Cell suspensions

Biull Eksp Biol Med, 1984 Dec, 98(12), 675 - 8
{Mechanism of the clinical effects of UV-irradiated blood: the stimulation of DNA synthetic activity of human cells in culture}; Belisheva NK et al.; Supernatants of UV-irradiated specimens of donor whole blood, leukocyte-platelet or red cell suspensions added to human embryonic cells in vitro produce a 1.4-1.6-fold increase in 3H-thymidine incorporation into human embryonic cells . Irradiation of blood plasma without the cells by the same doses as therapeutic ones is not followed by the effect indicated . Therefore the stimulation of the growth capacity of the blood after irradiation is connected with its cells . It is suggested that the effect under discussion is derived from the release of some active components from the blood cell surface (outer perimembranous layer) because of its photochemical destruction during UV-irradiation.

J Pharmacol Exp Ther, 1984 Dec, 231(3), 518 - 26
Suppression of humoral antibody production by exposure to 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin; Holsapple MP et al.; The day 4 IgM antibody (AB) response to sheep red blood cells was suppressed in adult female B6C3F1 mice subchronically (14 day) exposed to 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (1,2,3,6,7,8-HCDD) at concentrations reflecting the levels of this dioxin isomer as a contaminant in technical-grade pentachlorophenol . Hepatic microsomal parameters were also measured in these mice and indicated the characteristic induction of mixed-function oxidase enzyme activities, particularly aryl hydrocarbon hydroxylase . The response to sheep red blood cells was also suppressed when measured in vitro by culturing the antigen with spleen cells from a second group of mice subchronically exposed to 1,2,3,6,7,8-HCDD . Enumeration studies with fluorescent-labeled antisera in these animals indicated a dose-related suppression in the number of T-lymphocytes with no effect on B-lymphocytes . 1,2,3,6,7,8-HCDD directly suppressed several in vitro AB responses when added to cultures of spleen cell suspensions from untreated B6C3F1 mice . The rank order of sensitivity was determined to be: polyclonal AB response to LPS greater than or equal to T-independent AB response to DNP-Ficoll greater than T-dependent AB response to sheep red blood cells . Subsequent studies indicated that the suppression by 1,2,3,6,7,8-HCDD could be produced after only a 30-min preincubation . The suppression of all in vitro AB responses was abolished when the dioxin was preincubated with a metabolic activation system: crude liver homogenate (B6C3F1 mice pretreated with the mixed-function oxidase inducer; Aroclor 1254) plus NADP and isocitrate . The suppression of the T-dependent AB response was still evident when the metabolic activation system was heat-inactivated (as verified by an inability to activate cyclophosphamide) prior to the preincubation.

Neurochem Res, 1984 Dec, 9(12), 1689 - 98
Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures; Frangakis MV et al.; We describe the use of the neutral protease Dispase for the dissociation of neonatal rat brain tissue for the preparation of primary monolayer astrocyte cultures . The method involves 5 to 6 successive extractions with careful separation of sedimenting, undissociated tissue . This method gives an initial cell suspension of high viability (93.7 +/- 1.7% cells exclude trypan blue) . In comparison trypsin (0.25%) dissociated tissue gave a cell suspension that showed a lower viability of 58.2 +/- 7.6% . Identical saturation densities of 1.1 to 1.2 X 10(4) cells/cm2 of the Dispase dissociated tissue . Staining for glial fibrillary acidic protein showed that 90-100% cells were positive for this astroglial marker . Thus, the use of Dispase for the initial dissociation of rat brain tissue seems to give primary astrocyte cultures which are very reproducible and homogeneous.

Immunobiology, 1984 Dec, 168(3-5), 285 - 300
Langerhans cells: antigen presenting cells of the epidermis; Streilein JW et al.; While epidermis in the skin provides an excellent barrier to the environment, it is an incomplete one . Some antigenic material can penetrate through the stratum corneum (or be introduced pathologically) where strategically placed epidermal Langerhans cells reside . In this review, we have assembled relevant data concerning the antigen presenting potential of epidermal Langerhans cells . Strong circumstantial evidence derived from in vitro studies of epidermal cell suspensions enriched for Langerhans cells indicates that Langerhans cells possess this capability . In vivo studies with intact skin indicate that critical numbers of functioning Langerhans cells are essential for successful induction of contact hypersensitivity by epicutaneously applied haptens . And within the past several months, experiments with purified preparations of epidermal Langerhans cells have proven that these cells, and perhaps they alone among epidermal cells, possess the capacity of processing and presenting haptenic determinants to the immune system . The challenge for the future is to determine the extent to which this unique property of Langerhans cells affords physiologic protection to the skin and under what pathologic circumstances altered Langerhans cell function leads to disease.

J Clin Chem Clin Biochem, 1984 Dec, 22(12), 935 - 42
Automated flow-cytometric identification of colo-rectal tumour cells by simultaneous DNA, CEA-antibody and cell volume measurements; Valet G et al.; A new method for the automated flow-cytometric identification of colo-rectal tumour cells was developed . Fresh tissue is cut mechanically to obtain single cell suspensions . The cells are then incubated with antibodies in an indirect immunofluorescence assay for CEA (carcino-embryonic antigen) on the cell surface, and counterstained with the DNA stain propidium iodide . Monosized latex particles are added as internal standard, then cell volume, antibody fluorescence and DNA are measured simultaneously in a FLUVO-METRICELL flow cytometer . A FORTRAN IV computer program was used to determine whether aneuploid cells or cells with high density of CEA on their surface were present in the sample . All relevant data were stored automatically in a self updating data base, which is important for quality control and automated thresholding . The samples were taken from 120 different patients . A tumour sample and a sample of healthy adjacent mucosa of the same patient were available in 88 patients . 97.5% of all tumours and 88.6% of the normal mucosa samples were correctly identified . This shows for the first time that the majority of colo-rectal tumour samples can be identified by a flow cytometric measurement with automated data evaluation . The identification of tumour samples was substantially better when based on the measurement of the three parameters, compared with identification by aneuploidy (59%) or by the CEA antibody alone (91%) . It will be possible to automate the measurement of the samples.

J Bacteriol, 1984 Dec, 160(3), 943 - 8
Spectrophotometric determination of affinities of peptides for their transport systems in Escherichia coli; Perry D et al.; The use of novel synthetic peptides to measure peptide transport by spectrophotometric means is described . These peptides contain glycine residues alpha-substituted with thiophenol and are recognized as substrates by both peptide transport systems and intracellular peptidases of Escherichia coli (Kingsbury et al., Gilvarg, C., Proc . Natl . Acad . Sci . U.S.A . 81:4573-4576, 1984) . Transport and peptidase cleavage results in the intracellular release of thiophenol, which exits rapidly from the cell . The release of thiophenol from these peptides by cell suspensions can be measured with Ellman sulfhydryl reagent {5,5'-dithiobis(2-nitrobenzoic acid)} and provides a direct determination of the rate of peptide transport . The reductions in thiophenol release from these peptides resulting from the addition of peptide competitors enable the affinities of the competitors for their transport systems to be determined . By this method, it is shown that the dipeptide transport system is more restrictive with respect to changes in the amino acid sidechains of its substrates than those of the oligopeptide transport system.

Immunobiology, 1984 Dec, 168(3-5), 313 - 24
The Langerhans cell, as a representative of the accessory cell system, in health and disease; Thorbecke GJ et al.; Cell surface receptors and antigens present on Langerhans' cells (LC) suggest a close relationship between LC and interdigitating cells (IDC) in lymph node sections or lymphoid dendritic cells (LDC) in cell suspensions . One of the reported differences is that Fc receptors (FcR) which are present on LC, are absent on LDC . This difference can at least partly be explained by a laboratory artefact: FcR on murine LC are irreversibly modulated by the Ig that contaminates the bovine albumin commonly used for gradients . Furthermore, the demonstration of FcR on murine LC requires prolonged exposure to EA at 4 degrees C . A significant percentage of nonadherent, low density, Ig- lymph node cells also express such FcR . These cells can stimulate syngeneic mixed lymphocyte reactions as can LC isolated from epidermis . Consideration of LC as cutaneous representatives of the accessory cell system led to an investigation of their numbers in epidermal sheets of patients with acquired immunodeficiency syndrome (AIDS) . A marked reduction in Ia+, ATPase+, OKT6+ LC was found as compared to controls, suggesting that accessory cell deficiency may play a role in the extreme immunodeficiency seen in AIDS patients.

Lab Invest, 1984 Dec, 51(6), 635 - 42
Induction of neovascularization in vivo and endothelial proliferation in vitro by tumor-associated macrophages; Polverini PJ et al.; The role of macrophages in neovascularization of tumors was investigated by examining the ability of tumor-associated macrophages (TAM) and their conditioned culture media to induce neovascularization in the cornea of syngeneic rats and proliferation of bovine aortic endothelial cells in culture . TAM were isolated from a 3-methycholanthrene-induced fibrosarcoma propagated in F344 male rats by enzymatic dissociation and were purified by centrifugation through continuous Percoll density gradients, followed by adherence to fibronectin-coated dermal collagen gels . The angiogenic potential of (a) TAM and their 72-hour conditioned culture media, (b) whole tumor cell suspensions (WTCS), (c) tumor cell suspensions depleted of TAM (TCS), and (d) macrophage-depleted tumor cell suspensions reconstituted with TAM (TCS + TAM) were compared . Cells were injected directly; conditioned media were concentrated 10-fold, incorporated into slow-release Hydron pellets, and implanted intracorneally . Stimulation of bovine aortic endothelial cell growth by TAM was assayed in culture with TAM-conditioned media and compared with responses induced by conditioned media from peptone-elicited rat peritoneal exudate macrophages . TAM and their conditioned media induced neovascularization in 38 of 40 corneas (95%) and 15 of 17 corneas (88%), respectively . Maximal vessel ingrowth occurred by the 5th day of implantation . Neovascular responses induced by WTCS (24 of 26 corneas, 92%) and TCS (17 of 24 corneas, 71%) occurred on the 7th and 10th day, respectively . TCS + TAM induced neovascular responses comparable to those elicited by WTCS (19 of 20 corneas, 95%) . Addition of TAM-conditioned media to bovine aortic endothelial cell cultures stimulated a 10-fold increase in cell number within 10 days . This growth stimulatory effect was comparable to or greater than responses induced by conditioned media from rat peritoneal macrophages . Our results demonstrate that TAM are potent stimulators of neovascularization and endothelial cell proliferation and that depletion of macrophages from tumor cell suspensions significantly decreased their angiogenic potential . This suggests that neovascularization of this tumor is mediated in part by macrophages.

Cancer Res, 1984 Dec, 44(12 Pt 1), 5718 - 24
Isolation of preneoplastic rat liver cells by centrifugal elutriation and binding to asialofetuin; Evarts RP et al.; Putative preneoplastic hepatocytes were isolated from male Fischer 344 rats treated with a single dose of diethylnitrosamine, 2-acetylaminofluorene feeding, and partial hepatectomy (Solt-Farber model) . The isolation procedure involved, after collagenase dispersion of the liver, separation of the hepatocytes into small- and large-cell fractions by centrifugal elutriation, and subsequent selection of cells deficient in asialoglycoprotein receptor(s) by plating onto asialofetuin (ASF)-coated plates . The number of cell surface binding sites for the asialoglycoprotein receptor was measured with both asialoorosomucoid and ASF as ligands . There was a 50% reduction of binding sites for both ligands in the original cell suspensions obtained from preneoplastic livers . The reduction in receptor binding sites was most pronounced in the large cell fraction (less than or equal to 30% of control value) after separating the original cell suspension by elutriation into small and large cell fractions . Immunohistochemical studies showed a lack of asialoglycoprotein receptor in preneoplastic (i.e., hyperplastic foci) areas . These areas were entirely super-imposable with glucose-6-phosphatase-deficient areas and partially overlapped the gamma-glutamyltranspeptidase-positive areas in serial liver sections . The attachment of preneoplastic hepatocytes to ASF-coated tissue culture dishes was greatly impaired, and the number of gamma-glutamyltranspeptidase-positive cells on the ASF dishes was reduced to less than 7% as compared to 45 to 70% on the collagen-coated plates . Thus, the lack of asialoglycoprotein (asialofetuin) surface receptors and the increased size of the early preneoplastic hepatocytes are characteristics that can be used to separate the preneoplastic cell population from normal liver cells.

Southeast Asian J Trop Med Public Health, 1984 Dec, 15(4), 547 - 53
Application of peroxidase-antiperoxidase (PAP) staining for detection and localization of dengue-2 antigen . I . In an endogenous peroxidase containing cell systems; Churdboonchart V et al.; The unlabelled immunoperoxidase, peroxidase-anti-peroxidase (PAP), technique was used to detect dengue type-2 viral antigen in several cell systems including the endogenous peroxidase containing cells . These cells are the mosquito cell line (C6/36), continuous cell line of rhesus monkey kidney (LLC-MK2), human monocyte culture both cell suspension and monolayer, and human peripheral blood leukocytes . All of these specimens gave the same results that dengue-2 viral antigen presented in cytoplasm only and the patterns of marker presentation in positive cells varied depending on the duration after infection . The sensitivity of this method is extremely high since it can detect dengue-2 antigen after its attachment on mosquito cells (15 min) as seen in experiments with mosquito cell line, C6/36 . False positive was not observed in all cell systems tested.

J Hypertens Suppl, 1984 Dec, 2(3), S293 - 5
Inhibition of aldosterone production by atrial natriuretic factor; Atarashi K et al.; Extracts of rat atrial muscle lowered basal aldosterone release from rat adrenal glomerulosa cell suspensions, and partially inhibited the stimulation of aldosterone release by adrenocorticotropic hormone (ACTH) and angiotensin II . Atriopeptin I, an atrial peptide with natriuretic, diuretic and smooth muscle relaxant activities, significantly decreased basal aldosterone release at 1 pM concentrations . Also, atriopeptin I decreased the sensitivity of the glomerulosa cells to adrenocorticotropin and angiotensin II . These data suggest that peptides contained in mammalian atria affect sodium excretion not only by a direct effect on the kidney, but also indirectly through inhibition of aldosterone production.

J Reprod Immunol, 1984 Dec, 6(6), 377 - 91
Evaluation of human chorionic trophoblast cells and placental macrophages as stimulators of maternal lymphocyte proliferation in vitro; Hunt JS et al.; Dispersed cell suspensions of human chorion membrane and placentae were obtained by enzyme digestion and the cells examined for HLA expression and for the ability to stimulate immune cell proliferation in vitro . Chorion cells with the characteristics of trophoblast were HLA-A, B, C and Ia negative following tissue digestion whereas placental cells, primarily Fc gamma R positive macrophages, were HLA-A, B, C positive and were frequently Ia positive . When chorion and placental cell suspensions were used as stimulator cells in one-way mixed cell cultures (MCC) with maternal mononuclear leukocytes (MNL) as responder cells, chorion cells were not normally stimulatory (mean stimulation index (SI), 2.7) whereas placental cells usually were (mean SI, 11.5) . No evidence for active suppression by chorion cells was obtained in a group of experiments designed to detect suppressive activity . The results support the concept of the trophoblast layer as an immunologically inert barrier between the mother and the fetus.

Surgery, 1984 Dec, 96(6), 1019 - 26
Failure of secretin to stimulate gastrin release and adenylate cyclase activity in gastrinoma in vitro; Ellison EC et al.; It has been hypothesized that secretin may act directly on gastrinoma through the adenylate cyclase system to cause stimulation of gastrin release . We studied gastrinoma cells in vitro to determine whether secretin would stimulate gastrin release directly and whether the gastrinoma cell membrane had a functional secretin receptor adenylate cyclase system . Fresh tumor was prepared in cell suspensions containing 1.5 X 10(6) viable cells and incubated for 2 hours with either 2 mM CaCl2 alone (control) or 2 mM CaCL2 and 0.025 U/ml secretin . The gastrin content of the cells in each incubation chamber and the medium were determined by radioimmunoassay and results were expressed as mean gastrin pg/microgram protein +/- SD . Under basal conditions the cellular gastrin content was 39.9 +/- 6.4 (control) compared with 16.7 +/- 2.1 (secretin) . After 2 hours of incubation, cellular gastrin content increased in both groups: 68.5 +/- 11.9 (control) to 68.3 +/- 5.5 (secretin) . However, the percent of gastrin released into the medium during incubation decreased by one half in both groups (control 37.3% +/- 4.0% to 22.2% +/- 3.0%; secretin 42.8% +/- 7.0% to 18.9% +/- 1.8%) . Adenylate cyclase activity was assessed by measuring cAMP generation in fresh-frozen gastrinoma and cultured gastrinoma cell membranes . Isoproterenol (10(-5) M), PGE1 (10(-4) M), and GppNHp (guanine nucleotide) (10(-5) M) caused fivefold to 25-fold increases in cAMP generation . Secretin did not stimulate adenylate cyclase activity above basal (21.73 +/- 4.07 and 2.29 +/- 1.2 pmol cAMP/mg protein/min) for frozen and cultured gastrinoma, respectively . Secretin failed to stimulate gastrin release and adenylate cyclase in vitro . This suggests that secretin-stimulated gastrin release in vivo may not be due to a direct effect of secretin on the gastrinoma.

J Pharmacol Exp Ther, 1984 Dec, 231(3), 527 - 31
Kinetics of the enhancing effect produced by norepinephrine and terbutaline on the murine primary antibody response in vitro; Sanders VM et al.; Experiments were performed to determine the minimal drug exposure time required for norepinephrine and a relatively selective beta-2 adrenoceptor agonist to produce a maximal enhancing effect on the murine primary antibody response in vitro . The antibody response was determined by counting the number of spleen cells producing immunoglobulin M antibody directed against sheep erythrocytes 5 days after exposure of spleen cells to antigen and drugs . Norepinephrine (10(-5) M) or terbutaline (10(-5) M) were added to mouse spleen cell suspensions at the time of immunization with sheep erythrocytes on day 0 . Phentolamine (10(-5) M) and/or propranolol (10(-6) M) were added to the spleen cell cultures to block adrenoceptor activation either at the time of agonist exposure (time = 0 hr) or at various times after agonist exposure (time = 15 min-96 hr) . The addition of adrenoceptor antagonists at times after agonist exposure allowed for determination of the minimal time required for adrenoceptor activation to produce a maximally enhanced antibody response . Norepinephrine and terbutaline produced a maximally enhanced antibody response, as measured 5 days later, after 5 to 6 hr of agonist exposure before the addition of the antagonists . Addition of the antagonists at times later than 5 to 6 hr after agonist had no effect on the maximal antibody response . When antagonists were added before this time, the magnitude of the enhancing effect attained was time-dependent . These results indicate that early adrenoceptor activation in vitro is responsible for initiating events which culminate in an enhanced primary antibody response measured a number of days after drug exposure.

Endocrinology, 1984 Dec, 115(6), 2464 - 72
Self-suppression of corticosteroidogenesis: evidence for a role of adrenal 5 alpha-reductase; Carsia RV et al.; Exogenous corticosterone (B), the natural glucocorticoid product of rats, suppressed endogenous B production of isolated rat adrenocortical cells induced by alpha ACTH-(1-24), {9-tryptophan (O-nitrophenylsulfenyl)}ACTH-(1-24) {( Trp (Nps)9}ACTH-(1-24}, and cAMP as well as pregnenolone supported-steroidogenesis . This self-suppression occurred within 2 h . It was dependent on the concentration of exogenous B . However, self-suppression did not alter the half-maximal steroidogenic concentration (ED50) of each steroidogenic agent . In addition, exogenous B did not suppress ACTH-induced cAMP production or gross protein synthesis, as measured by leucine incorporation into bulk cellular proteins . These results with isolated cells suggested at least two mechanisms for self-suppression: 1) exogenous B inhibited steroidogenic steps in a noncompetitive manner, and/or 2) exogenous B induced B degradation . In this study we examined the effect of exogenous B on the degradation of B . Accordingly, we measured the adrenal 5 alpha-reductase activity (5 alpha RA) of cell homogenates prepared from treated cells . Isolated adrenocortical cells were incubated for 2 h with alpha ACTH-(1-24), ovine PRL (oPRL), and B . They were then homogenized and assayed for 5 alpha RA, as indicated by the disappearance of exogenous B, as shown by RIA . In addition, the percentage of exogenous tritium-labeled B {( 3H}B) converted to 5 alpha-dihydrocorticosterone (DHB), the principal reduced metabolite of B, was determined by TLC . Isolated adrenocortical cells from intact rats showed insignificant 5 alpha RA and DHB formation when incubated with or without alpha ACTH-(1-24) and with or without oPRL . However, with exogenous B, there was significant 5 alpha RA and DHB formation . oPRL plus B decreased DHB formation . The effects of B and oPRL were more demonstrable with cells from hypophysectomized rats . These cells exhibited high 5 alpha RA and DHB formation; exogenous B increased these values, whereas oPRL acutely reversed the effects of hypophysectomy and exogenous B . In other work avoiding cell homogenization, exogenous B suppressed ACTH-induced B accumulation and increased DHB formation in intact cell suspensions from intact rats and intact male domestic fowl . Furthermore, exogenous B increased the conversion of {3H} pregnenolone to DHB in intact cell suspensions from intact rats, showing that B synthesized de novo as well as exogenous B can be degraded during self-suppression . These data indicate that acute self-suppression of corticosteroidogenesis is at least partly mediated by an increase in 5 alpha RA.

Biol Reprod, 1984 Dec, 31(5), 1037 - 48
Serological and biochemical identification of a plasma membrane antigen specific to Leydig cells; Millette CF et al.; Purified mouse Leydig cells have been prepared from interstitial cell suspensions using a Percoll gradient procedure . The isolated cells are 88-95% pure as determined by light microscopy . Staining for 3-beta-hydroxysteroid dehydrogenase indicates that over 85% of the Leydig cells recognized by differential interference microscopy are also positive for this enzyme . After separation, the Leydig cells are viable by dye exclusion assays and exhibit normal in situ morphology when examined at the ultrastructural level . Leydig cell suspensions have been used to raise a polyclonal antiserum in rabbits . This antiserum, prior to absorption, reacts in indirect immunofluorescent studies with the surfaces of isolated Leydig cells, with testicular germ cells, with spermatozoa and with somatic cells such as splenocytes . Following absorption with lymphocytes and spermatogenic cells, the antiserum binds only to Leydig cell plasma membranes . Quantitative measurements with 125I-protein A confirm the specific labeling of Leydig cells by the absorbed antiserum . Biochemical identification of the Leydig cell plasma membrane antigen has been accomplished by immunoblotting polyacrylamide gel nitrocellulose transfers . A single major band of Mr approximately 40,000 is detected on one-dimensional transfers; two weakly reactive spots of Mr 43,000 and 45,000 are detected using two-dimensional immunoblots . Blotting experiments conducted using concanavalin A have identified the major Leydig cell constituents reactive with this lectin . The Leydig cell plasma membrane antigen(s) does not bind concanavalin A.

J Immunol Methods, 1984 Nov 30, 74(2), 375 - 84
A simple rapid method for detection of IL-2 in a physiological medium; De Vos C et al.; The Con A-activated proliferation of C57BL/6 mice thymus cell suspension is completely blocked by a low concentration of hydrocortisone . This inhibition is reversed by the addition of a 24 h Con A-activated splenic cell supernatant to the thymic cell culture . The restoring activity is completely destroyed by heating . Restoration is not obtained by addition of IL-1-rich supernatant from an LPS-activated peritoneal adherent cell population . The supernatant of a 24 h splenic cell culture activated by Con A in the presence of indomethacin not only restores hydrocortisone-blocked thymocyte reactivity but also enhances thymic cell stimulation . Addition of PGE to the splenic cell culture inhibits the restoring potency of the supernatant . These results are in accord with the proposition that IL-2 is responsible for the restoring phenomenon . These properties have been used to develop a semi-quantitative method for detection of IL-2 in a physiological medium.

J Immunol Methods, 1984 Nov 30, 74(2), 317 - 25
Removal of rat dendritic cells from single cell suspensions by passage through columns of Sephadex G-10; Bowers WE et al.; Rat T lymphocytes require dendritic cells as accessory cells in order to respond to the mitogen sodium periodate . Passage of a lymph node cell suspension through a column of Sephadex G-10 reduced the mitogenic response by greater than 90%, despite a cell recovery of 75% . An even greater reduction in the response occurred after a second passage over Sephadex G-10; addition of purified dendritic cells restored the response . Lymph node cell suspensions and preparations enriched in dendritic cells were nearly depleted of these cells by passage over Sephadex G-10 . After passage of lymph node cells over Sephadex G-10, a limited number of retained cells could be recovered; these included both dendritic cells and macrophages . Enumeration of macrophages in the various passed and retained fractions by non-specific esterase staining of smears confirmed that macrophages were effectively removed from lymph node cell suspensions by repeated passage over Sephadex G-10 . Cell preparations that pass through Sephadex G-10 are therefore depleted of both dendritic cells and macrophages.

Biochim Biophys Acta, 1984 Nov 21, 778(1), 185 - 90
Effect of thiourea on PCMBS inhibition of osmotic water transport in human red cells; Chasan B et al.; The organomercurial reagent p-chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane . We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether . For a 2 mM PCMBS concentration Ki = 3 +/- 1 mM . When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time . Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when {thiourea} = 50 mM . Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.

J Immunol Methods, 1984 Nov 16, 74(1), 31 - 8
Intracellular fluorescent labelling of cells for analysis of lymphocyte migration; Brenan M et al.; A procedure for analysing in vivo migration of lymphocytes labelled in vitro using intracellular fluorochromes is described . Comparison of carboxyfluorescein diacetate, a cytoplasmic label, with Hoechst dye No . 33342 (H33342), a DNA-binding fluorochrome, indicated that H33342 is superior . The concentration of H33342 used for labelling does not significantly affect viability or lymphocyte migration and permits long-term visualization . H33342 allows quantitation of in vivo migration in cell suspension and histological localization in frozen sections . Fluorescence is retained in fixed frozen sections for at least 3 months . This method can be used for analyses of lymphocyte migration and maturation.

Eur J Biochem, 1984 Nov 15, 145(1), 195 - 202
Induction of chalcone isomerase in elicitor-treated bean cells . Comparison of rates of synthesis and appearance of immunodetectable enzyme; Robbins MP et al.; Chalcone isomerase, an enzyme involved in the formation of flavonoid-derived compounds in plants, has been purified nearly 600-fold from cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.) . Chromatofocussing yielded a single form of the enzyme of apparent pI 5.0 . This preparation was used to raise rabbit anti-(chalcone isomerase) serum . Changes in the rate of synthesis of chalcone isomerase have been investigated by indirect immunoprecipitation of enzyme labelled in vivo with {35S}methionine in elicitor-treated cultures of P . vulgaris . Elicitor, heat-released from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes increased synthesis of the isomerase, with maximum synthetic rate occurring 11-12 h after exposure to elicitor . Immune blotting studies indicate that the elicitor-mediated increase in extractable activity of the isomerase is associated with increased appearance of immunodetactable isomerase protein of Mr 27 000 . However, the maximum level of immunodetectable isomerase was attained approximately 6 h earlier than maximum extractable activity . Furthermore, a 2.8-fold increase in enzyme activity above basal levels at 12 h after elicitor-treatment was associated with a corresponding 5.8-fold increase in immunodetectable enzyme . It is concluded that elicitor induces the synthesis of both active and inactive chalcone isomerase of Mr 27 000, and that some activation of inactive enzyme occurs during the elicitor-mediated increase in isomerase activity . The presence of a pool of inactive chalcone isomerase in bean cell cultures has recently been suggested on the basis of density labelling experiments utilising 2H from 2H2O {Dixon et al . (1983) Planta (Berl.) 159, 561-569}.

Biochemistry, 1984 Nov 6, 23(23), 5549 - 55
Measurement of intracellular pH and deoxyhemoglobin concentration in deoxygenated erythrocytes by phosphorus-31 nuclear magnetic resonance; Labotka RJ; Deoxygenation of erythrocytes produced marked changes in their 31P nuclear magnetic resonance spectra in the superconducting spectrometer . Most significantly, all intracellular and extracellular phosphates underwent downfield shifts . In fully deoxygenated blood the extracellular phosphates showed downfield shifts that were dependent upon packed cell volume, when added pyrophosphate was used as a measure of extracellular chemical shift behavior . This effect on extracellular signals was attributed to the paramagnetic contribution of deoxyhemoglobin to the "bulk" magnetic susceptibility of the red cell suspension . Line broadening was observed in deoxygenated whole cell systems but not in hemolysates, as a result of paramagnetic susceptibility gradients across the cell membrane . The degree of downfield shift upon deoxygenation was of different magnitude for each intracellular phosphate {2-P of 2,3-diphosphoglycerate (2,3-DPG) greater than 3-P of 2,3-DPG greater than inorganic phosphate greater than ATP phosphates}, independent of packed cell volume but dependent on the degree of deoxygenation of hemoglobin . When deoxygenation shift effects in adult cells were compared to those of cord blood cells containing 70% fetal hemoglobin, it was found that 45% of the 2,3-DPG shift effects were attributable to binding of that compound to hemoglobin . By use of a nonphysiologic phosphate analogue, methylphosphonate, as an internal reference, it was found that an increase in pH of deoxy cells contributed to the downfield shift of inorganic phosphate . In hemolysates, the methylphosphonate - inorganic phosphate chemical shift difference was found to be pH dependent, with a sensitivity of (-) 0.39 pH unit/ppm, independent of the hemoglobin oxygenation state.

J Natl Cancer Inst, 1984 Nov, 73(5), 1119 - 24
Mammary carcinoma arising in mice undergoing a chronic graft-versus-host reaction; Gartner JG et al.; A graft-versus-host reaction (GVHR) was induced in 30 (CBA X A)F1 mice by the iv injection of 50 X 10(6) parental strain A lymphoid cells . Solid tumors emerged in 5 of 10 experimental animals that survived beyond 14 months after the GVHR was initiated . The neoplasms were judged to be mammary carcinomas by light and electron microscopic examinations . C-type RNA viral structures were observed in some tumor cells . The neoplasms were successfully transplanted into syngeneic animals by either sc or ip injections of tumor cell suspensions . Tumor transfer to syngeneic mice was not possible if only spleen cells from tumor-bearing animals were transferred . No tumors developed in an age-matched control group that received no treatment.

Hokkaido Igaku Zasshi, 1984 Nov, 59(6), 762 - 74
{Studies on the sensitivity test of anticancer agents with special reference to double layer colony forming assay}; Ohsaki Y et al.; The necessity to assess the effect of chemotherapeutic agents on the patients beforehand treatment has been stressed . In 1977, Salmon & Hamburger developed a new method to evaluate the drug sensitivity using cell suspension derived from the patients . They called the method as "human tumor stem cell assay" . Since then, many reports were published about the correlation between the clinical outcomes and the results of human tumor stem cell assay, and high relationship between them was documented . The present study was undertaken to evaluate the method using the modified system; double layer colony forming assay, cell growth curve, tritiated thymidine uptake and flow cytometry . The drugs used in this study were adriamycin (ADR) and cis-platinum (C-DDP) . We chose the human small cell lung cancer cell line PC-6 as a target cell . The conclusions are follows . The effects of ADR and C-DDP were concentration dependent respectively . The time dependent effect of C-DDP on PC-6 was revealed by enhancement of lethal effect, when they were exposed to the drug for 24 hours . A possibility exists that reproductive death would take place among PC-6 by ADR, along with DNA injury . It is one of the explanations for reduction of colony formation in contrast to increase in cell number . The effect of C-DDP on PC-6 was diminished with time . This phenomenon was reflected on double layer colony forming assay, and was possible explanation for reduction of colony formation which was greater than that of expected from cell growth curve.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1984 Nov, 130 ( Pt 11), 2865 - 71
Transport of alpha-aminoisobutyrate into Trypanosoma brucei brucei; Coolbear KP et al.; The uptake of alpha-aminoisobutyrate (AIB) by washed cell suspensions of bloodstream forms of Trypanosoma brucei brucei has been shown to be an energy-dependent process . No metabolism of AIB was detected under conditions leading to a 100-fold accumulation of AIB within the organism . Kinetic studies revealed that AIB uptake involved two components; that operating at low substrate concentrations had an apparent Km of 4.6 mM . Experiments with ionophores such as gramicidin and carbonylcyanide m-chlorophenylhydrazone were consistent with the AIB uptake system operating as a H+-symporter responding to the electrochemical gradient of H+, the major component of which was the membrane potential.

Andrologia, 1984 Nov-Dec, 16(6), 517 - 24
Human testicular cell suspensions in vitro using post mortem material; Dietrich AJ; A method is described for culturing human testicular cells from post mortem material for at least 10 days in a serum free medium . The success of the technique is based on the fact that a layer of testicular cells remains undisturbed on the bottom of a culture flask during culturing and the medium is constantly renewed by a perfusion flow . It is shown that cells form junctions within several hours allowing metabolic coupling, indicating active survival of cells during culture period.

Br J Haematol, 1984 Nov, 58(3), 465 - 72
Isolation of normal human megakaryocytes; Sitar G; This paper reports a simple procedure for obtaining human megakaryocytes with a high purification and high recovery yield . Bone marrow cells, obtained from surgically removed ribs, were separated by a two-step procedure . Initially, a single cell suspension was enriched in megakaryocytes by equilibrium density centrifugation, the low density cell fraction was subsequently layered over a shallow continuous albumin gradient in a glass sedimentation chamber . Megakaryocytes averaged 0.03 +/- 0.02% of all nucleated cells in the starting marrow cell suspension, after this procedure an average 80 +/- 15% of the initial megakaryocyte population was recovered with a purity of 94 +/- 4% . Previous methods, based upon the use of a two-step procedure, are reviewed . The theory of velocity sedimentation is discussed with regard to the differences in the methodology used, which account for the different results I obtained.

Radiat Res, 1984 Nov, 100(2), 328 - 39
Reduced oxygen enhancement ratio at low doses of ionizing radiation; Palcic B et al.; A decreased oxygen enhancement ratio (OER) at lower radiation doses has been previously reported (B . Palcic, J . W . Brosing, and L . D . Skarsgard, Br . J . Cancer 46, 980-984 (1984} . The question remained whether or not this effect is due to a possible oxygen contamination at low doses, which was not the case at high doses . To ensure a sufficient degree of hypoxia prior to the start of irradiation, Chinese hamster cells (CHO) were made hypoxic by gas exchange combined with metabolic consumption of oxygen at 37 degrees C . At the same time oxygen levels in cell suspension were measured using a Clark electrode . It was found that under experimental conditions used in this laboratory for hypoxic irradiations, the oxygen levels before the start of irradiation are always below the levels which could give any significant enhancement to radiation inactivation by X rays . Full survival curves were determined in the dose range 0-30 Gy using the conventional survival assay and in the dose range 0-3 Gy using the low dose survival assay . The results confirmed the earlier finding that the OER decreases at low doses . It is therefore believed that the dose-dependent OER is a true radiobiological phenomenon and not an artifact of the experimental method used in the low dose survival assay.

J Surg Res, 1984 Nov, 37(5), 354 - 60
The use of pig mononuclear cells to inhibit pulmonary tumor formation in isogenic mice; Khezri AA et al.; A-strain mice received 1 X 10(5) A-strain mammary carcinoma, B72, cells iv on Day 0 . At the same time fragments of the same tumor were implanted into the ileal mesentery of a pig together with fragments of cellulose sponge . The middle third of the pig mesenteric lymph node chain was resected . Sponge fragments of equivalent size, shape, and total weight were implanted alone into the jejunal mesentery . Seven days later, separate cell suspensions were made from the (disconnected) proximal (nonimmune) and distal (tumor-immune) segments of lymph node chain . Cells were also expressed by digital pressure from the sponges removed from the jejunal (nonimmune) and ileal (immune) segments of mesentery . A comparison was made between the antitumor action of immune lymph node cells and the two categories of sponge cell . Cells (4 X 10(6} were injected iv into the mice on Day 7 and the number of pulmonary tumors counted after killing the mice on Day 14 . Cells obtained from the "immune" sponges were significantly more effective in reducing the number of pulmonary tumors than cells from the "nonimmune" sponge, or from the immune lymph nodes . The antitumor action of all cell suspension was abolished when 4 X 10(6) cells from the "interface" layer, following centrifugation on a Ficoll-triosil column, were injected into the mice . Antitumor activity was found to be correlated with the presence of "blast" cells in the pig mononuclear cell suspensions.

J Exp Med, 1984 Nov 1, 160(5), 1273 - 83
Dual origin of mouse spleen macrophages; van Furth R et al.; The present study concerns the isolation, characterization, origin, and kinetics of spleen macrophages . The spleen was first perfused in situ to remove monocytes from the vascular bed and then dissected and treated with collagenase . The macrophages in the cell suspension thus obtained were characterized morphologically and cytochemically and then quantitated . The spleen cell suspension was incubated for 24 h in Leighton tubes to obtain an enriched glass-adherent population of macrophages for characterization and {3H}thymidine-labeling studies . Almost all of the adhering macrophages were esterase positive, had Fc and C3b receptors, and ingested EIgG and opsonized bacteria . In vitro labeling with {3H}thymidine showed that approximately 5% of the mononuclear phagocytes in the spleen synthesize DNA and must be considered to be dividing cells . The course of the number of labeled monocytes and macrophages after a single injection of {3H}thymidine indicates migration of monocytes into the spleen, where they become macrophages . Calculation of the influx of monocytes into the spleen and of the local production of macrophages by DNA-synthesizing mononuclear phagocytes showed that under steady-state conditions, 55% of the population of spleen macrophages is supplied by monocyte influx and 45% by local production . This means that there is a dual origin of spleen macrophages . The mean turnover time calculated with the value for the efflux of spleen macrophages is 6.0 d.

