.1% (mean 0.03%) CD1-reactive Langerhans cells in the depleted cell fraction . The alloantigen presenting capacity of the depleted cell fraction, measured by {3H}thymidine incorporation, was abolished.

Cancer Res, 1987 Dec 15, 47(24 Pt 1), 6528 - 31
Antibody-guided localization of intraperitoneal tumors following intraperitoneal or intravenous antibody administration; Rowlinson G et al.; Intraperitoneal tumors from a human cancer cell line (LoVo) were established in nude mice by i.p . inoculation of a single cell suspension . Two preparations of the same monoclonal antibody, radiolabeled with 125I and 131I were injected i.p . and i.v . into the same animals . Localization was assessed by dissection and counting the activity in tumors and normal tissues . Tumor/tissue ratios 1 h after i.p . injection of antibody were approximately 50 times higher than after i.v . administration . This i.p./i.v . advantage fell to around 4 by 8 h and was just greater than 1 by 24 h . This effect was observed with both specific and nonspecific antibody, indicating that it is due to the route of administration . However, the absolute amounts of antibody bound to tumors depended on the specificity of the antibody . Twenty % of the injected dose of specific antibody was bound per gram to tumor 1 to 2 h after i.p . injection, falling to 10%/g by 24 h and remaining at this level up to 5 days after antibody administration . In contrast, less than 10%/g of nonspecific antibody was detected in tumors after 1 h; this fell rapidly to normal organ levels of less than 5%/g by 8 h . This study demonstrates a major advantage when administering radiolabeled monoclonal antibodies i.p . for targeting intraperitoneal tumors.

Cancer, 1987 Dec 15, 60(12), 2965 - 70
The effect of tumor necrosis factor on normal human hematopoietic progenitors; Abboud SL et al.; Tumor necrosis factor-alpha (TNF-alpha), a product of activated macrophages that is cytotoxic to tumor cells, could be used to purge tumor cells from bone marrow before autologous bone marrow transplantation for hematologic malignancies and/or solid tumors . To determine whether exposure to TNF-alpha would have an inhibitory effect on hematopoietic progenitors, we incubated normal human bone marrow with a wide range of concentrations of recombinant human TNF . In order to mimic the conditions that would be used in bone marrow purging, bone marrow cell suspensions were incubated with TNF in doses ranging from 500 to 100,000 U/ml for 24 hours, and were assayed for colony formation in agar . We noted a dose-dependent inhibition of total colony-forming units (CFU) at days 7 and 14, with 50% inhibition occurring at 60,000 U/ml of TNF . TNF exerted a differential effect on CFU so that colony formation by erythroid (CFU-E), multipotential (CFU-GEMM), and macrophage (CFU-M) progenitors was suppressed to a greater extent than that by granulocyte progenitors (CFU-G) . However, even after preincubation with TNF at high doses such as 100,000 U/ml, the inhibitory effects of TNF could be abolished by washing cells before culturing . This study demonstrates that hematopoietic precursors survive treatment with TNF at doses that have been shown to be cytotoxic to tumor cells . Although TNF has a significant inhibitory effect on the growth of erythroid, multipotential, and macrophage progenitors in vitro, this effect depends on continuous exposure to TNF for more than 24 hours . Thus, TNF may be useful as a bone marrow purging agent against tumor cells, with relative sparing of normal marrow elements.

Biochim Biophys Acta, 1987 Dec 11, 905(2), 231 - 9
Properties of a Na+-coupled serine-threonine transport system in Escherichia coli; Hama H et al.; Based on the following experimental results we conclude that the serine-threonine transport system in Escherichia coli is a Na+-coupled cotransport system . (1) Addition of serine to cell suspensions induced H+ efflux in the presence of Na+ . (2) Addition of serine to cell suspensions induced Na+ uptake by cells . (3) Imposition of an artificial electrochemical potential of Na+ in starved cells induced serine uptake . Some of these phenomena were observed when threonine was added instead of serine or inhibited when cells were preincubated with threonine . The Na+/serine (threonine) cotransport system was considerably repressed when cells were grown on a mixture of amino acids . Serine transport in cells grown in the absence of amino acids mixture was stimulated by Na+ . The half maximum concentration of Na+ was 21 microM . Sodium ion increased the Vmax of serine transport without affecting the Km.

Virology, 1987 Dec, 161(2), 527 - 32
Characterization of Tm-1 gene action on replication of common isolates and a resistance-breaking isolate of TMV; Watanabe Y et al.; Tm-1 is a gene which confers resistance to infection, in tomatoes, by tobacco mosaic virus (TMV) . To investigate the biochemical mechanism of the resistance, we have established cell suspensions of three lines of tomatoes, i.e., +/+ (susceptible, wild-type, no Tm-1 gene), Tm-1/+ (heterozygous for the Tm-1 gene), and Tm-1/Tm-1 (homozygous for the Tm-1 gene) . Protoplasts isolated from these cells were inoculated with RNA of the tomato strain L and Lta1 (a resistance-breaking strain which was recently isolated spontaneously from L) of TMV by means of electrophoration . The syntheses of all viral-coded proteins and TMV-specific RNAs could be detected in L-inoculated +/+ and Lta1-inoculated +/+, Tm-1/+, Tm-1/Tm-1 protoplasts, while their production was markedly reduced in L-inoculated Tm-1/+ protoplasts . L strain could multiply in Tm-1/+ protoplasts to a greater extent with less delay when a large amount of inoculum RNA was used . However, viral production was completely blocked in Tm-1/Tm-1 protoplasts even when a large amount of L-RNA was used for inoculation.

