Cell suspensions

J Immunol, 1986 Apr 15, 136(8), 2776 - 86
Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2; Nixon-Fulton JL et al.; Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC) . Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes . To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2 . Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture . Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin . Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period . Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population . Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2 . Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells . Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)

J Cell Biol, 1986 Apr, 102(4), 1298 - 303
Liver endothelium mediates the hepatocyte's uptake of ceruloplasmin; Tavassoli M et al.; The mode of transport of ceruloplasmin (CP) into the liver was investigated in fractionated liver cell suspensions . Incubation of 125I-CP at 4 degrees C with these different fractions led to its binding only to endothelial cells but not Kupffer cells and hepatocytes . Incubation at 37 degrees C led to rapid uptake of 125I-CP by endothelium, but cell-associated radioactivity declined after 15 min, which suggests the release of the labeled substance . Internalization was confirmed by fractionation of surface-bound and internalized ligand . The released label now acquired binding potential for fresh target hepatocytes, and the binding was inhibitable with asialoceruloplasmin but not native CP . This suggested that the released molecule was modified in the endothelium by desialation . Desialation was confirmed by incubation of endothelium with double-labeled CP (3H label on sialic acid and 125I on the protein part) . We conclude that in the liver, CP is first recognized and taken up by endothelial cells that are endowed with appropriate surface receptors for the protein . Endothelium then modifies the molecule by desialation to expose the penultimate galactosyl residues . The modified molecule is then released, recognized, and taken up by hepatocytes through their membrane galactosyl-recognition system . These findings are consistent with the role of endothelium as an active mediator of molecular transport between blood and tissue, and further assign a biological role for the galactosyl-recognition system in hepatocytes.

Blood, 1986 Apr, 67(4), 1110 - 8
Red cell rheology in stomatocyte-echinocyte transformation: roles of cell geometry and cell shape; Reinhart WH et al.; The influence of the shape of the red blood cell during stomatocyte-echinocyte transformation on its deformability was studied by microsieving through pores with diameters of 2.6, 4.5, and 6.9 micron . A stomatocytic transformation was produced by chlorpromazine (0.02, 0.1, and 0.5 mmol/L) and an echinocytic transformation by sodium salicylate (7.5, 30, and 120 mmol/L) . For spherostomatocytes, an increase in filtration resistance through 2.6 and 4.5 micron pores was observed, whereas for spheroechinocytes, a decrease in filtration resistance through 2.6 micron pores was found . Larger pores (6.9 micron) were not sensitive to those shape changes . The changes in deformability can be explained by the fact that the surface area of (sphero)-stomatocytes decreased, whereas that of (sphero)-echinocytes increased; the cell volume remained essentially constant . Echinocytes produced by 24-hour adenosine triphosphate depletion differed from drug-induced echinocytes: they had an increased cell volume at constant surface area and consequently an increased filtration resistance through 2.6- and 4.5-micron filter pores . Shape changes with spicule formation are therefore not a homogeneous entity, and cell geometric factors (eg, surface area and volume) must be assessed with care . The viscosity of red cell suspensions at a hematocrit level of 45% was higher for drug-induced echinocytes than discocytes or stomatocytes at all shear rates tested . We conclude that the normal discocyte represents an optimum shape for the flow in vivo since a stomatocytic transformation could impair the passage through the microcirculation (decrease in cell filterability) and an echinocytic transformation could impair the flow in larger vessels (increase in blood viscosity).

Blood, 1986 Apr, 67(4), 1090 - 7
Radiobiological properties of the human hematopoietic microenvironment: contrasting sensitivities of proliferative capacity and hematopoietic function to in vitro irradiation; Laver J et al.; We describe the effects of in vitro irradiation on the proliferative capacity and hematopoietic supportive function of human marrow stromal cells . To assess the effects on the proliferative capacity of stromal progenitors and differentiated fibroblasts, marrow cell suspensions and trypsin-dispersed marrow fibroblasts were treated with a single dose of gamma radiation at 100 rad/min . Fibroblastic progenitors (CFU-F) showed an exponential decrease in colony formation with increasing doses of irradiation, with a Do slightly higher than that of granulomonopoietic progenitors (CFU-GM); Do values for CFU-F and CFU-GM were 130 and 115, respectively . However, although the CFU-F survival curve exhibited a shoulder (n = 1.3), the CFU-GM curve did not (n = 1.0), indicating that only fibroblastic progenitors have the potential to repair irradiation-induced damage . Passaged marrow fibroblast colony-forming cells also showed a shouldered exponential survival curve with a Do of 110 and n value of 1.4 . Marrow stromal progenitors giving rise to adherent layers in long-term marrow cultures also demonstrated a highly radiosensitive proliferative capacity . Stromal layers derived from irradiated marrow suspensions failed to establish adherent layers after relatively low doses of irradiation (over 240 rad) in a dose-response manner . To assess any functional damage in stromal progenitors surviving irradiation, stromal layers derived from marrow suspensions irradiated up to 240 rad were cocultured with freshly isolated autologous hematopoietic cells and assayed for their capacity to support prolonged CFU-GM production . Confluent stromal layers derived from irradiated marrow suspensions sustained CFU-GM production as well as controls . To study the effects of irradiation on the hematopoietic supportive capacity of established marrow-derived stromal layers, 4 to 6-week-old adherent layers were irradiated as described and cocultured with autologous marrow cells enriched for colony-forming cells . Stromal layers irradiated up to 1,320 rad sustained prolonged CFU-GM production, indicating that the hematopoietic supportive function remained intact at this dose of irradiation . In conclusion, we demonstrated that the proliferative capacity of human marrow stromal progenitors, as well as that of their differentiated descendants, is quite sensitive to in vitro radiation, while the hematopoietic supportive function of differentiated stromal cells is relatively resistant to the effects of radiation.