J Invest Dermatol, 1984 Nov, 83(5), 370 - 6
Selective cultivation of human melanocytes from newborn and adult epidermis; Gilchrest BA et al.; Development of adequate culture systems for the human epidermal melanocyte is critical to further advances in pigment cell biology . We now report selective growth and long-term maintenance of melanocytes derived from both newborn and adult skin specimens . Disaggregated epidermal cell suspensions were plated in a hormone-supplemented medium containing cholera toxin and a hypothalamic extract treated to remove keratinocyte growth-promoting activity . After 3-4 weeks, pure melanocyte populations could be harvested and serially passaged up to 6 times over several months for a total of 10 or more cumulative population doublings in vitro . Electron microscopic studies revealed metabolically active cells with abundant melanosomes in various stages of melanization throughout the culture lifespan . Differences in size and number of melanosomes attributable to race of the tissue donor were readily apparent, and pigment content of melanocytes from both black and Caucasian donors appeared to increase with time in culture . Newborn melanocytes proliferated more rapidly and survived longer than did adult melanocytes, but there were no consistent morphologic differences as a function of donor age . Comparison of growth potential for the 3 major skin-derived cell types in this hormone-supplemented medium revealed striking specificity for melanocytes, with total elimination of keratinocytes over 1-2 weeks, and no fibroblast proliferation whatever in the absence of serum supplementation . This system promises to facilitate in vitro investigation of epidermal melanocytes in normal and diseased human skin.

Int J Radiat Oncol Biol Phys, 1984 Nov, 10(11), 2125 - 30
Development of a rat lung cancer model; Byhardt RW et al.; A rat lung cancer model based on intrabronchial instillation of a tumor cell suspension has been developed for use in therapy and toxicity testing . Two tumors were used in this study, a sarcoma and an adenocarcinoma, both of which were of spontaneous origin in the strain of rats used . The inoculated tumor cells implant on the bronchiolar mucosa, forming a detectable single "primary" tumor resembling the spontaneous lung cancers arising in humans . The tumor growth is detectable by use of diagnostic radiographs, weight loss and other "clinical" signs . The tumors appear on chest radiographs 3 to 5 weeks after inoculation, and the implant rate is proportional to the number of tumor cells inoculated . Untreated animals have a median survival (after radiographic detection of the tumor) of 8 days, and die of local complications of tumor growth . When a slow growing transplantable tumor line of lung origin is developed, this model will be used to evaluate radiotherapy and chemotherapy schedules.

Dev Biol, 1984 Nov, 106(1), 53 - 60
Two glial cell lineages diverge prenatally in rat optic nerve; Raff MC et al.; Three types of glial cells have been previously described in cultures of neonatal rat optic nerve--oligodendrocytes, type 1 astrocytes, and type 2 astrocytes--which can be distinguished using three different antibodies: antigalactocerebroside antibodies recognize oligodendrocytes; antibodies against glial fibrillary acidic protein recognize both types of astrocytes, while the A2B5 monoclonal antibody distinguishes between the two, binding to type 2 but not type 1 astrocytes . It was subsequently shown that oligodendrocytes and type 2 astrocytes, but not type 1 astrocytes, develop in cultures of 7 day optic nerve from a common, A2B5+ progenitor cell . In the present study, the distribution of rat neural antigen-2 (Ran-2), a cell-surface antigen defined by a monoclonal antibody, has been examined on optic nerve cells . It is demonstrated that, in contrast to A2B5, Ran-2 is present on type 1 but not type 2 astrocytes in optic nerve cultures . More importantly, it is shown that Ran-2 and A2B5 antibodies react with largely nonoverlapping populations of cells in cell suspensions of embryonic Day 17 (E17) and postnatal Day 1 (P1) optic nerve, and that the Ran-2+, A2B5- population contains type 1 astrocytes and their precursors while the A2B5+,Ran-2- population contains the progenitor cells for oligodendrocytes and type 2 astrocytes . These findings provide strong evidence that the glial cells of the rat optic nerve develop as two distinct lineages--one giving rise to type 1 astrocytes and the other to oligodendrocytes and type 2 astrocytes--and that the two lineages diverge as early as E17.

Clin Immunol Immunopathol, 1984 Nov, 33(2), 191 - 8
The effect of NPT 15392, 9-(erythro-2-hydroxy-3-nonyl)-6-hydroxypurine, on the phytohemagglutinin of OKT3+, OKT4+, OKT8+, and OKM1+ cell-depleted and undepleted peripheral blood mononuclear cells; De Simone C et al.; NPT 15392, 9-(erythro-2-hydroxy-3-nonyl)-6-hydroxypurine, has been reported to influence immunological responses involving different cell types . Herein data are obtained by studying the influence of NPT 15392 on the phytohemagglutinin (PHA)-induced proliferative responses of unfractionated peripheral blood lymphocytes (PBLs) and of cell suspensions, which have been depleted of the cell subsets recognized by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1, in an attempt to identify which cell types respond to NPT 15392 in the PHA-driven blast transformation assay . The proliferative responses of unfractionated peripheral blood lymphocytes are potentiated when the drug is employed at the concentration of 0.1 microgram/ml and inhibited when NPT 15392 is added to the cell suspensions at concentrations over 5 micrograms/ml . The data reported here suggest that this phenomenon is a composite effect, made up of a combination of the counteracting effects caused by the OKT4+ cells on the one hand, and the OKT8+ and OKM1+ cells on the other.

Clin Immunol Immunopathol, 1984 Nov, 33(2), 144 - 53
Natural killer (NK) cell activity in murine muscular dystrophy . II . Age-related tissue distribution and enhanced NK activity in the thymus of dystrophic mice; Semple JW et al.; The age-related tissue distribution of natural killer (NK) cell activity in murine muscular dystrophy was investigated . Lymphoid tissues including the spleen, thymus, mediastinal (or bronchial) lymph nodes (BLN), mesenteric lymph nodes (MLN), inguinal/popliteal lymph nodes (PLN1), and axillary/brachial lymph nodes (PLN2) were obtained from various aged normal (+/+) and dystrophic (dy2J/dy2J) C57BL/6J mice . Cell suspensions were incubated with 51Cr-labeled YAC-1 lymphoma target cells in a 4-hr 51Cr-release assay . The data indicated that dystrophic mice, at all ages studied, had elevated levels of NK activity in the spleen, BLN, MLN, PLN1, and PLN2 as compared with the normal age- and sex-matched control group . The NK activity in the thymocyte population from dystrophic mice at 2 weeks of age was found to be negligible, while at 8 weeks of age it was two-fold higher than that for the normal control group . In addition, dystrophic mice had an age-related decline in NK activity in all tissues after 10 weeks of age but the activity was still elevated at 40 weeks of age as compared with the normal control group . Target cell binding studies revealed that the number of conjugate-forming cells in thymocytes from 8-week-old dystrophic mice were found to be significantly higher than that found in normal mouse thymocytes using NK-sensitive YAC-1 tumor target cells . The number of cells bound per YAC-1 target cell ranged from 1 to 3 for dystrophic mouse thymocytes as compared with 1 to 2 for the normal control group . Thus, the data indicate an elevated NK activity in all lymphoid tissues studied from dystrophic mice of different ages . In addition, the thymus from dystrophic mice at 8 weeks of age contains an enhanced number of conjugate-forming NK cells and NK activity.

Cancer Res, 1984 Nov, 44(11), 5369 - 75
Cytotoxicity of adriamycin in MGH-U1 cells grown as monolayer cultures, spheroids, and xenografts in immune-deprived mice; Erlichman C et al.; The cytotoxic activity of Adriamycin was examined in the MGH-U1 human bladder carcinoma line, grown as monolayer culture, as spheroids, and as xenografts in immune-deprived mice . The MGH-U1 cells grown as spheroids were much more resistant to Adriamycin (concentration of drug resulting in 37% cell survival, 4.5 micrograms/ml) than when treated as monolayer cultures (concentration of drug resulting in 37% cell survival, 0.9 microgram/ml) . Adriamycin fluorescence was demonstrated only in the outer two layers of cells forming the spheroids, suggesting that limited drug penetration is an important factor in the resistance of spheroids to Adriamycin . Sequential trypsinization of spheroids 750 micron in diameter allowed us to determine the cytotoxic effects of Adriamycin in MGH-U1 cells derived from different depths of the spheroid . We found that cells near the surface of the spheroid had a survival similar to those of exponentially growing monolayer cells treated with Adriamycin . Cells located in the middle of the viable rim were more resistant to Adriamycin, and those found near the necrotic center were most resistant to Adriamycin . The effects of Adriamycin treatment on spheroid growth delay were determined also . In spite of a small cytotoxic effect on the clonogenic fraction of cells in MGH-U1 spheroids, the growth delay effect of Adriamycin in intact spheroids was marked . This observation is consistent with Adriamycin killing primarily the cells in the outer layers of the spheroid, where most of the proliferation in the spheroid occurs . In vivo treatment of MGH-U1 xenografts with Adriamycin followed by assessment of cell survival in vitro showed minimal evidence of cytotoxicity, consistent with the poor drug penetration observed in the spheroid model . These studies suggest that: (a) Adriamycin penetrates poorly into solid tissues; (b) in vitro clonogenic survival following Adriamycin exposure of a cell suspension may predict falsely for drug sensitivity to chemotherapy; (c) a small decrease in clonogenic survival can be translated into a long growth delay but, ultimately, the tumor regrows because some clonogenic cells are spared; and (d) for Adriamycin, the spheroid model more closely parallels the in vivo effects than does monolayer culture . The use of the spheroid model for the study of Adriamycin cytotoxicity gives further insight into the action of this drug in solid tumors.

J Pharmacol Exp Ther, 1984 Nov, 231(2), 441 - 8
Arachidonic acid metabolism in a cell suspension isolated from rabbit renal outer medulla; Ferreri NR et al.; We studied arachidonic acid (AA) metabolism by a cell suspension containing principally cells of the thick ascending limb of the loop of Henle (TALH) obtained from the inner stripe of the outer medulla of the rabbit kidney . Based on comparison of specific activities of enzymes before and after separation, alkaline phosphatase, Na+-K+-adenosine triphosphatase, as well as Tamm-Horsfall glycoprotein and electron microscopic appearance, 80% of these cells were estimated to be TALH in origin . TALH cells had low activity of cyclooxygenase and did not show evidence of lipoxygenase activity . However, they selectively converted exogenous AA to oxygenated metabolites by a cytochrome P-450 related mechanism . AA metabolites were produced in large amounts (30-40% conversion of {14C}AA, 1 to 5 micrograms/mg of protein/30 min) and were increased 5-fold after separation of TALH cells from a suspension of outer medullary cells, suggesting that TALH cells synthesized these metabolites . Induction of cytochrome P-450 by pretreatment of rabbits with beta-naphthoflavone and 3-methylcholanthrene increased formation of the AA metabolites by almost 2-fold in the separated cells and correlated with cytochrome P-450 content of the renal outer medulla . Additionally, SKF 525A and carbon monoxide inhibited product formation in these renomedullary cells, supporting a role for a cytochrome P-450-like monooxygenase in TALH cell function.

Cell Tissue Kinet, 1984 Nov, 17(6), 593 - 600
Two-step cell-death kinetics in vitro during cis-platinum, hydroxyurea and mitomycin incubation; Bohmer RM; A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest . At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer . Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss . The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2) . The kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea . The subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration . Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.

Semin Diagn Pathol, 1984 Nov, 1(4), 272 - 84
Immunohistology of lymphoproliferative disorders; Tubbs RR et al.; Malignant lymphomas have come to be recognized as neoplasms of the immune system . These lymphoproliferative disorders demonstrate surface and cytoplasmic antigenic phenotypes that reflect qualitative and quantitative alterations or aberrant expression of genetic material . Traditionally, cell suspension studies have been used for phenotypic analysis . Alternative immunohistologic methods can be used to profile immunophenotypes in situ . Most lymphoproliferative disorders can be readily classified as T or B cell malignancies, and criteria have been evolved to differentiate neoplastic from reactive/physiologic expansions of lymphoid clones . However, antigenic phenotypic expression does not always correspond to known immunophenotypes of subsets of T or B cells and probably reflects the complexity of neoplastic transformation . Currently, frozen tissue sections, preferably in combination with cell suspension analysis using cytocentrifuge preparations and/or flow cytometry, can provide information to phenotype lymphomas classified by the International Formulation or other nomenclature . Their continued utility depends on development of and adherence to strict quality assurance programs.

Biochem J, 1984 Nov 1, 223(3), 723 - 31
The transient kinetics of uptake of galactosides into Escherichia coli; Page MG et al.; The uptake of galactosides into Escherichia coli via the lactose permease was studied in the time range 0.01-10s by rapid mixing and quenched flow . An initial transient was observed under two conditions . Firstly, a lag in the approach to the steady state was observed at low galactoside concentrations (less than Km) . Secondly, a burst of uptake was observed when anaerobic cell suspensions were mixed with aerobic substrate solutions . However, the cause of the burst of uptake appears to be a burst in the rate of respiration . The rate of galactoside uptake during this phase is 10-fold greater than during the steady state.

Brain Res, 1984 Nov, 318(2), 203 - 17
Some immature tetanus toxin-positive cells share antigenic properties with subclasses of glial cells . An immunofluorescence study in the developing nervous system of the mouse using a new monoclonal antibody S1; Schnitzer J et al.; Monoclonal antibody S1 reacts in monolayer cultures with the cell surfaces of oligodendrocytes and a subclass of astrocytes derived from early postnatal mouse cerebellum, cerebrum and spinal cord, as well as with some glial cells in mouse retina but not in dorsal root ganglia . At earlier developmental stages S1 antigen is present in addition to oligodendrocytes and astrocytes on some tetanus toxin-positive neurons . S1 antigen is a developmentally early marker, detectable already in freshly trypsinized single cell suspensions from cerebella of 13-day-old embryos . Immunocytolysis of S1 antigen-bearing cells leads to reappearance of S1 positive glial cells but not tetanus toxin receptor-positive neurons . S1 antigen is also expressed in rat, rabbit, chicken and human . When cultured cells are permeabilized with denaturing agents, S1 antibody not only labels cell surfaces of some glial cells and, depending on the developmental stage, some neuronal cells but also intracellular components of all astrocytes, oligodendrocytes, neurons and fibroblasts.

Lab Invest, 1984 Nov, 51(5), 504 - 14
Identification of two major B cell forms of nodular mixed lymphoma; Grogan TM et al.; To resolve the controversy over the immunologic nature of nodular mixed lymphoma (NM), we examined nine cases of NM for surface antigens using both tissue section and cell suspension methods . These were contrasted with 12 cases of nodular poorly differentiated lymphocytic lymphoma . We found two major B cell types of NM, those with monoclonal immunoglobulin (SIg+)-positive nodules with an SIg+B1+B2+Ia+T- phenotype (four cases) and those with nodules devoid of immunoglobulin with an SIg-B1+B2-Ia+T- phenotype (five cases) . Our SIg+ NM cases appear similar to nodular poorly differentiated lymphocytic lymphoma (SIg+B1+B2+Ia+T-), except suspension assay indicates fewer SIg+ cells in NM . In our SIg- NM cases, the neoplastic nodules consistently expressed B1 and Ia-like antigens and lacked T cells, indicating a B cell neoplasm similar to many large cell lymphomas . By demonstrating a B cell antigen in SIg- nodules, we substantially resolve the controversial NM cases previously called "null" or T cell . The two distinct immunotypes indicate the complexity of B cell antigenic expression in NM and might also explain the variable response to therapy in NM described in previous studies . Finally, we describe NM cases with the simultaneous occurrence of several stages of B cell differentiation . This suggests that some NM cases are not frozen in a single stage of B cell development but may express a range of B cell antigens . NM, then, may be a paradigm of variable, simultaneous B cell maturation.

Exp Hematol, 1984 Nov, 12(10), 794 - 9
Effect of serum from mice treated with lipopolysaccharide on cycling of CFUs in vitro; Molendijk WJ et al.; Serum of lipopolysaccharide(LPS)-treated LPS-high-responder C3H/He mice was shown to increase survival of low-responder C3H/HeJ CFUs in an otherwise serum-free suspension culture by initiating cell cycling . Post-LPS serum of low-responder mice and serum of phosphate-buffered-saline-injected high-responder mice was significantly less effective in this respect . Since prolonged maintenance of CFUs was also found when cell suspensions highly enriched for stem cells were used, it seems unlikely that assessory cells mediated the effect of the post-LPS serum activity on CFUs maintenance . The serum activity did not enhance the stimulatory effect of saturating levels of highly purified stem-cell-activating factor (SAF) on CFUs maintenance in vitro . Upon injection of post-LPS serum from C3H/He mice a relatively small splenic CFUs accumulation in C3H/HeJ mice was observed.

Am J Pathol, 1984 Nov, 117(2), 184 - 94
The immunohistology of the thymus in myasthenia gravis; Kornstein MJ et al.; We have investigated cell subpopulations in frozen sections of thymus tissue obtained from myasthenic (MG) and control subjects . With the use of an avidin-biotin immunoperoxidase system with monoclonal antibodies, the following cell surface antigens were studied on frozen sections (12 MG and 3 control thymus); T11, T4, T6, T8, IgM, IgD, and Ia . The pattern of T cell phenotypes in MG thymus is similar to that of normal control thymus when examined by immunohistologic techniques . MG cortical thymocytes are virtually all T11+, T4+, T8+, and T6+ . In the medulla, at least 45% of thymocytes are T11+, with T4+ cells predominating over T8+ cells . Approximately 10% of medullary thymocytes are T6+ . Scattered medullary cells expressing surface IgM and IgD are identified in both MG and normal thymuses . However, unlike the normal thymus, the MG thymus has numerous secondary follicles containing IgM- and IgD-bearing cells . This finding supports the hypothesis that the MG thymus microenvironment is aberrant . The Ia antigen is found in similar tissue section localization patterns in MG and control thymus . Ultramicroscopic studies show the Ia antigen predominantly on epithelial and interdigitating dendritic cells . By immunoperoxidase techniques, numerous keratin-positive cells are demonstrated in MG and control thymus . This suggests that thymic epithelial cells, like epithelial cells elsewhere, contain keratin . Because these data differ in degree from our previous findings in suspensions of MG thymocytes, this study emphasizes the importance of examining tissue sections as well as cell suspensions when one is studying lymphocyte surface markers.

Res Commun Chem Pathol Pharmacol, 1984 Nov, 46(2), 187 - 205
Effect of calcium, sodium and isoproterenol on renin secretion from disaggregated rat renal cortical cells; O'Dea RF Jr et al.; A disaggregated cell system prepared from rat renal cortices has been developed to study the regulation of renin release in vitro . Cell suspensions were prepared by incubating minced renal cortical tissue in collagenase . The digested tissue was filtered sequentially through graded Teflon meshes of 125 and 44 mu, respectively . This preparation contained less than 2% intact glomeruli or tubules . Incubation of cell suspensions with the beta-adrenergic agonist L-isoproterenol (ISO) significantly increased the activity of renin released into the incubation media . Peak levels of renin activity were detected 10 to 15 min after the addition of ISO . The apparent ED50 for stimulation of renin release by ISO was 6 X 10(-8)M and the response was antagonized by the beta-adrenergic antagonist dl-propranolol . The spontaneous release of renin was also suppressed by increasing the concentration of extracellular calcium, whereas sodium ions had no effect on this process . These data have demonstrated that disaggregated renal cortical cell models are useful for studying the in vitro effects of pharmacologic agents and ions on renin secretion.

Exp Cell Res, 1984 Nov, 155(1), 232 - 40
Ceruloplasmin receptors in liver cell suspensions are limited to the endothelium; Kataoka M et al.; The interaction of ceruloplasmin (CP) with isolated liver cell suspensions was studied using 125I-labeled and latex minibead-derivatized CP . Fractionation of liver cell suspensions was done using metrizamide gradient centrifugation . In crude liver cell suspensions only endothelial cells, but not hepatocytes and Kupffer cells bound the minibead probe . The binding was specific and inhibited by excess native CP . These results were confirmed using 125I-CP combined with cell fractionation technique . Kinetic data, obtained from the latter system, indicated a dissociation constant (Kd) of 1 X 10(-7) M and the number of receptors to be 5.7 X 10(5) per endothelial cell . The exclusive binding of CP to liver endothelium suggests that this cell may mediate the hepatocytes uptake of CP and is, therefore, a crucial element of the tissue-blood barrier.

Proc Soc Exp Biol Med, 1984 Nov, 177(2), 278 - 82
Lymphoblastoid cell-induced suppression of human peripheral blood leukocyte mitogenic responses; Zhang JL et al.; Two lymphoblastoid tumor cell lines, the Burkitt lymphoma derived BJAB cell line which is free of Epstein-Barr virus (EBV) and B95-8 cells, which are marmoset lymphocytes transformed by EBV isolated from an infectious mononucleosis patient, were studied in regards to their effects on the blastogenic responsiveness of normal human peripheral blood leukocytes stimulated in vitro with mitogens . Mitomycin C treated tumor cell suspensions, when cocultured with normal human blood leukocytes, markedly depressed the expected blastogenic responses in vitro to concanavalin A, pokeweed mitogen, and phytohemagglutin . In addition, cell-free sonicates from the cell lines also depressed blastogenic responsiveness of the leukocytes in vitro . Heating the sonicates for 10 min at 100 degrees C markedly diminished the suppressive properties of the sonicates, as did ultraviolet light irradiation . The suppressive activity of the B95-8 sonicates was pelleted by high speed centrifugation as compared to the activity of sonicates derived from the BJAB cells . Further studies are warranted to determine the nature and mechanism of suppression of blastogenic responsiveness of normal human leukocytes by soluble components derived from such lymphoblastoid cell lines.

Biochem Biophys Res Commun, 1984 Oct 30, 124(2), 393 - 9
Rapid polyphosphoinositide decrease is an early event in the steroidogenic response of bovine adrenocortical fasciculata cells to angiotensin II; Hadjian AJ et al.; Addition of angiotensin II (0.3 microM) to bovine adrenal fasciculata cell suspensions prelabeled with {32P} induced a rapid (15 seconds) and marked decrease of the radioactivity from phosphatidylinositol 4,5-biphosphate (62%) and phosphatidylinositol 4-monophosphate (35%) . This effect was concentration-dependent and specifically inhibited in the presence of (Sar1-Ala8)-angiotensin II; it was also completely prevented in the absence of extracellular calcium . The present data appear to illustrate the earliest biological response detectable in bovine fasciculata cells under angiotensin II challenge.

J Biol Chem, 1984 Oct 25, 259(20), 12619 - 27
Morphological and physiological factors affecting oxygen uptake and release by red blood cells; Vandegriff KD et al.; The kinetics of oxygen uptake and release by human, salamander (Amphiuma means), and artificially constructed red cells were measured under a variety of physiological conditions using stopped-flow, rapid mixing techniques . The results were analyzed quantitatively using the generalized, three-dimensional disc model that was developed in two previous publications (Vandegriff, K . D., and Olson, J . S . (1984) Biophys . J . 45, 825-835 and Vandegriff, K . D., and Olson, J . S . (1984) J . Biol . Chem . 259, 12609-12618) . The apparent rate of gas exchange is governed primarily by the oxygen flux at the red cell surface . In the case of uptake, this flux is roughly independent of intracellular chemical reaction parameters and inversely proportional to the thickness of the unstirred solvent layer which is adjacent to the red cell surface . For release experiments in the presence of high concentrations of sodium dithionite, the flux at the cell surface is inversely proportional to the oxygen affinity of the intracellular hemoglobin and roughly independent of the thickness of the external unstirred solvent layer . As a result, the effects of cell size, internal heme concentration, and pH are expressed differently in the two types of kinetic experiments . The rate of oxygen uptake depends on roughly the second power of the surface area to volume ratio of the erythrocyte, whereas the rate of release is much less dependent on the size and shape of the red cell . The half-time of oxygen uptake is directly proportional to intracellular heme concentration for cells of equivalent geometries; the half-time of oxygen release is linearly dependent on internal heme concentration but, at low heme concentrations, is determined primarily by the rate of oxygen dissociation from hemoglobin . The rate of cellular oxygenation is roughly independent of pH and internal 2,3-diphosphoglycerate concentration; in contrast, the rate of deoxygenation depends markedly on these conditions . As the pH is lowered or the internal diphosphoglycerate concentration is raised, the overall oxygen affinity of the cell suspension decreases severalfold, and the rate of oxygen release increases by roughly the same extent.

FEBS Lett, 1984 Oct 15, 176(1), 55 - 60
Pyrimidine metabolism in Trichomonas vaginalis; Heyworth PG et al.; Pyrimidine metabolism in Trichomonas vaginalis was investigated using washed cell suspensions of the organism with radiolabelled pyrimidine ring precursors and preformed pyrimidines . The precursors {14C}orotate, {14C}bicarbonate and {14C}aspartate were not incorporated into the pyrimidine bases of trichomonal nucleic acids, indicating that the protozoan is unable to synthesise the pyrimidine ring and is dependent on the salvage of exogenous pyrimidines . {3H}uracil, {3H}uridine, {3H}cytidine, deoxy{3H}cytidine and {3H}thymidine were all efficiently salvaged, and interconversion between cytosine and uracil nucleotides was detected . Thymidylate synthase activity was not detected, suggesting that T . vaginalis is dependent upon an exogenous supply of thymidine for TMP synthesis.

Cell Immunol, 1984 Oct 15, 88(2), 401 - 10
Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant; Mulder AH et al.; The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay . This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate . Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence . Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension . This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days . The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration . These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S . The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated . Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte) . During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.

Tsitologiia, 1984 Oct, 26(10), 1203 - 8
{Sedimentation analysis of cell suspensions}; Doronin IuK; Simple methods of measurements of the velocity sedimentation at unit gravity of cells obtained by disaggregation procedure from some fixed tissues are described . The relationship between the velocity sedimentation and cellular volume is discussed.

Eur J Cancer Clin Oncol, 1984 Oct, 20(10), 1249 - 59
Flow cytofluorometric analysis of serial biopsies of tumours of the uterine cervix; Dyson JE et al.; The technique of flow cytofluorometry has been employed to assess, by means of cell suspensions prepared from serial biopsies, the radioresponsiveness of tumours of the uterine cervix . This enables DNA profiles and content of proliferating cells to be determined prior to treatment and during external beam and intracavitary therapy . Results show that elimination of hyperdiploid and hypertetraploid cells and reduction in the proliferating fraction of cells can readily be monitored by this method during therapy . This information, quickly available during treatment, may assist in estimating radioresponsiveness of the tumour and possible prognosis for the patient . Dose fractionation schedules may also be adjusted according to tumour response to therapy . Our results, however, show no relationship between histopathological classification of a tumour (WHO) and its ploidy state . The advanced stages of the disease (II and III) do, however, show an increased content of hypertetraploid cells in the tumour biopsies.

Radiologe, 1984 Oct, 24(10), 478 - 87
{Hemorheologic effects of ioxaglate: a contribution to an interpretation of the effect of hyperosmolar roentgen contrast media on the fluidity of erythrocytes}; Schmid-Schonbein H et al.; Almen and Aspelin have shown that the use of non-ionic radio contrast media allows the iodine concentration to be increased (which is desirable because of its effect on radio opacity) without a very large increase in osmolarity (which is undesirable because it impairs the fluidity of erythrocytes) . This latter effect can also be diminished by reducing the osmolarity of a dimeric contrast medium as has been achieved by incorporating more iodine atoms into the molecule in the case of Ioxaglate (Hexabrix) . In various microrheological tests systems, the fluidity of packed red cell suspension, the corrected filtration rate though 5 micron pores and the relative apparent viscosity of blood-contrast media mixtures (1 to 50% concentration) were determined in experiments comparing this compound with Urografin 76 of the same iodine content . In all systems, the former showed fewer rheological effects . In whole blood viscometry, this can be detected only after appropriate corrections for the effects of the two contrast media on hematocrit and plasma viscosity . As there is a more pronounced water shift from the cells to the plasma, Urografin tends to reduce the viscosity of the plasma-contrast media mixture . The concomitant reduction in MCV and hematocrit level tends to conceal the macrorheological influence of strong cell stiffening . This microrheological effect of the dehydrated cells becomes immediately obvious when the viscometric data are corrected for hematocrit value and plasma viscosity effects.

J Embryol Exp Morphol, 1984 Oct, 83, 43 - 61
Intercellular relationships during cavitation of aggregates of extraembryonic endoderm cells from gastrulating chick embryos; Milos N et al.; Extraembryonic endoderm cells from gastrulating chick embryos undergo epiboly and change from a multilayered cell group to a single cell layer surrounding the yolk . Single cell suspensions from this cell layer can aggregate in vitro to form aggregates that cavitate . To study the stages of cavitation aggregates were harvested after different times in culture, and fixed and processed for light and electron microscopy . In aggregates harvested at 75 min of culture cell contact consisted of areas of parallel and close membrane apposition and interdigitation . Desmosomes were occasionally observed . Aggregates in the early stages of cavitation (24 h) contained numerous intercellular spaces bordered by irregularly shaped cells which appeared to be digesting their yolk and releasing material extracellularly . Long cytoplasmic projections were extended into these spaces . In addition to regions of parallel membrane apposition and interdigitation, desmosomes and adherens junctions were observed . Cells closer to the periphery of the aggregates displayed fewer cell projections and also showed signs of release of material extracellularly . After 48 h of culture, a single smooth-walled central cavity was present and cells still exhibited signs of extracellular release of material . These same cell shapes and intercellular junctions were also observed when area opaca tissue dissected from gastrulating embryos was examined . Aggregates of different sizes were created and cultured . The results suggest that a critical tissue mass may be important for cavitation.

Transplantation, 1984 Oct, 38(4), 392 - 5
Minor antigen graft-versus-host reactions revealed in irradiated spleen and popliteal lymph node assays; Claman HN et al.; The graft-versus-hot (GVH) reaction across minor (non-H-2) histocompatibility barriers was studied in mice, in vivo . To increase GVH potential and to mimic clinical bone marrow transplantation protocols, we modified the popliteal lymph node (PLN) and the splenomegaly assays by irradiating the recipients before they received allogeneic lymphoid cell suspensions . In several combinations across major (H-2), minor (non-H-2) and multiple minor (non-H-2 plus minor lymphocyte stimulation) barriers, increased recipient organ weight (a measure of GVH activity) was seen with irradiated F1 recipients of parental cells . The irradiated splenomegaly (x-splenomegaly) assay was more sensitive than the (x-PLN) assay, but both correlated with in vivo GVH experiments of the P----F1 variety . The x-splenomegaly test indicated histoincompatibility in a system (B10.D2----BALB/c) in which the primary in vitro mixed leukocyte reactions is nonreactive, but in which systemic GVH can be induced . The x-splenomegaly test should be useful in analyzing complex reactions involving minor histocompatibility antigens in vivo.

J Neurosurg, 1984 Oct, 61(4), 761 - 6
Intramedullary canine spinal cord tumor model; Salcman M et al.; The development of a transplantable model brain tumor in the neonatal dog, the adaptation of the tumor to tissue culture, and the successful growth of the tumor in adult mongrel dogs has been adapted to producing similar tumors in the thoracic spinal cord of the adult dog . Ten adult dogs, weighing 4 to 25.4 kg each, were subjected to formal laminectomy . The tumor cell suspension was injected by hand with a Hamilton syringe at two or three sites over a distance of 1 cm; each site received an injection volume to 0.02 to 0.05 cc of the cell suspension after the dura had been opened . Immediately after injection the field was copiously irrigated and the puncture area sealed with a single drop of ethyl cyanoacrylate . Tumor cells for injection were obtained by thawing ampules stored at -195 degrees C in a mixture of 10% dimethyl sulfoxide and RPMI 1640 culture medium . Cells were resuspended in Hank's balanced salt solution and 15% fetal calf serum on ice . Solutions had 90% cell viability, and animals received a dose in the range of 3 to 13 X 10(6) cells . Eight animals developed tumors and became paraparetic on the 9th to 14th postinjection day . Metrizamide myelography in three animals revealed complete blocks; two animals underwent spinal computerized tomography (CT) and demonstrated syringohydromyelia . Histology revealed the tumors to be highly vascular primitive neoplasms that invaded the surrounding cord . This spinal cord tumor model is large enough to be operated on, studied by CT and myelography, and subjected to pharmacological, electrophysiological, and blood flow study.

J Infect Dis, 1984 Oct, 150(4), 508 - 12
Comparison of strains of Legionella pneumophila serogroup 1 isolated in four Amsterdam hospitals from patients and hot-water supplies; Zanen-Lim OG et al.; Legionella pneumophila serogroup 1 was isolated from patients (14 strains) and hot-water taps (49 strains) in four hospitals in Amsterdam . Precipitation patterns obtained by reaction of heated cell suspensions and rabbit antisera in the double diffusion test revealed antigenic properties of serogroup 1 strains from water unique to each of the four hospitals . By this method similarity could be demonstrated between strains from patients and hot water in three of the four hospitals . No such relationship was present in the fourth hospital, a fact that could be explained by the case histories of the patients . Comparison of strain antigens in the double diffusion test is simple and suitable for epidemiological studies of L . pneumophila.

Endocrinology, 1984 Oct, 115(4), 1406 - 11
Preparation of thyrotroph cells from adult male rat pituitary glands by centrifugal elutriation; Chamras H et al.; To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs . For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells . Trypsin-dispersed cells (4-8 X 10(7} were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm . Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min) . Cell recovery was 77-98% . The viability of the cells after elutriation was 90-95% based on trypan blue exclusion . Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA . Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA . The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs . Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5-fold; there was no significant increase in TSH release in fractions 3-6 . Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells . This technique should be valuable for study of the metabolism of thyrotrophs.

Int J Radiat Oncol Biol Phys, 1984 Oct, 10(10), 1913 - 22
Characterization of the biophysical properties of human tumor and bone marrow cells as a preliminary step to the use of centrifugal elutriation in autologous bone marrow transplantation; Keng PC et al.; The principle of centrifugal elutriation (CE) depends on a balance of an outwardly directed centrifugal force and inwardly directed fluid flow and buoyant forces . This method (CE) can be used effectively to separate cells on the basis of size . In the murine model, neoplastic cells from different tumors are generally larger than bone marrow cells and can be removed from bone marrow almost completely with centrifugal elutriation . In order to determine if CE is capable of eliminating human tumor cells from harvested bone marrow (BM), the biophysical characteristics of a variety of human tumor cells and bone marrow cells were determined . Human tumor cells were dispersed into single cell suspensions by several enzymatic digestion and mechanical dissociation methods . The size and density characteristics of these cells were determined with an electronic particle counter and channelyzer and density gradients . Of 40 solid tumors studied, 29 tumors had cell size distributions distinctively larger than BM, as was found in the experimental animal model . The cell size distributions of tumor cells from 11 solid tumors and 7 leukemias were not substantially different from that of BM . Mixtures of BM and cultured human hypernephroma, ovarian, and neuroblastoma cells, were separated into BM and tumor fractions by CE . The separation results as indicated by the labeling index and colony forming efficiency of tumor cells in each fraction showed that a BM fraction virtually free of tumor cells could be obtained . Thus, CE should be able to separate BM cells from most tumor cells metastatic to BM.

Am Rev Respir Dis, 1984 Oct, 130(4), 650 - 8
Accurate quantification of cells recovered by bronchoalveolar lavage; Saltini C et al.; Quantification of the differential cell count and total number of cells recovered from the lower respiratory tract by bronchoalveolar lavage is a valuable technique for evaluating the alveolitis of patients with inflammatory disorders of the lower respiratory tract . The most commonly used technique for the evaluation of cells recovered by lavage has been to concentrate cells by centrifugation and then to determine total cell number using a hemocytometer and differential cell count from a Wright-Glemsa-stained cytocentrifuge preparation . However, we have noted that the percentage of small cells present in the original cell suspension recovered by lavage is greater than the percentage of lymphocytes identified on cytocentrifuge preparations . Therefore, we developed procedures for determining differential cell counts on lavage cells collected on Millipore filters and stained with hematoxylin-eosin (filter preparations) and compared the results of differential cell counts performed on filter preparations with those obtained using cytocentrifuge preparations . When cells recovered by lavage were collected on filter preparations, accurate differential cell counts were obtained, as confirmed by performing differential cell counts on cell mixtures of known composition, and by comparing differential cell counts obtained using filter preparations stained with hematoxylin-eosin with those obtained using filter preparations stained with a peroxidase cytochemical stain . The morphology of cells displayed on filter preparations was excellent, and interobserver variability in quantitating cell types recovered by lavage was less than 3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood, 1984 Oct, 64(4), 768 - 73
Studies of lymph nodes from patients with classical hemophilia; Andes WA et al.; Within the last 18 months, we have noted the development of unexplained lymph node enlargement in otherwise asymptomatic patients with hemophilia . Because such changes are poorly understood and, in some patient groups, may be related to the acquired immunodeficiency syndrome (AIDS), we studied the enlarged lymph nodes in four patients with severe factor VIII deficiency and abnormally low peripheral blood helper-inducer/suppressor cell (OKT4/OKT8) ratios . Surgically excised lymph nodes were studied for histopathologic, electron microscopic, and chromosomal changes . Cell suspensions from these and normal nodes were also studied using monoclonal antibodies . Excised lymph nodes showed follicular hyperplasia . Electron microscopy revealed no viral particles or vesicular rosettes . Chromosomal aberrations included an acrocentric marker chromosome in one patient and monosomy 21 in another . T lymphocyte ratios (OKT4/OKT8) in lymph node suspensions were lower than those in nodes from normal controls (1.2 v 6.1) and reflected the lymphocyte ratio in peripheral blood . Mature B cell percentages were increased in the lymph nodes from patients with hemophilia (38% v 27% in controls) . Patients treated with factor VIII concentrates and male homosexuals have similarities in persistent lymph node enlargement, histologic features of follicular hyperplasia, and changes in lymph node and circulating lymphocyte subpopulations.

J Virol, 1984 Oct, 52(1), 290 - 2
Monoclonal antibodies to a monkeypox virus polypeptide determinant; Roumillat LF et al.; Three monkeypox virus (MPV) antibody-secreting murine monoclones were characterized as being of the immunoglobulin G1 isotype, gave a 4+ reaction in the indirect fluorescent-antibody test, gave a positive reaction in the enzyme immunoassay, and did not neutralize MPV . These monoclonal antibodies were determined by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis transblot method to react to a 15,500-molecular-weight MPV polypeptide . This reactivity could not be removed by adsorption to a vaccinia virus-infected cell suspension . The three monoclonal antibodies were specific for MPV when tested against epidemiologically unrelated isolates of cowpox virus, variola virus, vaccinia virus, and MPV.