Surgery, 1987 Dec, 102(6), 932 - 40
The effect of phorbol ester on in vitro release of parathyroid hormone from abnormal human parathyroid cells; Saxe AW; Intracellular events that regulate parathyroid hormone (PTH) release are not well understood . Cyclic AMP (cAMP) and cAMP-dependent protein kinases play a role in the regulation of release due to several agonists, but these factors do not fully explain PTH release that is mediated by extracellular ionized calcium (Ca++) . A calcium-phospholipid-dependent (non-cAMP-dependent) protein kinase can be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) . To determine whether this protein kinase regulates PTH release, we examined the effect of TPA on PTH release from human parathyroid tissue . Cell suspensions of abnormal parathyroid tissue removed at surgery were prepared by enzymatic dispersion and incubated for several hours with and without 10(-7) mol/L TPA at low and high calcium levels . In ten preparations in the absence of TPA, increasing Ca++ from 0.25 to 2.5 mmol/L reduced PTH release to an average of 39% of maximal release (range, 11% to 67%) . The effect on TPA on Ca++-regulated PTH release appeared biphasic . At low (0.25 mmol/L) Ca++ level, TPA suppressed PTH release to an average of 78% of maximal release without TPA (95% confidence interval, 67% to 88%) (p less than 0.01 compared to cells incubated without TPA) . At high (2.25 mmol/L) Ca++ level, TPA augmented PTH release from an average of 39% of maximal release without TPA to 62% of maximal release without TPA (95% confidence level, 48% to 78%), an average augmentation of 22% (95% confidence level, 9% to 36%) (p less than 0.01 compared with cells incubated without TPA) . TPA appeared to make PTH release independent of Ca++ . Both inhibitory and stimulatory effects were dose dependent . Incubations with TPA demonstrated no toxicity as judged by trypan blue dye exclusion, linearity of PTH release, and cellular incorporation of tritiated leucine . TPA had no effect on the radioimmunoassay for PTH . We conclude that a calcium/phospholipid-dependent, non-cAMP-dependent protein kinase may play a role in mediating Ca++-regulated PTH release from abnormal human parathyroid cells . Its site of action and integration with other regulatory pathways remain to be determined.

J Clin Invest, 1987 Dec, 80(6), 1698 - 705
Interaction of murine granulocyte-macrophage progenitors and supporting stroma involves a recognition mechanism with galactosyl and mannosyl specificities; Aizawa S et al.; To study the molecular basis of "homing" of granulocyte-macrophage progenitors (CFU-C), we synthesized probes by covalent linking of sugars to bovine serum albumin . Long-term marrow cultures were established in the presence and absence of these probes . In the presence of galactosyl and mannosyl probes, total cell and CFU-C production in the supernate as well as the adherent layer were halted, and cobblestones (representing CFU-C bound to stroma) disappeared . Fucosyl probe and diffusible sugars had no effect on these parameters . These studies suggested membrane lectins with specificity for galactosyl and mannosyl residues may be responsible for the binding of CFU-C to supporting stroma . To determine if CFU-C possesses homing receptors with these specificities, we induced agglutination in marrow cell suspensions with these neoglycoprotein probes . Selective agglutination was observed only by galactosyl and mannosyl probes . The results suggest that CFU-C homing receptors are membrane lectins with specificity for galactosyl and mannosyl residues.

Clin Immunol Immunopathol, 1987 Dec, 45(3), 491 - 8
Peripheral and intestinal lymphocyte activation after in vitro exposure to cow's milk antigens in normal subjects and in patients with Crohn's disease; Biancone L et al.; We studied in Crohn's disease and controls the in vitro activation of either peripheral (PBMNC) or intestinal (LPMNC) mononuclear cells in response to the cow's milk antigen beta-lactoglobulin (Blg) . The activation of mononuclear cells was investigated by analyzing the kinetics of the transferrin receptor (T9 antigen) and interleukin 2 receptor (Tac antigen) expression . In both controls and Crohn's disease patients Blg (1 microgram/ml) induced a significant (P less than 0.01) increase of T9 and Tac expression by both PBMNC and LPMNC cultured for 3 days . After Blg exposure the counts of T9- and Tac-bearing cells were significantly (P less than 0.05) higher in PBMNC cultures from healthy controls than in those from patients with Crohn's disease . In both groups peripheral T-enriched cell suspensions did not express T9 and Tac when stimulated with Blg but were restored to express either antigen by the addition of 10% autologous adherent cells . In LPMNC cultures from patients with Crohn's disease the Tac expression after 3 days of Blg stimulation was significantly (P less than 0.05) higher than in the autologous PBMNC . Data from this study indicate that in both controls and patients with Crohn's disease lymphocytes sensitized to Blg occur in the circulation as well as in the gut lamina propria . Our data also suggest that in Crohn's disease an increased proportion of lymphocytes sensitized to Blg are recruited from the circulation into the site of the intestinal lesion.

Mol Pharmacol, 1987 Dec, 32(6), 807 - 12
Hydroquinone inhibits bone marrow pre-B cell maturation in vitro; King AG et al.; Environmental exposure to benzene results in both myelotoxicity and immunotoxicity . Although benzene-induced immunotoxicity has been well documented, no studies to date have addressed the possibility that benzene toxicity is due in part to altered differentiation of marrow lymphoid cells . We investigated the effect of acute exposure to the benzene metabolite, hydroquinone, on murine bone marrow B-lymphopoiesis . Bone marrow cell suspensions from B6C3F1 (C57BL/6J x C3H/HeJ) mice were depleted of mature surface IgM+ (sIgM+) B cells and cultured for 0, 24, 48, or 72 hr and production of newly formed B cells was assayed both by sIgM expression and colony formation in soft agar cultures . One hr exposure of bone marrow cells to hydroquinone before culture reduced the number of sIgM+ cells generated in liquid cultures . Small pre-B cells (cytoplasmic mu heavy chain+, sIgM-) were numerically elevated as compared with control cultures . Hydroquinone exposure also decreased the number of adherent cells found in cultures of bone marrow cells . These results suggest that short-term exposure to hydroquinone, an oxidative metabolite of benzene, may in some way block the final maturation stages of B cell differentiation . This apparent differentiation block resulted in reduced numbers of B cells generated in culture and a corresponding accumulation of pre-B cells . Reduction of adherent cells in treated cultures may also suggest that toxicity to regulatory cells for the B lineage may be in part responsible for this aspect of hydroquinone myelotoxicity.

Am J Clin Pathol, 1987 Dec, 88(6), 707 - 12
Gastrointestinal lymphomas . Immunohistologic study of 23 cases; Berger F et al.; Twenty-three primary gastrointestinal lymphomas were studied morphologically and immunologically on fresh frozen tissue, and on cell suspension for 16 of them . Polyclonal antibodies reactive with immunoglobulin chains and a panel of 16 monoclonal antibodies reactive with B- and T-cells, histiocytes, and epithelial cells were used . According to the Working Formulation, 5 cases were low grade, 12 intermediate grade, and 5 high grade; 1 case was an extramedullary plasmocytoma . Forty-seven percent were large cell lymphomas and 13% follicular lymphomas . There were 20 (86%) B-cell lymphomas and 2 T-cell lymphomas; one case lacked detectable markers for B-, T-, or histiocytic cells . Monoclonality was demonstrated in 13 out of the 20 B-cell lymphomas, whereas the other 7 expressed pan-B antigens . It is concluded that immunologic studies on frozen surgical material are of precise diagnostic value in gastrointestinal lymphomas, whereas fixed endoscopic biopsies only permit the distinction between lymphomas and undifferentiated carcinomas.

Exp Clin Endocrinol, 1987 Dec, 90(3), 331 - 6
The effect of oxotremorine on in-vitro secretion of parathyroid hormone; Wagner PK et al.; The effect of the muscarine-like acting parasympathomimetic drug oxotremorine and its antagonist atropine on PTH release was tested using single cell suspensions obtained from a) primary parathyroid adenomas, and b) secondary hyperplastic parathyroid tissue from patients undergoing chronic hemodialysis . For both cell-types we found a dose-dependent suppression of PTH release with oxotremorine . The suppressive activity could be antagonized with atropine, suggesting that parasympathomimetic nerval impulses play a suppressive role in the regulation of PTH release . Muscarine-receptors appear to be responsible for this mechanism.

Cell Calcium, 1987 Dec, 8(6), 413 - 27
Nifedipine is an antagonist to cyclotron resonance enhancement of 45Ca incorporation in human lymphocytes; Rozek RJ et al.; The incorporation of 45Ca in mixed human lymphocytes was measured following one-hour exposures of the cells to combined steady and periodic magnetic fields designed to probe for cyclotron resonance response in calcium incorporation . Measurements were made as a function of magnetic field frequency, up to 30 Hz, and as a function of magnetic field amplitude, up to 1.5 x 10(-4) Trms . The amplitude measurements demonstrated that the relative 45Ca uptake at resonance follows different mechanisms of interaction above and below 0.2 x 10(-4) Trms . After adjusting the magnetic field configuration for maximum incorporation, we then determined the effects of the calcium influx blocker nifedipine on 45Ca incorporation, with and without simultaneous exposure to this specific magnetic field combination . The presence of nifedipine in both unexposed and exposed cell suspensions resulted in decreased 45Ca uptake, presumably through the slow inward calcium channels . Evidence was found suggesting that nifedipine acts antagonistically to the 45Ca cyclotron resonance tuning signal.

Scand J Gastroenterol, 1987 Dec, 22(10), 1270 - 6
Flow cytometric DNA studies in human gastric cancer and polyps; Odegaard S et al.; Flow cytometric DNA studies were performed on cell suspensions of biopsy specimens from gastric tumors and neutral gastric mucosa in 18 patients with gastric cancer and 9 patients with gastric polyps . In cancer, aneuploidy was found in two tumors in the antral and five in the body part of the stomach (39%) . The mean DNA index for aneuploid cancers was 1.57 . In patients with aneuploid carcinomas three biopsy specimens from uninvolved mucosa also showed aneuploidy . In diploid carcinomas in the antral part of the stomach, the proliferative index (PI) was increased when compared with that of antral mucosa in controls (p less than 0.05) . Increased PI was found in uninvolved mucosa in aneuploid carcinomas of the body part of the stomach when compared with that in diploid carcinomas (p less than 0.001) . In uninvolved body mucosa in aneuploid carcinomas of the body part significantly reduced levels of acid-beta-glucuronidase (p less than 0.0001) were found when compared with diploid carcinomas . No polyps showed aneuploidy, and the PI in biopsy specimens from patients with gastric polyps did not differ from that in controls.

J Pediatr Surg, 1987 Dec, 22(12), 1212 - 5
Reversal of ischemic liver failure by intrasplenic liver cell transplantation; Flye MW et al.; A model of ischemic hepatic failure in Sprague-Dawley rats (SD) with 100% mortality has been developed by one-hour occlusion of the right portal vein and hepatic artery followed by left 70% hepatectomy . The intrasplenic injection of 40 x 10(6) syngeneic adult or three-day neonatal single liver cell suspensions decreased the mortality from 100% to 50% and 36%, respectively . Mortality decreased with increasing time from the intrasplenic injection of neonatal liver cells to the time of acute hepatic ischemia . Mortality also decreased with increasing interval between hepatic ischemia and removal of the transplanted liver cells by splenectomy . Intrasplenic injection of graded doses of neonatal liver cells decreased mortality from 75% at a dose of 10 x 10(6) cells, to 36% at 160 x 10(6) cells . Treatment of neonatal liver cells with metabolic inhibitors did not significantly affect their ability to reverse acute hepatic ischemia.