J Histochem Cytochem, 1984 Oct, 32(10), 1028 - 34
Immunocytochemical assays of amylase and chymotrypsinogen in rat pancreas secretory granules . Efficacy of using immunogold-labeled ultrathin cryosections to estimate relative protein concentrations; Posthuma G et al.; An exploration was conducted as to whether the relative concentration of two intracellular proteins could be evaluated quantitatively from their labeling densities in ultrathin cryosections labeled with the immunogold technique . As a model rat pancreatic cells were used in which the content of amylase (Am) and chymotrypsinogen (Ch) was experimentally altered . Rats were fed either normal laboratory chow or food containing soybean trypsin inhibitor (STI), which affects the Am/Ch ratio in the tissues . The changes in Am and Ch protein levels and enzyme activities were measured biochemically in cell suspension homogenates or in zymogen granule fractions . Within 5 days a maximal change in the Am/Ch was observed as a result of adaptation to the STI diet . The Am/Ch ratio determined biochemically was compared with that from counts of gold particles bound to the respective protein in immunogold-labeled cryosections . The two data sets matched fairly well, indicating that the intensity of the immunoreaction is a reliable reflection of antigen concentration in this system.

Am J Clin Pathol, 1984 Oct, 82(4), 439 - 41
Use of a modified procedure for treating small amounts of red blood cells with 2-aminoethylisothiouronium bromide; Ellisor SS et al.; Kell null red blood cell samples can be prepared using 2-aminoethylisothiouronium bromide (AET) . This article describes a modification whereby only three drops of a 5% red blood cell suspension may be AET treated . This procedure has been used routinely in the authors' laboratory for more than a year . One patient's serum contained anti-Kpb plus anti-C and anti-D . Tests with panel cells pretreated with AET made it possible to identify underlying Rh antibodies without using a panel of genetic Kpb negative red blood cells . Of 24 red blood cell eluates from patient sample with warm autoantibodies, one had specificity within the Kell blood group system . This small volume AET-treatment method is a quick screen for the differentiation of a Kell-related specificity from a non-Kell specificity of both warm autoantibodies and alloantibodies to high-incidence antigens.

Carcinogenesis, 1984 Oct, 5(10), 1267 - 75
Emergence of a population of small, diploid hepatocytes during hepatocarcinogenesis; Schwarze PE et al.; The sequential treatment of young Wistar rats with two different carcinogens (diethylnitrosamine - plus partial hepatectomy - as an initiator, and 2-acetylaminofluorene as a cytotoxic selection pressure) induces the appearance of foci and nodules of liver cells which are phenotypically altered . By means of an algorithm which takes into account binuclearity as well as cell-to-cell aggregation it is possible to compute cellular ploidy distributions from flow-cytometric analysis of either hepatocyte suspensions or suspensions of hepatocytic nuclei . Cell suspensions isolated from carcinogen-treated rats can be shown to contain, already after 8 weeks, approximately 70% small, diploid hepatocytes, whereas suspensions from normal or partially hepatectomized control livers contain only approximately 10% diploid cells (the remainder being mostly tetraploid) . Isolated nodules, i.e., expanding clones of proliferating cells, believed to be neoplastic precursor lesions, contained almost only diploid cells . These observations suggest that the selective outgrowth of a population of small, diploid hepatocytes may be a significant early step in the development of liver cancer.

Proc Natl Sci Counc Repub China B, 1984 Oct, 8(4), 275 - 81
An improved dispersed adrenal cell assay for corticotropin in rat plasma; Lin JH et al.; The present study was designed to improve the dispersed adrenal cell technique for determining adrenocorticotrophic hormone (ACTH) concentrations in small amounts of rat plasma . Priming with ACTH, incubation with methyl-isobutylxanthine, or dexamethasone pre-treatment were employed as modifications . Of these, only dexamethasone pre-treatment increased the sensitivity of the assay . The adrenal fragments obtained from 10-12 adult male rats pre-treated with dexamethasone (100 micrograms/kg B.W.) one hour before sacrifice, were digested with collagenase and deoxyribonuclease solution for 30 minutes . The dispersed cells were collected by centrifugation and resuspended in Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.5% bovine serum albumin . Aliquots of cell suspension (3-4 X 10(4)/tube) were incubated with various doses of ACTH1-24 or the eluate of plasma samples at 37 degrees C for 2 hours in an atmosphere of 95% O2/5% CO2 in a Dubnoff shaker . The quantity of corticosterone produced was measured fluorimetrically . The assay is precise (lambda = 0.06), extremely sensitive (10 fg/tube), and convenient . One skilled technician can handle 15 to 20 plasma samples per day using 10 rats as the source of assay cells . ACTH can be measured in as little as 10-50 microliters of eluate.

Biochim Biophys Acta, 1984 Sep 19, 776(1), 1 - 9
K+-valinomycin and chloride conductance of the human red cell membrane . Influence of the membrane protonophore carbonylcyanide m-chlorophenylhydrazone; Bennekou P; Chloride ion conductance of the human red cell membrane has been calculated, as the ratio between ion net charge flux and driving potential . The proton carrier CCCP was used to monitor changes in membrane potential following addition of valinomycin in sufficient quantities to raise the K+ conductance to a level comparable to the Cl- conductance . A K+-specific electrode was used to monitor changes in extracellular K+ concentration, and an H+-sensitive glass electrode for changes in extracellular pH, reflecting changes in membrane potential . The effects of varied concentrations of valinomycin and CCCP upon K+ and Cl- conductances were studied . It was found that, within an experimental error of about 10% S.D., the chloride conductance was constant for valinomycin concentrations in the range 1.0 X 10(-8)-1.0 X 10(-6), and for CCCP-concentrations in the range 2.0 X 10(-7)-2.0 X 10(-5) mol per litre cell suspension, while at a constant concentration of valinomycin the induced K+ conductance was considerably augmented by addition of CCCP.

Dtsch Med Wochenschr, 1984 Sep 7, 109(36), 1356 - 61
{Clinical significance of the short-term incubation test for the therapy of metastatic breast cancer}; von Matthiessen H et al.; Between January 1978 and October 1980 97 tissue samples of histologically verified carcinoma of the breast were received for performance of the short-term incubation test . 25 tumour samples could not be prepared as cell suspension sufficient for testing . Among the 72 performed tests there were 9 with stimulation of 3H-uridine uptake by doxorubicin . Thus only 63 tests could be evaluated . Results were correlated with clinical data of the patients . No significant correlations could be established between tumour stage at time of diagnosis, age, menopausal state, receptor state, and the test result . There was also no differentiation between favourable and unfavourable prognoses as regards free interval and rate of survival . A correlation between results of tests and success or failure of cytostatic treatment could not be ascertained.

J Bacteriol, 1984 Sep, 159(3), 843 - 9
Physiological function of hydrogen metabolism during growth of sulfidogenic bacteria on organic substrates; Lupton FS et al.; Desulfovibrio vulgaris Madison and Thermodesulfobacterium commune contained functionally distinct hydrogenase activities, one which exchanged 3H2 into 3H2O and was inhibited by carbon monoxide and a second activity which produced H2 in the presence of CO . Cell suspensions of D . vulgaris used either lactate, pyruvate, or CO as the electron donor for H2 production in the absence of sulfate . Both sulfidogenic species produced and consumed hydrogen as a trace gas during growth on lactate or pyruvate as electron donors and on thiosulfate or sulfate as electron acceptors . Higher initial levels of hydrogen were detected during growth on lactate-sulfate than on pyruvate-sulfate . D . vulgaris but not T . commune also produced and then consumed CO during growth on organic electron donors and sulfate or thiosulfate . High partial pressures of exogenous H2 inhibited growth and substrate consumption when D . vulgaris was cultured on pyruvate alone but not when it was metabolizing pyruvate plus sulfate or lactate plus sulfate . The data are discussed in relation to supporting two different models for the physiological function of H2 metabolism during growth of sulfidogenic bacteria on organic electron donors plus sulfate . A trace H2 transformation model is proposed for control of redox processes during growth on either pyruvate or lactate plus sulfate, and an obligate H2 cycling model is proposed for chemiosmotic energy coupling during growth on CO plus sulfate.

Br J Surg, 1984 Sep, 71(9), 659 - 63
Viability of exfoliated colorectal carcinoma cells; Umpleby HC et al.; The viability of tumour cells shed into the intestinal lumen was determined in 49 patients with carcinoma of the large bowel . Preoperative colorectal lavage was performed in 19 patients and irrigation of the cut ends of the operative specimen in 30 patients . The resulting cell suspensions were centrifuged on a Nycodenz linear density gradient column so that tumour cells, being larger, were concentrated in a band at the top . In 14 of 19 colorectal lavage cases viable tumour cells were recovered, as assessed by their characteristic morphology and ability to exclude trypan blue . A median of 0.78 X 10(6) viable tumour cells was recovered . The median percentage cell viability in the suspension was 92, i.e . 8 per cent of the tumour cells were dead (stained with trypan blue) . In eight specimens viability was confirmed by the ability of tumour cells to hydrolyse fluorescein diacetate . In 17 of 30 proximal resection margin irrigations a median of 0.55 X 10(5) viable tumour cells was recovered, with a median percentage viability of 92.5 . In 15 specimens the neoplastic cells showed fluorescence . In 21 of 25 distal resection margin irrigations a median of 1.92 X 10(5) viable tumour cells was recovered with a median percentage cell viability of 79.3, and fluorescence was observed in all specimens . The number of viable tumour cells did not correlate with the stage, differentiation, diameter or fixity of the tumour . However, the number of tumour cells recovered from the distal resection margin was inversely related to the distance of the tumour from that margin (Rank Difference Coefficient R = -0.6) . Thus viable exfoliated tumour cells were demonstrated in 52 of 74 specimens (70 per cent) . Their presence in large numbers at the site of intestinal anastomoses supports a potential role in the aetiology of suture-line recurrence.

J Cell Biol, 1984 Sep, 99(3), 1173 - 8
Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures; Adler R et al.; We report here that cells present in embryonic chick retinal monolayer cultures express differentiated properties characteristic of chick cones developing in vivo . Cell suspensions from 8-d chick embryo retina (a stage when photoreceptor differentiation has not yet started) were cultured for up to 7 d in low density, glial-free monolayers . Under these conditions, monopolar cells represent approximately 40% of the total number of process-bearing neurons . After 6 d in vitro, most of these monopolar cells showed morphological features reminiscent of developing chick cones . These features could be detected with phase-contrast microscopy, lectin cytochemistry, and transmission and scanning electron microscopy . Characteristic cone traits expressed by cultured monopolar cells included the following: (a) a highly polarized organization; (b) a single, short, usually unbranched neurite; (c) the polarized position of the nucleus close to the origin of the neurite; (d) characteristic cone inner segment features such as abundant free ribosomes, a polarized Golgi apparatus, a cluster of mitochondria distal to the nucleus, a big, membrane-bound, pigment-containing vacuole reminiscent of the "lipid droplet" characteristic of chick cones, and at least in some cases, a well-developed paraboloid; (e) the presence of a complex of apical differentiations including abundant microvilli and in some cases also a cilium-like process; and (f) the staining of the apical region of the cell with peanut lectin, which has been shown to be selective for chick embryo cones (Blanks, J.C., and L.V . Johnson, 1983, J . Comp . Neurol., 221:31-41; and Blanks, J.C., and L.V . Johnson, 1984, Invest . Ophthalmol . Visual Sci., 25:546-557) . This pattern of differentiation achieved by 8-d chick retina cells after 6 d in vitro is similar to that shown by 14-d-old chick embryo cones in vivo . Outer segments are not present at this stage of development either in vivo or in vitro . This experimental system is now being used to search for cellular and molecular signals controlling survival and differentiation of cone cells.

Cell Immunol, 1984 Sep, 87(2), 674 - 7
Natural killer activity of Kurloff cells: a direct demonstration on purified Kurloff cell suspensions; Debout C et al.; In order to study their natural killer effect, guinea pig splenic Kurloff cells were fractionated by Percoll discontinuous density gradient centrifugation . Kurloff cells were collected and tested for cytotoxicity in a 24-hr chromium-release test . Comparison of different splenic cellular samples (of males or estrogenized females) with increasing percentage of Kurloff cells, revealed a highly significant positive correlation (r = 0.93, alpha less than 0.01) with the cellular cytotoxicity developed against the K 562 target cells.

Blood, 1984 Sep, 64(3), 649 - 55
Light scattering by polymorphonuclear leukocytes stimulated to aggregate under various pharmacologic conditions; Yuli I et al.; Enhancement of light transmission has been widely accepted as an empirical measure of cell aggregation in suspension . Several years ago, this measurement was introduced to the study of polymorphonuclear leukocyte (PMN) aggregation by adapting a hypothesis originally developed for platelets . Accordingly, an increase in light transmission is attributed to cell aggregation and a decrease in transmission below baseline level is indicative of increased cell symmetry . We tested this hypothesis for human PMNs by comparing the whole cell shape or the cells' aggregation state with the light transmission under particular experimental conditions . The PMN light response to the chemoattractant, f-Met-Leu-Phe, in the presence of low doses of aliphatic alcohols was associated with transient enhanced transmission, followed by a rapid decrease below baseline . In contrast to the platelet hypothesis, the below-baseline effect coincided with a decrease in PMN symmetry from spheres to wedge-shaped (polarized) cells . PMNs fixed mildly with various doses of formaldehyde (0.1% to 0.3%) were completely aggregated by the addition of 50 micrograms/mL phytohemagglutinin (PHA) . Despite the complete aggregation of the PMNs, there was a dose-dependent inhibition of the above-baseline level transmission response by the fixative, demonstrating a clear dichotomy between aggregation and increased light transmission . However, PMN aggregation could be monitored by observing the pattern of enhanced light transmission coupled with decreased perpendicular light scattering immediately after the stirring of the cell suspensions was stopped . PMNs aggregated by PHA cleared from suspension very rapidly (t1/2 less than or equal to one minute), whether or not they were formalin-fixed . In contrast, unaggregated cells revealed constant transmission and perpendicular scattering intensities for as long as five minutes after the stirring was stopped . The clearance patterns of f-Met-Leu-Phe-stimulated PMNs initiated even at the time of maximally increased light transmission were indistinguishable from those of the unstimulated cells, indicating the absence of aggregation . The lack of correlation between light output and changes in cell shape or degree of aggregation of PMNs causes us to reject the hypothesis that attributes enhanced light transmission to PMN aggregation . We suggest that modulation of light transmission by PMNs stimulated with chemoattractants is due to changes in light output from subcellular objects.

Cancer Res, 1984 Sep, 44(9), 3870 - 2
Lymphokine-induced migration inhibition of murine tumor cells derived from solid neoplasms; Donskoy M et al.; We have previously described a noncytotoxic lymphokine, tumor migration inhibition factor, with the capacity of inhibiting the in vitro migration of a variety of tumor cells maintained by animal passage in ascitic form . In the present study, we demonstrate that it is possible to prepare viable, motile tumor cell suspensions from solid tumors and that those cells migrate better than cells that had been propagated in ascitic form . Such preparations derived from solid tumors are inhibited by tumor migration inhibition factor to a degree comparable to that achieved with cells from ascitic tumors . Comparative studies utilizing a methylcholanthrene-induced fibrosarcoma as well as solid and ascitic forms of P815 mastocytoma and Ehrlich tumor demonstrate that responsiveness to tumor migration inhibition factor is not merely a property conferred upon tumor cells by prior animal passage in suspension . These results provide a further suggestion that the capacity for lymphokine-induced migration inhibition is a general property of tumor cells . This in turn raises the possibility that this capacity might vary in a predictable manner with malignant potential.

Tsitologiia, 1984 Sep, 26(9), 983 - 95
{Cell electrophoresis}; Sungurov AIu; The physical principle of cell electrophoresis, the role of media pH and ionic strength, and the nature of cell coat electric charge are considered . The advantages and defects of analytic and preparative cell electrophoresis variants are analyzed . The results of use of cell electrophoresis for studying and separation of erythrocyte, leukocyte and bone marrow cell suspension are presented.

Parasite Immunol, 1984 Sep, 6(5), 435 - 42
Antigen-specific and concanavalin A-induced lymphocyte blastogenesis responses during the course of Plasmodium berghei infection in rats; Raybourne R et al.; Antigen specific and concanavalin A (Con A)-induced lymphocyte blastogenesis responses were monitored in the spleens of rats infected with Plasmodium berghei . Con A responses were depressed only at the time of peak parasitaemia . The antigen specific blastogenesis response was either not in evidence, or at a low level during the periods of patent or subpatent infection (up to 8 weeks after infection) . Higher levels of blastogenesis were seen from 8 to 12 weeks after infection, which correlates with clearance of subpatent infection . Fractionation of immune spleen cell suspensions on nylon wool columns indicated that purified T cells did not respond to parasite antigen.

Exp Cell Res, 1984 Sep, 154(1), 125 - 35
Isolation and characterization of vitamin-A-storing lung cells; Okabe T et al.; Vitamin A-storing cells have been shown to be distributed among various organs and tissues, including the lungs . In order to investigate this unique type of cell, the in vitro isolation has been carried out from rat lungs . Lungs were perfused with EGTA and collagenase solution in situ, and were digested with trypsin-collagenase solution at 60-min intervals for 2 h . Then, the cell suspensions obtained were incubated at 37 degrees C in F-10 medium supplemented with 10% fetal bovine serum (FBS) for 72 h . Non-adherent cells were then removed by vigorous washing with medium, and the resultant cell monolayer was harvested with trypsin to remove the contaminating macrophages . These cell fractions were shown to contain more than 96% of vitamin A-storing cells, judged by electron and fluorescence microscopic examinations . The cells grown in vitro retained well the overall morphology characteristic of the vitamin A-storing cells found in lung tissues . The isolated cells grew well in vitro and the growth was inhibited by D-valine or cis-hydroxyproline . The progeny of the cells still contained vitamin A lipid droplets after several transfer generations . Characteristic networks of fibronectin were also demonstrated around the cells . These results have shown that vitamin A-storing cells in the lung was successfully isolated from rat lungs and the cells possessed fibroblast-like characters storing vitamin A in small lipid droplets.

Clin Exp Immunol, 1984 Sep, 57(3), 614 - 20
Peripolesis followed by cytotoxicity in chronic idiopathic inflammatory bowel disease; Wilders MM et al.; Antigen presenting veiled cells have recently been described in cell suspensions prepared from the gut wall of patients with chronic idiopathic inflammatory bowel disease (CIBD) . The normal gut wall is virtually devoid of these cells . In this report we describe a phenomenon known as peripolesis studied by phase contrast cinematography . This is a process in which lymphocytes are seen to wander around larger target cells . These could be identified ultrastructurally as Ia positive veiled cells . In most cases peripolesis was followed by lysis of the target cell . Peripolesis was recorded in cell suspensions of three out of seven patients with ulcerative colitis and in three out of nine patients with Crohn's disease; furthermore peripolesis was observed in one out of two patients with non-classifiable CIBD . In four cell suspensions showing peripolesis, cell lysis could be recorded and was especially striking in ulcerative colitis . Peripolesis involving veiled cells was previously described in delayed hypersensitivity reactions . This study lends support to the concept that delayed allergic reactivity plays a part in chronic inflammatory bowel disease . The antigens involved are, however, completely unknown.

Clin Exp Immunol, 1984 Sep, 57(3), 626 - 32
Characterization of lymphoid cells in the thyroid of patients with Graves' disease; Warford A et al.; The distribution and function of lymphoid cells has been investigated in thyroid glands obtained at operation from 16 patients with Graves' disease (GD) using a peroxidase technique to enumerate total T and B lymphocytes as well as helper and suppressor T cell subsets in tissue sections . A spectrum of lymphocytic infiltration was observed and the increase from minimal numbers of immune cells in some GD thyroids to focal thyroiditis in others appeared to be due to a rise in all the lymphoid cell types analysed and was not the result of major change in any one lymphoid compartment . T cells were diffusely distributed whereas B cells tended to occur in aggregates . Small numbers of OKT6+ cells (possibly antigen presenting cells) were observed although these were less numerous than in lymphoid organs such as tonsil . Lymphoid cell suspensions prepared from the thyroid tissue of five of seven GD individuals treated pre-operatively with propranolol synthesized thyroid autoantibodies spontaneously in culture and this synthesis was decreased in the presence of pokeweed mitogen . Since the OKT8+ T cell subset has been shown to suppress immunoglobulin production by lymphocyte cultures containing mitogen, it appears that the suppressor T cells, which are readily demonstrable in GD thyroid sections, are functional . It seems unlikely, therefore, that a defect in this type of suppression is responsible for the initiation or perpetuation of the autoimmune response to thyroid antigens in GD.

Cell Immunol, 1984 Sep, 87(2), 366 - 78
T and B cells induce macrophages which suppress proliferation but not lymphokine secretion; Li W et al.; In vitro culture of mouse spleen cells for 2 days or more leads to the production of adherent, phagocytic, Thy-1-, Ia+, Lyt-2- cells ("suppressor macrophages") which strongly inhibit the proliferative response of T and B lymphocytes to a variety of stimuli: mitogens, specific antigens, and antigen-nonspecific growth factors . Suppressive activity fails to develop, however, in cultured spleen cells from which nonadherent cells have been removed before the initial 48-hr incubation, and only partial suppression is obtained from cell suspensions from which T cells have been depleted before culture . We find that the requirement for nonadherent cells can be replaced by graded doses of lymphocytes . Lyt-2- and Lyt-2+ T cells are about equally potent in inducing suppressive activity in nonadherent cells . Surprisingly, B cells (containing fewer than 0.1% contaminating T cells) are also able to induce suppression in this system . The suppression induced includes both indomethacin-sensitive and indomethacin-resistant components . Interestingly, not all stages of mitogen-induced T-cell activation are blocked by these adherent cells: proliferation is inhibited, but production of interleukin 2 (IL-2) and interleukin 3 (IL-3) is unaffected.

Blood, 1984 Sep, 64(3), 662 - 6
Purification of common acute lymphoblastic leukemia antigen positive cells from normal human bone marrow; Hokland P et al.; Mononuclear cells expressing the common acute lymphoblastic leukemia antigen (CALLA) were purified from normal adult human bone marrow, where they constitute a small fraction of the total population . This was accomplished by a two-step purification from Ficoll-Hypaque-isolated mononuclear cells . Isolated mononuclear cells were first labeled with a mixture of monoclonal antibodies (MoAb) specific for myeloid and erythroid precursor cells, and immune rosettes were then formed with sheep erythrocytes coated with rabbit anti-mouse antibodies (R/M-SRBC) . Sedimentation through Ficoll-Hypaque then eliminated the majority of mature myeloid cells . The second step consisted of labeling the remaining rosette-negative cells with CALLA-specific MoAb and purifying CALLA+ cells by fluorescence activated cell sorting . Alternatively, CALLA+ cells were purified in a second R/M-SRBC rosette sedimentation step . The purified CALLA+ cells, which morphologically were medium to large lymphoid cells, were subsequently studied using dual fluorescence techniques to identify surface markers as well as intracytoplasmic staining to detect terminal deoxynucleotidyl transferase enzyme (TdT) and intracytoplasmic mu . While the CALLA+ cell suspensions contained very few mature myeloid cells or T lymphocytes, the finding that 5% to 11% of them were cyto-mu+ and 13% to 22% expressed the B1 differentiation antigen clearly indicated that at least some of these cells were B cell precursors . Because 48% to 63% of the cells were TdT+ and practically all of them expressed Ia antigen, it appears that these cells are a mixture of very early lymphoid precursor cells as well as more differentiated pre-B cells . The phenotype of these normal cells is very similar to that of common ALL cells . Differences in the surface marker phenotypes between adult and fetal CALLA+ cells that have previously been purified were also identified.

J Exp Med, 1984 Sep 1, 160(3), 633 - 51
Transcriptional control of HLA-A,B,C antigen in human placental cytotrophoblast isolated using trophoblast- and HLA-specific monoclonal antibodies and the fluorescence-activated cell sorter; Kawata M et al.; Human placental cell suspensions prepared by trypsin digestion were analyzed with several monoclonal antibodies on a multiparameter fluorescence-activated cell sorter (FACS) . Five distinct cell populations were isolated on the basis of size and quantitative differences in the coordinate expression of cell surface antigens detected by monoclonal antibodies against an HLA-A,B,C monomorphic determinant (MB40.5) and against human trophoblasts (anti-Trop-2) . By FACS analysis and after sorting we clearly identified the major cell population as cytotrophoblasts based on several independent criteria, including presence of trophoblast-specific surface antigens, Trop-1, and Trop-2; absence of all HLA class I, class II, and beta 2-microglobulin (beta 2m) antigens; absence of the pan-leucocyte and monocyte antigens, HLe1 and LeuM1, respectively; presence of Y-chromatin in a male placenta; presence of placental and not liver alkaline phosphatase; and a large, mononuclear morphology . These procedures provide a reproducible method for obtaining highly purified human cytotrophoblast populations for further studies . We measured by molecular hybridization (RNA or Northern blots) the HLA-A,B,C and beta 2m mRNA in total RNA extracted from sorted cytotrophoblasts . We find that normal human cytotrophoblasts have extremely small amounts of HLA-A,B,C mRNA: approximately 300 times less than that in the lymphoid cell line LCL-721 or normal lymphocytes . In contrast, they have approximately 11% the level of beta 2m mRNA present in LCL-721 cells . Thus, HLA-A,B,C antigen expression on human cytotrophoblasts is limited by the level of HLA heavy chain mRNA.

J Invest Dermatol, 1984 Sep, 83(3), 210 - 3
Monoclonal antibody 4F2 reactive with basal layer keratinocytes: studies in the normal and a hyperproliferative state; Patterson JA et al.; To establish a method for separating different keratinocyte subpopulations in the epidermis, we studied the specificity of monoclonal antibody 4F2 for keratinocytes . Preliminary screening experiments had previously demonstrated 4F2 reactivity with the epidermis . 4F2 reacted with a subpopulation (19.29 +/- 5.23%) of human epidermal cells in suspension . The membrane antigen identified by 4F2 continues to be expressed by cultured keratinocytes . In frozen tissue section using an indirect immunofluorescence technique, the 4F2-positive cells in the basal layer are sharply demarcated from the negative suprabasilar layers . Even in the hyperproliferative state of psoriasis, the 4F2 reactivity is confined to the basal layer . Cell suspensions of psoriatic epidermis demonstrated a greater percentage of reactivity with 4F2 (49.51% +/- 6.50%), probably reflecting the expanded population of basal layer cells . Monoclonal 4F2, therefore, reacts with a membrane antigen present on basal keratinocytes, and provides a probe for use in the isolation of the basal keratinocyte subpopulation . Thus, this antibody should be useful in studies of normal and aberrant differentiation of the epidermis.

Clin Chem, 1984 Sep, 30(9), 1462 - 6
Immunogold staining: adaptation of a cell-labeling system for analysis of human leukocyte subsets; Rosenberg JS et al.; We have assessed the Immunogold Cell-Labeling System (IGS) for potential use in the clinical laboratory . In this technique, cell suspensions incubated with monoclonal mouse antibodies are reacted with anti-mouse antibodies labeled with colloidal gold . Surface marker cells, bearing dark blue-black granules, are easily distinguished by light microscopy . The percentages of T cells, T cell subsets, B cells, monocytes, or granulocytes identified by IGS corresponds with numbers obtained by flow cytometry analysis or immunofluorescence studies . Results by IGS and flow cytometry were similar for samples from patients with aberrant lymphocyte populations (e.g., leukemias) or from transplant recipients . IGS may thus be a useful diagnostic technique for studying neoplasias or other immunologically mediated disorders . This technique can also be used to characterize the surface phenotype of leukemic cell lines . The sensitivity and accuracy of IGS can be evaluated by measurements of different cell lines mixed in predetermined ratios.

Fed Proc, 1984 Sep, 43(12), 2709 - 13
Regulation of retinal dopamine biosynthesis and tyrosine hydroxylase activity by light; Iuvone PM; Dopamine (DA)-containing neurons of the rat retina are apparently activated transsynaptically by photic stimulation . Exposure of dark-adapted rats to light increases retinal DA biosynthesis and metabolism . Associated with the light-evoked increase of DA biosynthesis is a rapid activation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis . The activation of TH is characterized by an increased affinity of the enzyme for the pteridine cofactor . Because TH in dark-adapted retinas is apparently not saturated with cofactor, the light-evoked increase of affinity is probably responsible for the observed stimulation of DA biosynthesis . Cyclic AMP (cAMP)-dependent protein phosphorylation in vitro activates TH extracted from dark-adapted retinas, and phosphorylation-induced TH activation is very similar and not additive with light-evoked activation of the enzyme . Incubation of viable cell suspensions of dissociated retinas with 8-bromo cAMP also activates TH, which indicates the availability of sufficient cAMP-dependent protein kinase in the proper subcellular compartment to regulate the enzyme in situ . The DA-containing neurons of the rat retina are tonically inhibited in darkness, and evidence is presented that this tonic inhibition involves direct synaptic input to the DA neurons from gamma-aminobutyric acid-containing amacrine cells . The DA-containing neurons are also subject to feedback inhibition through DA receptors, and to modulation by alpha 2-adrenergic receptors.

Cell Immunol, 1984 Sep, 87(2), 692 - 7
T-cell subsets in the thyroids of mice developing autoimmune thyroiditis; Creemers P et al.; To examine the role of T-cell subsets in the development of thyroid lesions, female CBA/J mice were immunized with 60 micrograms mouse thyroglobulin (MTg) in 0.1 ml complete Freund's adjuvant in both hind footpads . The thyroids were removed 12-21 days later, pooled, and dispersed . The cell suspension was examined by membrane immunofluorescence for the distribution of Thy-1+, Lyt-1+, Lyt-2+, and sIg+ lymphocytes . For comparison, peripheral blood leukocytes (PBL) from the same animals were similarly examined . Throughout this 10-day interval, B cells in the thyroid were consistently below 5%, whereas B cells represented 19-24% of PBL . Thy-1+ cells in PBL ranged from 45 to 59%, whereas Thy-1+ cells in the thyroid were 37-50% . However, only thyroidal T cells showed a consistent decline with time and were replaced gradually by cells without T or B cell markers . In particular, there was a clear shift in the Lyt-1+:Lyt-2+ ratio from about 7 down to 2 in the thyroid as the early predominance of Lyt-1+ cells was followed by a relative increase in Lyt-2+ cells . Our results show that there is an accumulation of Lyt-1+ and Lyt-2+ cells in the infiltrated thyroid . These cells may include MTg-reactive, helper, and cytotoxic T cells which localize (or differentiate) in the thyroid and initiate the lesions.

Cell Immunol, 1984 Sep, 87(2), 379 - 88
Heterogeneity of bone marrow lymphocytes: radioautographic detection of pre-B cells bearing cytoplasmic mu chains, and of B and T lymphocytes, and characterization of null lymphoid cells; Rahal MD et al.; A radioautographic immunolabeling technique has been developed to detect pre-B cells bearing cytoplasmic mu chains among populations of bone marrow lymphoid cells identified by conventional hematologic stains . 125I-Anti-mu antibody was applied either to fixed marrow smears, labeling total mu chains both in the cytoplasm (c mu) and at the cell surface (s mu), or to cell suspensions, labeling s mu alone . In stained radioautographs the incidence of c mu+ s mu- pre-B cells was derived both indirectly by subtracting values for s mu+ cells from those for total mu+ cells of various sizes in normal mice and directly by the total mu chain labeling in mice depleted of s mu+ cells by anti-IgM treatment in vivo . Binding specificity was demonstrated by the displacement of labeling by nonradioactive anti-mu antibody . The c mu+ s mu- cells showed a bimodal size distribution . They accounted for 40% of the large lymphoid cells and 30% of the small lymphocytes in the marrow . A further 50% of the small lymphocytes were B lymphocytes (s mu+) and 8% were T lymphocytes (Thy 1.2+) . Thus, the technique demonstrates the presence of c mu+ s mu- pre-B cells among both proliferating large lymphoid cells and nondividing small lymphocytes, as classically defined in marrow smears . In addition, the results reveal a broad size distribution of mu- lymphoid cells, including a subset of small lymphocytes which lack c mu, s mu, and Thy 1.2 and thus cannot be assigned to either B or T lineage by these criteria . The findings suggest that in addition to B cells the marrow may produce other types of lymphoid cells, yet to be defined.

Farmakol Toksikol, 1984 Sep-Oct, 47(5), 63 - 7
{Mechanism of the cytotoxic action of the natural zeolite clinoptilolite}; Korkina LG et al.; Interaction between the natural ceolite clinoptilolite and cell suspensions has been investigated using rat peritoneal macrophages and erythrocytes . The ceolite under study has been demonstrated to exhibit a high hemolytic activity and cytotoxicity . The viability of macrophages was evaluated from the incorporation of trypane blue . The ability of macrophages to phagocytosis was measured by chemiluminescence with luminol . The modification of clinoptilolite surface by ammonia ions led to a decrease in its cytotoxic properties . Ethanol, mannit and sodium azide did not affect whereas catalase appreciably reduced the ability of CPT to damage the membranes of macrophages and red cells . The role of hydrogen peroxide in the mechanism of cell membrane damage is discussed.

Thromb Haemost, 1984 Aug 31, 52(1), 45 - 9
Platelet accumulation on collagen: drugs which inhibit arachidonic acid metabolism and affect intracellular cyclic AMP levels; Adams GA et al.; We have studied the accumulation of washed platelets on collagen-coated glass from flowing platelet-red blood cell suspensions in the presence and absence of drugs . Glass tubes were 10 cm long and the flow rate was 1 ml/min, 80 s-1 . For all experiments, platelet accumulation was greatest near the tube's inlet with a continuous decrease to the exit . A common feature, of those drug treatments which lead to reduced accumulation at the inlet, was an increase in outlet accumulation when compared with outlet control values . Platelet-collagen adhesion resulted in maximal release of 3H-serotonin in the presence of agents that prevent platelet aggregation on collagen . Only drug treatment known to raise cAMP levels (PGE1 and dipyridamole) or prevent the formation of prostaglandins and thromboxanes (sulfinpyrazone, indomethacin and ASA) were found to inhibit aggregate growth . Platelet aggregation on collagen in the absence of thrombin likely proceeds through the liberation of prostaglandins and thromboxanes from surface-bound platelets into the flow where they may stimulate flow-born cells . An alternate hypothesis is that such treatments affect the release of alpha-granule components necessary for aggregation.

Biochem Biophys Res Commun, 1984 Aug 30, 123(1), 358 - 64
Vanadate and dicyclohexylcarbodiimide insensitive proton extrusion from oxygen pulsed cells of the cyanobacterium Anacystis nidulans; Nitschmann WH et al.; Oxygen pulses applied to dark anaerobic suspensions of Anacystis nidulans provoked immediate acidification of the external medium . The reaction was inhibited only 75% by dicyclohexylcarbodiimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole at concentrations which completely arrested all oxidative phosphorylation . Carbonyl cyanide m-chlorophenylhydrazone eliminated the acidification of oxygen pulsed cell suspensions while ortho-vanadate and diethylstilbestrol had no effect . No lag occurred between the onset of respiration and proton extrusion . H+/O ratios were 4.1 +/- 0.5 in the absence, and 1.9 +/- 0.4 in the presence, of dicyclohexylcarbodiimide . These results are consistent with a recently described proton-translocating aa3-type cytochrome c oxidase (H+/O = 1.6 +/- 0.4) in the cell membrane of A . nidulans (G.A . Peschek, J . Bacteriol . 153 (1983) 539-542).

Biochim Biophys Acta, 1984 Aug 17, 804(4), 427 - 33
Stimulation of phosphatidylinositol turnover by acetylcholine, angiotensin II and ACTH in bovine adrenal fasciculata cells; Hadjian AJ et al.; The effect of acetylcholine, angiotensin II and adrenocorticotropin (ACTH) on phosphatidylinositol (PI) metabolism was examined using bovine adrenocortical fasciculata cell suspensions . The three agents, which acutely stimulate glucocorticoid production by these cells, were all able to increase {32P}Pi incorporation into cellular PI . However, whereas the relative steroidogenic potency (at maximally active concentrations) was ACTH greater than or equal to angiotensin II greater than acetylcholine, the effect on PI labeling was in the order angiotensin II greater than acetylcholine greater than ACTH . The dose-response curves for steroidogenesis and that for PI labeling were superimposable in the case of angiotensin II (ED50 = 1 X 10(-8) M) and of acetylcholine (ED50 = 5 X 10(-7) M), while the two responses were dissociated under graded ACTH challenge . Both steroidogenic response and increased PI labeling elicited by angiotensin II and acetylcholine were respectively inhibited by (Sar1-Ala8)-angiotensin II and muscarinic antagonists . Time-course study showed that in the case of angiotensin II and acetylcholine, the sequence of events was: increased phosphatidic acid labeling, increased PI labeling, activated steroidogenesis . By sharp contrast, under ACTH stimulation, increased steroidogenesis was detected well before activation of PI metabolism . These data suggest that in bovine adrenocortical fasciculata cell, steroidogenesis may be activated by two different pathways . The first one would act mainly through cyclic AMP-dependent intracellular events and is usually accepted in the mechanism of action of ACTH . The other, cyclic AMP-independent pathway, as in the case of angiotensin II and acetylcholine actions, may involve phospholipid-mediated intracellular processes.

Anal Biochem, 1984 Aug 15, 141(1), 74 - 8
Simple and small-scale breakdown of yeast; Naganuma T et al.; A simple and small-scale method for the preparation of homogenate from yeast was developed . The principle of this new method involves the shaking of a small amount of yeast-cell suspension with glass beads on a tower-shaped mixer . When this method is used the cells in 1 ml of cell suspension are broken down and 14 samples can be simultaneously processed under controlled conditions . The degree of cell breakdown, the amount of soluble protein liberated from the cells, and the increase in each enzyme activity in prepared homogenate correlated to the shaking time: maximum values were obtained at 20 min . Enzyme activities in the homogenate were equivalent to or higher than that procured with former methods . This new method is applicable to various species of yeasts.