Am J Pathol, 1987 Dec, 129(3), 441 - 7
Analysis of prolactin and growth hormone production in the MtT/F4 transplantable pituitary tumor by the reverse hemolytic plaque assay; Lloyd RV; The reverse hemolytic plaque assay (RHPA) was used to detect hormone secretion from normal pituitary cells and from the transplantable MtT/F4 pituitary tumor cells . Aliquots of the same cell suspensions were analyzed by immunocytochemistry (ICC) . Normal pituitaries had more growth hormone (GH)-producing cells than tumors when analyzed by both the RHPA and ICC . However, the MtT/F4 tumor had significantly more prolactin (PRL)-secreting cells . Mammosomatotropic (MS) cells, which produced both PRL and GH, were identified in both normal and tumorous pituitaries with the RHPA and ICC . A combined procedure of RHPA followed by ICC staining on the same slide also revealed MS cells in both normal and tumorous pituitary cells, although the percentage of MS with this technique was less than with the other two methods . These results show that MS cells from a significant population of cells in the MtT/F4 tumor and that the RHPA and ICC can be used to study the regulation of this cell type.

Pathol Biol (Paris), 1987 Dec, 35(10), 1285 - 91
{Anti-CD3 monoclonal antibodies . Characterization and function}; Bourel D et al.; Four anti-T lymphocyte monoclonal antibodies were produced by the hybridoma technique . Percentages of miscellaneous labelled cell suspensions, as profiles of fluorescent histograms, showed a reactivity of these 4 reagents quite comparable to that observed with anti-CD3 monoclonal antibodies . Labelling summation studies, cell-sorting studies and blocking experiments gave similar results as gave other anti-CD3 reagents . Biochemical study and functional tests: modulation effects and mitogenic properties, argued for the belonging of these 4 antibodies to the CD3 cluster . The actual data concerning the CD3 molecule are briefly resumed to underline the interest of such antibodies in the understanding of T-lymphocyte activation mechanisms during the immune response.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8712 - 5
Grafted noradrenergic neurons suppress seizure development in kindling-induced epilepsy; Barry DI et al.; Norepinephrine-rich cell suspensions, prepared from the locus coeruleus region of rat fetuses, were grafted bilaterally into the hippocampus of rats made hypersensitive to hippocampal kindling by a neurotoxic lesion of the central catecholamine system . The animals with grafts showed a marked suppression of the onset and progression of kindling-induced epilepsy, and this effect was correlated with the degree of graft-derived noradrenergic innervation of the host hippocampal formation . We conclude that grafted neurons can modulate the excitability of epileptic brain regions.

Blood, 1987 Dec, 70(6), 1784 - 9
Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo; Niskanen E et al.; In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice . This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1) . After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents . Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells . Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells . BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF . The addition of BCL1 CM to multi-CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)-containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity . On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo . Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments . It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile . These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.

Immunol Cell Biol, 1987 Dec, 65 ( Pt 6), 517 - 27
Further studies on the heterogeneity of antigens recognised by CD-1 monoclonal antibodies: distribution of epitopes and analysis of serological binding patterns; Favaloro EJ et al.; The binding patterns of 28 monoclonal antibodies (MAB) recognizing antigens belonging to Cluster of Differentiation One (CD-1) were analyzed in order to investigate heterogeneity within this cluster . Competitive binding assays using radiolabelled MAB provided detailed information on CD-1 antigenic heterogeneity, and demonstrated that at lease six epitopic regions are recognised as CD-1 MAB . Further studies, based on single cell suspension immunofluorescence assays (using thymocytes and subclones on the cell line Molt-4), suggested that the majority of MAB studied could be serologically separated into three groups . In view of the most recent information that CD-1 MAB recognize at least three different early T-cell differentiation molecules, our results indicate that there are three or more distinct epitopes on the 'gp49'(HTA-1/CD-1a) molecule, two on the 'gp45'(HTA-3/CD-1b) molecule and one on 'gp43'(HTA-2/CD-1c).

J Leukoc Biol, 1987 Dec, 42(6), 689 - 96
New method for the measurement of eosinophil migration; Hakansson L et al.; A new application of the Boyden chamber method for the measurement of eosinophil migration, without the need of eosinophil isolation, has been developed . A cell suspension containing a mixture of granulocytes, neutrophils to the greater part, was used . The eosinophils were identified by staining their granules with Chromotrope 2R . The method made it possible to study the migration of eosinophils from normal individuals without eosinophilia . Control experiments demonstrated that the chemotactic and chemokinetic response of eosinophils in the granulocyte mixture was in accordance with the response of isolated eosinophils from the same donor . Normal eosinophils demonstrated a significant chemotactic response to C5f, platelet-activating factor (PAF), leukotriene B4 (LTB4), and f-meth-leu-phe (f-MLP) . Furthermore, PAF was demonstrated to be significantly more eosinophil chemotactic than neutrophil chemotactic.