Nature, 1984 Aug 30-Sep 5, 310(5980), 792 - 4
Monoclonal antibody production by receptor-mediated electrically induced cell fusion; Lo MM et al.; Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies . High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination, but it is not possible to identify B-cells destined to secrete relevant antibodies . However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies . Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells . Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells . The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells . This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells . An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody . We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.

Science, 1984 Aug 3, 225(4661), 533 - 6
Intrahippocampal septal grafts ameliorate learning impairments in aged rats; Gage FH et al.; Grafts of fetal septal tissue rich in cholinergic neurons were implanted as a dissociated cell suspension into the depth of the hippocampal formation in aged rats with severe impairments in spatial learning abilities . After 2 1/2 to 3 months, the rats with grafts, but not the controls, had improved their performance in a spatial learning test . Their improvement was due, at least in part, to an increased ability to use spatial cues in the task . In all animals the grafts had produced an extensive acetylcholinesterase-positive terminal network in the surrounding host hippocampal formation . Thus, the action of cholinergic neurons in the graft onto elements in the host hippocampal circuitry may be a necessary, but perhaps not sufficient, prerequisite for the observed functional recovery.

Blood, 1984 Aug, 64(2), 526 - 33
The effects of tumor-promoting phorbol esters on human granulopoiesis in vitro; Sullivan R et al.; In order to determine whether the tumor-promoting phorbol esters are capable of inducing normal human committed granulocytic-monocytic progenitor cells (CFUc) to proliferate and differentiate in the absence of granulocyte-monocyte colony-stimulating activity (CSA), we studied the effects of these compounds on human granulopoiesis in vitro . We found that when light-density human marrow cells or peripheral blood leukocytes were depleted of adherent cells and then incubated in semisolid tissue culture medium under conditions optimal for CFUc growth, phorbol myristate acetate (PMA) and its congeners produced no measurable stimulatory effect on the proliferation of CFUc in the absence of added CSA . Likewise, when light-density marrow cells that had not been depleted of adherent cells were plated in the cultures, no stimulation of CFUc colony growth resulted from the addition of PMA . However, when light-density peripheral blood leukocytes were used as a target source of CFUc without first subjecting them to adherence separation, enhanced proliferation and differentiation of CFUc were noted in cultures that contained PMA . To investigate the possibility that CSA production by monocytes in these cultures in response to activation by PMA might account for the enhanced colony formation that we observed, we incubated isolated peripheral blood monocytes in short-term liquid suspension cultures and found that in the presence of PMA, large quantities of CSA were secreted into the surrounding medium . Finally, we noted that when marrow cell suspensions were suboptimally stimulated by low concentrations of CSA added to the cultures, the effects of PMA on CFUc proliferation were unpredictable, enhancing colony formation in some cases and inhibiting it in others . Our data indicate that although the tumor-promoting phorbol esters do not appear capable of directly stimulating the proliferation or differentiation of human CFUc in the absence of CSA, they may do so indirectly by causing auxiliary cells such as monocytes to secrete CSA.

J Steroid Biochem, 1984 Aug, 21(2), 169 - 72
Relative cellular distribution of particulate androgen binding between Sertoli and germ cells in rat testis; Cigorraga S et al.; An androgen binding activity with characteristics similar to ABP is present in a particulate fraction (105,000 g pellet) obtained by differential centrifugation of seminiferous tubules, impure Sertoli cells and impure germ cells homogenates . Purification of germ cells through albumin gradients, results in almost a complete loss of androgen binding activity in the purified germ cell suspensions . Furthermore, Sertoli cell enriched testes from 22-, 32- and 40-day old rats showed increases in particulate androgen binding, when compared to matched controls, parallel to increments in the activity of a Sertoli cell marker enzyme (beta-glucuronidase) . These results suggest that particulate androgen binding activity is only present in Sertoli cells and this protein may play a role in the compartmentalization of androgens in the testis.

Toxicol Appl Pharmacol, 1984 Aug, 75(1), 25 - 34
Partial characterization of bone marrow hemopoiesis in mice after cisplatin administration; Wierda D et al.; After cessation of cisplatin (cis-dichlorodiammineplatinum) chemotherapy, selective recovery of certain cell lineages occurs during bone marrow hemopoiesis . To further investigate the process of selective hemopoiesis, we combined the use of buoyant density gradient separation, morphology, and lymphocyte function assays to characterize changes in the hemopoiesis of immature and mature marrow cells after exposure to cisplatin . A single, cytotoxic dose of cisplatin was administered to B6D2F1 male mice, and marrow cell suspensions were taken from these mice 3 and 7 days later to characterize hemopoietic recovery . Morphology and buoyant density separation of marrow cells revealed that the most significant changes in cellular composition occurred at Day 3 . At this time, there was an increase in immature white blood cells (WBCs) and immature polymorphoneutrophils (PMNs) with a concomitant reduction in immature red blood cells (RBCs) . By Day 7, the normal proportion of immature RBCs, immature WBCs, and PMNs was restored; however, the buoyant distribution patterns for PMNs indicated that a greater proportion of immature PMNs was still present relative to marrow suspensions from normal mice . Fewer lymphocytes were also observed in marrows from the Day 7 group when compared with controls . Lymphocyte function tests indicated reduced mitogen responsiveness of lymphocytes from both Day 3 and Day 7; however, more immature lymphocytes were present after 7 days than were seen with either normal or Day 3 marrow suspensions . Overall, the results indicated that hemopoiesis proceeded through a specific hierarchy which began with the restoration of the erythrocyte line followed by the leukocyte cell lines . Lymphocyte recovery lagged behind the restoration of all the cell lineages examined.

J Appl Toxicol, 1984 Aug, 4(4), 211 - 8
Quantitation of specific myeloid cells in rat bone marrow measured by in vitro 35S-sulphate incorporation; Wright AF et al.; A biochemical measurement which can be used for quantitation of specific early myeloid cells in rat bone marrow has been developed . This measurement consists of a rapid, simple assay for the in vitro quantitation of 35S-sulphate incorporation into rat bone marrow cells . Incubation of bone marrow cells with 35S-sulphate led to a time-dependent increase in radioactivity obtained in perchloric acid insoluble fractions of bone marrow cell suspensions . This incorporation was inhibited by cyanide and puromycin . Autoradiography has demonstrated the radiolabel to be specifically associated with immature cells of the myeloid series . The cells most active in this respect were eosinophils . When rats were treated with endotoxin, the rate of 35S-sulphate incorporation was increased . Cell number measurements, using conventional histopathology and a Coulter Counter, demonstrated that endotoxin caused an initial release of mature granulocytes from the bone marrow . The regeneration of this mature population in the marrow was rapid, and was characterized by an increase in the number of immature cells and a concomitant increase in the rate of 35S-sulphate incorporation measured in preparations of bone marrow cells in vitro . Furthermore, this response to endotoxin has demonstrated that Coulter Counting techniques can be used to distinguish specific populations of cells (e.g . mature granulocytes) within the bone marrow.

J Clin Microbiol, 1984 Aug, 20(2), 209 - 13
Specific enzyme-linked immunosorbent assay for detection of bovine antibody to Brucella abortus; Tabatabai LB et al.; Six soluble antigens prepared from Brucella abortus were compared with a salt-extractable protein (CSP) antigen in an enzyme-linked immunosorbent assay for the detection of antibody to B . abortus in cattle sera . Of seven preparations tested, antigens from B . abortus soluble antigen (prepared from an autoclaved cell suspension) and CSP were stable on frozen storage . Enzyme-linked immunosorbent assay with CSP antigen under optimal conditions was from 100- to 700-fold more sensitive than the standard agglutination, card, Rivanol precipitation-plate agglutination, and the complement fixation tests in detecting immunoglobulin G antibody . From a practical point of view, however, using the most stringent criteria for determining an "upper negative" value, the enzyme-linked immunosorbent assay with CSP was at least 12-fold more sensitive than the standard agglutination test and any of the other serological tests . Furthermore, the enzyme-linked immunosorbent assay with CSP was specific for antibody to B . abortus.

Transplantation, 1984 Aug, 38(2), 107 - 11
Intracerebral allotransplantation of purified pancreatic endocrine cells and pancreatic islets in diabetic rats; Tze WJ et al.; Allogeneic pancreatic endocrine cells (PEC) and whole islets from inbred Lewis (AgB 1/1) and outbred Wistar rats were implanted intracerebrally (i.c.) into two designated areas of streptozotocin-induced diabetic ACI (AgB 4/4) rats across the major histocompatibility barrier . All the transplants of PEC from Lewis (n = 12) and Wistar (n = 7) donors remained functional for an observation period in excess of 200 days . In contrast, only 3/6 Lewis and 3/9 Wistar whole-islet transplants were able to maintain function for a prolonged period . Recipients with functional PEC or islet allografts had normalized nonfasting blood glucose (BG) in the 24-hr . BG profile, and they maintained a steady body weight gain . ACI recipients of PEC from Lewis rats had glucose disappearance K rates of 1.3 +/- 0.3 (mean +/- SE) and a normal basal BG level in 4 hr following the i.v . glucose load . Histological section of the brain tissues with successful i.c . islet or PEC grafts up to a duration of 5 1/2 months revealed healthy endocrine cells in the cortex and the subarachnoid space . These grafts were permeated with capillaries but devoid of exocrine tissues or lymphoid cell infiltration . These observations suggest that the brain is an immunologically privileged site, and that it is a hospitable site for the pancreatic endocrine cell suspension . However, the immunological protection offered to allogeneic transplants by the brain is incomplete, and purified PEC must be employed to ensure consistent long-term allograft survival.

No Shinkei Geka, 1984 Aug, 12(9), 1007 - 18
{A review of cell kinetic studies on brain tumors with special reference to anti-bromodeoxyuridine monoclonal antibody method}; Nagashima T et al.; Cell kinetic studies on various human brain tumors were reviewed . Most studies have been carried out by means of 3H-thymidine and autoradiography in the past two decades . The average labeling index (LI) obtained from a pulse of 3H-thymidine is very high in medulloblastomas and glioblastoma multiforme (5-15%), low in well-differentiated gliomas (less than 1%), and intermediate in anaplastic astrocytomas . The higher the LI, the faster the tumor grows, probably reflecting a larger growth fraction . Therefore, measurement of the LI appears to be very helpful in predicting the prognosis of the patient as well as potentially helpful in the design of chemotherapy . However, isotopic studies not only take a long time to complete, but also have severe limitations because of potential radiation hazard to patients and environmental pollution . Development of an anti-BUdR (or BrdUrd) monoclonal antibody to detect nuclei which incorporate BUdR is a breakthrough that can expand this line of research, since: 1) BUdR is non-radioactive and doses needed for this purpose are virtually non-toxic; 2) use of flow cytometric analysis of FITC conjugated anti-BUdR antibody facilitates rapid acquisition and data analysis and, 3) it will be technically easier to study the proliferative capacity of patients with brain tumors . A 30 min exposure of 9L monolayer cells to 10 X 2-10 X 2(-4) microM BUdR produced satisfactory results against a 1: 60 dilution of FITC conjugated antibody (Becton-Dickinson, Mt . View, CA) . Also 9L cells which were exposed various concentrations of BUdR (10 X 2-10 X 2(-4) microM) for 30 min were harvested in single cell suspension and reacted with the antibody and analysed with flow cytometry . Fluorescent nuclei (48.6%) were similar to the fraction of cells in S phase analysed from DNA histograms as well as the LI obtained from autoradiographic study after a pulse of 3H-thymidine . After 1-40 mg/kg of BUdR were injected into the peritoneal cavity of rats with 9L brain tumors, tumors were removed 1 hr later and digested with enzyme cocktail, fixed with 70% ETOH, denatured with HCl, and stained with FITC conjugated anti-BUdR antibody . The nuclei which reacted with the antibody were discriminated well with flow system at 488 nm light through LP 515 and SP 560 filters . 10.0-15.5% of the cells were fluorescent in each group and the intensity of fluorescence was dose dependent although not strictly proportional to the amount of BUdR administered.(ABSTRACT TRUNCATED AT 400 WORDS)

Tsitologiia, 1984 Aug, 26(8), 863 - 73
{Partition of cell suspensions in 2-phase systems}; Sungurov AIu; The physical principles of cell suspension partition in aqueous two-polymer phase systems are considered . The partition procedures and phase composition are analyzed . The results of application of analytic and preparative two-phase partition are presented.

Biol Reprod, 1984 Aug, 31(1), 165 - 74
Immunocytochemical localization and determination of hormone-induced synthesis of the sulfated oviductal glycoproteins; Oliphant G et al.; Secretory products of the oviduct provide part of the milieu for the critical events of fertilization and embryo development . Past work from this laboratory has indicated that three large sulfated glycoproteins can be isolated from rabbit oviductal fluid and are synthesized by oviductal epithelium incubated in vitro . These three glycoproteins are antigenically similar . This paper presents evidence for their localization within the oviductal tissue and their hormonal control of synthesis . Utilizing goat antiserum to these oviductal glycoproteins and the immunoglobulin-horseradish peroxidase bridge method, these macromolecules have been localized in the ampulla and isthmus of the oviduct . Ten days after ovariectomy an oviduct was removed for immunolocalization . The does were then given estradiol for the next 4 days and the second oviduct was removed . Oviducts treated with estradiol showed immunostaining of virtually all of the secretory granules within the secretory cells of the isthmus . While light level immunocytochemistry suggested the possibility of two populations of secretory granules within the ampulla because some of the granules did not show immunocytochemical staining, the more sensitive immunocytochemistry at the electron microscopic level showed staining of all granules of the ampulla and isthmus . Absorption of the antiserum with pure antigen prevented all staining . After ovariectomy and hormone withdrawal, most of the immunostaining was lost in the isthmus and virtually no staining in the ampulla was observed . Oviductal cell suspensions were made to evaluate incorporation of {35S} sulfate and {3H} leucine as a function of hormonal priming of the tissue . Estrogen-primed oviductal cells incorporated the sulfate and leucine into these specific glycoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

J Natl Cancer Inst, 1984 Aug, 73(2), 363 - 9
Human renal antigen defined by a murine monoclonal antibody; Tagliabue E et al.; Fusion of spleen cells from a mouse immunized with a surgical specimen of a human renal carcinoma with murine P3 myeloma cells resulted in the establishment of a hybridoma cell line that secreted a monoclonal antibody (MKi-1), of IgG1 subclass, which preferentially reacted on kidney crude membrane (CM) preparations . This monoclonal antibody was tested by solid-phase radioimmunometric assay and immunofluorescence (IF) on a panel of tumor cell lines and on CM preparations and cell suspensions from surgical specimens of normal and neoplastic tissues . In addition, cryosections of normal and cancer tissues of various histologic types were tested by IF . The expression of the MKi-1 antigen was limited to normal kidney epithelium, renal cancers, some areas in the pancreas, the apical region of some breast ducts, and a proportion (5-50%) of activated lymphocytes . Electron microscopic study by the immunoperoxidase technique on fixed sections from normal kidney showed that MKi-1 stained the brush border of almost all proximal tubules . The molecule recognized by MKi-1 was a single polypeptide chain with a molecular weight of 140,000.

Scand J Immunol, 1984 Aug, 20(2), 105 - 11
Modulation of pokeweed mitogen-induced human B-cell differentiation by aggregated IgG; Le Thi Bich-Thuy et al.; The modulatory effect of human heat-aggregated IgG on human B-cell differentiation induced by pokeweed mitogen was investigated with three experimental protocols . Pulse exposure to aggregated IgG, followed by extensive washings before culture, of peripheral blood mononuclear cell suspensions rigorously depleted of platelets and containing less than 4% monocytes resulted in a selective decrease of the numbers of IgG-containing cells and IgG-secreting cells, whereas a simultaneous decrease of the numbers of cells producing IgG and, to a lesser extent, of those producing IgM or IgA was observed when the pulsed suspensions contained platelets and more than 4% monocytes . This non-isotype-specific suppression was shown to be more pronounced when aggregated IgG and platelets were present in the cell suspensions throughout the cultures . The results suggest that two distinct suppressor pathways can be triggered by aggregated IgG . The first one is restricted to cells producing the matching isotype, in the absence of platelets, with few monocytes in the cell suspensions . The second one leads to a nonspecific suppression of the three major Ig classes . It requires the presence of platelets and/or a high percentage of monocytes and, although it remains to be demonstrated, is probably mediated by prostaglandin E2.

Clin Exp Immunol, 1984 Aug, 57(2), 487 - 94
Activated lymphocyte killer cells derived from melanoma tissue or peripheral blood; Burns GF et al.; Lymphoid cells infiltrating metastatic melanomas were grown directly from cell suspensions of tumour tissue by the addition of T cell growth factor . Lymphoid cells grew out at the expense of tumour cells in six of seven freshly excised tumours, and cells from two cultures were expanded for in vitro testing of cytolytic function against different target cells . Early in culture the tumour derived lymphocytes killed fresh autologous melanoma cells and, particularly later in culture, were highly and non-specifically cytolytic for cultured melanoma and non-melanoma cells . Cultured peripheral blood lymphocytes from patients with melanoma, and from normal subjects, were cytolytic to the same degree as tumour derived lymphocytes, and also resembled cells grown from tumour tissue in possessing acid phosphatase activity which was resistant to tartrate . Cultured lymphoblasts from both tumour and peripheral blood had a T cell phenotype when analysed with monoclonal antibodies . An in vitro co-culture system was employed to study the kinetics and the precursors of these non-specific killer cells among blood mononuclear cells . Blood mononuclear cells cultured with irradiated B lymphoblasts led to the generation of non-specific cytolytic cells, referred to as activated lymphocyte killer (ALK) cells, after 7-10 days of culture and the progenitors of these ALK cells were demonstrated to be distinct from those of specific cytolytic T cells.

Blut, 1984 Aug, 49(2), 69 - 73
B cells in chronic lymphocytic leukaemia . Comparative analysis of blood and bone marrow; Pizzolo G et al.; A study was performed on cell suspension from peripheral blood and bone marrow aspirates and on cryostat sections from bone marrow biopsies in order to investigate the membrane phenotype of neoplastic B cells in chronic lymphocytic leukaemia (B-CLL) . The immunological analyses, performed on 43 patients, included rosetting ability with sheep and mouse erythrocytes, evaluation of surface immunoglobulins and reactivity with anti-HLA-DR, UCHT 1 (OKT-3 like) and RFA-1 (OKT-1 like) monoclonal antibodies . The results demonstrate that neoplastic B lymphocytes in B-CLL display an identical phenotype in peripheral blood and bone marrow . Possible interpretations on the origin of proliferating cells in B-CLL are discussed.

Surgery, 1984 Aug, 96(2), 315 - 20
Inhibition of host immunity by fluid and mononuclear cells from healing wounds; Barbul A et al.; Severe trauma impairs host immunity, which in turn renders the host susceptible to infection often terminating in death . This impairment occurs 7 to 14 days after injury, a time when wound healing is at its maximum . We examined the interactions of wound healing to host immunity by studying the in vitro and in vivo immune effects of wound components (i.e., wound fluid {WF} and wound mononuclear cells {WMNC}) . Lewis male rats (RT-1(1} weighing 300 to 350 gm underwent 7 cm dorsal skin incisions and subcutaneous placement of polyvinyl alcohol sponges . At 7 and 10 days after wounding, sponges were removed and WF was separated from the cellular elements . The cell suspension was purified to contain 80% to 90% WMNC . Ten percent WF from 7- and 10-day-old wounds inhibits normal thymic lymphocyte blastogenesis to concanavalin A and phytohemagglutinin . Addition of 5 X 10(4) WMNC leads to similar inhibition . WF and WMNC from 10-day-old wounds also inhibit in vitro allogeneic responses tested in one way MLR of Lewis splenocytes with inactivated ACI (RT-1a) spleen cells by 75% to 96% and 85% to 98%, respectively . The inhibitory action of WF is heat resistant (56 degrees C for 30 minutes) and noncytotoxic . In vivo allogeneic responses, tested by grafting ACI skin onto Lewis recipients, were inhibited by intraperitoneal administration of 10-day-old WF (p less than 0.01) . We conclude that WF contains factor(s) that inhibit in vitro and in vivo immune responses . WMNC exhibits the same action, suggesting that they may be the source of the WF inhibitory factor(s) . These findings may explain host immunosuppression after severe trauma.

Am J Pathol, 1984 Aug, 116(2), 245 - 52
Distribution and kinetics of mononuclear phagocytes in granulomas elicited by eggs of Schistosoma mansoni; Stadecker MJ et al.; Previous work has shown that cell suspensions from egg granulomas of Schistosoma mansoni-infected mice contain populations of both I-A-positive and -negative granuloma macrophages (GMs) . The present study was undertaken to investigate the distribution of I-A-bearing macrophages within the granulomas, as well as the kinetics of I-A antigen expression by these cells in vivo . Cryostat sections of liver tissue from infected animals, stained with monoclonal anti-I-A antibodies, demonstrated the presence of I-A-positive GMs in peripheral areas of the granulomas, whereas I-A-negative cells concentrated in their centers . For investigation of their expression of I-A antigen, dispersed GMs were studied at time intervals after subjecting infected mice to lethal doses of total body irradiation . I-A half-life on GMs in vivo was estimated to be 2 days, based both on visual detection by immunofluorescence and functionally on the ability of GMs to perform as antigen-presenting cells . Autoradiographic studies, performed on infected liver tissue obtained 1 hour after in vivo administration of tritiated thymidine, showed that macrophages predominantly replicated in peripheral areas of the schistosomal egg granulomas . After longer intervals, however, labeled cells were seen in more central areas of the granuloma, suggesting an overall cell flux from the periphery to the center . These findings indicate that macrophages express I-A antigens in peripheral areas of the granulomas, where macrophage replication and recruitment from the bone marrow take place . They suggest that I-A expression occurs during a limited period of time in "young" macrophages, which later may convert to an I-A-negative phenotype.

Cell Immunol, 1984 Aug, 87(1), 101 - 9
Immunohistology of thymic nurse cells; van Vliet E et al.; The demonstration of thymic nurse cells (TNC), complexes between stromal cells and thymocytes, in cell suspensions of murine thymuses, prompted us to investigate (1) the relationship of TNC to other thymic stromal cell types defined in situ, and (2) the maturation stage of the enclosed thymocytes . To this purpose we incubated frozen sections of TNC suspensions with various monoclonal antisera directed to T cells and stromal cell types, using immunohistology . This approach enabled us to study antigen expression on the "nursing" cell itself and to analyze the phenotype of the enclosed lymphocytes in cross sections of TNC . The results show that lymphocytes enveloped by TNC express high levels of Thy-1, moderate levels of T200, and variable amounts of Lyt-1 . Due to enzymatic degradation Lyt-2 expression could not be studied . The enveloped cells also bear PNA receptors, but no detectable I-A/E antigens . Expression of H-2K antigens on enclosed thymocytes varied from weak to absent . The "nursing" cells react with ER-TR4, a monoclonal antibody which detects cortical epithelial-reticular cells . In addition TNC express I-A/E and H-2K antigens . In contrast, TNC do not react with ER-TR 5 and 7, monoclonal antibodies, which detect medullary epithelial cells and reticular fibroblasts, respectively . TNC do not express the macrophage antigens Mac-1 and Mac-2 . We conclude that TNC in vitro represent the in vivo association of epithelial-reticular cells with cortical thymocytes . However, the enclosed thymocytes do not constitute a phenotypically distinct subset of subcapsular or outer cortical cells.

Clin Exp Immunol, 1984 Aug, 57(2), 331 - 7
Evidence of cells bearing interleukin-2 receptor at sites of disease activity in sarcoid patients; Semenzato G et al.; The frequency of cells reactive with anti-Tac monoclonal antibody (MoAb), which recognizes the interleukin-2 (IL-2) receptor, has been evaluated in cell suspensions from peripheral blood and bronchoalveolar lavage (BAL), and in frozen sections from involved tissues in 18 patients with active sarcoidosis . Peripheral blood lymphocytes of sarcoid patients do not bear Tac determinant and reduced numbers of Tac+ cells are inducible following PHA stimulation . On the other hand, significant numbers of lymphocytes reactive with anti-TacMoAb are present in the cells obtained from the BAL and a number of Tac+ cells infiltrate the lung, lymph node and conjunctiva . The finding of Tac+ cells in the BAL fluid and in other organs in patients with sarcoidosis provides evidence that some T cells in these involved tissues have the characteristics of IL-2 responder cells and thus the potential to absorb IL-2, supporting the hypothesis that T lymphocytes replicate in situ at sites of disease activity.

J Nucl Med, 1984 Aug, 25(8), 913 - 6
Evaluation of neutrophil labeling techniques using the chemotaxis radioassay; English D et al.; Neutrophils isolated from human blood were labeled by various methods and exposed to a chemotactic gradient . The chemotactically functional cells that migrated into the gradient were isolated . The portion of radioactivity of the original cell suspension carried with the chemotactically responsive cells was related to the relative number of migrating cells as determined microscopically . Of the radionuclides used, P-32 diisopropylfluorophosphate (DFP), In-111 oxine, and Tc-99m sulfur colloid provided cell preparations with the highest relative portion of radioactivity confined to functionally intact (chemotactic) neutrophils . Results with Na2(51)CrO4 and with SnCl2-reduced 99mTcO4- were less than optimal . Neutrophils exposed to Ga-67 citrate apparently took up the label and retained chemotactic responsiveness . However, little or no radioactivity was detected in the neutrophils that migrated from the suspensions of Ga-67-labeled cells . The results indicate that the chemotaxis radioassay can yield unique information pertaining to the extent to which a radiotracer is specifically associated with viable neutrophils in a suspension of labeled cells.

Boll Soc Ital Biol Sper, 1984 Jul 31, 60(7), 1369 - 75
{In vitro evaluation of the chemosensitivity of an experimental murine leukemia rendered resistant in vivo to adriamycin}; Lepri E et al.; The results obtained using a rapid assay for in vitro chemosensitivity detection of leukemias are presented . The assay, performed according to the technique already described, involves in vitro incubation of a tumor cell suspension with various concentrations of antitumor drugs for 1 h and evaluation of drug-induced cell damage by addition to the cultured cells of 125I-deoxyuridine 48 h after pharmacological treatment . Results are expressed as percent inhibition of the isotope incorporation with respect to untreated controls . Preliminary results demonstrated that this assay is able to evidence differential chemosensitivity exhibited in vivo by murine leukemias . The present study reports the results obtained using comparatively P388 and P388/ADR, a subline of P388 murine leukemia with acquired resistance to Adriamycin in vivo . We found that P388/ADR exhibited resistance to ADR and DNR at all the concentrations tested, whereas P388 was highly sensitive . Cross-resistance of P388/ADR was also found to some structurally dissimilar agents, i.e . VCR and Act-D . These in vitro results correlate well with much data in the literature concerning the characteristics of resistance and cross-resistance exhibited in vivo by P388/ADR . These results suggest the possibility of using a similar in vitro assay for predicting the in vivo drug resistance of human leukemias.

FEBS Lett, 1984 Jul 23, 173(1), 129 - 33
Chromatin-bound and free RNA polymerase A activities in rat thymus cells following glucocorticoid treatment; Dembinski TC; Treatment of rat thymus cell suspensions with dexamethasone resulted in inhibition of engaged RNA polymerase A, without significant change in free pool activity . Studies with the re-initiation inhibitor, rifamycin AF/0-13, and measurements of numbers of RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis resulted from a decrease in elongation rate . This effect was selectively abolished by mild proteolysis of nuclei . It is concluded that glucocorticoid treatment of rat thymus cells suppresses 45 S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate, rather than by effecting a change in enzyme redistribution or concentration.

Eur J Biochem, 1984 Jul 2, 142(1), 127 - 31
Induction of phytoalexin synthesis in soybean . Stereospecific 3,9-dihydroxypterocarpan 6a-hydroxylase from elicitor-induced soybean cell cultures; Hagmann ML et al.; A microsomal preparation from elicitor-challenged soybean cell suspension cultures catalyzes an NADPH-dependent and dioxygen-dependent 6a-hydroxylation of 3,9-dihydroxypterocarpan to 3,6a,9-trihydroxypterocarpan . The latter is a precursor for the soybean phytoalexin glyceollin . No reaction is observed with NADH . The 6a-hydroxylase is inhibited by cytochrome c . Optical rotatory dispersion spectra of the enzymatic product formed from racemic dihydroxypterocarpan and of the remaining unreacted substrate proved that the product has the natural (6aS, 11aS)-configuration and that hydroxylation proceeds with retention of configuration . The 6a-hydroxylase was also found in elicitor-challenged soybean seedlings . The results indicate that the 6a-hydroxylase is specifically involved in the biosynthesis of glyceollin.

Dev Biol, 1984 Jul, 104(1), 247 - 54
A flow cytometric analysis of the embryonic origin of lymphocytes in diploid/triploid chimeric Xenopus laevis; Flajnik MF et al.; Quantitative flow cytometry was used to examine the embryonic origin of lymphocytes in Xenopus laevis . Reciprocal head/body transplants were made between diploid (2N) and triploid (3N) embryos of the same developmental stages ranging from neural plate to tail bud stages . Thymuses and spleens were removed from postmetamorphic chimeras . Cell suspensions were stained with the fluorescent DNA stain, mithramycin, and the ploidy (relative fluorescence intensity) of the cells was then determined by flow cytometry . All lymphocytes in the chimeras were derived from the posterior portion of the embryo . In other experiments, various regions of the lateral plate or ventral mesoderm were grafted from triploid to diploid embryos . Only transplants that included middorsal mesoderm gave rise to lymphocytes.

Cell Tissue Kinet, 1984 Jul, 17(4), 351 - 65
Subpopulations of slowly cycling cells in S and G2 phase in mouse epidermis; Clausen OP et al.; Evidence has been presented supporting the existence of heterogeneity in cell-cycle progression in mouse epidermis, The present study was undertaken to characterize this heterogeneity in more detail . Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days . Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry . Cell-cycle analysis was combined with sorting of cells from windows in G1, S and G2 phase, and the proportion of labelled cells within each window was determined in autoradiographs . Reanalysis and resorting to control the purity of of sorted fractions were performed . Computer simulations of the data were made using a mathematical model assuming different S and G2 phase characteristics . A good fit to the data was only obtained when heterogeneity in mouse epidermal cell-cycle progression was assumed, indicating the existence of slowly traversing, distinct subpopulations of cells in G2 and S phase . These cells are assumed to contribute to about 40% of all cells in S phase and to about 70% of all in G2 phase . The estimated residence times in the resting states were 38 and 32 hr in S and G2 phase, respectively . Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells were larger than the average . There is no evidence of significant subpopulations of permanently non-proliferating keratinocytes in any of the cell-cycle phases.

J Immunol, 1984 Jul, 133(1), 495 - 501
Strategies for production of monoclonal anti-idiotype antibodies against human B cell lymphomas; Thielemans K et al.; Murine monoclonal antibodies (MAB) against the idiotype (Id) of B lymphocyte malignancies are powerful reagents for the study of these diseases, and are potentially useful for treatment . Different strategies for the production of these anti-Id MAB have been compared . Initially, the Id Ig from nonsecreting B cell tumors was "rescued" by human X mouse or human X human hybridization . These somatic cell hybridizations resulted in the secretion of human Ig in 10 and 100% of the fusions, respectively . In a second step, anti-Id MAB were produced by using the "rescued" Id Ig as immunogen . A more streamlined approach is based on a one-step procedure in which the tumor cell suspension is used as immunogen . This method of immunization, coupled with a four-layer ELISA, results in the detection of anti-Id MAB in a frequency of approximately 1% of the total hybrids . By using a pool of 10 different anti-Id MAB, each reactive with the tumor of one patient, we searched for idiotypic relatedness among a panel of 50 additional tumors . No cross-reactions were found, indicating that our current strategy results in the identification of unique idiotypic determinants among human B cell tumors . Idiotypic Ig can be found in the serum of patients with B cell tumors . Among groups of patients, there is a wide spectrum of serum Id levels, ranging from less than 0.01 microgram/ml to greater than 500 micrograms/ml.

Clin Exp Metastasis, 1984 Jul-Sep, 2(3), 223 - 33
Effectiveness of AMSA alone or in combination with radiation on murine fibrosarcoma pulmonary nodules; Grdina DJ et al.; The cytotoxic effects in vivo of 4'-(9-acridinylamino) methanesulfon-m-anisidide (AMSA), radiation or both modalities in combination on murine fibrosarcoma (FSa) cells grown as pulmonary tumors were determined . Fourteen days following the i.v . injection of viable FSa cells, recipient mice developed between 100 and 150 visible pulmonary nodules . At that time, tumor-bearing animals were exposed to either single or combined modality treatments, as well as single and fractionated dose regimens . Animals were sacrificed 1 hour after the last treatment . Tumor nodules were excised, made into a single cell suspension and separated on the basis of cell size by centrifugal elutriation . Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, as well as the percentage contamination by normal diploid cells in each of the tumor cell populations . Known numbers of viable cells from each elutriator fraction were injected into recipient mice to determine their colony-forming efficiency (CFE) . Surviving fractions were determined by comparing the CFEs of treated FSa cells from each of the separated elutriator fractions with those of appropriate untreated controls . Following a single i.v . dose of AMSA (30 mg/kg), populations of cells enriched in S phase were the most sensitive . When a single dose of AMSA was combined with a single dose of radiation (100 rad), there was a marked schedule dependence with the more effective sequence, especially if a 12 hour interval was chosen between doses, being AMSA followed by irradiation . No schedule dependence was observed if both modalities were combined and administered under a fractionated protocol of four fractions of AMSA (5 mg/kg per fraction) and four fractions of radiation (300 rad per fraction) . Under these conditions the greatest reduction in CFE was in cell subpopulations most enriched in S and G2 + M phase cells.

Clin Exp Metastasis, 1984 Jul-Sep, 2(3), 213 - 22
Involvement of cell-cell adhesion molecules in liver colonization by metastatic murine lymphoma/lymphosarcoma variants; McGuire EJ et al.; Metastatic variant sublines of the murine RAW117 large cell lymphoma or lymphosarcoma have been established in vitro by sequential cycles of harvesting of liver tumor nodules after intravenous inoculation of tumor cell suspensions into syngeneic BALB/c mice . After five and ten in vivo selections for liver colonization, variant sublines RAW117-H5 and -H10, respectively, were established, and these formed significantly more surface liver tumors than the parental RAW117-P line . RAW117 sublines were tested for their abilities to adhere to embryonic mouse liver or brain cells in an in vitro cell-cell adhesion assay . Liver colonizing RAW117-H10 cells adhered with greater selectivity to liver cells than to brain cells . Parental RAW117-P cells were more homotypically adhesive, but they were nonselective in their organ cell adhesion properties . We examined RAW117 cells for the presence of liver cross-reactive antigens using polyclonal xenoantibody preparations directed against embryonic murine liver cells . These antibody preparations block organ-specific homotypic adhesion of embryonic murine liver cells in vitro . The amount of fetal liver antigen(s) expressed on RAW117 sublines correlated with liver colonization potentials (H10 greater than H5 greater than P) in quantitative absorption assays . Treatment of the highly metastatic RAW117-H10 subline with polyclonal anti-embryonic murine liver F(ab')2 or Fab' antibody fragments had no effect on RAW117-H10 cell viability or growth in vitro or in vivo, but inhibited liver colonization (median liver tumor colonies reduced from greater than 200 to 0) and prolonged life expectancy . In contrast, pretreatment of RAW117-H10 cells with polyclonal anti-H-2 did not modify the in vivo biologic properties of these metastatic cells.

Transfusion, 1984 Jul-Aug, 24(4), 353 - 6
Red cell phenotyping using hexadimethrine bromide (Polybrene) in a microplate system; Anderson HJ et al.; To provide rapidly phenotyped units of blood, we adapted the hexadimethrine bromide (Polybrene) technique to microplate technology . Pilot samples from 282 donor units were phenotyped for antigens in the Rh, Kidd, Kell, Duffy, and Ss systems with a standard tube-testing method and a Polybrene-microplate (P-MP) technique . Diluted antisera and a 1 percent red cell suspension were used to give P-MP reactions that were accurate and easy to interpret . One microplate, containing 96 tests, was prepared and read within 15 minutes for P-MP tests yielding direct agglutination (Rh, Jkb, Fya, Fyb), or within 19 minutes for P-MP tests requiring an antiglobulin phase (Jka, K, S, s) . No false-positive results were found . No false-negative reactions were found in typing for Rh, K, S, s, or Jka antigens . Phenotyping for Jkb and Duffy antigens gave a false-negative rate of less than 0.015 . Considerable savings in reagents were obtained through microplate miniaturization and through the enhancement of apparent antibody avidity in the Polybrene/low ionic medium which permitted dilution of reagent antisera . The P-MP technique affords a rapid, accurate, simple, and inexpensive means of phenotyping large numbers of donor units.

Am J Physiol, 1984 Jul, 247(1 Pt 1), G95 - 104
Pepsinogen secretion from dispersed chief cells from guinea pig stomach; Raufman JP et al.; In the present study we examined the actions of various secretagogues on pepsinogen secretion from freshly dispersed chief cells prepared from guinea pig stomach . Chief cells were obtained by preparing dispersed gastric glands, subjecting the glands to mechanical disruption in the presence of EGTA, and fractionating the resulting mucosal cells on a Percoll density gradient . Chief cells constituted 90% of the final cell suspension and cell viability was 99% . In these cells, pepsinogen secretion was stimulated by agents whose actions are probably mediated by calcium: carbachol, cholecystokinin, and A23187 . Pepsinogen secretion was also stimulated by agents whose actions are probably mediated by cAMP: secretin, vasoactive intestinal peptide, and 8-bromo-cAMP . Reducing the incubation temperature from 37 degrees to 4 degrees C or adding carbonyl cyanide m-chlorophenylhydrazone abolished secretagogue-induced pepsinogen secretion . These results indicate that freshly dispersed chief cells from guinea pig stomach are responsive to secretagogues and provide a suitable model for investigating cellular mechanisms of secretagogue-induced pepsinogen secretion.

Hematol Oncol, 1984 Jul-Sep, 2(3), 221 - 37
In situ immunologic characterization of follicular lymphomas; Ancelin E et al.; Surface markers were studied in a series of follicular lymphomas with immunofluorescence on frozen sections (39 cases) and on cell suspensions (21 cases), and with immunoperoxidase on frozen sections using a panel of 15 monoclonal antibodies (17 cases) . With immunofluorescence on frozen sections, 22/39 cases showed monotypic sIg (IgMK: 14 cases, IgML: 7 cases, M: 1 case) . In the remaining 17 cases the neoplastic follicles were negative . Nevertheless, even if sIg is not detected, the absence of an extracellular immunoglobulin network is indicative of the neoplastic, and not of the reactive nature of lymphoid follicles . The results obtained with immunofluorescence on frozen sections and on cell suspensions were identical in about half of the cases . In 9/21 cases monotypic sIg were detected by only one of these two methods . All the 17 cases studied with immunoperoxidase on frozen sections using monoclonal antibodies demonstrated monotypic sIg . On low magnification 6/17 sIg+ exhibited a nodular staining pattern while 7/17 cases this staining was diffuse . In 4/17 cases the staining pattern for heavy and light chains was different . A thin mantle zone, with sIgM plus sIgD cells, was observed in only 4 cases . Anti-HLA-DR and Leu-10 were positive in all cases . T cells positive for OKT3 were mainly distributed in the interfollicular areas; OKT4+ cells outnumbered OKT8+ cells . Within the neoplastic follicles, T cells stained mainly for OKT4 and OKT8+ cells were scarce . Leu-7+ cells predominated within the neoplastic nodules in 5 cases . With the anti-dendritic reticulum cell monoclonal antibody, all 17 cases showed a network, usually more loosely arranged than in reactive follicles . In 4 cases, of follicular and diffuse lymphoma, this network was extremely dissociated and in some areas these cells were scanty or lacking . We concluded that immunoperoxidase on frozen sections, using monoclonal antibodies, appears to be the most reliable method for the immunological phenotyping of follicular lymphomas.

Brain Res, 1984 Jul, 317(1), 21 - 32
Specificity of monoclonal antibody N1 for cell surfaces of mouse central nervous system neurons; Schnitzer J et al.; Monoclonal antibody N1 reacts by indirect immunofluorescence with the cell surface of tetanus toxin-positive neurons from early postnatal mouse cerebellum . In freshly trypsinized single cell suspensions from early postnatal mouse cerebellum, 5-10% of all viable cells express N1 antigen on their surface . After 3-24 h of maintenance in vitro all N1 antigen-positive cells are tetanus toxin-positive . After culture periods of 3-4 days, most (approximately 90%) tetanus toxin-positive cells express N1 antigen on their surface . When horse serum-supplemented medium (HSSM) is used for cultivation, neurons begin to lose N1 antigen from their surface after about one week in vitro, until after two weeks in vitro, N1 antigen is no longer detectable, although some tetanus toxin-positive neurons can be shown to survive in culture . In defined medium, however, N1 antigen-positive neurons can still be detected after 34 days in vitro, the longest culture period examined so far . Complement-dependent immunocytolysis deletes all N1 antigen-positive and approximately 90% of all tetanus toxin-positive neurons from cultures . The remaining neurons reveal a morphology different from the one of the majority of small neurons, the granule cells . They have slightly larger cell bodies and several branched and unbranched cellular processes . Neonatal cerebellar cells show the same temporal sequence of appearance and disappearance of N1 antigen on most tetanus toxin-positive neurons in HSSM, and a persistence of N1 antigen on neurons in defined medium . N1 antigen becomes first detectable at embryonic day 17, and never becomes detectable in cell cultures derived from cerebella of younger mice . At all stages studied, N1 antigen expression is restricted to tetanus toxin-positive neurons, while it is absent from the cell surfaces of astrocytes, oligodendrocytes and fibroblasts . N1 antigen is also found in cultures derived from early postnatal mouse cerebrum, but is not detected in cultures derived from mouse retina, spinal cord, dorsal root ganglion, and embryonic telencephalon . It is also not detectable in cerebellar cultures from rabbit, rat, chicken and human . When N1 antibody is applied to fixed cultures where intracellular antigens are accessible, all cell types are labeled intracellularly, with astrocytes and fibroblasts revealing a fibrillary, vimentin-like staining pattern.

J Clin Pathol, 1984 Jul, 37(7), 767 - 71
Reed-Sternberg/lymphocyte rosette: lymphocyte subpopulations as defined by monoclonal antibodies; Morris CS et al.; The Reed-Sternberg cell/lymphocyte rosette characteristic of Hodgkin's disease tissue and cell suspensions was investigated with monoclonal antibodies on fresh viable cell suspensions prepared from nine cases of Hodgkin's disease . The biopsy material comprised six spleens and three lymph nodes . The majority of the rosetting lymphocytes were T cells, primarily of the helper subset . Some of the attached lymphocytes were suppressor T cells . In addition, a few of the rosetting lymphocytes around Reed-Sternberg cells were B cells.

Br J Haematol, 1984 Jul, 57(3), 373 - 82
Leu-10 (HLA-DC/DS) antigen distribution in human leukaemic disorders as detected by a monoclonal antibody: correlation with HLA-DR expression; Al-Katib A et al.; Reactivity of the monoclonal antibody anti-Leu-10 that detects the human equivalent of the murine 1-A subregion antigen(s) was studied and correlated with anti-HLA-DR expression on 83 cases of acute and chronic leukaemias, leukaemic non-Hodgkin's lymphomas (NHL) and seven human cell lines . Peripheral blood and/or bone marrow leukaemic cell suspensions were stained by indirect immunofluorescence for both monoclonal antibodies and analysed by flow cytometry . Leu-10, like HLA-DR, was absent from T-cell acute lymphoblastic leukaemia (ALL) (two cases) . It was expressed on: 33% of TdT +, CALLA + ALL cases (8/24); 27% (4/15) of acute non-lymphocytic leukaemia (ANLL); 85% (24/28) of HLA-DR + B-cell chronic lymphocytic leukaemia (CLL); and on 9/14 (64%) B-cell NHL cases . There were no differences in clinical characteristics between Leu-10 + and Leu-10--patient subgroups . We were able to induce Leu-10 expression on 'Josh' cell line by culturing it with 12-0-tetra-decanoylphorbol-13-acetate (TPA) . Our data indicate that Leu-10 expression on leukaemic cells is more restricted than HLA-DR and is likely to be differentiation related, since it can be induced to be expressed at a later stage than HLA-DR.

J Exp Med, 1984 Jul 1, 160(1), 138 - 51
Proliferation of peritoneal mast cells in the skin of W/Wv mice that genetically lack mast cells; Sonoda T et al.; Presence of mast cell precursors in the mouse peritoneal cavity was demonstrated, and the precursors were characterized . When a cell suspension, containing mast cell precursor(s), was directly injected into the skin of genetically mast cell-deficient WBB6F1 (WB X C57BL/6)-W/Wv mice, a cluster composed of approximately 2,000 mast cells appeared at the injection site . By determining the proportion of injection sites at which the mast cell cluster appeared, the concentration of mast cell precursors can be calculated by limiting dilution analysis . The concentration in the peritoneal cavity was about five times as great as the concentration in the bone marrow . Although peritoneal mast cell precursors were shown to originate from the bone marrow, physical characterization revealed that the peritoneal precursors differed from the marrow precursors . The peritoneal precursors were less susceptible to irradiation than the marrow precursors; the former were heavier than the latter . When a 95% pure mast cell suspension was prepared from the peritoneal cells by the removal of phagocytes and the density gradient centrifugation, 1 out of 16 cells had the potentiality to make a mast cell cluster in the skin of the W/Wv mice . Moreover, when a single mast cell was identified under the phase contrast microscope and picked up with the micromanipulator, 1 out of 17 mast cells made the cluster . This indicated that some peritoneal mast cells kept extensive proliferative potentiality even after morphological differentiation . In other words, some peritoneal mast cells themselves may function as the committed precursors.

J Leukoc Biol, 1984 Jul, 36(1), 1 - 15
Impaired motility of neonatal PMN leukocytes: relationship to abnormalities of cell orientation and assembly of microtubules in chemotactic gradients; Anderson DC et al.; To allow a further understanding of the pathogenesis of impaired stimulated locomotion by polymorphonuclear leukocytes (PMNs) in human neonates, we studied cellular orientation by neonatal PMNs in response to well-defined chemotactic gradients (Zigmond orientation chambers) and characterized the cytoplasmic microtubule (MT) complex of neonatal PMNs during cell orientation and movement . PMN suspensions obtained from 52 neonates demonstrated a diminished capacity to undergo orientation at all time intervals after exposure to gradients of N-formyl-methionyl-leucyl phenylalanine (f-Met-Leu-Phe) or C5a . Among responding (orienting) neonatal PMNs observed, only 70% (f-Met-Leu-Phe) or 59% (C5a) oriented accurately (toward chemotactic gradients) as compared to values of 96% (f-Met-Leu-Phe) or 92% (C5a) for adult controls . Furthermore, neonatal PMNs failed to alter their direction of orientation/migration when chemotactic gradients were reversed . Similar abnormalities were observed when 10-fold gradients of f-Met-Leu-Phe were employed over a concentration range between 10(-7) and 10(-11) M . Employing tubulin immunofluorescence, the cytoplasmic MT complex of-neonatal PMNs was assessed prior to and after cell exposure to uniform concentrations or gradients of chemotactic factors (CFs) . MT assembly by neonatal PMNs studied under these experimental conditions was significantly diminished . Neonatal cell suspensions demonstrated 26 +/- 5 (f-Met-Leu-Phe) or 27 +/- 6 (C5a) MT/cell as compared to respective values of 36 +/- 6 or 35 +/- 5 for adult suspensions (P less than .001) . MT lengths of neonatal PMNs increased from 6.7 +/- 1 micron (PBS) to 7.5 +/- 1 micron (f-Met-Leu-Phe) or 7.3 +/- 1 micron (C5a) as compared to values of 6.5 +/- 1 micron (PBS), 11.1 +/- 1 micron (f-Met-Leu-Phe), and 10.9 +/- 1 micron (C5a) for adult PMNs exposed to gradients or uniform concentrations of CFs (P less than .01 for both f-Met-Leu-Phe and C5a) . Thus, the polymerized tubulin mass product of chemotactically stimulated neonatal PMNs (202 micron) was significantly (P less than .001) diminished as compared to adult PMNs (360 micron) . As shown by a {3H}colchicine binding assay, impaired MT assembly could not be attributed to diminished cytoplasmic tubulin content of neonatal PMNs, which was comparable to adult PMNs.

Am J Anat, 1984 Jul, 170(3), 491 - 9
Cellular basis of graft-versus-host reactions; Clancy J Jr; The biologic basis of Graft-Versus-Host Disease (GVHD) is presented as an extremely complex immunopathologic syndrome that involves interaction between many different donor and host cell types . A model of acute lethal GVHD was employed where adult unirradiated (DA X LEW)F1 rats were injected with LEW spleen and lymph node cells . Controls received the same dose of syngeneic cells . At intervals from 2 to 21 days after cell injection, GVHD and control animals were killed and nonadherent cell suspensions prepared from their lymph nodes, spleen and peripheral blood . Cell suspensions were treated with LEW-anti-DA-alloantiserum or normal LEW serum and then analyzed for sIgM+ (B cells), W 3/13+ (T cells), and IgG-Fc receptors (FcR) . Evidence is discussed for the selective removal of host cells with the alloantiserum . In addition, the level of naturally cytolytic (NK/NC) cells was assessed by adding GVHD and control nonadherent lymphoid cells to heterologous lymphoma and sarcoma target cells . Evidence is presented that during acute GVHD, in this parental----F1 combination, there is an early increase within most compartments of donor as well as host W 3/13+ and W 3/13+FcR+ cells . NK/NC cells are increased as well at day 7 . During middle stages of acute GVHD, host sIgM+ cells predominate . Late-stage acute GVHD rats contain few donor and host W 3/13+, W 3/13+FcR+, and NK/NC cells but many null cells most of which are FcR- . The importance of unraveling the nature of donor- and host-cell interactions occurring during acute GVHD, which result in rats whose lymphoid tissues are severely depleted of all nonadherent lymphoid cells but FcR- null cells, is discussed.

Radiat Res, 1984 Jul, 99(1), 151 - 64
Effect of sublethal ionizing radiation on rat Peyer's patch lymphocytes; Hale ML et al.; After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes . Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source . On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets) . Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes . The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined . From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation . In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation . These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.

Drug Metab Dispos, 1984 Jul-Aug, 12(4), 427 - 31
Oxidative and conjugative metabolism of xenobiotics by isolated rat and hamster acinar cells; Wiebkin P et al.; Isolated rat and hamster acinar cell suspensions possess the ability to carry out the cytochrome P-450-dependent O-deethylation of 7-ethoxycoumarin, 2-,3-, and 4-hydroxylation of biphenyl and 3-hydroxylation of benzo(a)pyrene . Rat and hamster acinar cells isolated from 5,6-benzoflavone-pretreated animals oxidize all three substrates at measurable rates . These rates are considerably lower (16-210-fold in the rat and 290-2670-fold in the hamster) than those in incubations using hepatocytes isolated from 5,6-benzoflavone-pretreated animals . Hydroxylation of biphenyl at the 2-, 3-, and 4-positions proceeds at similar rates in rat acinar cells . The rate of 3-hydroxybiphenyl formation is barely detectable in hamster acinar cells where the rates of 2- and 4-hydroxybiphenyl formation are slower than in the rat . No detectable oxidation products of 7-ethoxycoumarin, biphenyl, or benzo(a)pyrene are found with acinar cells of either species isolated from untreated and phenobarbital-pretreated animals . The O-deethylation of 7-ethoxycoumarin in rat and hamster acinar cells is decreased in the presence of inhibitors of the cytochrome P-450-dependent monooxygenase system, 7,8-benzoflavone being much more effective than metyrapone . The deethylation product of 7-ethoxycoumarin, 7-hydroxycoumarin, is conjugated with sulfate and glucuronic acid moieties at appreciable rates by acinar cells isolated from both rat and hamster . Pretreatment of rats and hamsters with either 5,6-benzoflavone or phenobarbital has little effect on the rates of conjugation in isolated acinar cell preparations.

J Gen Virol, 1984 Jul, 65 ( Pt 7), 1221 - 4
Rapid differentiation of herpes simplex virus types 1 and 2 by cytopathic effect in BS-C-1 cells; Hills RA et al.; Conditions are defined under which herpes simplex virus (HSV) type 2 produces a characteristic cytopathic effect (c.p.e.) in African green monkey kidney (BS-C-1) cells . The BS-C-1 microplate c.p.e . test was simultaneously evaluated against immunological and biological marker procedures on 359 HSV strains . A BS-C-1 cell suspension was added to serial dilutions of HSV strains in Falcon Micro Test II plates and incubated at 36.5 degrees C in a 4% CO2 atmosphere . When the infected cultures were examined microscopically, distinctive pseudopod-like extensions were seen in cultures infected with type 2 strains but not seen when the infecting virus was type 1, forming the basis for a differentiating test . The test was 100% accurate in differentiating between HSV-1 and -2 on a one test per specimen basis . The method is simple, rapid and requires no specialized reagents.

Boll Soc Ital Biol Sper, 1984 Jun 30, 60(6), 1283 - 5
A monoclonal antibody to human transferrin receptor which inhibits erythroid differentiation of human leukemic K-562 cells; Gambari R; In this paper we demonstrate that the 42/6 monoclonal antibody to human transferrin receptor (1) inhibits erythroid differentiation of human leukemic K-562 cells without affecting cell proliferation . Erythroid induction was monitored by benzidine-staining of K-562 cell suspensions and hemoglobin accumulation by cellulose acetate gel electrophoresis of post-mitochondrial cell lysates (4,5) . Our results suggest that erythroid differentiation and cell growth require a different number of transferrin receptors.

Eur J Biochem, 1984 Jun 15, 141(3), 527 - 9
Changes of glycolipids dependent on cell density of Ehrlich ascites carcinoma cells; Prokazova NV et al.; The glycolipid composition of Ehrlich ascites carcinoma cells was found to depend strongly on the cell density of the suspension . The general trend observed upon dilution of the cell suspension was a reduction of the less complex gangliosides GM3 and GM2 with concomitant increase of the more complex gangliosides, especially GM1 . The increase of the content of ganglioside GM1 upon dilution was accompanied by a comparable decrease of the content of its immediate precursor, asialo-GM1, whereas the content of other neutral glycosphingolipids did not change very much . When the cell suspension was diluted with medium conditioned by dense cells the ganglioside profile of the diluted suspension remained similar to that of the dense cell suspension . It is postulated that the medium conditioned with dense cells contains a transferable factor inhibiting sialylation of asialo-GM1.

Biochem Biophys Res Commun, 1984 Jun 15, 121(2), 499 - 506
Steroidogenic properties of phorbol ester and a Ca2+ ionophore in bovine adrenocortical cell suspensions; Culty M et al.; When added independently to bovine adrenocortical fasciculata cell suspensions, 12 tetradecanoyl-phorbol-13 acetate (TPA) and the Ca2+ ionophore A23187 activated net cortisol production in a time and dose-dependent manner during one hour incubation . When added together (each at 1 microM concentration), the drugs appeared synergistic and mimicked the steroidogenic effect of suboptimal concentration of angiotensin II or acetylcholine on these cells, with no detectable variation of cellular cyclic nucleotide levels . In addition, the drug mixture markedly enhanced the steroidogenic effect of acetylcholine . These observations suggest that Ca2+-activated, phospholipid dependent protein kinase, which is present in adrenal cortex, might be considered as a possible target in the mechanism of action of steroidogenic agents such as angiotensin and acetylcholine, acting in adrenocortical cell through cyclic AMP independent processes.

Biochim Biophys Acta, 1984 Jun 13, 773(1), 143 - 56
Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells; Tiffert T et al.; Calcium chelators which can be incorporated inside small cells without disruption have become useful tools to investigate the role of intracellular ionized calcium in the processes of cell activation and signal-effect mediation . In experiments designed to investigate further Ca2+ pump function in chelator-loaded human red cells we found that the chelator-loading procedure itself caused delayed Ca2+-pump inhibition when pump function was explored by increasing the intracellular Ca2+ levels with the aid of the divalent cation ionophore A23187 . Ca2+-pump inhibition was found to be secondary to ATP-depletion, and ATP-depletion, in turn, could be attributed to formaldehyde, which was released during the hydrolytic incorporation of free chelator, from the cleavage of the four ester groups which anchor it to cell membranes on addition to cell suspensions . The evidence suggests that the formaldehyde released stays largely within the cells . Formaldehyde, in concentrations of up to 20 mmol/l cells had no direct effects on Ca2+ transport in red cells, other than through ATP depletion . Procedures to circumvent the difficulties arising from the formaldehyde effects are outlined and discussed.

Nucleic Acids Res, 1984 Jun 11, 12(11), 4621 - 4
Nucleotide sequences of chloroplast 5S ribosomal RNA from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata; Yamano Y et al.; The nucleotide sequences of chloroplast 5S rRNAs from cell suspension cultures of the liverworts Marchantia polymorpha and Jungermannia subulata were determined . Their nucleotide sequences, 119 nucleotides long, were highly homologous to each other (96% identity) and had high homology with those from chloroplast 5S rRNAs of two higher plants, tobacco (92% identity) and spinach (92-91% identity), but less homology (87-85% identity) with that from a lower plant, the fern Dryopteris acuminata.

J Biol Chem, 1984 Jun 10, 259(11), 6806 - 11
Purification and properties of a stilbene synthase from induced cell suspension cultures of peanut; Schoppner A et al.; Stilbene synthase ( resveratrol -forming) converts one molecule of rho- coumaroyl -CoA and three molecules of malonyl-CoA into 3,4',5- trihydroxystilbene . Following selective induction of stilbene synthesis in cell suspension cultures of peanut (Arachis hypogaea), the enzyme was extracted and purified to apparent homogeneity by chromatography on DEAE-cellulose and hydroxylapatite . The enzyme was found to be a dimer of estimated Mr = 90,000 exhibiting under denaturing conditions a subunit Mr of approximately 45,000 . The isoelectric point was determined with pI = 4.8 . The enzyme's high selectivity towards rho- coumaroyl -CoA (Km = 2 microM) as substrate qualified it as resveratrol -forming stilbene synthase . Structurally related CoA esters, e.g . dihydro-rho- coumaroyl -CoA and cinnamoyl-CoA, were converted less than 1/10 as efficiently as rho- coumaroyl -CoA . Malonyl-CoA (Km = 10 microM) could not be substituted by acetyl-CoA . The purified enzyme was free of chalcone synthase activity . Antibodies raised against stilbene synthase were shown to be monospecific and not to cross-react with chalcone synthase.

Am J Vet Res, 1984 Jun, 45(6), 1225 - 9
Autotransplantation of bovine ocular squamous cell carcinoma and effects on primary tumor growth; Dennis MW et al.; Autologous transplantation of ocular squamous cell carcinoma was done in 7 Hereford cows in 17 trials . Three preparations of tumor were used in orthotopic transplantation to 5 sites on the eye and eyelid . None of the transplants was successful . However, in 2 of 5 cows given autografts of a pure, viable tumor cell suspension, marked regression of the primary tumor was observed after transplantation.

Br J Cancer, 1984 Jun, 49(6), 787 - 93
Hypoxic cells and in situ chemopotentiation of the nitrosoureas by misonidazole; Wheeler KT et al.; Intracerebral (i.c.) and subcutaneous (s.c.) 9L tumours were treated simultaneously with various doses of the nitrosoureas, BCNU or CCNU, and 2.5 mmol kg-1 of misonidazole (MISO) . After 24 h, tumours were removed, dissociated into single cell suspensions and the cells plated for colony formation . In both i.c . and s.c . tumours, no cell kill was observed after exposure to MISO alone, and no additional cell kill was observed when MISO was combined with either nitrosourea . If s.c . 9L tumours were clamped 30 min after i.p . injection of 2.5 mmol kg-1 MISO, then 2 h later the clamps were removed and the nitrosourea injected, an increase in cell kill was observed . This increase in cell kill was statistically significant (P less than 0.01) for each dose of BCNU administered, but not statistically significant (P greater than 0.05) for the moderate dose of CCNU administered . Clamping did not alter the colony forming efficiency of cells from untreated 9L s.c . tumours or from those treated with each drug alone . These data demonstrate that hypoxic cells are required for misonidazole to potentiate the cell-killing effects of the nitrosoureas and that s.c . 9L tumours contain no such cells.

Biochem Pharmacol, 1984 Jun 1, 33(11), 1715 - 8
Phenylhydrazine-induced lipid peroxidation of red blood cells in vitro and in vivo: monitoring by the production of volatile hydrocarbons; Clemens MR et al.; Human red blood cells and male Sprague-Dawley rats were treated in vitro and in vivo, respectively, with phenylhydrazine in order to determine whether the release of volatile hydrocarbons can serve as a suitable index for phenylhydrazine-induced red blood cell peroxidation . Lipid peroxidation following phenylhydrazine administration (in vitro experiments: dosage calculated at 0.5-50 mM; in vivo experiments: intraperitoneal injection of 2.8 mg/100 g body wt) was monitored by the release of ethane and pentane measured by gas chromatography . Further hydrocarbons such as ethylene, propane, n-butane, iso-butane and iso-butene were monitored to form a basis of comparison . In vitro haemolysis was also determined during the course of incubation . Red blood cell suspensions yielded more than 15-fold concentrations of propane and more than 2-fold concentrations of iso-butane compared to pentane and ethane yields . Haemoglobin solutions also produced propane and iso-butane in the presence of phenylhydrazine, whereas pentane and ethane were not detectable . Time-course studies revealed that ethane and pentane reached maximum in vitro levels after red blood cell suspensions had been incubated for 2 hr whereas the maximum degree of haemolysis (approximately 60%) was attained between 60 and 90 min following the beginning of phenylhydrazine treatment . The dosage did not affect the final degree of haemolysis . Rats treated with phenylhydrazine exhaled greater concentrations of ethane (6-fold increase) and pentane (2-fold increase) compared to control animals . Exhaled propane showed a 30-fold increase in concentration following drug treatment . Our results suggest that the release of pentane and ethane may be useful in assessing red blood cell lipid peroxidation in the presence of phenylhydrazine in vitro and in vivo.

Cancer, 1984 Jun 1, 53(11), 2444 - 9
Monocyte-mediated-antibody-dependent cellular cytotoxicity in malignant lymphoma and solid tumors; de Mulder PH et al.; Monocyte-mediated-antibody-dependent cellular cytotoxicity (MO-ADCC) was studied in 21 patients with Hodgkin's disease (HD), 15 patients with a long-lasting remission of HD, 11 patients with non-Hodgkin's lymphoma (NHL), 11 patients with solid tumors, and 15 normal controls . Lymphocyte ADCC (LY-ADCC) was evaluated in 12 patients with HD and 9 normal controls . Monocytes lymphocytes were isolated with cell-scatter monitored counterflow centrifugation providing high purity and yield . Antibody-dependent cellular cytotoxicity was evaluated by means of DNA flowcytometry, using antibody-coated chicken erythrocyte targets (CRBC) . In comparison with normal controls MO-ADCC was significantly increased in HD (P less than 0.0005), NHL (P less than 0.005), and solid tumors (P less than 0.005) . In patients in long-lasting complete remission of HD, MO-ADCC was in the normal range . Lymphocyte-ADCC of 12 patients with HD was similar to that of 9 normal controls . In all experiments LY-ADCC was invariably lower than MO-ADCC of the same donor, indicating the monocyte as the most potent effector cell towards CRBC targets . Results indicate the following: (1) purified cell suspensions of both lymphocytes and monocytes are essential to unravel their role as effector cells; (2) LY-ADCC in HD is similar to normal controls; (3) MO-ADCC enhancement is not uncommon in malignant lymphoma and several solid tumors; (4) normal MO-ADCC in a group of successfully treated patients with HD suggests a disease-related induction of enhanced MO-ADCC.

J Immunol, 1984 Jun, 132(6), 3142 - 8
Fibroblast stimulation in schistosomiasis . V . Egg granuloma macrophages spontaneously secrete a fibroblast-stimulating factor; Wyler DJ et al.; Isolated intact egg granulomas from the liver of Schistosoma mansoni-infected mice have been previously shown to elaborate factors in vitro that can stimulate fibroblasts for biological functions that are of potential importance in the pathogenesis of hepatic fibrosis in schistosomiasis . We report here that cell cultures obtained from monodispersed granuloma cell suspensions, and specifically enriched for macrophages (95% to 100%) spontaneously elaborated fibroblast proliferation-stimulating activity in vitro . These cells possessed functional and phenotyptic characteristics of activated macrophages . In contrast, control peritoneal macrophages from uninfected mice lacked such phenotypic characteristics, and did not spontaneously elaborate fibrogenic activity in vitro . The granuloma macrophage activity was present, pre-formed within the isolated cells, and was continuously elaborated during 72 hr of incubation . By gel infiltration chromatography (Sephacryl S-200 sf), fibroblast-stimulating activity was identified in two pooled fractions, one with estimated molecular radius (Mr) of 46 kd to 57 kd and the other with Mr of 10 kd to 16 kd . Preparative isoelectric focusing in granular gel of crude macrophage culture supernatants identified peak activity in fractions with pI approximately 5 . Two different serine esterase inhibitors had no effect on the ability of crude granuloma macrophage supernatants to stimulate fibroblast proliferation . Whereas crude and chromatographed fractions of granuloma macrophage supernatant were active for fibroblasts, they had minimal or no interleukin 1 (IL 1) activity when tested in a thymocyte proliferation assay . In contrast, resident peritoneal macrophages from the same infected mice spontaneously secreted substantial IL 1 and fibroblast-stimulating activity in vitro . We conclude that egg granuloma macrophages are activated in vivo to secrete fibrogenic molecules functionally distinct from IL 1, which might contribute to the pathogenesis of hepatic fibrosis in schistosomiasis.

J Leukoc Biol, 1984 Jun, 35(6), 561 - 72
In situ proliferation of intratumor macrophages; Evans R et al.; This investigation was carried out to assess whether the progressive increase in the number of macrophages associated with growing tumors was the result of an influx of monocytes from the circulation as well as proliferation of macrophages in situ . Tumor-associated macrophages (TAM) were identified in cell suspensions prepared from three C57BL/6J (B6), methylcholanthrene-induced sarcomas, designated MCA/76-9, 76-64, and 77-23, growing in normal or irradiated mice . When the sarcomas were implanted in B6 mice exposed to a sublethal dose (800 R) of whole body irradiation ( WBI ), tumors grew very slowly compared with control tumors . The TAM numbers were small and the percentages of macrophages were very low . The levels of TAM corresponded with the low number of circulating blood monocytes . However, compared with the number of TAM in the initial inoculum of tumor cells injected into the WBI or control mice, the TAM numbers over a period of 14 days increased several fold in WBI mice . That this increase was initially due to infiltration from the circulation was shown by injecting macrophage-free, cultured tumor cells into WBI or control mice . Proliferation in situ was demonstrated by autoradiography experiments . When 3H-thymidine was injected 1 hr before excision of MCA/76-9 tumors, labeled cells were seen in histological sections and in cell suspensions . The labeling indices for TAM from tumors growing in WBI or normal mice were not significantly different from tumor cell labeling indices . The overall data indicated that the progressive increase in TAM in these sarcomas was the combined result of monocyte infiltration and proliferation of macrophages in situ.

Eur J Cancer Clin Oncol, 1984 Jun, 20(6), 807 - 15
The effect of tamoxifen on the growth of human malignant melanoma in vitro; Gill PG et al.; The effect of tamoxifen on the growth of malignant melanoma was investigated using human cell lines and single-cell suspensions prepared from patients' tumours cultured in soft agar . Tamoxifen stimulated both {3H}-thymidine incorporation and cell numbers in all of the cell lines tested . Cytoplasmic oestrogen receptor (ER) was detected in one of the responding lines and progesterone receptor (PR) in another . Tumour colony formation in soft agar culture was satisfactorily established from tumour cell suspensions from 13 of 21 patients, only one of which had detectable cytoplasmic ER . Greater than 50% reduction in colony formation with 5 X 10(-7) M tamoxifen occurred in two tumours, neither of which contained ER . These results indicate that tamoxifen has the potential to either retard or accelerate the growth of human malignant melanoma.

Cell Struct Funct, 1984 Jun, 9(2), 193 - 6
Somatic hybridization between human and mouse lymphoblast cells produced by an electric pulse-induced fusion technique; Ohno-Shosaku T et al.; The technique of electric pulse-induced cell fusion (electro-fusion) was used to obtain heterokaryons between normal human lymphoblasts (HSC93) and mouse leukemic lymphoblasts (MCN151) . The two types of cells were brought into contact in the cell suspension by dielectrophoresis with an alternating electric field (0.8 kV/cm, 100 kHz) in the presence of calcium ions and pronase E . Cell fusion was induced by giving two successive electric pulses (3.3 and 5 kV/cm, 10 microsec) . Prior treatment of human (but not mouse) lymphoblasts with neuraminidase improved fusion efficiency . Differential staining of the two types of cells with Janus Green and Neutral Red showed that about 40% of the viable fused cells underwent heterokaryonic fusion . We concluded that electrofusion is an efficient method for obtaining heterokaryons from human and mouse lymphoblasts.

Biochem Int, 1984 Jun, 8(6), 821 - 30
Visible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensions; Videla LA et al.; Red blood cells from Wistar rats were exposed to milimolar concentrations of t-butyl hydroperoxide . Extensive hemoglobin oxidation (methemoglobin formation), t-butyl hydroperoxide cleavage (t-butanol formation) and peroxidation (measured by oxygen consumption and thiobarbituric acid reactive substances) was observed . Significant chemiluminescence was emitted by the system . Hemoglobin oxidation and t-butanol production were independent of oxygen pressure and free radical scavengers, however, luminescence was enhanced as oxygen pressure increased and it was reduced by addition of free radical scavengers . The spectral distribution of the light emitted suggests that the luminescence detected is not due to singlet oxygen dimol emission . The results are in agreement with a lipid peroxidative mechanism initiated by t-butoxy radicals produced in the interaction of hemoglobin and t-butyl hydroperoxide.

Lab Invest, 1984 Jun, 50(6), 733 - 41
Methods in laboratory investigation . Immunoelectron microscopic methods for demonstration of antigens on normal human melanocytes and other epidermal cells; van Duinen SG et al.; Different procedures for fixation and processing were evaluated in order to examine the antigenic profile of melanocytes and other epidermal cells for immunoelectron microscopy . For this purpose the monoclonal antibodies anti-HLA-A, B, C, anti-HLA-DR, anti-T6, and the melanoma-associated monoclonal antibody NKI /C-3 were used as markers . Fixation with periodate-lysine-paraformaldehyde yielded better antigenic and ultrastructural preservation than 3% paraformaldehyde or picric acid-paraformaldehyde did . Skin was further processed by five different methods: (a) 15-micron frozen sections; (b) 75-micron agar-embedded, tissue chopper sections; (c) 15-micron polyethylene glycol-embedded sections; (d) epidermal cells in suspension; and (e) epoxy sections (postembedding staining) were prepared for the immunoperoxidase procedure . Antigenicity was best preserved in the cell suspension method and somewhat less, but with a similar staining distribution, with the first three methods . Staining with the polyethylene glycol-embedded sections was only achieved if they were left free-floating in buffer; no staining was observed when the sections were mounted on glass slides and left to dry overnight at 37 degrees C . Epidermal cells remained unreactive in the postembedding method, even after etching . Ultrastructural preservation of the agar-embedded sections and the cells in suspension was superior to the other preembedding methods . Melanocytes mostly showed moderate staining for HLA-A, B, C and slight staining for the antigen that is recognized by NKI /C-3 . The latter was further demonstrated on Langerhans cells and indeterminate cells which also expressed HLA-A, B, C, T6, and HLA-DR antigens.

J Invest Dermatol, 1984 Jun, 82(6), 602 - 4
Epidermal Langerhans cells contain intermediate-sized filaments of the vimentin type: an immunocytologic study; de Waal RM et al.; In epidermal cell suspensions, prepared from healthy human and murine skin, Langerhans cells (LC) were identified by indirect immunofluorescence microscopy using monoclonal antibodies directed against human T6 and Ia markers, and against a murine Iak determinant, respectively . The cells were double-labeled on cytospin slides with antibodies against a cytoskeletal protein, either vimentin or keratin . In this way it was shown that epidermal LC contain intermediate-sized filaments of the vimentin type.

J Neurocytol, 1984 Jun, 13(3), 329 - 38
A4: an antigenic marker of neural tube-derived cells; Miller RH et al.; The A4 monoclonal antibody was originally found to bind to the surface of the majority of neurons in rat CNS cultures, but not to PNS neurons or non-neural cells . It was subsequently shown to bind to immature oligodendrocytes and their precursor cells but not to the most mature oligodendrocytes . In the present study, we have used immunofluorescence assays on cell suspensions and cultures and on semi-thin, frozen tissue sections to show that protoplasmic and fibrous astrocytes and most ependymal cells are also A4+ . Taken together, these results suggest that in adult rats A4 is expressed exclusively by cells of the CNS and that all cell types derived from the neural tube are A4+, at least at some time in their development . While neurons, astrocytes and ependymal cells continue to express the antigen in adults, most oligodendrocytes appear to lose it as they mature . The finding that macrophages in CNS cell suspensions and cultures are A4- suggests that microglial cells are not derived from the neural tube.

Biull Eksp Biol Med, 1984 Jun, 97(6), 762 - 4
{Method of determining the activity of cell suspensions as determined by their natural motility}; Es'kov AP et al.; A new method and setup for determination of motile cell suspension activity are offered . A possibility of measuring the activity with regard to the quota of motile cells and their mean velocity has been shown . Bovine sperm in two diluents was applied as a test object.

J Invest Dermatol, 1984 Jun, 82(6), 605 - 7
Langerhans cell production of interleukin-1; Sauder DN et al.; Epidermal Langerhans cells (LC) are related to certain cells of the monocyte-macrophage lineage and to other dendritic cells . While epidermal LC and macrophages bear receptors for the Fc portion of IgG, other dendritic cells do not . However, unlike human dendritic cells from peripheral blood, LC bear the antigen against which the OKT6 antibody is directed . Within the skin this antibody binds only to LC or indeterminate cells . Functionally LC and dendritic cells can present antigen to sensitized T cells and are capable of stimulating allogenic T cells . Since lymphokines are thought to play an important role in T-cell activation and since LC are potent stimulators of antigen-specific T-cell proliferation, we investigated whether LC could produce interleukin-1 (IL-1) . Our initial studies revealed that whole epidermal cell suspensions comprised of LC, keratinocytes, and melanocytes produce a factor that is similar to macrophage-derived IL-1 . We termed this factor epidermal cell-derived thymocyte-activating factor ( ETAF ) . Using a panning technique we obtained a highly enriched (up to 97%) population of LC which constitutively produced significant IL-1-like activity . Using an antibody against the monocyte-derived leukocytic pyrogen (LP) which has been shown to inhibit IL-1 activity, we were able to inhibit IL-1 activity from LC-enriched cultures . The results of this study indicate that within the epidermis there are at least two cell populations, keratinocytes and LC, that can constitutively secrete potentially important soluble immunostimulatory factors.