J Chromatogr, 1987 Nov 27, 422, 175 - 85
Determination of amsacrine in human nucleated hematopoietic cells; Brons PP et al.; A new method has been developed for the determination of amsacrine (AMSA) in human nucleated hematopoietic cells . In order to prevent efflux during the cell separation procedure, white blood cells (WBCs) were separated from red blood cells by dextran sedimentation, leaving the WBCs in their natural environment . After cell counting, pelletting the cell suspension and correcting for the admixture of supernatant, AMSA was extracted from the WBCs and determined by high-performance liquid chromatography . Linearity of extraction was observed up to 40.10(6) cells . The inter-assay variation was 4.7% . Plasma and cellular concentrations were measured in five patients at the end of a 3-h infusion of 100 mg/m2 AMSA . A pharmacokinetic study of plasma and cellular AMSA concentrations up to 19 h after infusion was carried out . AMSA concentrations in WBCs correlated well with the plasma levels (n = 20, r = 0.967) with an accumulation factor compared to the plasma concentration of 2.6-9.8 in the patients studied . The method described is useful for studying cellular pharmacokinetics of AMSA in man.

J Immunol Methods, 1987 Nov 23, 104(1-2), 231 - 6
Extraction of leucocytes from human decidua . A comparison of dispersal techniques; Ritson A et al.; Functional studies of human utero-placental tissues have been limited by poor characterisation of the morphology and antigenic phenotype of the cells under investigation . The present study documents the effect of dispersal methods on the viability and cellular composition of cell suspensions prepared from decidualised endometrium in early human pregnancy . First trimester decidua was subjected to both mechanical disaggregation and digestion with various enzyme combinations in an attempt to optimise the yield of infiltrating decidual leucocytes . Cell types were characterised with monoclonal antibodies using an alkaline phosphatase immunolabelling method . Mechanical disaggregation resulted in suspensions containing many large decidual cells and much cell debris but few leucocytic cells . Overall viability was low, although the viability of small leucocytes common antigen-bearing cells remained high . Enzymic digestion yielded cell suspensions rich in leucocytes with high viability and minimal contamination by other cell types . Collagenase produced a high yield of leucocytes with high viability and minimal disruption of surface antigens in contrast with pronase which caused extensive antigenic loss . The disaggregation method determines the yield of bone marrow derived cells in decidual cell suspensions and the extent of contamination by true decidual cells and epithelial cells.

J Immunol Methods, 1987 Nov 23, 104(1-2), 81 - 6
The use of rat mixed-thymocyte culture-conditioned medium for hybridoma production, cloning and revival; Micklem LR et al.; Allogeneic rat mixed-thymocyte 48 h culture-conditioned medium (MTM) was used successfully in place of feeder cells for hybridoma production with the NS-1 and NS-0 plasmacytoma lines . It permitted lower concentrations of fused cells to be seeded, and supported the transition from 96 to 24 well plates . MTM improved the performance of poor sera during cloning . It also assisted the survival of cells that were sensitive to thawing from liquid nitrogen storage, and cells that had inadvertently been allowed to overgrow . Two rats could produce the equivalent of 1500-5000 ml feeder cell suspension according to the dilution used; 150-500 mice would be required to produce such a quantity of cells . Thus use of MTM entailed a considerable saving in mice and provided a secure supply of 'reagent', since a batch could be prepared, checked for sterility, frozen and stored indefinitely.

Biochim Biophys Acta, 1987 Nov 21, 922(2), 214 - 20
Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey neutrophils; Chabot MC et al.; Platelet activating factor (PAF) is rapidly metabolized via a deacetylation: reacylation pathway which shows striking specificity for arachidonate at the sn-2 position of the 1-O-alkyl-2-acyl-GPC thus formed . We have now examined the effects of a diet enriched in fish oils on the metabolism of PAF and specificity for arachidonate in the reacylation reaction . {3H}PAF was incubated for various lengths of time with neutrophils from monkeys fed a control diet or one enriched in fish oils . The {3H}PAF added to the cell suspension was rapidly converted to 1-O-alkyl-2-acyl-GPC . Reverse-phase HPLC analysis of the acyl chains added at the sn-2 position revealed that arachidonate was the major fatty acid incorporated into the 1-O-alkyl-2-acyl-GPC formed by neutrophils from monkeys on the control diet . In contrast, both 1-O-alkyl-2-arachidonoyl-GPC and 1-O-alkyl-2-eicosapentaenoyl-GPC were formed by the fish-oil-enriched neutrophils . We also report on the fatty acid composition of neutrophil phospholipids during such a diet.

Biochemistry, 1987 Nov 17, 26(23), 7363 - 71
Aspergillus niger sulfhydryl oxidase; de la Motte RS et al.; A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000 . Sedimentation equilibrium experiments indicated a native molecular weight of 106,000 . Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight . This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2 . The ratio of GSH consumed to H2O2 produced was determined to be 2:1 . At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5 . Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH . Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH . The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups . The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1987 Nov 16, 169(1), 73 - 7
Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis; Grand C et al.; A fivefold increase in the extractable activity of cinnamyl-alcohol dehydrogenase, an enzyme of phenylpropanoid metabolism specific for lignin synthesis, was observed within 10 h of treatment of cell-suspension cultures of bean (Phaseolus vulgaris L.) with a high-molecular-mass elicitor preparation heat-released from mycelial cell walls of the bean pathogen Colletotrichum lindemuthianum . Elicitor caused a rapid, marked but transient increase in the synthesis of cinnamyl-alcohol dehydrogenase with maximum rates 2-3 h after elicitation, concomitant with the phase of rapid increase in enzyme activity . There is a close correspondence between increased polysomal mRNA activity encoding cinnamyl-alcohol dehydrogenase, as measured by incorporation of {35S}methionine into immunoprecipitable enzyme subunits in vitro, and the stimulation of enzyme synthesis in vivo in response to elicitor . This marked increase in polysomal mRNA activity represents an increase as a proportion of total cellular mRNA activity, indicating that elicitor does not stimulate synthesis of this enzyme by selective recruitment from the total pool of cellular mRNA . Elicitor stimulation of cinnamyl-alcohol dehydrogenase activity and enzyme synthesis is more rapid than previously observed for other proteins involved inducible defense mechanisms, such as enzymes of phytoalexin biosynthesis or the apoproteins of cell-wall hydroxyproline-rich glycoproteins.