Cancer Res, 1984 Jun, 44(6), 2530 - 3
Comparison of growth of human bladder cancer in tissue culture or as xenografts with clinical and pathological characteristics; Kovnat A et al.; Seventy-four biopsies of human bladder carcinoma were assessed by implantation as xenografts in immune-deprived mice and/or by culture of cell suspensions in agar or methylcellulose . The quality of the cell suspensions was assessed immediately after plating in vitro . The results were compared with the pathological stage and grade of the biopsies and with the clinical course of the disease in patients from whom the biopsies were obtained . We found that (a) progressively growing xenografts were generated from 20 of 53 biopsies (38%) . These xenografts grew with mean volume doubling times in the range of 1 to 3 weeks; all of them examined histologically were consistent with transitional cell carcinoma . (b) Colony formation occurred from 21 of 49 cell suspensions (43%), and plating efficiency was in the range of 0.0004 to 1.7% . The majority of cell suspensions were found to have residual small clusters of cells . Colony formation sometimes originated from these clusters, an effect that would be expected to introduce artifacts when the in vivo cloning assay is used for chemosensitivity testing . (c) There was no evident correlation between expression of clonal growth in vitro and success of xenografting, and no correlation between the results of either of these experimental procedures with stage, grade, or clinical course of the disease . Further improvements in tissue culture and xenograft technology will be required before these methods can be used as a guide to patient management.

J Neurosci, 1984 Jun, 4(6), 1598 - 606
Combination of immunocytochemistry and radioligand receptor assay to identify beta-adrenergic receptor subtypes on astroglia in vitro; Trimmer PA et al.; There is an increasing need to assess the distribution of receptors for neuroactive substances on specific neural cell types . This study describes the establishment of methodology that combines the quantification of beta-adrenergic receptor subtypes by radioligand binding assays with immunocytochemical analysis of the contribution of astroglia (identified by the presence of glial fibrillary acidic protein) and fibroblasts (identified by the presence of fibronectin) to cultures prepared from neonatal rat cerebral cortex . The effects of subtle changes in culture methodology on the cellular composition of cerebral cortical cultures and the distribution of beta-adrenergic receptor subtypes were examined . The data indicate that (1) a decrease in the density of the initial plating suspension, (2) an increase in the age of the animals, or (3) supplementation of the cortical cell suspension with meningeal fibroblasts all result in an increase in fibronectin staining and a decrease in glial fibrillary acidic protein antibody staining . This change in the cellular composition of the cortical cultures correlated with an increase in the number of beta 2-adrenergic receptors and a corresponding decrease in the number of beta 1-adrenergic receptors . These observations point out the care which must be exercised when preparing primary astroglial cultures of sufficient purity for large-scale biochemical and pharmacological studies.

Immunology, 1984 Jun, 52(2), 325 - 30
Differential effect of two monoclonal anti-Lyt-1 alloantibodies on lymphocyte distribution in vivo; Carroll AM et al.; The effect of anti-Lyt-1 monoclonal antibody treatment on the in vivo migration properties of mouse T lymphocytes was examined . Cells were treated with antibody in the presence or absence of complement . T cells of the Lyt-2 cytotoxic/suppressor phenotype were selected from B6 spleen cell suspensions by cytotoxic elimination with an allospecific anti-Lyt-1.2 antibody . These cells were labelled with 51Cr and transferred to syngeneic recipients which were killed 1 or 24 hr later for assessment of radiolabel tissue distribution . In vivo migration of anti-Lyt-1.2 selected cells was similar to that of unselected T cells . By contrast, a functionally and phenotypically identical population selected from C3H spleen T cells with an anti-Lyt-1.1 monoclonal antibody showed increased accumulation in recipient liver and a decline in recovery from lymph nodes and spleen . Treatment with anti-Lyt-1.1 in the absence of complement caused even greater localization to the liver, with a simultaneous poor recovery from lymphoid tissues, similar treatment of cells from B6 Lyt-1.1 . congenic mice also resulted in altered migration . Treatment of B6 lymphocytes with anti-Lyt-1.2 in the absence of complement caused little change in the normal T cell tissue localization . These results suggest that selected Lyt-2 cells, reported to express low levels of Lyt-1 antigen undetectable by conventional u.v light microscopy immunofluorescence and serological methods, may contain residual bound antibody which can result in opsonization and sequestration of the cells by recipient stromal cells after in vivo transfer . Binding of the antibody directed to the Lyt-1.2 allospecificity of B6, although of the same immunoglobulin class and selecting for a similar functional set by cytotoxic elimination, does not cause such sequestration in vivo.

Endocrinology, 1984 Jun, 114(6), 2344 - 8
Down-regulation by 17 beta-estradiol of D2 dopamine receptors in the MtTF4 pituitary tumor; Albaladejo V et al.; We have recently reported that 17 beta-estradiol (E2) paradoxically inhibits the growth of the rat MtTF4 pituitary tumor which has been induced by estrogen administration . While looking for a molecular explanation for these divergent effects, we observed that E2 treatment resulted in a marked decrease of D2 dopamine receptors (RDA) in the tumor but not in normal pituitary glands . Herein, we characterize further the effect of E2 on RDA concentration in the tumor . Three weeks after a sc injection of a MtTF4 -cell suspension, adult male Fischer rats were treated, or not, either with E2 or with various other steroids . The number of dopamine-binding sites (Bmax) was determined on crude membranes by Scatchard analyses with the dopamine antagonist {3H}spiroperidol . Only one kind of binding site was observed, and the affinity constant for {3H}spiroperidol was not significantly modified by any of the various treatments used . The decrease of Bmax after 8 days of treatment was dose dependent and was maximal with 5-micrograms daily doses of E2 . With 10 micrograms E, daily, Bmax decreased exponentially with the duration of the treatment; t 1/2 was approximately 5 days . Treatment for 8 days with progesterone (50 micrograms/day), dihydrotestosterone (50 micrograms/day) or 17 alpha-estradiol (10 micrograms/day), known to be inactive on tumor growth, did not alter Bmax, whereas diethylstilbestrol (10 micrograms/day) or dexamethasone (50 micrograms/day), which inhibit tumor growth, were as efficient as E2 in decreasing Bmax . In conclusion, the number of dopamine-binding sites in the membranes of MtTF4 tumor is decreased by E2 in a time- and dose-dependent fashion . Circumstantial evidence suggests that this decrease is due to a loss of RDA per cell rather than the loss of RDA-bearing cells . The relationship between the control of dopamine-binding sites and cell growth is not clear; however, this model may be useful for the elucidation of the mechanism by which E2 modulates cell membrane properties.

Cancer Res, 1984 Jun, 44(6), 2309 - 12
Comparison of drug sensitivity among tumor cells within a tumor, between primary tumor and metastases, and between different metastases in the human tumor colony-forming assay; Tanigawa N et al.; The human tumor colony-forming assay was used to compare chemosensitivity among tumor cells within a primary tumor, between primary tumor and metastases, and between different metastases . No significant differences in cloning efficiency were found in any of the three comparison studies . However, considerable differences in chemosensitivities were observed between different parts of the same tumor and between the primary tumor and metastases . Two different parts of the same tumor were comparably assayed for nine primary tumors . In nine paired samples which allowed in vitro drug sensitivity testing, there was no satisfactory correlation of sensitivity to cytostatic drugs . Cell suspensions were prepared from 28 primary tumors and from metastases taken from the same patient . In 14 paired samples which formed sufficient colonies for determination of drug effect, the data showed no satisfactory correlation of chemosensitivity between a primary tumor and its metastases . Both tumor samples from different metastatic sites of the same patient formed sufficient colonies in seven of eight instances . In the seven paired samples, there was strong association of chemosensitivity (p less than 0.005) . The results indicate that the reported discrepancies of in vitro and in vivo results in clinical trials using the tumor colony-forming assay for predicting resistance or sensitivity to cytostatic drugs may be due to therapeutic heterogeneity among tumor colony-forming units within a primary tumor and between a primary tumor and its metastases.

Clin Exp Immunol, 1984 Jun, 56(3), 716 - 22
Preferential generation of leukotriene C4 by human eosinophils; Shaw RJ et al.; The leukotriene generating capacities of ionophore stimulated human eosinophils and neutrophils were compared using specific radioimmunoassays for LTB4 and LTC4 . Mixed granulocyte preparations (neutrophils and eosinophils) produced both LTB4 and LTC4 in a time-dependent fashion which was maximal at 10 and 15 min, respectively . Following the separation of eosinophils (greater than 75%) and neutrophils (greater than 90%) by metrizamide gradients, LTC4 production was predominantly from eosinophils, whereas neutrophils were the principal source of LTB4 . The concentrations of leukotrienes produced by the eosinophil and neutrophil rich cell preparations were directly proportional to the concentration of ionophore . Following purification of eosinophil derived products by RP-HPLC the LTC4 immunoreactivity corresponded to the elution profile of a synthetic LTC4 marker . Furthermore, in 32 atopic subjects (21 bronchial asthmatics and 11 non-asthmatics) the amounts of LTC4 produced by unseparated leucocytes were directly proportional to the percentage of eosinophils in the total cell suspension . Preferential generation of LTB4 by neutrophils was also demonstrated by immunoreactivity of ionophore stimulated supernatants subjected to RP-HPLC, as well as by its characteristic u.v . absorbance and GC-MS profile and the ability to promote directional neutrophil locomotion (chemotaxis) . These experiments support the concept that eosinophils accumulate in tissues partly as a result of the response to neutrophil derived LTB4, and that these cells contribute to the production of sulphidopeptide leukotrienes with subsequent amplification of the acute allergic response.

Biochim Biophys Acta, 1984 May 30, 772(3), 407 - 10
Magneto-electro-fusion of human erythrocytes; Kramer I et al.; In inhomogeneous (static) magnetic fields close contact between 'magnetic' human erythrocytes was established . The cells were made magnetic by incubating them in a medium containing small Fe3O4 -particles which adsorbed to the outer membrane surface . Fusion was induced by applying two electric field pulses (field strength: 8.5 kV X cm-1; duration: 60 microseconds) to the magnetically collected cells . This procedure allowed the use of electrically conductive media (3 X 10(-3) omega -1 X cm-1) . Fusion of red blood cells occurred very often . If cell suspensions of high density were used fusion resulted in the formation of giant red blood cells with osmotically intact membranes.

J Biol Chem, 1984 May 25, 259(10), 6612 - 5
Potentiation of oxidative cell injury in hepatocytes which have accumulated Ca2+; Thor H et al.; Incubation of freshly isolated rat hepatocytes with exogenous ATP, but not with succinate, resulted in intracellular Ca2+ accumulation which was partly prevented when the inhibitor of mitochondrial Ca2+ sequestration, ruthenium red, was also present in the medium . Although the bulk of the accumulated Ca2+ was sequestered by the mitochondria, formation of surface blebs and stimulation of phosphorylase alpha activity during incubation of the hepatocytes with ATP indicate that this treatment was also associated with an increase in cytosolic free Ca2+ concentration . When hepatocytes loaded with Ca2+ by preincubation with ATP were exposed to either 2-methyl-1,4-naphthoquinone or t-butyl hydroperoxide, the cytotoxicity of both agents was markedly potentiated . Our results suggest that ATP-induced Ca2+ accumulation in hepatocytes is not due to contamination of the cell suspension with damaged cells or free intracellular organelles and that the intracellular Ca2+ concentration can affect the response to toxic agents.

FEBS Lett, 1984 May 21, 170(2), 277 - 80
Fluctuations in phosphoribosyl pyrophosphate levels in monolayer tumor cell lines . Effects of drugs; Peters GJ et al.; The concentration of PRPP (phosphoribosyl pyrophosphate) measured in tumor cells grown in monolayer showed a large variation with the various harvesting methods examined, including trypsinization . This variation could be reduced by a 1-h incubation of trypsinized cells as a suspension in Dulbecco's medium . After this preincubation these cell suspensions were suitable for the study of modulation of PRPP . One microM methotrexate caused a 2-3-fold increase and 1 mM N- phosphonoacetyl -L-aspartate a slight increase, but inosine and deoxyinosine drastically reduced PRPP concentrations . 5-Fluorouracil had no effect . This study demonstrates that metabolic parameters such as PRPP concentrations can be studied conveniently in suspensions of cells which are commonly cultivated as monolayers.

Biochem Pharmacol, 1984 May 15, 33(10), 1671 - 7
Nitroheterocycle metabolism in mammalian cells . Stimulation of the hexose monophosphate shunt; Varnes ME et al.; Misonidazole, SR-2508, nitrofurazone and other nitroheterocycles stimulated release of 14CO2 from {1-14C}glucose but not from {6-14C}glucose when incubated with mouse Ehrlich ascites cells or human A549 lung carcinoma cells in vitro . This demonstrated that the nitro compounds activated the hexose monophosphate shunt and is evidence that an important pathway of nitro reduction in these cell lines is electron transfer from NADPH-dependent cytochrome c reductase to the nitro group . Shunt activity was stimulated under both aerobic and anaerobic conditions . For catalase-free Ehrlich cells, aerobic effects were greater than anaerobic, indicating that NADPH was used for reduction of H2O2, via GSH peroxidase and reductase, as well as for one-electron nitro reduction, under aerobic conditions . Several of the compounds tested stimulated 14CO2 release from {2-14C}glucose as well as from {1-14C}-glucose . This shows that the cellular requirement for NADPH, in the presence of nitro drug, was great enough to cause recycling of pentose phosphates . Recycling could decrease the availability of ribose-5-P needed for nucleic acid synthesis, which could partly explain the inhibition of DNA synthesis observed upon prolonged aerobic incubation of cells with nitro compounds . Comparison of the rate of disappearance of nitrofurazone from anaerobic A549 cell suspensions with the rate of 14CO2 release suggests that the drug reduction in this cell line was catalyzed almost entirely by NADPH-requiring enzymes.

Eur J Biochem, 1984 May 15, 141(1), 217 - 22
Coupling of ATP synthesis and methane formation from methanol and molecular hydrogen in Methanosarcina barkeri; Blaut M et al.; The addition of methanol to a cell suspension of Methanosarcina barkeri resulted in an increase of the intracellular ATP concentration from 1 nmol/mg to 10 nmol/mg protein and in the formation of a proton-motive force delta p of -130 mV . delta p consisted of more than 90% of the membrane potential delta psi . These values were similar under N2 and under H2 . The addition of the uncoupler tetrachlorosalicylanilide to the above system under N2 led to a drastic decrease of both, the ATP concentration and the delta p and to a stop of methanogenesis . With methanol and H2, however, methane formation continued, although the effect of the uncoupler on the ATP pool and on delta p was a under N2 . The proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide caused a rapid exhaustion of the ATP pool and a discontinuation of methane synthesis, whereas delta p was unaffected . Inhibition of methane formation under these conditions could be relieved by the addition of the uncoupler tetrachlorosalicylanilide . These results demonstrate that methane formation according to the equation CH3OH + H2----H2----CH4 + H2O was coupled to ATP synthesis by a chemiosmotic mechanism and was under the control of delta psi: Methane formation only proceeded if the delta psi generated was used for ATP synthesis or if an uncoupler was present . Under N2, methane formation according to the equation 4CH3OH ----CO2 + 3CH4 + 2H2O was abolished by an uncoupler, because one step in the oxidation of methanol to 1 CO2 apparently depended on an energized state of the membrane.

J Immunol Methods, 1984 May 11, 70(1), 23 - 30
Lymphocyte isolation from human spleen by counterflow centrifugation employing two different flow chambers on line; Janssen JT et al.; Studies on splenic lymphocytes have hitherto been performed on single cell suspensions depleted of phagocytic cells by adherence to plastic or incubation with carbonyl iron . These techniques have the disadvantages of selective cell loss, suboptimal cell purification and cell activation . This paper describes purification of splenic lymphocytes by the use of counterflow centrifugation (CFC) . The method was adapted to overcome pelleting of cells in the separation chamber to form a plug at the inlet and impede adequate flow . By combining 2 different separation chambers on line in 1 rotor this problem was overcome . Of all lymphocytes recovered after CFC 88.8 +/- 1.4% were collected in 2 pooled fractions with a purity of greater than or equal to 98% and a cell viability of 95% . After CFC, 80.8 +/- 12.1% of the viable cells loaded were recovered.

J Immunol Methods, 1984 May 11, 70(1), 119 - 25
A simple reliable assay for myeloperoxidase activity in mixed neutrophil-eosinophil cell suspensions: application to detection of myeloperoxidase deficiency; Cramer R et al.; Quantitation of myeloperoxidase (MPO) activity by guaiacol peroxidation (GP) assay is profoundly affected by the peroxidase present in eosinophils (EPO) that contaminate the granulocyte suspensions . Inclusion of 3-amino-1,2,4-triazole (AMT) in the GP assay permits quantitation of MPO activity in mixed neutrophil-eosinophil suspension because of the differential inhibition of EPO and MPO by AMT . Results show that: (1) the peroxidase activity of eosinophil-free granulocyte suspensions is not appreciably affected by AMT; (2) in the presence of AMT the peroxidase activities of granulocyte preparations containing different numbers of eosinophils are similar on a neutrophil basis, regardless of the number of eosinophils and correspond with the activity of eosinophil-free granulocyte suspensions; (3) AMT almost completely inhibits the activity of partially purified EPO, only slightly affecting the catalytic activity of partially purified MPO; (4) AMT completely inhibits the residual peroxidase activity of granulocyte suspensions from MPO-deficient subjects contributed by contaminating eosinophils . The GP assay in the presence of AMT was used to study the pattern of hereditary transmission of MPO deficiency . The genealogy derived on the basis of this assay was compatible with an autosomal recessive inheritance, in agreement with previously reported results, while no definite pattern of inheritance could be established by use of the GP assay without AMT . We suggest that the GP assay supplemented with AMT is the method of choice for detection of MPO deficiency, particularly partial deficiency.

Can J Microbiol, 1984 May, 30(5), 526 - 31
Glucose metabolism of Treponema bryantii, an anaerobic rumen spirochete; Stanton TB; The pathway of glucose metabolism by Treponema bryantii, an obligately anaerobic spirochete isolated from bovine rumen contents, was studied . Washed cell suspensions of the spirochete consumed glucose and CO2 and produced equimolar amounts of acetate, formate, and succinate . Carbon dioxide was essential for glucose metabolism . Determination of radioactivity in products formed from 14C-labelled glucose and NaH14CO3 and assays of enzyme activities in cell-free extracts were used to determine the pathway of glucose metabolism . Treponema bryantii catabolized glucose to pyruvate via the Embden--Meyerhof--Parnas pathway . The spirochete used a coliform pyruvate-formate lyase to degrade pyruvate and produce formate and acetate . Succinate was formed by a pathway which involved the condensation of CO2 with pyruvate (or phospho(enol)pyruvate) formed from the breakdown of glucose.

Bull Eur Physiopathol Respir, 1984 May-Jun, 20(3), 229 - 35
Suppression of bronchoalveolar lymphocyte antibody secretion by alveolar macrophage: an in vitro study in the rabbit; Fournier M et al.; The effect of rabbit alveolar macrophage (AM) on the antibody secretion of bronchoalveolar lymphocytes was investigated in vitro, using a plaque-forming cell assay . Animals were intratracheally primed and reimmunized with 10(10) sheep red blood cells (SRBC) . Free alveolar cells were obtained by lung lavage and broncho-alveolar lymphocyte-enrichment was achieved through Sephadex G-10 columns . Cell suspensions with various macrophage-to-lymphocyte ratios (AM:L from 1:25 to 3:1) were prepared using appropriate numbers of plastic adherent alveolar cells . After a 4 day in vitro co-culture of 5 X 10(6) alveolar cells (AM plus lymphocytes) with 3 X 10(6) SRBC, IgM and IgG antibody-forming cells (AFC) were counted . A significant suppressive effect (p less than 0.05) of AM on both IgM and IgG-AFC was observed when AM:L ratio was increased from 1:25 to 1:10 . This effect was slightly accentuated with higher AM:L values, required viable AM, and was not affected by adding indomethacin in the culture medium . Moreover, a preliminary co-culture of AM with bronchoalveolar lymphocytes and the antigen was necessary for the expression of this suppressive effect . These data suggest that the suppressive activity of primed AM on alveolar lymphocytes may be of physiologic significance in vivo in the regulation of one of the pulmonary immune responses to airborne antigens, namely local antibody production.

Cytometry, 1984 May, 5(3), 263 - 7
Comparative flow DNA analysis of different cell suspensions in breast carcinoma; Chassevent A et al.; This study compared three methods of dissociation of breast lesions for DNA flow cytometry . Eleven benign lesions and 66 cancers were dissociated using mechanical, Ficoll, or enzymatic methods . DNA flow analysis showed that the DNA index did not vary from one method of dissociation to another . All benign lesions were diploid and 67% of all cancers were aneuploid . Enzymatic dissociation gave a lower percentage of aneuploid cells with a diminution of the proportion of cells in the G2 + M phase (13.2% enzymatic against 17.6% Fi-coll); on the other hand, it provided cell populations of greater viability than the other methods (32.6% enzymatic, 17.2% Fi-coll; P less than 0.01) . The mechanical and Ficoll suspensions did not differ significantly when they were analyzed on the basis of their DNA content and their cellular viability . When compared with mechanical preparation, Ficoll suspension showed a lower recovery of tumor cells, but this inconvenience was compensated for by a more homogeneous aspect where the contribution of aggregates and debris was clearly lessened . Therefore, this study led us to choose Ficoll suspension for subsequent flow analysis of breast tumors.

Biochem Pharmacol, 1984 May 1, 33(9), 1417 - 21
Glucose metabolism of oxidatively stressed human red blood cells incubated in plasma or medium containing physiologic concentrations of lactate, pyruvate and ascorbate; Sullivan SG et al.; Red cells suspended in either defined medium or buffered plasma were oxidatively stressed by incubation in the presence of 1,4-naphthoquinone-2-sulfonate at concentrations which caused less than 50% methemoglobin accumulation, stimulation of the hexose monophosphate shunt to less than 15% of capacity, and about a 30% increase in flux through glycolysis . Normal plasma concentrations of lactate and pyruvate in either defined medium or buffered plasma allowed increased contribution of reducing equivalents from glycolysis in response to oxidative stress . Increased utilization of reducing equivalents by the red cell was observed as increased accumulation of pyruvate, whereas accumulation of lactate represented storage of reducing equivalents . Exogenous lactate or pyruvate did not serve as a net electron source or sink since the total content in red cell suspensions of both lactate and pyruvate was increased during exposure to oxidative stress . If exogenous lactate had been used as a net source of reducing equivalents, the lactate concentration would have decreased during incubation of red cell suspensions . Plasma ascorbate or other constituents did not alter the qualitative response of glycolysis to oxidative stress (decreased lactate accumulation, increased pyruvate accumulation, and increased total flux through glycolysis), but plasma constituents did raise significantly the dose of oxidant agent required to elicit a given quantitative response . At levels of oxidative stress likely to be encountered in vivo, glycolysis and the hexose monophosphate shunt may be equal in importance as aerobic/antioxidant pathways.

J Pharmacol Exp Ther, 1984 May, 229(2), 493 - 500
Effects of N-nitrosodimethylamine on humoral immunity; Holsapple MP et al.; Female B6C3F1 mice were given i.p . injections with 1.5, 3.0 and 5.0 mg/kg N-nitrosodimethylamine (DMN) daily for 14 days and evaluated on day 15 . The day 4 immunoglobulin M (peak day) antibody response to sheep red blood cells (sRBC) was inhibited by 20, 53 and 81%, respectively . The day 5 immunoglobulin G (peak day) antibody response to sRBC was only inhibited significantly (60%) at the highest dose . Recovery studies indicated that the IgM antibody response was still significantly inhibited (48%) 30 days after the completion of the exposure to 5 mg/kg of DMN . The peak response (day 3) to 100 micrograms of the B-cell mitogen, lipopolysaccharide, was inhibited by 15, 26 and 32%, respectively, indicating that a portion of the suppression of the antibody response by DMN may be due to an effect on the ability of the lymphocytes to proliferate . Concentrations of DMN up to 100 mM added directly to untreated spleen cell suspensions had no effect on the in vitro antibody responses to lipopolysaccharide and sRBC . Preincubating DMN (100 mM) with either phenobarbital-induced or 3-methylcholanthrene-induced liver proteins (postmitochondrial supernatant from a 9000 X g liver homogenate) was still ineffective . The activation of DMN by either preparation was verified by measuring formaldehyde production, which reflects demethylation . In contrast to the results with DMN added directly to untreated spleen cell suspensions, the most sensitive indicator of suppression by DMN was the in vitro antibody responses to lipopolysaccharide and sRBC by spleen cell suspensions from DMN-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue Antigens, 1984 May, 23(5), 274 - 9
Anti-HLA-A2 and -A28 monoclonal antibody: production and study of the cross-reaction; Bourel D et al.; An anti-HLA-A2 and -A28 monoclonal antibody, XV.17, has been prepared by immunizing a Balb/c mouse with PBL . This XV.17 monoclonal antibody is a cytotoxic IgM . Its reactivity was tested by lymphocytotoxicity test and indirect immunofluorescence technique, in parallel with an alloantiserum ORA having the same anti-HLA-A2, -A28 reactivity pattern, against different panels . Family studies were undertaken . Absorptions-elutions and cytofluorometry experiments were performed to study the cross-reaction . The XV.17 monoclonal antibody is cytotoxic against all the HLA-A2 and -A28 tested cells, and is absorbed by HLA-Aw23 and -Aw24 cell suspensions.

Clin Exp Immunol, 1984 May, 56(2), 407 - 14
Development of pre-B and B lymphocytes in the human fetus; Asma GE et al.; Cell suspensions from human fetal liver, bone marrow and spleen were systematically studied at between the fetal ages of 8 and 20 weeks by the direct immunofluorescence technique for the presence of pre-B and B cells . Pre-B cells were characterized as lymphoid cells containing cytoplasmic mu heavy chains but lacking surface IgM . Based on their size and morphological appearance, these cells were subdivided into large and small pre-B cells . In the livers of 8 week old fetuses, more than 90% of the total pre-B plus B cell population consisted of pre-B cells; the relative number of liver pre-B cells gradually decreased with increasing gestational age and, after the 14th week, B cells outnumbered pre-B cells . At 20 weeks, the ratio of pre-B to B cells was only 0.25 . In contrast, the number of pre-B cells in fetal bone marrow (12-20 weeks) was always greater than that of the B cells . Large and small pre-B cells were present in the liver and bone marrow . Small pre-B cells outnumbered the large ones in both organs and with increasing gestational age the ratio of small to large pre-B cells increased four-fold . In fetal spleen (12-20 weeks), no large pre-B cells were seen and the small ones comprised only a minor fraction of the total B-cell population . It can be concluded from these data that during early human fetal life the liver is an important site of pre-B cell production . From 12 weeks onwards, this function is gradually taken over by the bone marrow . During the second half of pregnancy, pre-B cell production in fetal liver becomes very much less as compared with the bone marrow . No generation of pre-B cells takes place in the fetal spleen, but a certain amount of maturation of cells of the B cell line may take place in this organ.

Eur J Cancer Clin Oncol, 1984 May, 20(5), 673 - 7
Discrimination between human melanoma cell lines by fluorescence anisotropy; Weinreb A et al.; The fluorescence polarization of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) was measured when these markers were imbedded in cells of the human melanoma cell lines IGR37, IGR39, IGR3 and IGR4, as well as in cells of the mouse melanoma cell lines B16F1 and B16 F10 . These measurements were performed on cell cultures which were grown on quartz plates as well as on cell suspensions . Considerable differences are found between the polarization values of the human cell lines that are related to their different origins . Differences for the plated cells are considerably greater than those for the suspensions . No differences in the polarization values were found for the two mouse melanoma lines . It is concluded that differences in lipid structural order can be found between cell types endowed with different metastasizing capabilities.

Blood, 1984 May, 63(5), 1080 - 7
Cytofluorometric detection of B cell clonal excess: a new approach to the diagnosis of B cell lymphoma; Weinberg DS et al.; A sensitive cytofluorometric technique, the "kappa-lambda test," permits detection of small numbers of monoclonal B lymphocytes (clonal excess) . Such a method might represent a new diagnostic tool for diagnosis of non-Hodgkin's lymphomas, potentially providing definitive evidence of lymphomatous involvement in cases equivocal by standard immunologic methods . To determine the significance of detecting B cell clonal excess in lymphoid tissues, we applied the kappa-lambda test to cell suspensions from 60 consecutive specimens suspected to involvement by malignant lymphoma . Results were correlated with the pathologic diagnosis and with standard cell marker studies in each case . B cell clonal excess was observed in 24 of the 25 cases of non-Hodgkin's lymphoma of B cell origin, including a single case involving early detection of recurrence . None of the remaining cases, including benign reactive hyperplasia, T cell lymphoma, and Hodgkin's disease, showed evidence of B cell clonal excess . Selective examination of cell subpopulations was also achieved using this cytofluorometric method . We conclude that the detection of B cell clonal excess by the kappa-lambda test represents a new approach to the diagnosis of B cell lymphoma, which provides certain advantages over more standard methods of cell marker analysis.

J Neurosci Methods, 1984 May, 11(1), 39 - 45
A procedure for small-volume brain grafting; vasopressin cells in neonatal and adult Brattleboro rats; Boer GJ et al.; A simple and reliable technique is described for the transplantation of fetal vasopressin (VP) neurons in the third ventricle of the brain of homozygous Brattleboro neonates . Small-volume grafting is introduced by microdissection of paraventricular and supraoptic areas and by pelleting the minced tissue for insertion into the transplantation cannula . Morphological and immunocytochemical evaluation yielded results in both neonatal and adult host brain that were similar to those described for anterior hypothalamic grafts in adult Brattleboro brain . The present protocol circumvents some of the general problems encountered when the use of small grafts is imperative, and is also applicable to the implantation of pelleted cell suspensions.

Neuroscience, 1984 May, 12(1), 33 - 43
Use of central neuronal cultures for the detection of neuronotrophic agents; Barbin G et al.; Neuronotrophic factors, a class of macromolecules thought to be present within the neuronal environment are required to support the survival in vitro of peripheral neurons . In the present study we have established bioassay culture systems suitable for the identification of similar agents for intrinsic neurons of the central nervous system . The striatum, hippocampus and septum of 18 day fetal rats were dissociated and plated in a serum-free medium on a neurite conducive substratum which allows an easy recognition of neurons under phase contrast microscopy . These cultures contain predominantly neurons as assessed by tetanus toxin labelling, a well recognized neuronal marker . Seeding the cell suspensions at decreasing densities yields after 24 h a density dependent survival of the neuronal population . Thus a low seeding density could be chosen where survival of these neurons required an exogenous source of trophic factors . Survival of central neurons was promoted by several conditioned media derived from rodent glial cell cultures, both primary (astroglia, Schwann) and clonal (C6 glioma, Schwannoma) . Serial dilutions of these media allowed the titration of their respective neuronotrophic activities . In addition, conditioned media derived from the central neuronal cultures themselves, when seeded at a high density, were also able to support the survival of low density seeded central neurons.

Immunobiology, 1984 May, 166(3), 296 - 307
Morphology, kinetics and secretory activity of antibody-forming cells; Geldof AA et al.; Specific antibody-forming cells from spleen, bone marrow and popliteal lymph nodes were studied in mice after subcutaneous priming and intravenous boosting with horseradish peroxidase (HRP) . Functional antibody-secreting capacity of these cells was correlated with their morphology at the cell population level . For this purpose, cells synthesizing anti-HRP antibody from the same cell suspensions were studied simultaneously by light and electron microscopy and by a plaque assay . It appeared that the population of cells responsible for antibody synthesis as well as antibody secretion was morphologically heterogeneous: besides plasma cells, considerable numbers of antibody-forming lymphocytes, antibody-forming plasmablasts and antibody-forming immature plasma cells were observed . Immature plasma cells constituted the majority of antibody-forming as well as antibody-secreting cells . Among the immature plasma cells in the popliteal lymph nodes proliferation occurred . Evidence is presented that the light-microscopically identified mature plasma cell is not the main antibody-forming cell . It does not show 3H-Thymidine incorporation and should be considered as a non-dividing end-cell.

Am Rev Respir Dis, 1984 May, 129(5), 827 - 32
Lymphocyte subpopulations in rat lungs and Peyer's patches; Crawford JM et al.; The distribution of lymphocyte subpopulations in rat lungs and Peyer's patches was examined by immunofluorescent microscopy of frozen sections and single cell suspensions . The results showed that there are differences in the lymphocyte subpopulations in these tissues . More B cells were present in Peyer's patches (56.5%) than in lung (15.2%), and, conversely, there were more T cells (W3/13+) in lung (61.7%) than in Peyer's patches (20.7%) . In addition, the great majority of T cells found in Peyer's patches were W3/25+ cells (the phenotypic marker of helper T cells in the rat) . This was not the case in lung, where a maximum of 46% of T cells were W3/25+ . Alveolar macrophages (defined by adherence to plastic and nonspecific esterase activity) did not express W3/13 or W3/25 antigens.

J Lab Clin Med, 1984 May, 103(5), 768 - 75
Cryopreservation of pancreatic islet cells; Yokogawa Y et al.; Our study describes a useful procedure for cryopreservation of pancreatic islet cells . The pancreatic islets of adult hamsters were collected by collagenase digestion succeeded by gradient centrifugation and were dispersed by EDTA-Dispase treatment . The dispersed cells were suspended in medium consisting of 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum, supplemented with 10% dimethyl sulfoxide or glycerol . One milliliter each of the cell suspensions containing 10(6) cells was distributed into 2 ml polypropylene tubes, processed for freezing under six cooling conditions, and stored in liquid nitrogen (-196 degrees C) . Of the various conditions tested, the cells suspended in dimethyl sulfoxide-containing medium and cooled in a program freezer at a rate of 0.5 degrees C/min down to -40 degrees C, succeeded by a rate of 5 degrees C/min down to -70 degrees C, resulted in the highest recovery rate of cells, 61.2% +/- 3.1% . This rate was comparable to those of several tissue culture cell lines frozen under similar conditions . Recovered cells preserved their morphologic characteristics under light and phase-contrast microscopy and formed cell sheets in culture . Response of insulin secretion to 3 mg/ml glucose appeared 6 hours after thawing, and the response to both 3 mg/ml glucose and 10 mmol/L theophylline was recovery to the same level as nonfrozen islet cells after 12 hours . The applicability of cryopreserved cells for the detection of islet cell surface antibody was demonstrated by the indirect method of immunofluorescence using islet cell surface antibody-positive human sera.

Endocrinology, 1984 May, 114(5), 1665 - 71
Insulin regulation of lipoprotein lipase in cultured isolated rat adipocytes; Eckel RH et al.; The cellular regulation of adipose tissue lipoprotein lipase by insulin was investigated using cultured isolated rat adipocytes . Evidence for sustained cell viability over 3 days included stability of specific {125I}insulin binding and adipocyte number . Lipoprotein lipase was measured in three functional compartments: 1) enzyme activity secreted into the culture medium, 2) activity releasable from cell suspensions by heparin, and 3) activity extractable from cells (after maximal heparin release) in deoxycholate and detergent . One day after preparation, these activities stabilized and were 1.3 +/- 0.2, 1.4 +/- 0.2, and 7.7 +/- 0.9 neq/10(6) cells X min, respectively (n = 24, mean +/- SEM) . Insulin, added the day after preparation, produced a dose-dependent (1-400 ng/ml) increase in lipoprotein lipase releasable from cells by heparin at 2, 4, and 24 h . Insulin also increased intracellular enzyme measured as deoxycholate-detergent-solubilized activity extracted from previously heparin-released cells . However, insulin-mediated increases in culture medium enzyme only occurred subsequent to cellular effects . All insulin-mediated effects were prevented by cycloheximide (1 microgram/ml) . Thus, insulin increased two cellular pools of adipocyte lipoprotein lipase in a dose-dependent manner, but had no direct effect on enzyme secretion . Overall, cultured isolated rat adipocytes appear to be a valuable system for the study of lipoprotein lipase regulation at the level of the adipocyte.

Blood, 1984 May, 63(5), 1147 - 52
Multiinstitution study of non-Hodgkin's lymphomas using frozen section immunoperoxidase: the Southeastern Cancer Study Group experience; Borowitz MJ et al.; This report describes the experience of the Southeastern Cancer Study Group (SECSG) with a transport medium used for immunologic phenotyping of non-Hodgkin's lymphomas . In a 2-mo pilot study, portions of 53 specimens of non-Hodgkin's lymphoma from four member institutions of the SECSG and affiliated community hospitals were sent by regular mail to a central laboratory . Immunologic phenotyping was carried out using a frozen section immunoperoxidase technique . In 48 of the cases, a clear-cut immunologic phenotype was obtained . Thirty-four tumors were of B cell origin and 7 had T cell markers . Six of the remaining lymphomas had neither B nor T cell markers, and the seventh had both . In 12 cases, phenotyping was also carried out at the originating institution using conventional cell suspension techniques; agreement between the two methods was excellent . The immunologic results were correlated with histopathologic diagnosis standardized using the Working Formulation for non-Hodgkin's lymphomas . It was found that the low grade tumors were all B cell, but that the intermediate grade tumors were very heterogeneous immunologically . About one-fourth of the diffuse, intermediate grade or miscellaneous tumors had T cell markers . Our results indicate that immunologic phenotyping may be performed satisfactorily on transported material, making multiinstitution studies on the prognostic significance of immunologic phenotype in non-Hodgkin's lymphomas feasible.

J Steroid Biochem, 1984 May, 20(5), 1187 - 94
ACTH sensitivity of isolated human pathological adrenocortical cells: variability of responses in aldosterone, corticosterone, deoxycorticosterone and cortisol production; Racz K et al.; In vitro aldosterone, deoxycorticosterone, corticosterone and cortisol production of human adrenocortical cells derived from adenomas (Conn's syndrome, Cushing's syndrome), from hyperplastic adrenals (Cushing's syndrome) and from adrenals surrounding aldosteronoma are described . Cells from adenomas causing either Cushing's syndrome or Conn's syndrome harboured the highest basal and ACTH-stimulated corticosteroid production . Adrenocortical cells derived from micronodular hyperplasia causing Cushing's syndrome and cells from cortisol producing adenoma displayed predominantly cortisol and corticosterone secretion both under basal conditions and following stimulation with ACTH . Aldosteronoma cells showed highly variable aldosterone, deoxycorticosterone, corticosterone and cortisol response to ACTH . However, in aldosteronoma cell suspensions, the basal and ACTH-stimulated ratios of aldosterone to cortisol were increased when compared to ratios of steroids produced by cells from other adrenal tissues . Chronic treatment with spironolactone of patients with Conn's syndrome before surgery was associated with a decreased ratio of aldosterone to corticosterone, revealing that 18-hydroxylase in aldosteronoma cells may be inhibited during long-term therapy . Non-tumorous cells isolated from adrenals surrounding aldosteronoma displayed less aldosterone prior to and after stimulation with ACTH than aldosteronoma cells.

Cell Immunol, 1984 May, 85(2), 499 - 510
Macrophages in murine uterus are immunosuppressive; Hunt JS et al.; The mechanisms by which the fetal allograft is protected from a maternal anti-fetal immune response are not understood . This study was designed to examine the possibility that tissues near the developing fetus contain immunoregulatory cells and to begin the process of identification of those cells . Dispersed uterine cell suspensions from pregnant Swiss/Webster mice consistently inhibited the responses of normal murine spleen cells to the polyclonal mitogen phytohemagglutinin (PHA) . These suspensions contained few lymphocytes (mean 1%), but abundant macrophages (mean 28%), identified by morphology and Fc gamma-receptor expression . Depletion of Fc gamma-receptor-positive cells restored spleen cell (SC) responses to PHA to near normal levels and partial depletion of adherent cells provided varying degrees of relief of the observed suppression . Adherent cells (greater than 95% macrophages) recovered from plastic surfaces were highly immunosuppressive . Suppressor cells appeared to interfere with both early and late stages of spleen cell proliferative responses . The results suggest that cells with some characteristics of macrophages within tissues near the maternal-fetal interface may create a local environment prohibitive to maternal lymphocyte proliferation.

Cell Biol Int Rep, 1984 May, 8(5), 363 - 71
Determination of the growth rate of human prostatic cells in primary culture by a morphometric technique; Romijn JC et al.; A morphometric technique, based on the measurement of the area of individual cell colonies and of its increase in time, was applied to study the rate of proliferation of human prostatic cells in vitro . The reliability of the method was checked by determination of the growth rate of cultures of the continuous cell line PC93 by the morphometrical technique as well as by counting of the cell number . No significant difference was found in the population doubling times measured by either of these methods . It was therefore concluded that the morphometrical technique could be applied also to study the growth rate of primary cultures of prostatic epithelial cells, in which counting of the cell number is generally impossible . The results showed that, with primary cultures derived from hyperplastic prostates and prostatic carcinomas as well as from the prostatic tumor line PC82, rapid growth occurred during the first two or three days of culture; measurements performed at a later time appeared to be less reliable . It was demonstrated by the effect of serum deprivation on the growth of PC82 cells that the technique described here is, in principle, suitable to monitor the effect of various agents on the growth of cells in primary culture . The method is non-destructive and requires minimal amounts of tissue; it may be applied especially to cultures that cannot be dispersed easily into single cell suspensions.

Am J Pathol, 1984 May, 115(2), 266 - 74
Immunohistochemical studies of the spleen in hairy-cell leukemia; Meijer CJ et al.; Immunohistochemical studies of 10 spleens from patients with hairy-cell leukemia (HCL) with a battery of monoclonal and heterologous antibodies were performed in order to obtain information on the phenotype of the neoplastic cells and the admixture of reactive cells . The hairy cells (HCs) were shown to react with several anti-B-cell antibodies (Y29/55, Leu10, HLA-DR), but only a minority of cases showed reactivity with antibody BA-1 . The cells of all cases were nonreactive with anti-T and/or anti-macrophage antibodies . In contrast with cell-suspension studies, in immunohistochemical studies the HCs did not react with antibody OKM1 . The reactive T-lymphocytes showed a shift toward T helper cells in the red pulp, but not in the white pulp . It was confirmed that "pseudosinuses" and "bloodlakes" are outlined by HCs and not by sinus-lining cells: the lining cells of the blood lakes were Y29/55+ and Leu10+, but negative with antibodies known to react with sinus-lining cells (Leu2a, BA-2) . These data suggest that immunohistologic studies of the spleen in patients with HCL can be helpful in differential diagnosis and can provide information concerning the reaction of splenic tissue to the infiltration by hairy cells.

J Invest Dermatol, 1984 May, 82(5), 496 - 500
Rodent epidermal Langerhans cells demonstrate greater histochemical specificity for ADP than for ATP and AMP; Chaker MB et al.; Langerhans cells (LCs) in mammalian epidermis possess the ectoenzyme Ca++/Mg++-dependent adenosine triphosphatase (ATPase), which has served as a useful histochemical marker for these dendritic cells in a variety of tissue preparations . Since ATPase represents only one of several potential cell surface polyphosphatases, we investigated the capacities of 3 related adenine nucleotide substrates to identify rodent epidermal LCs . Cell surface ATPase activity was not inhibited in the presence of ouabain and was observed to be strictly divalent cation-dependent, with complete interchangeability between Ca++ and Mg++ . Optimal staining in the presence of either cation occurred at a 20 mM concentration . Substrate concentration dependence was also observed, with optimal staining at 0.33 mM adenosine 5'-triphosphate (ATP) . On an equimolar basis, however, adenosine 5'-diphosphate (ADP) was superior to ATP for the identification of LCs both in whole mounts of epidermis and in suspensions of disaggregated epidermal cells . The substrate adenosine 5'-monophosphate (AMP) stained follicular epithelial cells in both rodent species but failed to identify epidermal LCs in the mouse and only weakly stained these dendritic cells in rat epidermis . We conclude from these studies that ADP demonstrates greater specificity for LC surface polyphosphatase activity than ATP and that the inadvertent inclusion of AMP during identification procedures for epidermal cell suspensions will falsely identify cells other than LCs.

J Cell Biol, 1984 May, 98(5), 1757 - 62
Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6; Murphy RF et al.; The acidification of various ligands was measured on a cell by cell basis for cell suspensions by correlated dual fluorescence flow cytometry . Mouse 3T3 cells were incubated with a mixture of fluorescein- and rhodamine-conjugated ligands, and the ratio of fluorescein and rhodamine fluorescence was used as a measure of endosome pH . The calibration of this ratio by both fluorometry and flow cytometry is described . Dual parameter histograms of average endosome pH per cell versus amount of internalization were calculated from this data, for samples in the absence and presence of chloroquine added to neutralize acidic cellular vesicles . The kinetics of acidification of insulin were measured and compared with previous results obtained with the chloroquine ratio technique . Rapid acidification of internalized ligand was observed both for insulin, which was mostly internalized via nonspecific pathways, and for alpha 2-macroglobulin, which was mainly internalized by specific receptor-mediated endocytosis . The average pH observed for internalized insulin was less than pH 6 within 10 min after addition of insulin . At 30 min, the average pH began to decrease to approximately pH 5, presumably because of fusion of endosomes with lysosomes.

Thromb Res, 1984 Apr 15, 34(2), 147 - 57
Activation of platelet prostaglandin biosynthesis pathway during neoplastic cell-induced platelet aggregation; Grignani G et al.; In a previous study we found a correlation between metastatic potential and platelet aggregating activity in sublines of a benzopyrene-induced murine fibrosarcoma ( mFS6 ); the purpose of the present work was to elucidate the role of thromboxane biosynthesis by platelets and/or by neoplastic cells in the activation of platelets in this system . The cells of the more malignant subline induced higher aggregation and TxB2 production than those of the non metastasizing one . The supernatants of aggregating cell suspensions contained very few TxB2; furthermore, preincubation of platelets with ASA or Apyrase resulted in inhibition of aggregation and TxB2 production, while preincubation of the cells was ineffective; these results suggest the platelet origin of the measured TxB2 and indicate that platelet-derived ADP plays an important role in their activation, while the production of ADP by the cells does not seem to be relevant in this model . The involvement of platelet prostaglandin biosynthesis pathway in neoplastic cell induced platelet activation could play an important role in the development of platelet-dependent tumour metastasis.

Biochem J, 1984 Apr 15, 219(2), 619 - 24
Isolation and characterization of an intermediate steroid metabolite in diosgenin biosynthesis in suspension cultures of Dioscorea deltoidea cells; Tal B et al.; The aglycon form of the steroidal sapogenin furost -5-ene-3 beta, 22,26-triol, 3 beta- chacotrioside 26 beta-D-glucopyranoside was isolated from cell suspension cultures of Dioscorea deltoidea and its molecular structure was determined by mass spectrometry and 1H and 13C n.m.r . spectroscopy . From kinetic studies and incorporation experiments with {1-14C}acetate it was concluded that the steroidal compound (in the glycoside form) is an intermediate in vivo in diosgenin biosynthesis . It accumulated in growing cells of D . deltoidea and was metabolized to diosgenin (in the glycoside form, i.e . dioscin ) in non-dividing cells.

Cell Immunol, 1984 Apr 15, 85(1), 45 - 57
Relationships between suppressor macrophages and macrophage precursors in the spleens from tumor-bearing mice; Shibata Y et al.; Suppressor macrophages (M phi) which can inhibit mitogen-induced lymphocyte proliferation appeared in the spleens of mice bearing transplanted MC-A fibrosarcoma cells . An analysis of the ontogeny of such M phi revealed additional suppressor activity directed against macrophage stem cells . Treatment of spleen cell suspensions with carbonyl iron followed by centrifugation removed suppressor M phi but did not deplete M phi-colony forming cells (M-CFC) which could be demonstrated in soft agar culture in L-cell conditioned medium (LCM) . Untreated spleen cells had normal numbers of M-CFC; phagocyte-depleted mononuclear cells showed a threefold increase in M-CFC 14 days after subcutaneous inoculation of 10(6) MC-A cells per mouse . Further increases in M-CFC were also evident in similar preparations on Days 21 and 28 when the M-CFC concentration reached a maximum of eight times the normal level . The M phi which developed from the M-CFC grown in the presence of LCM were later shown to have indomethacin-sensitive suppressor activity suggesting the mediation of this phenomenon by prostaglandins . These observations suggest that locally produced phagocytic suppressor M phi from the spleens of tumor-bearing mice play important roles not only as inhibitors of lymphocyte proliferation as reported earlier, but also as regulators of monocyte-M phi production.

Cryobiology, 1984 Apr, 21(2), 148 - 56
A comparison of cryodestruction with excision or infarction of an implanted tumor in rat liver; Jacob G et al.; In this study a malignant tumor was implanted in rat livers and treated by infarction, excision, or cryodestruction . Survival and the pattern of metastases was studied in each group . Walker carcinomas were induced by the inoculation of a tumor cell suspension into the livers of male Sprague-Dawley rats . Ten days after inoculation a solitary tumor had formed . This was treated by (i) mobilization of the tumor-bearing lobe (controls); (ii) ischemic infarction by ligation of the vascular pedicle to the lobe; (iii) excision of the tumor-bearing lobe; or (iv) cryodestruction of the tumor and surrounding liver using a clinical liquid nitrogen probe . A double freeze/thaw cycle to - 180 degrees C at a mean cooling rate of 94 degrees C/min was performed . Autopsy was performed at death or after 110 days, when the experiment was terminated . In general, deaths within 5 weeks of treatment were from recurrent tumor growth in the liver and, after this time, from metastatic disease . A statistically significant increase in survival was noted in the cryotherapy group when compared with the other treatment groups (P less than 0.01 logrank ) and controls (P less than 0.001 logrank ) . No real difference in local tumor control was noted between the groups . The improved survival in the cryotherapy group was attributed to a statistically significant reduction in deaths from metastatic disease (P less than 0.05 Chi-square) . This finding may be related to an immunological response and warrants further study.

J Steroid Biochem, 1984 Apr, 20(4A), 935 - 9
Metabolism of {4-14C}androstenedione by cells cultured from human amniotic fluid; O'Shannessy DJ et al.; The conversion of {14C}androstenedione by human fibroblast (F) and amniotic fluid (AF) cells obtained by amniocentesis was investigated using human dermal fibroblasts (DF) as controls . Cell suspensions were incubated with {14C}androstenedione in the presence of a NADPH generating system . Steroid reaction products were separated from unreacted substrate by chromatography on micro-columns of magnesium oxide, partially resolved by partition chromatography on celite and further characterized by thin-layer chromatography and recrystallization to constant specific activity . In the case of F cells the pattern of metabolism was qualitatively similar to that of DF cells; the predominant metabolite was testosterone and several uncharacterized metabolites were detectable . However testosterone was the only metabolite isolated from incubations with AF cells . The results demonstrate distinct differences in the capacity of AF and F type cells to metabolize {14C}androstenedione and support the view that F cells resemble typical fibroblasts from dermis or other connective tissues.

Blood, 1984 Apr, 63(4), 768 - 78
Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions; Jackson CW et al.; The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis . Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis . We describe the measurement of cellular DNA distributions of megakaryocytes by two-color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days . Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution . The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded . We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.

J Exp Med, 1984 Apr 1, 159(4), 1277 - 82
Precocious and enhanced functional maturation of B lineage cells in New Zealand Black mice during embryonic development; Jyonouchi H et al.; Previous reports suggest that large numbers of immunoglobulin-secreting cells appear in tissues of NZB strain mice from the time of birth . In this study, we investigated the development of B lineage cells during embryonic life and found that they were present 2-3 d earlier and in higher numbers in NZB embryos than several other strains of mice . That is, liver cell suspensions from NZB embryos contained larger numbers of surface Ig (sIg)- cells that could form B cell colonies in mitogen-dependent semisolid agar culture . Sephadex G-10-adherent cell depletion diminished numbers of colonies and this was partially restored by addition of humoral factors . The latter were partially purified from serum of very young NZB mice . These findings document that abnormal changes take place in B lineage cells and possibly also in cells that regulate their maturation in NZB strain mice at a very early stage of development.

Arch Pathol Lab Med, 1984 Apr, 108(4), 305 - 7
Improved clonal growth of human solid tumors by isolation of viable cells; Katoh AK et al.; The potential for the establishment of predictive assays for cancer chemotherapy was generated recently by the development of a soft agar clonogenic assay for human tumor cells . Though the assay technique is promising, certain technical problems remain . One is the difficulty of preparing viable cell suspensions from solid human tumors . A method of separating viable and nonviable cells by Ficoll-Hypaque centrifugation was used . When this method was used to plate isolated viable single cells, an improvement in cloning was observed when compared with a nonseparated mixture of viable and nonviable cells.

Biophys J, 1984 Apr, 45(4), 767 - 76
Water exchange in plant tissue studied by proton NMR in the presence of paramagnetic centers; Bacic G et al.; The proton NMR relaxation of water in maize roots in the presence of paramagnetic centers, Mn2+, Mn- EDTA2 -, and dextran-magnetite was measured . It was shown that the NMR method of Conlon and Outhred (1972, Biochem . Biophys . Acta . 288:354-361) can be applied to a heterogenous multicellular system, and the water exchange time between cortical cells and the extracellular space can be calculated . The water exchange is presumably controlled by the intracellular unstirred layers . The Mn- EDTA2 - complex is a suitable paramagnetic compound for complex tissue, while the application of dextran-magnetite is probably restricted to studies of water exchange in cell suspensions . The water free space of the root and viscosity of the cells cytoplasm was estimated with the use of Mn- EDTA2 - . The convenience of proton NMR for studying the multiphase uptake of paramagnetic ions by plant root as well as their transport to leaves is demonstrated . A simple and rapid NMR technique (spin-echo recovery) for continuous measurement of the uptake process is presented.

Blood, 1984 Apr, 63(4), 886 - 96
Distribution of antigens defined by OKB monoclonal antibodies on benign and malignant lymphoid cells and on nonlymphoid tissues; Knowles DM 2nd et al.; Monoclonal antibodies OKB1, OKB2, OKB4 and OKB7 have been previously shown to detect distinctive antigens displayed on B, but not on T, lymphocytes . Benign and malignant lymphoid cells were investigated for their reactivity with these antibodies in cell suspension by indirect immunofluorescence and in cryostat tissue sections by the avidin-biotin complex immunoperoxidase technique . Fetal liver pre-B cells and pre-B and common type acute lymphoblastic leukemia cells isolated from 15 patients were OKB1-OKB2+OKB4-OKB7- . All mature lymphoid tissue B cells and the neoplastic cell surface immunoglobulin-positive (SIg+) B cells isolated from each of 47 B cell neoplasms were OKB2+ . OKB1 and OKB7 were expressed by interfollicular, follicular center, and many, but not all, mantle zone B cells . OKB4 was expressed by follicular center cells, but not by mantle zone or interfollicular B cells . The neoplastic SIg+ B cells isolated from 45 of 47 B cell malignancies were OKB1+OKB4+, and those isolated from 45 of 46 B cell malignancies were OKB7+ . The neoplastic B cells of one mantle zone lymphoma were OKB1-, of one small lymphocytic cell lymphoma were OKB7-, of one large cell lymphoma were OKB4-, and of one small lymphocytic cell lymphoma with a monoclonal gammopathy were OKB1-OKB4- . Normal and myeloma plasma cells were OKB- . The malignant T cells isolated from 12 T cell neoplasms were OKB2-OKB4-, but were OKB1+ and/or OKB7+ in 3 cases . Thus, the OKB antibodies appear to detect distinctive antigens that may be expressed at different stages of B cell differentiation . In addition, OKB4 reacted with selected renal and respiratory epithelium, and OKB2 reacted with a wide range of epithelial tissues . The OKB antibodies should prove useful in the investigation of B cell differentiation and may aid in the identification and characterization of lymphoproliferative malignancies with significant therapeutic and prognostic differences not identifiable by conventional histopathologic and immunologic methods.

J Invest Dermatol, 1984 Apr, 82(4), 322 - 5
Langerhans cells react with pan-leukocyte monoclonal antibody: ultrastructural documentation using a live cell suspension immunoperoxidase technique; Wood GS et al.; Langerhans cells are generally regarded as members of an Ia+ dendritic cell system capable of potent accessory cell function in immune responses . While it has been shown that murine Langerhans cells are bone marrow-derived, the ontogenic relationships among human Langerhans cells, other dendritic cells, macrophages, and leukocytes in general have yet to be fully clarified . Recently, several pan-leukocyte monoclonal antibodies have been produced which react with the human leukocyte common antigen . This antigen resembles the murine T200 antigen and is expressed by all leukocyte subtypes but not by nonhematopoietic cells . Using an immunoperoxidase technique for staining suspensions of live skin cells, we have documented Langerhans cell reactivity with pan-leukocyte monoclonal antibody L3B12 at the ultrastructural level . Reactivity with this highly sensitive and specific pan-leukocyte marker supports the concept of the human Langerhans cell as a specialized form of bone marrow-derived mononuclear leukocyte and defines an immunologic feature common to dendritic cells, macrophages, and leukocytes that is not shared by other cell types . This finding is discussed in the context of other recent data concerning the immunologic phenotype of Langerhans cells . Since the immunoultrastructural method employed does not require cell fixation of any kind prior to immunologic staining, it should prove particularly useful for studying cell surface antigens that are adversely affected by fixation.

Biophys J, 1984 Apr, 45(4), 693 - 8
Ca2+-activated K+ channels in human red cells . Comparison of single-channel currents with ion fluxes; Grygorczyk R et al.; Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches . The currents are preferentially carried by K+ (PK/PNa 17) and show rectification . Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts . Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability . Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced . These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents . Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions . At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential . This indicates a heterogeneity of the response to Ca2+ of the cells in the population . The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s . At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS).(ABSTRACT TRUNCATED AT 250 WORDS)

Ecotoxicol Environ Saf, 1984 Apr, 8(2), 167 - 82
Use of plant cell cultures to study the metabolism of environmental chemicals; Sandermann H Jr et al.; The metabolism of the following environmental chemicals has been studied in cell suspension cultures of wheat (Triticum aestivum L.) and soybean (Glycine max L.):2, 4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), hexachlorobenzene, pentachlorophenol, diethylhexylphthalate , benzo {alpha} pyrene, and DDT . All chemicals tested, including the persistent ones, were partially metabolized . Polar conjugates predominated in all cases . A covalent incorporation into lignin could be demonstrated for 2,4-D and pentachlorophenol . A specific deposition in the cellular vacuole could be demonstrated for the beta-D-glucopyranoside conjugates derived from 2,4-D . A rapid assay procedure to evaluate the metabolism of a given 14C-labeled chemical in plant cell suspension cultures is described . This procedure requires about 1 week, and the reproducibility of the results obtained has been assessed.

J Immunol, 1984 Apr, 132(4), 1924 - 30
The mechanism of intercellular aggregation . I . The kinetics of the Fc gamma receptor-mediated aggregation of P388D1 cells with antibody-coated lymphocytes at 4 degrees C; Segal DM et al.; The formation of specific, heterophilic conjugates between cells from the P388D1 mouse macrophage line and antibody-coated mouse spleen cells was followed in cell suspensions at 4 degrees C by dual parameter flow cytometry . Intercellular aggregation in this system is mediated by the binding of the Fc portions of IgG antibodies on the spleen cells with Fc receptors (Fc gamma R) on P388D1 . We show that the rate of aggregation reaches a plateau with increasing cell concentrations, suggesting that the initial collision between cells is not the rate limiting step of conjugate formation . The rates of aggregation are strongly dependent upon the cell surface densities of both Fc gamma R and antibody . In conjugates, however, only small fractions of available receptors or antibodies are utilized in bond formation . The rate-limiting step of aggregation, therefore, involves the formation of ligand-receptor bonds, and may be the diffusion of antibodies and receptors toward one another in small areas of intercellular contact . Inhibitor studies implicate microfilaments, but not microtubules, divalent cations, or energy-dependent processes as being important in aggregation . Finally, conjugates are stable when diluted into medium alone, but dissociate in media containing protein A, soluble immune complexes, or anti-Fc gamma R antibodies . This suggests that conjugates are stabilized by multiple intercellular ligand-receptor bonds, which constantly break and reform at the cell:cell interface, and that protein A, immune complexes, and anti-Fc gamma R disaggregate the conjugates by preventing the reformation of broken bonds.

J Immunol, 1984 Apr, 132(4), 1779 - 83
T cell regulation of polyclonally induced immunoglobulin secretion in humans; Rosenkoetter M et al.; We measured the pokeweed mitogen (PWM)-induced secretion of IgG by the unfractionated mononuclear cells (MNC) of young adult donors, and correlated the results with the functional activity of cell suspensions enriched for T helper (T4+) and T suppressor/cytotoxic (T8+) cells . The distribution of IgG levels secreted by MNC differs from a Gaussian curve, implying that the group is composed of distinct heterogeneous populations . When donors were compared who were judged to be very low responders or very high responders on the basis of IgG secretion levels by MNC (less than 700 ng/ml or greater than 2500 ng/ml), no differences were found in the capacity of T4+-enriched cells to support PWM-driven IgG secretion by a common B cell pool . In contrast, the addition of 0.2 X 10(5) T8+ cells from these low responders to PWM-stimulated cultures of 0.5 X 10(5) T4+ cells plus 0.5 X 10(5) B cells resulted in significantly less IgG secretion (389 +/- 121 ng/ml) than did the addition of the same number of T8+ cells from the high responders (2241 +/- 548 ng/ml, p less than 0.01) . Normalized percent suppression by T8+ cells was higher in low responders than in high responders (77.0 +/- 9.9% vs 33.0 +/- 8.5%, p less than 0.01) . Both high and low responders markedly suppressed IgG secretion when 0.5 X 10(5) T8+ cells were added . No correlation was found either between proportion of T3+, T8+, T4+, or M1+ cells within the MNC population and levels of IgG secretion by MNC or between T8+ numbers and levels of suppression induced by a constant number of T8+-enriched cells . Our data indicate that differences in the functional activity of T8+ cells, rather than quantitative differences, account for the wide range of PWM-induced IgG secretion by MNC.

Agents Actions, 1984 Apr, 14(3-4), 373 - 5
The interaction of cholinomimetics, peptides and compounds 48/80 on histamine secretion from the mast cell; Erjavec F et al.; The mechanism of selective, non-immunological histamine release from mast cells, caused by various endogenous substances, is not clearly understood . Since in vivo experiments indicate that the local control of secretory cells is influenced by acetylcholine and peptides, we investigated whether the secretion of histamine is similarly regulated in the mast cell . Experiments were performed with peritoneal cavity cell suspensions (PCS) of the rat . The endogenous polypeptide substance P, compound 48/80 and a non-hydrolysable cholinomimetic agonist, carbachol, were used . The concentrations of the drugs were kept low to avoid non-specific histamine release caused by morphological damage of mast cells . It was found that: (1) carbachol (2 X 10(-5) M) did not release histamine from PCS, (2) substance P (6.5 X 10(-6) M) released histamine and this effect was increased by the addition of carbachol (2 X 10(-5) M); the effect of carbachol was inhibited by atropine , (3) carbachol (2 X 10(-5)M) did not increase histamine release caused by compound 48/80 (0.02 micrograms/ml) . From these experiments it may be concluded that activation of peptidergic receptor(s) can cause histamine release from mast cells and that muscarinic agents may be involved in the regulation of the(se) receptor(s).

Neuroscience, 1984 Apr, 11(4), 847 - 55
Thy-1 antigen: a ganglion cell specific marker in rodent retina; Barnstable CJ et al.; A monoclonal antibody, 2G12 , has been produced against a rat cerebral cortex glycoprotein fraction . It interacts with Thy-1 based on both the tissue distribution of its reactivity and the blocking of its binding by pretreatment with a rabbit anti-Thy-1 serum . In rat retina this antibody labels only cell bodies in the ganglion cell layer, optic nerve fibres and the inner plexiform layer . That the cell-body labelling was confined to ganglion cells was confirmed by double-labelling experiments . Ganglion cells were distinguished by retrograde transport of fluorescent markers injected into the superior colliculi . The retinas were dissociated into single cells and the cell suspension was labelled with 2G12 . There was almost complete coincidence of the two labels . A monoclonal antibody against mouse Thy-1.2 gave an essentially identical pattern of labelling . In both rats and mice Thy-1 was also found on the vitreal surface of the inner limiting membrane in a pattern reminiscent of that formed by the Muller cell endfeet, although these cells do not express Thy-1.

Cell Biochem Funct, 1984 Apr, 2(2), 119 - 24
Cyclic nucleotides and haemoglobin concentration in rabbit bone marrow cell suspensions stimulated by purified erythropoietin; Fano G et al.; This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep) . The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions . The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively . The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture . (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions . (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions . This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.

Aust J Exp Biol Med Sci, 1984 Apr, 62 ( Pt 2), 167 - 80
Modulation of natural and acquired immunity of rats to tumour isografts; Inoue Y et al.; Syngeneic tumour cell suspensions were injected into the foot or intravenously in normal rats or rats bearing subcutaneous tumours . Resistance was assessed by measuring the swelling of the foot due to the growth of the tumours or by counting lung colonies or metastases . The tumours included two methyl-cholanthrene-induced fibrosarcomas (D7, D8), a metastatic variant of D8 (D8M) and a spontaneous adenocarcinoma (ST-2) . D8, D8M and ST-2 induced concomitant immunity to challenge in the foot . D8 and ST-2, but not D8M, induced concomitant immunity in the lung . Concomitant immunity was, in each case tested, non-specific and could be expressed against a tumour which itself failed to induce concomitant immunity . Irradiation significantly enhanced the growth of D7, D8 and ST-2 in the feet of non-immune rats . Irradiation, carrageenan and silica significantly reduced the growth of D8M in the feet of non-immune rats . Irradiation increased the growth of D8 and D8M and carrageenan increased the growth of D8 in the feet of concomitantly immune rats (i.e., depressed resistance) . Carrageenan decreased lung colony formation by D8 in normal and concomitantly immune rats and silica decreased colony formation by D8 in concomitantly immune rats (i.e., increased resistance) . Thymus-deprived rats allowed greater growth of D8 in the feet but still expressed concomitant immunity . It is concluded that: (1) both natural and acquired tumour immunity may be modulated non-specifically in rats; (2) concomitant immunity, which has a large non-specific component, may contribute to the suppression of metastases but may also have site specificity; (3) the exact nature of the rat host-tumour relationship differs from tumour to tumour.

Endocrinol Jpn, 1984 Apr, 31(2), 177 - 84
Cholesterol accumulation in adrenocortical mitochondria after ACTH-stimulation; Mukai S et al.; Rat adrenocortical cell suspensions (10(6) cells) were incubated with ACTH (40 nM) in 2 ml of Krebs-Ringer bicarbonate buffer for 90 min . About 42 nmol of corticosterone and 14 nmol of 18-hydroxydeoxycorticosterone were generated and released into the medium . Aminoglutethimide at 50 microM inhibited the steroidogenesis to 16% . Mitochondrial pellets were prepared from the cells incubated in the absence, or in the presence, of ACTH and aminoglutethimide, and cholesterol content was determined . The mitochondria of the cells incubated without the drugs contained 25.2 micrograms cholesterol/mg protein . Cholesterol content increased by 10% in the mitochondria of the ACTH-stimulated cells . The mitochondria of the cells incubated in the presence of both ACTH and aminoglutethimide contained 143% of cholesterol compared to those of the nontreated cells . When rats were subjected to ether stress after aminoglutethimide pretreatment, cholesterol content of the mitochondrial fraction increased to about 200% compared to that of the control rats . These results suggest that a cholesterol pool exists in the adrenocortical mitochondria and that the amount increases during the steroidogenic stimulation of the cells . The mitochondria were fixed with filipin-containing fixative and examined by freeze-fracture electron microscopy . Accumulations of filipin-cholesterol complexes were observed in the inner membrane of the mitochondria as protuberances or pits 25 nm in diameter.

J Immunol Methods, 1984 Mar 30, 68(1-2), 177 - 83
Detection of islet cell antibodies by a microcytotoxicity method; Vexiau P et al.; A microcytotoxicity test for antibodies against islet cells (ICA) is described . Sera from patients with insulin-dependent diabetes, their healthy first degree relatives, and normal controls, genotyped for HLA-A, -B and -DR, were tested by 4 different methods . Cytoplasmic ICA and complement fixing ICA were detected by indirect immunofluorescence with human pancreas sections, and cytotoxic complement dependent ICA and surface ICA were tested against murine beta cell suspensions . Strong correlation was found between cytotoxic and surface antibodies (P less than 10(-7) . The technique described is appropriate for use in the screening of large numbers of sera.

J Immunol Methods, 1984 Mar 30, 68(1-2), 45 - 53
Density separation of spleen cells increases fusion frequency and yield of Ig-producing hybridomas; Van Mourik P et al.; The efficiency of hybridoma formation and growth after cell fusion can be much improved by fractionation of the mouse splenocytes . A simple procedure is described in which splenocytes with a specific gravity of more than 1.065 g/cm3 are selected by centrifugation on a Percoll gradient . The resulting cell suspension is largely depleted of macrophages and fibroblasts while the cell viability is improved . In fusion experiments performed with these cells, overgrowth of hybridomas by macrophages, fibroblasts and P-cells is avoided . The fusion efficiency and the frequency of immunoglobulin-secreting hybridomas is increased compared with fusions carried out with unfractionated spleen cells.

FEBS Lett, 1984 Mar 26, 168(2), 327 - 30
Cotransport of proline and Li+ in Escherichia coli; Tsuchiya T et al.; Uptake of Li+ induced by the addition of proline to a cell suspension of Escherichia coli was detected using an Li+-selective electrode . This Li+ uptake was inhibited by L-azetidine 2-carboxylic acid, a competitive inhibitor of the proline transport system . Thus, direct evidence for Li+-proline cotransport via the proline transport system was obtained . Kinetic parameters of the Li+ uptake were determined.

Biochim Biophys Acta, 1984 Mar 23, 803(3), 137 - 44
Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions . The influence of in situ field gradients; Endre ZH et al.; The 1H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media . At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg . This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm . T2 values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with tau = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small . The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25-1.98 G/cm and 0.89-2.09 G/cm for intra- and extracellular compartments, respectively . The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique . Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.

Radiobiologiia, 1984 Mar-Apr, 24(2), 158 - 61
{Induction of double-strand DNA breaks in rat bone marrow cells by X-radiation}; Tronov VA et al.; The method of sedimentation in a neutral sucrose gradient was used to study double-stranded DNA in a total population of rat bone marrow cells . As a result of cell lysis in neutral conditions the fragments of double--stranded DNA were formed having the molecular mass of (3 +/- 0.3) X 10(9)D . A study was made of the dynamics of accumulation of DNA double-strand breaks after irradiation of a cell suspension . It was shown that the yield of double-strand breaks and the ratio between single- and double--strand breaks in bone marrow cells were similar to those of cultured L5178Y cells.

Nippon Sanka Fujinka Gakkai Zasshi, 1984 Mar, 36(3), 391 - 6
{Detection of early pregnancy factor in the sera of conceived women before nidation}; Bessho T et al.; In order to apply the early pregnancy factor (EPF) to early diagnosis of fertilization, the establishment of optimal conditions for assay of EPF was attempted, and then EPF in the sera of contracepted and conceived women 4 to 6 days after ovulation were measured . For assay of EPF, 0.25 ml of 1:2 step diluted anti-human lymphocyte serum (ALS), 0.05ml of guinea pig serum as complement and 0.1ml of lymphocytes suspension (1 X 10(7)/ml) pretreated with test serum were mixed and then incubated at 37 degrees C for 90 min . To this mixture 0.1ml of sheep red blood cell suspension (2 X 10(9)/ml) was added and the rosette formation was counted after centrifugation . The rosette inhibition titer (RIT) was expressed as reciprocal of ALS dilutions which resulted in less than 75% of rosette formation as compared with the control . RITs of the conceived women who were assayed on the 5 th day after ovulation were in the range from 16 to 32 X 10(3), while that of the control contraceptive women who were assayed on the same day was in the range from 2 to 4 X 10(3) . The sterile women who received AIH but failed to conceive all showed less than 4 X 10(3) as RIT . These results suggest that the assay of EPF is valuable in detecting the early stage of fertilization and possibly may help to differentiate the impairment of embryo implantation from non-fertilization of the ovum as a cause of sterility.

Ann Immunol (Paris), 1984 Mar-Apr, 135C(2), 205 - 18
Immunoactive products of murine placenta . II.--Afferent suppression of maternal cell-mediated immunity by supernatants from short-term cultures of murine trophoblast-enriched cell suspensions; Chaouat G et al.; Supernatants from short-term cultures of mid-term murine trophoblast cells were assayed for their in vitro regulatory potential . They markedly inhibited cell-mediated lympholysis and mixed lymphocyte reaction in a non-specific, non-restricted fashion . By contrast, the mitogenic response to optimal doses of ConA was unaffected, while the plaque-forming cell response to sheep red blood cells was either unmodified or slightly enhanced . These data suggest that placenta-derived cells secrete factors which selectively impair some cell-mediated immune responses . It is suggested that these factors play an important role in the lack of generation of cytolytic T lymphocytes towards paternal alloantigens expressed on the trophoblast during allopregnancy.

Microvasc Res, 1984 Mar, 27(2), 204 - 22
Margination of leukocytes in blood flow through small tubes; Goldsmith HL et al.; Leukocyte margination in the vessels of the microcirculation has been attributed to a flow-dependent interaction with red cells . To determine the extent of this effect, experiments with human blood were done in 100- to 180-micron tubes to detect changes in cell distribution as a function of hematocrit and flow rate . Using a flow visualization technique, the leukocyte concentration distribution was determined in 45% ghost cell suspensions . Migration of cells toward the wall was observed at centerline velocities greater than 1 mm sec-1 and increased with increasing flow rate . The effect was probably due to a more rapid inward migration of ghosts than leukocytes because of fluid inertia and cell density differences . Experiments were therefore carried out in whole blood at hematocrits from 20 to 60%, measuring the number concentration of leukocytes and erythrocytes within the tube, nt, and comparing it to that in the infusing reservoir, no, (Fahraeus effect) . At mean tube shear rates G less than 100 sec-1, nt/no less than 1 for both leukocytes and erythrocytes showing net migration of cells away from the wall, although at nearly all hematocrits there was an enrichment of leukocytes relative to erythrocytes in the tubes . At G less than 50 sec-1, nt/no remained less than 1 for erythrocytes but increased to greater than 1 for leukocytes showing migration toward the wall, the increase being greatest at 20% hematocrit in the 100-micron tubes . The nature of the effect was revealed by cine films which showed that, as the flow rate decreased, erythrocytes formed rouleaux which migrated inward creating a core and displacing leukocytes to the periphery . In control experiments using washed blood cells in phosphate buffer-albumin, nt/no less than 1 for both leukocytes and erythrocytes at all G and hematocrits, and leukocytes were now distributed . Cine films of washed blood confirmed that, in the absence of rouleaux, no significant inward migration of erythrocytes occurred.

Int J Radiat Oncol Biol Phys, 1984 Mar, 10(3), 379 - 83
Tumor sensitizing effect by misonidazole in a clinically relevant radiation dose range; Grdina DJ et al.; The survival of cells from two murine fibrosarcoma (FSa) subpopulations after exposure to radiation only or radiation in combination with misonidazole (0.2 mg/g/fraction) was determined using a lung colony assay . FSa tumors grown in the hind legs of C3Hf/Kam pathogen-free mice were irradiated in situ when the tumors were 8 to 10 mm in diameter . Single cell suspensions prepared from excised tumors were separated on linear density gradients of Renografin, and the clonogenicity of predominantly oxic Band 2 (density 1.08 g/cm3) and predominantly hypoxic Band 4 (density 1.14 g/cm3) cells were measured . The surviving fraction of cells after doses of 1, 2, and 3 Gy, alone or preceded 30 minutes earlier with an i.p . injection of misonidazole (0.2 mg/g) was estimated from that measured after total radiation doses of 5 Gy = 5 X 1 Gy, 10 Gy = 5 X 2 Gy, and 15 Gy = 5 X 3 Gy, with the misonidazole-treated groups receiving a total drug dose of 1 mg/g, under the assumption of an equal effect per fraction . Under these conditions the initial slopes of Band 2 cells following irradiation only or irradiation plus misonidazole were 1D0 = 3.6 Gy and 2.74 Gy, respectively, giving rise to a sensitizer enhancement ratio of 1.3 . Band 4 cells exhibited a 1D0 of 5.15 Gy to radiation only and 2.75 Gy to radiation plus misonidazole (SER of 1.9) . In addition, misonidazole when administered alone in a single dose or up to 5 fractions of 0.2 mg/g each separated by 4-hour intervals, was effective in killing 50% of the Band 4 cells . The target population at risk appeared to remain constant regardless of the number of dose fractions administered . In contrast, Band 2 cells were not affected by the cytotoxic action of misonidazole . These data suggest that misonidazole is effective in sensitizing hypoxic cells in the clinical dose range, and that it is directly cytotoxic to hypoxic tumor cells.