Biochem Pharmacol, 1987 Nov 15, 36(22), 3911 - 6
Drug-metabolizing enzymes in rat, mouse, pig and human macrophages and the effect of phagocytic activation; Peterson TC; Aryl hydrocarbon hydroxylase (AHH) activity mediated by cytochrome P-450 is present in pig hepatic microsomes {10 nmol.3 mg protein-1.hr-1} . AHH activity was detectable in both hepatocytes and Kupffer cells isolated from pig liver biopsy material . These cells were isolated from needle or wedge biopsy material by collagenase perfusion and incubation with collagenase at 37 degrees . The two cell types were separated from the resulting cell suspension as previously described for whole liver . Kupffer cells were enriched by adherence and were cultured for 24 hr prior to harvesting . Cells were harvested, and cell viability was determined . AHH activity was assayed in Kupffer cell and hepatocyte homogenates . Kupffer cell AHH activity was approximately one-eighth the level detected in hepatocytes . To determine whether this enzyme was present in other macrophages, monocytes were isolated from 10 ml of heparinized peripheral blood using Ficoll-Hypaque and were enriched by adherence . After 24 hr in culture, cell viability was assessed and monocytes were identified by by cytochemical staining . AHH activity was detectable in pig monocyte homogenates, and the AHH level was similar to that in pig Kupffer cells . AHH was also easily detectable in human monocytes . This macrophage AHH activity was compared with AHH activity in rat monocytes, mouse Kupffer cells and mouse peritoneal macrophages . Monocyte AHH was relatively stable in cell culture but decreased rapidly upon storage at -70 degrees . Macrophage AHH activity was depressed following phagocytic activation in vitro by latex beads with a concomitant increase in heme oxygenase activity.

Arch Biochem Biophys, 1987 Nov 15, 259(1), 46 - 51
Lipid peroxidation is not the cause of lysis of human erythrocytes exposed to inorganic or methylmercury; Ichikawa H et al.; Exposure of human erythrocytes in a 50% hematocrit to 0.5-1 mM Hg2+ initiated immediate hemolysis which proceeded at a constant rate without any formation of lipid hydroperoxides . Treatment of 0.03% hematocrits with 0.4 ppm of Hg2+ or 40 ppm of methylmercury caused rapid hemolysis after a short lag period . The kinetics of the process were unaltered by saturation of the cell suspensions with oxygen, by its replacement with He or CO, or by variation in the level of vitamin E in the membranes . The results show that peroxidation of erythrocyte membrane lipids is not the cause of hemolysis induced by either Hg2+ or methylmercury.

Schweiz Med Wochenschr, 1987 Nov 14, 117(46), 1832 - 4
{Quantitative detection and elimination of neuroblastoma cells in vitro . Comparative study of the efficacy of an immunomagnetic method and lymphokine-activated killer cells}; Kastli M et al.; Much effort is devoted to eliminating residual tumor cells - which escape detection by conventional methods - from remission bone marrow harvested for autologous transplantation . The quantitative determination of the efficacy of these purging methods is generally difficult . This report describes an in vitro reproducible tumor model . Normal mononuclear blood cells were deliberately contaminated with cells from the human neuroblastoma cell line SK-N-AS . The frequency of clonogenic SK-N-AS cells was determined in limiting dilution culture before and after treatment . Two methods of purging these tumor cells from the mixed cell suspension were compared, 1) an immunomagnetic method taking advantage of the existence of a monoclonal antibody (mAb) specific for 90-95% of the tumor cells, and 2) a method based on cell mediated cytotoxicity testing the activity of previously lymphokine activated killer (LAK) cells . Immunomagnetic purging eliminated 90% (1 log) of all SK-N-AS cells and 99% (2 log) of the mAb-binding clonogenic tumor cells . In contrast, LAK cell treatment removed only between 0.1 to 0.3 log (23-50%) of the clonogenic SK-N-AS cells.

J Immunol Methods, 1987 Nov 5, 103(2), 161 - 7
Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine; Van der Heijden PJ et al.; An isolation procedure for functionally active lamina propria lymphoid cells (LPL) from the murine intestine is described . The procedure involved EDTA-dithiothreitol incubation of intestinal tissue to remove epithelial and intraepithelial cells, followed by collagenase digestion of the basement membrane to liberate part of the LPL . The LPL were suspended by squeezing the remaining tissue strips through a nylon gauze filter . Functional activity was tested by enumeration of the immunoglobulin-secreting cells in the cell suspensions obtained by an isotype-specific protein A plaque-forming cell assay . On average 1-2 X 10(8) LPL were isolated from the intestine of C3H/He mice . 11% of these cells actively secreted Ig . From these Ig-secreting cells 99% produced IgA . The isolation procedure described in this paper permitted a higher recovery of viable cells than has previously been obtained with other methods.

Brain Res, 1987 Nov 3, 425(1), 34 - 44
Minimal connectivity between neostriatal transplants and the host brain; Walker PD et al.; The present study sought to determine if axonal connectivity is established between neostriatal transplants and the host brain during the first two months of graft development . Cell suspensions of embryonic neostriatum were transplanted into the adult rat neostriatum lesioned previously by kainic acid . After 1-2 months, injections of horseradish peroxidase conjugated with wheat germ agglutinin (HRP) were made either within the graft, into adjacent host neostriatum or the host ventral midbrain . In animals with HRP injection sites restricted to the graft no retrograde or anterograde label was found in the host brain . However, both anterograde axon label and retrogradely labelled neurons were found in areas within the transplant but distal to the injection site . Neither ventral midbrain nor host neostriatal HRP injections resulted in any significant anterograde or retrograde label within the graft . These results demonstrate a lack of connectivity between neostriatal grafts and the host brain 1-2 months post-transplantation but an ability of grafted neurons to project to different locations within the transplant . Therefore, transplanted neostriatal neurons develop for the first two months in the absence of normal neostriatal afferent and efferent connections.

J Cell Biol, 1987 Nov, 105(5), 2145 - 55
Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells; Malgaroli A et al.; The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ {( Ca2+}i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively . When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area . These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s) . When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h . Homogeneous distribution of the dye is a prerequisite for appropriate {Ca2+}i measurement . In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin . Additional problems were encountered when using fura-2 . Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions . This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport . Calibration of fluorescence signals in terms of absolute {Ca2+}i was complicated by the increased fluorescence of fura-2 in the intracellular environment . To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which {Ca2+}i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+) . These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in {Ca2+}i measurements.

J Urol, 1987 Nov, 138(5), 1318 - 20
Inactivation of bladder tumor cells and enzymes by methylene blue plus light; Gill WB et al.; Using a cystoscopic light source and methylene blue as the sensitizing dye, photoactivation was examined in two types of experiments . In the first, the in vitro study destruction of two enzymes (glucose-6-phosphate dehydrogenase and lactic dehydrogenase) was examined in suspensions of whole and homogenized tumor cells from a transplantable bladder tumor . In the second or in vivo study rats were used to demonstrate that tumor cell suspensions treated with methylene blue plus light, when inoculated into susceptible rats, failed to "take" and produce new tumors . These experiments suggest a possible therapeutic use in treatment of human bladder tumors, though further study would be required.

Int J Radiat Oncol Biol Phys, 1987 Nov, 13(11), 1725 - 33
Effect of tumor colony definition on ionizing radiation survival curves of melanoma-colony forming cells; Yohem KH et al.; Definition of survival and measurement of colony size in soft agar assays is important in establishing in vitro radiation survival curves . Conventionally, survival is assessed according to colony-forming ability . The distinction between small colonies that are abortive and those that are viable often involves a difficult and arbitrary choice for the investigator . We have examined the effect of different minimum colony sizes (greater than or equal to 25, greater than or equal to 50, greater than or equal to 75, and greater than or equal to 100 cells) on ionizing radiation survival curves for cells from established murine (CCL 53.1) and human (M1RW5) melanoma cell lines as well as from short-term human melanoma cell strains (C8146A, C8146C, C8161, C83-2C, C82-7A1, and C8442) and patient biopsy (83-4) . Single cell suspensions were plated in the upper layer of the agar bilayer and cells were irradiated by single dose X rays . Giant cells did not form in colonies containing 50 or more cells . D0 values were highest (D0 values, from 390 to 100 cGy) for cells forming smaller colonies (greater than or equal to 25 cells, greater than or equal to 4-5 doublings) and lowest (D0 values, from 190 to 50 cGy) for cells forming larger colonies (greater than or equal to 100 cells, greater than or equal to 6-7 doublings) . Therefore, apparent radiosensitivity was dependent on colony size selected for analysis . Precise measurement of colony size was important in establishing radiation survival curves because errors in determining the colony size will alter apparent radiosensitivity of cells . These results should help define the biological meaning of tumor colony growth in semisolid medium, and alter the interpretation of survival curves which measure sensitivity to agents using this assay.

Tokai J Exp Clin Med, 1987 Nov, 12(4), 253 - 61
Liposome mediated dissipation of valinomycin-imposed potassium potential across erythrocytes membrane; Matsudaira R et al.; Influence of liposomes made of phosphatidylcholine (PC) on the valinomycin-imposed potassium potential across erythrocyte membrane was examined by measuring the fluorescence change of the potential-sensitive cyanine dye . We concluded that the liposomes modulate ion selectivity of the membrane embedded valinomycin, on the basis of the following lines of evidence . (i) The valinomycin-imposed potassium potential across erythrocyte membrane (interior negative) was dissipated in the presence of PC-liposomes . (ii) When PC-liposomes were added to the cell suspension before the valinomycin, a membrane potential could not be imposed . (iii) Liposomes containing only the PC of saturated fatty acids were inactive in the potential dissipation, whereas the liposomes containing PC of unsaturated fatty acids were fully active . (iv) Liposome-mediated dissipation of the imposed-membrane potential was similarly observed in the resealed erythrocyte ghosts . (v) The liposomes did not show a detectable effect on the gramicidin-mediated proton potential . (vi) The effect of liposome was somewhat analogous to the nigericin-mediated dissipation of the valinomycin-imposed potassium potential.

J Pathol, 1987 Nov, 153(3), 225 - 32
Leu-M1 antigen expression in acute leukaemias; de Mascarel A et al.; Leu-M1 is a differentiation antigen present in human myelomonocytic cells . Seventy-seven acute leukaemias were retrospectively stained with anti-Leu-M1 using the immunoperoxidase technique on Bouin-fixed paraffin-embedded sections . The subjects were 44 acute lymphoblastic leukaemias (ALL) and 33 acute myeloid leukaemias (AML) previously characterized by cytochemical and immunologic (cell suspension) methods . Leu-M1 was positive in all the AML and in half of the ALL cases . These results suggest that Leu-M1 does not allow differentiation between AML and ALL . For the ALL cases Leu-M1 was positive in 15/28 B-cell types, 4/12 T-cell type and 3/4 'null'-cell type cases . Thus, this antibody is of no assistance in defining types B, T, or 'null' in ALL . Leu-M1 was also studied on paraffin sections of 34 high grade malignant lymphomas . The antibody was negative in all 13 B-cell lymphomas (lymphoblastic: 6; immunoblastic: 7) and in all 4 'null' cell lymphomas . It was positive in 4/9 peripheral T-cell type, the other T-cell lymphomas (lymphoblastic: 5; immunoblastic: 3) remaining negative . Thus, Leu-M1 may be positive in T-cell lymphomas but it is negative in B-cell lymphomas and is always negative in B or T lymphoblastic types . It seems that lymphoblasts are Leu-M1 negative in non-Hodgkin's lymphoma and may be Leu-M1 positive in leukaemias.