J Neurosurg, 1984 Mar, 60(3), 582 - 8
Intracerebral transplantation of a human glioma line in immunosuppressed rats; Saris SC et al.; A model was developed for in vivo study of the human glioma-derived D-54 MG cell line in the brains of immunosuppressed Fischer 344 rats . The rats were injected with horse anti-rat thymocyte serum before and after intracerebral inoculation with 5 or 10 microliters of a D-54 MG tumor cell suspension . Reproducible mortality distributions were obtained, with deaths occurring 18 to 34 days after intracerebral inoculation . Tumors grew as well circumscribed intracerebral masses with sheets of anaplastic cells, areas of necrosis bordered by concentrated nuclei, and minimal lymphocytic infiltration . Cytogenetic analysis revealed the same general chromosome distribution and markers in the heterotransplanted glioma cells as in the cultured line . Blood-brain barrier disruption was demonstrated by intracerebral tumor staining after intravenous injection of Evans blue dye . The in vivo growth of D-54 MG in immunosuppressed rats provides a reliable experimental model for the study of chemotherapy, immunodiagnosis, and immunotherapy of a human glioma-derived tumor in an animal sufficiently large to evaluate intracarotid or intratumoral injection of therapeutic agents.

Clin Immunol Immunopathol, 1984 Mar, 30(3), 337 - 45
T-lymphocyte subsets in human lymph nodes: relative increase of OKT-8+ cells in neoplastic and reactive B-cell proliferation; Ruco LP et al.; Cell suspensions obtained from 54 human lymph nodes involved by different pathological conditions were characterized by conventional markers and by the OKT-3, OKT-4, OKT-8, OKIa-1, and OKM-1 monoclonal antibodies . In 18 cases of reactive lymphoid hyperplasia, the majority of lymph node cells were mature T lymphocytes (E-RFC = 56 +/- 9%; OKT-3+ = 63 +/- 10%); among T-cell subsets, OKT-4+ cells were 49 +/- 8% whereas OKT-8+ cells were 21 +/- 8% (T4/T8 = 2.7 +/- 1.1) . This distribution of T-cell phenotypes was not similar in the different histological types of reactive lymphoid hyperplasia . In fact, an increase in the percentage of OKT-8+ cells (25 +/- 9%; P less than 0.05) and a decrease in the values of the T4/T8 ratio (2.1 +/- 1.0; P less than 0.05) were observed in 9 cases of reactive lymphoid hyperplasia of follicular type when they were compared to the mixed and sinus types . In 13 lymph nodes involved by B-cell lymphoma, the percentage of T lymphocytes was markedly reduced (E-RFC = 21 +/- 12%; OKT-3+ = 27 +/- 18%) and the percentage of OKIa-1+ cells (51 +/- 15%) was significantly (P less than 0.01) increased as compared to reactive nodes; in addition, in these cell suspensions, an increase in the relative proportion of OKT-8+ cells (T4/T8 = 1.4 +/- 0.7; P less than 0.01) could also be demonstrated . Finally, a clear prevalence of OKT-4+ cells on OKT-8+ cells was demonstrated in 5 cases of tuberculous lymphadenitis (T4/T8 = 3.9 +/- 1.5; NS) and in 18 cases of Hodgkin's disease (T4/T8 = 4.2 +/- 2.0; P less than 0.01) . In tuberculous lymphadenitis, a significant increase (P less than 0.01) in the percentage of OKM-1+ cells could also be demonstrated.

Am J Obstet Gynecol, 1984 Mar 1, 148(5), 663 - 9
The effects of adrenergic and cholinergic agents on progesterone production by human corpus luteum in vitro; Casper RF et al.; We investigated the effects of adrenergic and cholinergic agents on human corpus luteum production of progesterone in vitro . Luteinizing hormone (LH) (50 ng/ml), dibutyryl cyclic adenosine monophosphate (Bu2cAMP) (10(-3)M), and prostaglandin E2 (PGE2) (1 microgram/ml) significantly stimulated the production of progesterone in short-term (4-hour) cell suspensions of five early and middle luteal phase corpora lutea . The adrenergic agents isoproterenol, norepinephrine, and epinephrine, and the cholinergic agents acetylcholine and carbachol at concentrations up to 10(-4)M did not alter basal or stimulated production of progesterone . Similarly, in long-term (10-day) monolayer cultures of cells from four corpora lutea, human chorionic gonadotropin (hCG) (50 ng/ml) and PGE2 stimulated, but none of the adrenergic or cholinergic agents altered, the production of progesterone significantly, except for an inhibitory effect of norepinephrine and carbachol in the presence of 17 beta-estradiol (10(-7)M) added to the culture medium . These results differ strikingly from the consistent stimulatory effect of beta-adrenergic agents on the luteal production of progesterone in several animal species.

Mutat Res, 1984 Mar, 139(3), 107 - 10
Respiration shutoff in Escherichia coli K12 strains is induced by far ultraviolet radiations and by mitomycin C; Swenson PA et al.; Ultraviolet radiations (254 nm) (UV) cause respiration to shutoff in Escherichia coli B/r . It has been reported {P.A . Swenson, Photochem . Photobiol., 33 (1981) 855-859 and J . Barbe, A . Vericat and R . Guerrero, Mutation Res., 120 (1983) 1-5} that E . coli K12 strains do not shut off respiration after UV . The latter authors also reported that mitomycin C did not cause this 'SOS' response . In this paper we report that higher UV fluences than were previously used will cause respiration shutoff in K12 strain W3110 and that cyclic AMP increases the sensitivity of respiration shutoff of irradiated cell suspensions . We also report that mitomycin C shuts off respiration in this strain . Neither UV nor mitomycin C causes respiration shutoff in the recA56 derivative of W3110 . Thus respiration shutoff is a recA dependent response to UV and mitomycin C in E . coli K12 strains.

Biofizika, 1984 Mar-Apr, 29(2), 268 - 71
{Study of low temperature crystallization in E . coli cells using NMR}; Bizunok SN et al.; Freezing and thawing processes of the E . coli cell suspension have been studied by NMR . It was shown that the degree of the cell dehydration correlated with its freezing time . The effect of the recrystallization processes was evaluated and its temperature range was indicated . It was noted that nonfreezing water content increased during thawing of the cells as compared to this content at the same temperature during freezing.

Scand J Immunol, 1984 Mar, 19(3), 269 - 73
Vascular endothelial cells present alloantigens to unprimed lymphocytes; Groenewegen G et al.; Antigen-presenting cells (APC) were removed from canine peripheral blood by carbonyl iron treatment and adherence to plastic and to nylon-wool . This treatment resulted in low proliferation in mixed lymphocyte cultures (MLC) and lack of generation of cell-mediated cytotoxicity (CMC) in depleted cell suspensions compared with untreated cell suspension . The proliferative response could be restored to normal by the addition of low numbers of autologous arterial or venous endothelial cells to the MLC of depleted cell suspensions . Cytotoxicity against phytohaemagglutinin-stimulated lymphoblasts of the stimulator was generated in the untreated MLC and also in the MLC of APC-depleted cell suspensions with endothelial cells added . It is concluded that arterial and venous endothelial cells can substitute for APC in the proliferative response of autologous lymphocytes against alloantigen and in the generation of CMC . Therefore, endothelial cells have an alloantigen-presenting capacity.

Biochim Biophys Acta, 1984 Feb 29, 770(1), 73 - 8
The static head method for determining the charge stoichiometry of coupled transport systems . Applications to the sodium-coupled D-glucose transporters of the renal proximal tubule; Fukuhara Y et al.; The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented . The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient . The charge stoichiometry can then be calculated from thermodynamic principles . In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time . The charge stoichiometries of two renal sodium coupled D-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined . The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.

Biochim Biophys Acta, 1984 Feb 28, 785(1-2), 14 - 21
Structure and function of Hb Saint-Jacques (alpha 2 beta 2 140 (H18) Ala----Thr): a new high-oxygen-affinity variant with altered bisphosphoglycerate binding; Rochette J et al.; A low P50 value in a fresh red blood cell suspension was discovered in a polycythemic patient (Hb 19 g X dl-1) . Routine acid and alkaline electrophoreses of the hemolysate were identical to normal hemolysate . Isoelectrofocusing (pH gradient 6-8) did not reveal any abnormal band whether performed with the fully liganded or deoxygenated samples . Precise analyses of the oxygen dissociation curves of the propositus' red cells demonstrated a biphasic Hill plot, a normal Bohr effect and low interaction with 2,3-bisphosphoglycerate (2,3-DPG) . Studies on the unfractionated hemolysate confirmed these observations and the inhibition of the effect of organic phosphates . Structural studies were carried out on the mixture of beta A + beta X chains and revealed the presence of two beta Tp14 peptides . Sequencing the abnormal beta Tp14 peptide showed the substitution Ala----Thr of the beta 140 (H18) residue . This new variant was named Hb Saint-Jacques . Examination of the three dimensional model of HbAo indicates that the substitution beta 140 (H18) Ala----Thr induces van der Waals interactions with the nearby lysine-82 (EF6) and leucine-81 (EF5) and a displacement of the EF corner of the beta chains . This is likely to change the normal position of the lysine-82 (EF6), a major anionic binding site in the central cavity between the two beta chains . Functional studies confirm the interpretation of a steric hindrance inhibiting the binding of large organic phosphates to Hb Saint-Jacques.

J Biol Chem, 1984 Feb 25, 259(4), 2214 - 22
1,25-Dihydroxyvitamin D3 induces 25-hydroxyvitamin D3-24-hydroxylase in a cultured monkey kidney cell line (LLC-MK2) apparently deficient in the high affinity receptor for the hormone; Chandler JS et al.; A consequence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) action in kidney is the enhanced production of 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) . We have studied this apparent induction phenomenon in two established mammalian cell lines of renal origin . A porcine kidney cell line, LLC-PK1, was found to possess typical receptors for 1,25-(OH)2D3 which sediment at 3.3 S and bind to immobilized DNA . Saturation analysis of LLC-PK1 cell cytosol revealed an equilibrium binding constant (Kd) for 1,25-(OH)2D3 of 7.8 X 10(-11) M and a concentration of 5400 binding sites/cell . In the presence of serum, intact LLC-PK1 cells also internalize and bind 1,25-(OH)2D3 . In contrast, a monkey kidney cell line, LLC-MK2, was found to contain a negligible concentration of the 1,25-(OH)2D3 receptor by all criteria examined . However, both renal cell lines respond to 1,25-(OH)2D3 with a 2- to 20-fold increase in basal levels of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity . Incubation of viable cell suspensions with 25-hydroxy{26,27-3H}vitamin D3 (0.5 microM) at 37 degrees C for 30 min followed by subsequent analysis of lipid extracts via high performance liquid chromatography was carried out to assess 24,25-(OH)2{3H}D3 formation . Enzyme induction was found to be specific for 1,25-(OH)2D3 in both cell lines with half-maximal stimulation of 24-hydroxylase activity observed at 0.2 and greater than or equal to 1.0 nM 1,25-(OH)2D3 in LLC-PK1 and LLC-MK2, respectively . The response in LLC-PK1 was more rapid (1-4 h) than in LLC-MK2 (4-8 h) following 1,25-(OH)2D3 treatment of cultures in situ . In both cell lines, actinomycin D abolished the 1,25-(OH)2D3-dependent increase in 24-hydroxylase activity . Our results suggest that the high affinity 1,25-(OH)2D3 receptor may not be required for 1,25-(OH)2D3-dependent induction of renal 24-hydroxylase activity . Alternatively, LLC-MK2 cells could contain an atypical form of the 1,25-(OH)2D3 receptor protein which retains functionality but escapes detection by standard binding techniques.

Arch Biochem Biophys, 1984 Feb 15, 229(1), 136 - 44
Induction and suppression of phytoalexin biosynthesis in cultured cells of safflower, Carthamus tinctorius L., by metabolites of Alternaria carthami Chowdhury; Tietjen KG et al.; Cell suspension cultures derived from the safflower variety US-10 respond to treatment with cell wall elicitors from either Phytophthora megasperma f.sp.glycinea or Alternaria carthami Chowdhury by producing polyacetylenic phytoalexins . These polyacetylenes were absent from the uninduced cell cultures . Low concentrations of brefeldin A, a toxin produced by A . carthami, when added to suspension-cultured safflower cells, considerably diminished the accumulation of the phytoalexins following elicitor treatment . Suppression of the synthesis of polyacetylenic phytoalexins suggests a role for brefeldin A in limiting the host range of A . carthami, the causal agent of a leaf and head blight disease in safflower.

Int J Cancer, 1984 Feb 15, 33(2), 173 - 7
Transferrin receptor and B-lymphoblast antigen--their relationship to DNA synthesis, histology and survival in B-cell lymphomas; Kvaloy S et al.; The reactivity of two monoclonal antibodies identifying antigens related to B-cell activation, B3/25 (the transferrin receptor) and BB-I (the B-lymphoblast-I-antigen), was examined on cell suspensions from 75 cases of monoclonal B-cell lymphomas . The expression of B3/25 antigen was correlated to DNA synthesis as measured by spontaneous 3H-thymidine incorporation (p = 0.0003) and histopathologically high-grade malignancy (p = 0.00003) . Furthermore, B3/25 expression was associated with survival since the patients with B3/25-negative tumors survived longer than those with B3/25-positive tumors (p = 0.018) . B3/25 expression also defined a larger group of patients with shorter survival than did histopathology alone, 28 cases versus 16 cases, respectively . On the other hand, the BB-I antigen did not reveal an association with DNA synthesis, high-grade malignancy or survival . However, the findings indicated that BB-I may be related to B-cell maturation/differentiation.

Acta Pathol Microbiol Immunol Scand {C}, 1984 Feb, 92(1), 75 - 9
Granulocyte chemotaxis in ulcerative colitis; Belsheim J et al.; The chemotactic responsiveness of granulocytes obtained from patients with ulcerative colitis was compared to granulocytes from healthy individuals, using the LMAT (Leukocyte Migration Under Agarose Technique) . A depression of chemotaxis found in ulcerative colitis patients was seen to be related to the disease activity . This reduction in granulocyte migration could not be corrected by preincubation of cell suspensions with tinidazole . The pattern observed for granulocyte chemotaxis in ulcerative colitis was discussed with reference to use as an aid in the differential diagnosis of ulcerative colitis and Crohn's disease.

Can J Biochem Cell Biol, 1984 Feb-Mar, 62(2-3), 94 - 9
Specific binding of cholesterol to chromatin prepared from mouse spleen cells; Regenass-Klotz M et al.; Mouse spleen cell suspensions were incubated with tritiated cholesterol for various time intervals . The chromatin of these cells was then isolated, washed with Triton X-100, and fractionated on Sephadex columns . It was found that cholesterol binds specifically to the chromatin . The binding was saturated after 45 min of incubation and also displayed characteristics typical of dose-dependent binding . The number of cholesterol molecules bound per nucleus was estimated to be on the order of 10 000 . Oxygenated sterols, such as 25-hydroxycholesterol and 7-ketocholesterol, did not compete for the binding with {3H}cholesterol if added in 20-fold molar excess to the incubation medium of the cells . Chromatographic analyses on Sephadex columns displayed a distinct peak of radioactivity . The protein-sterol complex had an apparent molecular weight of 180 000 +/- 27 000 . Using extensive digestion with DNase I (EC 3.1.21.1) it could be concluded that DNA, binding to the complex, did not influence the estimate of the molecular weight, whereas digestion with pronase or treatment with sodium dodecyl sulfate destroyed the complex . Additional experiments using sucrose density gradients (5-20%) showed also, that {3H}cholesterol was bound to chromatin by one or several proteins.

Bioorg Khim, 1984 Feb, 10(2), 238 - 43
{Neutral glycosphingolipids in Ehrlich ascites carcinoma cells}; Prokazova NV et al.; The structure of the neutral glycosphingolipids of the Ehrlich ascite carcinoma (EAC) cells was studied . The main four components were identified as glycosylceramide, lastosylceramide, N-acetylgalactosyllactosylceramide and galactosyl-N-acetyllactosylceramide (asialo-GM1) . The neutral glycolipid pattern of the cells was found to depend on their density . Dilution of the cell suspension resulted in an increased content of asia-lo-GM1, whereas the content of the other neutral glycolipids remained unchanged . The possible connection between these changes and the earlier disclosed cell density dependence of the gangliosides in EAC cells is discussed.

Am J Clin Pathol, 1984 Feb, 81(2), 176 - 83
Pan-leukocyte monoclonal antibody L3B12 . Characterization and application to research and diagnostic problems; Wood GS et al.; In this report, the authors describe a murine anti-human monoclonal antibody, L3B12, which defines a pan-leukocyte cell surface antigen of approximately 180,000 m.w . Extensive screening against a variety of tissues indicates that L3B12 is sensitive and specific for leukocytes, related cells of bone marrow lineage, and their corresponding neoplasms . Unlike many lymphoid antigens that are not detectable following routine fixation and embedding, those recognized by L3B12 and related antibodies are variably preserved . L3B12 has proven useful in studying the antigen expression of normal leukocytic elements, lymphomas, and related disorders, and in enriching or depleting leukocytes from heterogeneous cell populations . From a diagnostic standpoint, L3B12 staining of tissue sections or cell suspensions is useful for distinguishing large cell lymphomas from undifferentiated carcinomas and in distinguishing lymphomas and leukemias from other small round cell tumors of childhood.

J Natl Cancer Inst, 1984 Feb, 72(2), 395 - 401
Lack of beta 2-microglobulin on the surface of canine transmissible venereal tumor cells; Cohen D et al.; beta 2-Microglobulin (beta 2m) expression on the cell surface of the naturally occurring, allotransplantable canine transmissible venereal tumor (TVT) was investigated by use of indirect membrane immunofluorescence and radioimmunoassay . Two cell populations were identified in animal-derived, collagenase-disaggregated TVT cell suspensions . About 80% of the cells lacked surface beta 2m expression, whereas about 20% of the cells strongly reacted with anti-dog beta 2m serum . With the use of a cell separation technique, beta 2m-negative cells were demonstrated to carry TVT markers on their surface, whereas the beta 2m-positive cells did not express the tumor markers . The beta 2m-positive cells seemed, therefore, to be tumor-infiltrating host cells . These findings were supported by fluorescence staining studies of frozen sections of the TVT . The lack of beta 2m expression on the surface of TVT cells might explain the allotransplantability of this neoplasm, since beta 2m expression on the cell surface appeared to be obligatory for the expression of class I major histocompatibility complex antigens.

Dev Biol, 1984 Feb, 101(2), 307 - 17
Identification and immunochemical characterization of spermatogenic cell surface antigens that appear during early meiotic prophase; O'Brien DA et al.; Three spermatogenic cell populations isolated from prepuberal mice--type B spermatogonia, preleptotene spermatocytes, and leptotene/zygotene spermatocytes--were used to elicit distinct polyclonal antisera . Surface binding specificities were determined for purified IgGs by indirect immunofluorescence and rosette assays on live cells . Binding activities were assayed both before and after absorptions with a variety of somatic and spermatogenic cells . Each of these antisera binds to surface antigens that are present on germ cells throughout spermatogenesis and are not shared by splenocytes, thymocytes, and erythrocytes . Only the antiserum raised against leptotene and zygotene spermatocytes (ALZ) recognizes a stage-specific subset of surface determinants . After appropriate absorptions, ALZ binds to the surface of early pachytene spermatocytes and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa . Antigens which react with this absorbed IgG are not detected on the surface of spermatogonia or meiotic cells prior to pachynema, including leptotene and zygotene spermatocytes . The observed binding specificities may result from the synthesis of one or more surface molecules during the early meiotic stages, followed by delayed insertion into the plasma membrane during the pachytene stage of meiotic prophase . Stage-specific antigens recognized by ALZ, including both protein and probably lipid, have been localized immunochemically on nitrocellulose blots from one-dimensional SDS gels . A dithiothreitol-sensitive constituent (Mr approximately 39,000) recognized by ALZ has been identified as the major protein determinant present in early meiotic cells but absent in 8-day-old seminiferous cell suspensions containing spermatogonia and Sertoli cells . This determinant is present in populations of preleptotene, leptotene/zygotene, and early pachytene spermatocytes isolated from 17-day-old animals, an observation consistent with the hypothesis of delayed insertion into the plasma membrane.

Blood, 1984 Feb, 63(2), 270 - 6
Liver endothelium and not hepatocytes or Kupffer cells have transferrin receptors; Soda R et al.; Using a visual probe, consisting of latex minibeads covalently linked to transferrin (TF), we found that, in rat liver cell suspensions, transferrin receptors were limited to endothelial cells . Neither hepatocytes nor Kupffer cells contained an appreciable number of TF receptors . Specificity of this reaction was demonstrated by preincubation with non-derivatized TF, which inhibited the binding . This was further confirmed by fractionation of liver cell suspensions on metrizamide gradients . The uptake of either the visual probe or 125I-labeled TF was again limited to the endothelium-rich fraction . Transferrin bound to endothelial membrane was internalized at 37 degrees C, but not at 4 degrees C, via a coated pit system . Again, hepatocytes and Kupffer cells did not internalize the probe . The findings suggest that iron may be first taken up by liver endothelium and then transmitted to parenchymal cells . These results emphasize the generally unappreciated role of endothelium in the transport across the tissue-blood barrier.

Transplantation, 1984 Feb, 37(2), 168 - 74
Perturbation of epidermal Langerhans cells in immunosuppressed human renal allograft recipients; Sontheimer RD et al.; As an initial attempt to gain a better understanding of the basis for the increased incidence of ultraviolet-light-related skin cancer in chronically immunosuppressed human renal allograft recipients, we have compared both morphological and functional characteristics of epidermal Langerhans cell (LC) populations present in the forearm skin of nine such patients with those of age, sex, and race-matched controls . The LC surface densities in vacuum-induced blister-derived epidermal sheets taken simultaneously from extensor and flexor forearm skin of the patients were significantly lower than those observed in the controls . The most abnormal LC densities seen were in the patients' extensor forearm skin . Likewise there were disturbances in LC distribution and morphology that were most marked in the extensor forearm skin of patients . Differences in the alloantigen-presenting capacity of LCs present in epidermal cell suspensions prepared from patient and control forearm skin were also noted--however, these differences were not as great as were the LC density differences . The alloantigen-presenting capacity of patients' LCs was depressed proportionately more than was the alloantigen presenting capacity of their peripheral blood mononuclear cells . These results demonstrate that the LC population is clearly perturbed in human renal allograft recipients and that this perturbation is greatest in a sun-exposed region of skin.

Br J Cancer, 1984 Feb, 49(2), 225 - 33
Improved optical detection of colony enlargement and drug cytotoxicity in primary soft agar cultures of human solid tumour cells; Alley MC et al.; The presence of cellular aggregates in cell suspensions derived from human solid tumours often complicates subsequent evaluation of colony formation in primary soft agar cultures (Agrez et al., 1982b) . In the present study, performance of a conventional colony formation assay was observed to lack sufficient sensitivity to identify growth and active chemotherapeutic agents in the majority of specimen cultures . Modification of conventional methodologies to include filtration of cell suspensions, use of "proliferation control" and "cytotoxicity control" cultures as well as vital staining were found to be essential for the valid assessment of primary soft agar cultures in our laboratory . In addition, application of drugs to culture surface in place of culture incorporation appeared to facilitate culture performance and drug sensitivity testing.

Cancer Res, 1984 Feb, 44(2), 852 - 6
Defects in natural killer cell activity and interferon response in human lung carcinoma and malignant melanoma; Sibbitt WL Jr et al.; The natural killer (NK) activity of peripheral blood mononuclear cells from 25 patients with squamous cell carcinoma of the lung, malignant melanoma, or epitheloid cancers of the gastrointestinal tract was measured by the lysis of 51Cr-labeled K562 target cells . NK activities of many patients with lung cancer or malignant melanoma were decreased relative to normal controls . This abnormality was significantly correlated with advancing stage of disease and the percentage of monocytes in the cell suspensions . Addition of indomethacin or removal of monocytes did not restore depressed NK function to normal levels . Abnormalities of NK function did not appear to be secondary to the presence of mononuclear suppressor cells . The response to interferon was also impaired in patients with advanced disease . The number of effector:target conjugates was normal even in patients with depressed NK function; however, the number of active lytic effectors was decreased . These results imply that the cells which bind tumor targets are present in patients with advanced cancers, but these cells are either immature or functionally inactive.

J Clin Endocrinol Metab, 1984 Feb, 58(2), 250 - 4
The interrelationship between the effects of somatostatin and human pancreatic growth hormone-releasing factor on growth hormone release by cultured pituitary tumor cells from patients with acromegaly; Lamberts SW et al.; The effects of somatostatin (SRIF) and human pancreatic tumor GRF on GH release by cultured pituitary tumor cells obtained during transsphenoidal operation from 15 acromegalic patients were investigated . In a study of the sensitivity of pathological GH release to SRIF, 1-10 nM SRIF induced maximal inhibition of hormone release in 3 consecutive tumors . In 12 of 15 tumor cell cultures, 10 nM SRIF produced statistically significant inhibition of basal GH release by 39 +/- 3% (mean +/- SEM) . In 2 of the 3 other tumors, SRIF inhibited GRF-stimulated GH release, while this was not investigated in the third tumor . A dose-response study of the effect of GRF on GH release by cultured pituitary tumor cells showed that doses of 0.1, 1, 10, and 100 nM induced similar maximal (35%) stimulation of hormone secretion . In four of five consecutive tumor cell suspensions, 1 and 10 nM GRF induced statistically significant GH stimulation by 18-300% . Preincubation of the tumor cells with 5 nM dexamethasone greatly increased the sensitivity and the maximal stimulation in response to GRF and made one tumor cell suspension, which did not react to GRF initially, sensitive to GRF . In the tumors of four patients, the interrelationship between the effects of SRIF and GRF on GH release were also studied . SRIF (10 nM) inhibited the stimulatory effects of GRF on GH release virtually completely . In conclusion, GH release by in vitro cell cultures of GH-secreting pituitary adenomas was inhibited by SRIF and stimulated by GRF . The interaction of GRF and SRIF on GH release by these pituitary tumor cells was similar to that in normal rat GH cells, as SRIF virtually completely overcame the GRF-induced GH release.

FEBS Lett, 1984 Jan 23, 166(1), 175 - 8
Increases in permeability of Escherichia coli outer membrane induced by polycations; Katsu T et al.; The action of polycations (such as polylysine and compound 48/80) on Escherichia coli was studied with use of Ca2+, K+ and TPP+ ion-selective electrodes . Rapid efflux of Ca2+ was observed when a polycation was added in cell suspension . The polycation treatment promoted a drug-inducing K+ release from the cytoplasmic membrane . TPP+ uptake was also increased by addition of a polycation . Without the polycation treatment, the uptake of TPP+ was largely suppressed due to a permeability barrier of the outer membrane . The results show that a polycation disrupted the permeability barrier of the outer membrane.

J Immunol Methods, 1984 Jan 20, 66(1), 149 - 59
Detection of antibody-coated sperm by 'panning' procedures; Hancock RJ et al.; The use of 'panning' procedures to detect immunoglobulin on the sperm surface are described . Wells on plastic plates are coated with anti-immunoglobulin molecules by either 1-step or 2-step procedures and the sperm under test are then incubated in these wells for up to 1 h and the wells washed . Antibody-coated sperm remain attached in large numbers while control sperm are washed out . These procedures have the advantages that they are cheap, simple, do not involve sperm fixation and may be used with relatively dilute cell suspensions and with sperm of low motility . The potential applications of the procedures are discussed.

J Biol Chem, 1984 Jan 10, 259(1), 662 - 8
A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum . II . Developmental regulation; Leichtling BH et al.; The cAMP-dependent protein kinase of the cellular slime mold, Dictyostelium discoideum, is developmentally regulated; there is an approximately 4-fold increase in activity during development . The incorporation of {3H}leucine into the enzyme demonstrates that there is de novo synthesis of the cAMP-dependent protein kinase . The activities of the catalytic and regulatory subunits increase in parallel . The maximal rate of increase of cAMP-dependent protein kinase activity precedes "tip" formation, a stage of development characterized by a sharp increase in mRNA complexity . The high level of cAMP-dependent protein kinase activity, attained at this stage of development, persists when aggregates are dispersed and the amoebae are kept in suspension without added cAMP . The synthesis of the developmentally regulated mRNAs under these conditions is dependent on exogenous cAMP . The increase in cAMP-dependent protein kinase activity during development does not require sustained cell-cell contact insofar as it occurs in single cell suspensions of amoebae . Furthermore, the increase does not require exogenous cAMP, although added cAMP stimulates the synthesis of the enzyme to a level higher than that found, when cAMP is not added . These observations support the hypothesis that in D . discoideum cAMP-dependent protein kinase mediates the effects of cAMP on development.

Int J Immunopharmacol, 1984, 6(3), 187 - 91
Carrageenan-mediated suppression or augmentation of mitogen-induced lymphocyte proliferation; Neveu PJ et al.; The effects of carrageenan on mitogen-induced lymphocyte proliferation were studied in the guinea pig . According to the dose used, carrageenan displayed opposite effects on thymidine uptake by spleen cells or peripheral blood leukocytes stimulated by Concanavalin A or phytohemagglutinin (25 micrograms ml-1 carrageenan increased whereas 0.25 microgram ml-1 depressed thymidine incorporation) . Carrageenan, at the high concentration which increased thymidine uptake by mitogen-stimulated spleen cells, potentiated the enhancing activity of macrophages that was observed with cell suspensions containing 20% macrophages . Conversely, low concentrations of carrageenan abolished the enhancing effect of macrophages . These effects of carrageenan on lymphocyte proliferation could be explained by its activities on a macrophage functional subset rather than on the whole macrophage population.

Bioelectromagnetics, 1984, 5(2), 173 - 91
Interaction between electromagnetic fields and cells: microelectrophoretic effect on ligands and surface receptors; Chiabrera A et al.; The aggregation between lectins and lymphocyte surface receptors can be affected strongly by a low-level electric field induced in the cell suspension by a time-varying magnetic field . One of the possible mechanisms is the microelectrophoretic effect due to the electric field, which influences the distance (in the mean square sense) between charged ligands and receptors when they are about to separate . On a purely theoretical basis, it is shown that, at low frequencies, an externally induced periodic electric field always decreases the mean lifetime of ligand-receptor complexes . As a consequence, the mitogenic gain obtained by lectin addition to cell suspension is decreased . These results suggest that such a mechanism, if effective, reduces the lectin mitogenic capability and offers a way of handling similar phenomena which have been described for other biological systems.

Arch Dermatol Res, 1984, 276(1), 41 - 4
The effect of an oral therapeutic single-dose of griseofulvin on polymorphonuclear leukocyte migration in a casein gradient; Bandmann U et al.; In a previous study, polymorphonuclear (PMN) leukocyte chemotaxis in vitro was inhibited by therapeutic concentrations of griseofulvin . In the present investigation, PMN migration in a casein gradient was studied in a cell suspension containing serum collected before and after a therapeutic peroral single-dose of griseofulvin . The PMN migration in vitro was not influenced significantly by the presence of griseofulvin-containing serum . It is suggested that peroral griseofulvin treatment has negligible effect on PMN chemotaxis in vitro.

Int J Biochem, 1984, 16(3), 327 - 31
Biochemical characterization of three hamster melanoma variants--II . Glycolysis and oxygen consumption; Scislowski PW et al.; Lactate production and oxygen consumption were studied in single cell suspension prepared from solid tumours of the black-melanotic (Ma), brown-melanotic (MI) and amelanotic (Ab) melanomas of hamster . Aerobic lactase production was about 5 times higher in the fast growing Ab melanoma than in the slow growing Ma and MI melanomas . Aerobic lactate production in both melanotic hamster melanomas was stimulated by 3-(3,4-dihydroxyphenyl)-L-alanine . This compound was without effect on the cells isolated from amelanotic hamster melanoma . L-Phenylalanine, a known competitive tyrosinase inhibitor reduced the stimulatory effect of L-DOPA on the the lactate production . Oxygen consumption was similar in all three melanomas . The oxygen consumption was inhibited completely by 1 mM potassium cyanide in the Ab melanoma but only in about 1/3 in the Ma and MI melanomas . The Pasteur effect was higher in relative terms and lower in absolute terms in the melanotic melanomas than in the Ab melanoma only . The Crabtree effect was present in the Ab melanoma only . Thus glycolysis measured by aerobic and anaerobic lactic acid formation, and cell respiration, measured by oxygen consumption sensitive to KCN, were both higher in the more malignant, less differentiated Ab melanoma than in the Ma and MI melanomas . The suggestion is presented that the process of melanogenesis influences both aerobic glycolysis and the KCN insensitive consumption in the melanotic hamster melanomas.

Exp Hematol, 1984 Jan, 12(1), 53 - 9
Enlargement and immune activity of chick embryo host spleen associated with transfer of B-haplotype-matched donor lymphoid cells; Seto F; Splenic enlargement was elicited in embryo hosts with the transfer of blood leukocytes, spleen cells, thymocytes, and bone marrow cells from B-haplotype-matched donors . The splenic growth profile of embryos grafted with B-haplotype-compatible donor cells, although elevated, was more like that of control embryos than hosts undergoing a graft-versus-host reaction (GVHR) . The spleens of compatible hosts, moreover, lacked the obvious signs of GVHR incompatibility . Even greater splenic enlargement and splenic formation of plaque-forming cells (PFCs) was associated with the transfer of blood leukocytes and spleen cells from mouse RBC-primed donor birds, but not with bone marrow or thymus cells . The residual splenic enlargement not attributable to the proliferation and maturation of PFC precursors is believed to be the consequence of the colonization and growth in the host spleen of other hemopoietic precursor cells in the cell suspensions.

Cytometry, 1984 Jan, 5(1), 55 - 62
Bivariate flow cytometric analysis of murine intestinal epithelial cells for cytokinetic studies; Pallavicini MG et al.; The heterogeneous nature of the small intestine and the lack of methods to obtain pure crypt populations has, in the past, limited the application of standard flow cytometric analysis for cytokinetic studies of the proliferating crypts . We describe a flow cytometric technique to discriminate crypt and villus cells in an epithelial cell suspension on the basis of cell length, and to measure the DNA content of the discriminated subpopulations . Our data indicate that bivariate analysis of a mixed epithelial cell suspension can be used to distinguish mature villus cells, G1 crypt cells, and S-phase crypt cells . In addition, continuous labeling studies suggest that the position of a cell on the cell length axis reflects epithelial cell maturity . We applied this flow cytometric technique to determine the cytokinetic nature of epithelial cells obtained by sequential digestion of the small intestine.

Nephron, 1984, 36(2), 89 - 93
Effect of cimetidine on basal and histamine-induced secretion of parathyroid hormone in vitro; Wagner PK et al.; The effect of cimetidine on basal and histamine-induced PTH secretion was tested using single cell suspensions obtained from (a) primary parathyroid adenomas, and (b) secondary hyperplastic parathyroid tissue from patients undergoing chronic hemodialysis . The histamine-stimulated hormone secretion was dose-dependent . Cimetidine suppressed both basal and histamine-stimulated hormone secretion . Within the therapeutic range the suppressive effect was identical for adenoma and hyperplasia . Both adenomata and secondary hyperplastic glands showed a histamine H2-receptor-related response . The role of histamine in the pathogenesis of hyperparathyroidism is not quite clear so that the possible benefits of cimetidine for medical treatment of primary and secondary hyperparathyroidism will have to be proven by careful clinical trials.

Cell Immunol, 1984 Jan, 83(1), 142 - 51
Leukemic transformation in hrs mice occurs in an immature-nonactivated thymocyte subpopulation; Tempelis LD; The murine leukemic strain HRS/J has an autosomal-recessive, mutant gene, hr, with homozygotes (hr/hr) having a 72% incidence of thymic leukemia at 18 months of age compared to 20% in heterozygotes (hr/+) . This study was done to (a) determine if expression of thymocyte differentiation and murine leukemia virus (MuLV) antigens during leukemic transformation were different in hr/hr compared to hr/+ mice, (b) define the subpopulations that were targets for leukemic transformation, and (c) compare the results to reports in other leukemic strains . Flow cytometry analysis of thymus cell suspensions was done with anti-T-cell and anti-H-2 monoclonal antibodies, peanut agglutinin (PNA), and heteroantisera to MuLV antigens . Thymocytes of 1- to 3-month-old HRS/J mice were Thy 1.2+, Lyt 1+2+, H-2Kk-, and MuLV- with an immature-nonactivated phenotype, i.e., PNA+, and Iak- . Preleukemic and leukemic thymocytes showed diversity in expression of Thy 1.2 and Ly antigens with increased H-2Kk and MuLV expression . No differences in phenotype patterns were noted between hr/+ and hr/hr mice during the time course of leukemogenesis . Persistently high PNA/low Iak expression of preleukemic and leukemic thymocytes indicated that the target for HRS leukemic transformation was an immature-nonactivated thymocyte subpopulation in contrast to AKR/J mice in which leukemic transformation involves a mature-activated thymocyte subpopulation . These findings suggest that spontaneously generated leukemogenic viruses in HRS mice have tropism for thymocytes of an immature-nonactivated phenotype.

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