J Natl Cancer Inst, 1987 Nov, 79(5), 983 - 90
Augmentation of interleukin-2 immunotherapeutic effects by lymphokine-activated killer cells and allogeneic stimulation in murine tumor cells; Eggermont AM et al.; Interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cells were used in intraperitoneal and pulmonary tumor models in C57BL/6 mice . To maintain the immunotherapeutic effects of IL-2 plus LAK treatment but reduce its toxicity, ways were sought to augment IL-2 effects . The investigation showed that the adoptive transfer of LAK cells was a prerequisite for successful therapy of intraperitoneal cancer . When LAK cells were given on consecutive days within one course of immunotherapy, antitumor efficacy was augmented with additional doses of LAK cells . However, with the reduction of 1 complete cycle of IL-2 + LAK cells, no further reduction in intraperitoneal tumor was observed as compared to the reduction after 2 or 4 cycles . LAK cells generated from splenocytes of mice that had received an allogeneic tumor challenge 1 week earlier exerted a highly increased cytotoxicity as compared to normal LAK cells . Furthermore, the potentiation effect of an allogeneic response of the host at the tumor site was demonstrated by decreased numbers of lung implants and improved survival in mice given mixtures of syngeneic and allogeneic tumor cell suspensions . An alloimmune response within the microenvironment of tumor tissue markedly enhanced the antitumor effect of IL-2 against the syngeneic tumor . It was concluded that there is a fundamental need to improve the recruitment of adoptively transferred LAK cells or LAK precursors into tumor tissue . This may be the next step required in the further development of IL-2 and LAK immunotherapy.

J Natl Cancer Inst, 1987 Nov, 79(5), 1067 - 75
In vivo antitumor activity of tumor-infiltrating lymphocytes expanded in recombinant interleukin-2; Spiess PJ et al.; A method was described for the generation of cells from tumor-bearing mice; these cells were capable of exhibiting significant antitumor reactivity when adoptively transferred into tumor-bearing hosts . Tumor cell suspensions from a variety of tumors were able to be separated using enzymatic techniques and they were cultured in medium containing recombinant interleukin-2 . Activated infiltrating lymphocytes within these tumors expanded; and, by 6-8 days after initiation of culture, lymphocytes predominated and were able to grow to large numbers . The adoptive transfer of these tumor-infiltrating lymphocytes (TILs) made possible mediation of the reduction of established 3-day pulmonary micrometastases from 5 of 7 tumors tested, including two 3-methylcholanthrene (CAS: 56-49-5)-induced sarcomas, one 1,2-dimethylhydrazine (CAS: 540-73-8)-induced colon carcinoma, and the B16 melanoma, all in C57BL/6 mice, as well as the 1660 bladder carcinoma in BALB/c mice . Approximately 2-4 X 10(6) transferred cells were capable of totally eliminating 3-day established metastases . These cells were thus 50 to 100 times more effective than lymphokine-activated killer cells in reducing established metastases; however, they could not be generated from all tumors . The concomitant administration of recombinant interleukin-2 enhanced, by approximately fivefold, the in vivo activity of these cells . The specificity of action of TILs in vivo was different from that determined by classic amputation rechallenge experiments . The tumor-infiltrating lymphocytes that developed this antitumor reactivity appeared to be Thy-1+ and did not bear the asialo GM1 antigen . The potent antitumor effect of these TILs, when transferred in vivo to tumor-bearing hosts, raises the possibility of utilizing similar approaches for the isolation and therapeutic use of lymphocytes with antitumor reactivity from human tumors.

Lab Invest, 1987 Nov, 57(5), 592 - 9
Rapid microwave fixation of rat mast cells . I . Localization of granule chymase with an ultrastructural postembedding immunogold technique; Login GR et al.; We defined the ultrastructural localization of chymase in rat peritoneal mast cells using standard aldehyde fixation and a newly described microwave fixation method (Login GR, Dvorak AM: Microwave energy fixation for electron microscopy . Am J Pathol 120: 230, 1985; Login GR, Stavinoha WB, Dvorak AM: Ultrafast microwave energy fixation for electron microscopy . J Histochem Cytochem 34:381, 1986) and postembedding immunogold labeling . Thin sections were exposed first to goat IgG anti-rat chymase and second to gold-conjugated rabbit Ig directed against goat IgG . By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules . Control sections treated with nonimmune sera did not exhibit labeling of mast cells . Thin sections treated simultaneously with purified rat mast cell chymase and anti-chymase antibody in competition studies, showed a marked reduction in granule staining . These findings demonstrate that a microwave fixation method can be used to rapidly fix cell suspensions for postembedding immunocytochemical studies.

Blood, 1987 Nov, 70(5), 1543 - 9
Limiting-dilution analysis for the determination of leukemic cell frequencies after bone marrow decontamination with mafosfamide or merocyanine 540; Porcellini A et al.; To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines . The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540) . A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added . Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents . Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively . Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540 . Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow . MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.

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