J Immunogenet, 1986 Apr-Jun, 13(2-3), 211 - 7
Expression of HLA molecules on cells from fresh explants of human digestive tract cancer; Garcia-Espejo R et al.; It has been recently established that there is a correlation between the lack of MHC class I gene expression on murine tumour cells and their ability to grow and metastasize . We have studied the expression of HLA-ABC and HLA-DR products on human malignant tumours from the digestive tract using monoclonal antibodies, by indirect immunofluorescence on the cell suspensions obtained from 29 freshly explanted digestive tumours . Our results show that digestive tract cancers have an heterogeneous expression of HLA class I molecules on their surface . Whereas 50% have high levels of expression of these molecules (more than 60% positive cells), 25% have a moderate level of expression (20-60% positive cells) and 25% have weak expression (less than 20% positive cells) . It has been found that there is a correlation between the level of HLA class I molecule expression and the degree of histological differentiation of a tumour . The absence of MHC class I antigens on human tumour cells, detected in this study, may play a relevant role in oncogenesis, as has been established in experimental models.

Biochem Pharmacol, 1986 Apr 1, 35(7), 1091 - 8
Oxidative activity of hydroxylated primaquine analogs . Non-toxicity to glucose-6-phosphate dehydrogenase-deficient human red blood cells in vitro; Baird JK et al.; The individual effects of two putative metabolites of primaquine (5,6-dihydroxyprimaquine and 5,6-dihydroxy-8-aminoquinoline) on the hexose monophosphate shunt (HMS) and on the ATP-dependent proteolytic system which rapidly degrades oxidized erythrocyte protein were measured in intact red blood cells in vitro from two blood donors . In red cells treated with nitrite (1-40 mM) or phenylhydrazine (0.01-10 mM), proteolytic activity was detected only with concentrations (7.5 mM NaNO2 and 0.25 mM phenylhydrazine) causing greater than 15-fold elevation of HMS activity, and glucose-6-phosphate dehydrogenase (G6PD)-deficient (25% of normal activity) red cell suspensions thus treated showed approximately 30% greater proteolysis . G6PD-normal and deficient red cells treated with the primaquine analogs, however, did not experience proteolysis with concentrations (0.25 mM) in excess of those causing 17-fold elevation of HMS activity . Stimulation of the HMS by the primaquine analogs thus appears unrelated to an erythrotoxic oxidative stress . Methylene blue is known to cause an elevation of HMS activity through direct and diaphorase II-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) which is independent of injurious oxidative stress . It was found that the putative primaquine metabolites also caused direct and diaphorase II-dependent oxidation of NADPH in dilute hemolysate, thus suggesting that the putative primaquine metabolites have a methylene blue-like redox disposition in red blood cells . Results obtained in this study suggest that the hemolytic toxicity of primaquine may be unrelated to processes which lead to oxidative deterioration of red cell protein.

Gan To Kagaku Ryoho, 1986 Apr, 13(4 Pt 2), 1194 - 200
{Experimental study of soft agarose colony formation assay of renal carcinoma}; Tsukamoto T et al.; Several problems have been pointed out in colony formation assay (CFA) developed by Hamburger and Salmon . We studied those using 87 fresh renal carcinoma, comprising of 72 renal cell carcinoma and 15 transitional cell carcinoma of renal pelvis . Results are as follows: Pure single cell suspension was difficult to be yielded from these carcinoma . Our result suggests that cell suspension contains various sized growth unit as well as greater than or equal to 60 microns growth unit . Presence of substantial number of greater than or equal to 60 microns growth unit in plating cell suspension was revealed to influence on the cell growth, ie, the increase of newly formed greater than 60 microns growth unit . This indicates that initial growth unit count is mandatory for "quality control" in CFA . The current study in extent of growth for renal carcinoma revealed three different patterns, one of which did not seem suitable for in vitro chemosensitivity test with CFA . This finding brings for us the fact to prepare "proliferation control" which can check periodically the growth extent of renal carcinoma.

Clin Exp Metastasis, 1986 Apr-Jun, 4(2), 73 - 89
Quantitative analysis of cancer invasion in vitro: comparison of two new assays and of tumour sublines with different metastatic capacity; Waller CA et al.; Various murine tumour sublines which differed considerably in their in vivo metastatic capacity were tested in vitro for their ability to invade normal tissue . For this purpose we developed two quantitative tests, a Boyden chamber endothelial cell invasion assay and a brain tissue microsphere invasion assay . The invasion of {75Se}methionine-prelabelled tumour cells into the normal tissues was followed by measuring the percentage of tumour-associated label in the brain microspheres or the endothelial monolayers after 12-48 h of co-cultivation . Clear and comparable differences existed in both assays between the amount of radiolabel found in the normal tissues after a co-cultivation with the different tumour lines . In three of the four tumour lines invasiveness correlated with metastatic capacity . The fourth line, a plastic adherent variant, was highly invasive but low metastatic . The ability of tumour cells to invade normal tissue, therefore, while necessary for the generation of metastases, is not in itself sufficient . Since both assays are independent of time-consuming histological sectioning and staining and allow a quantitative determination of invasive capacity of tumour cells grown as single cell suspensions they appear well suited for experimental manipulation and for screening of anti-invasive drugs.

Ultrasound Med Biol, 1986 Apr, 12(4), 297 - 305
Acoustic emission as a measure of exposure of suspended cells in vitro; Edmonds PD et al.; Cell viability, survival, and growth of C1300 mouse neuroblastoma cells were assayed by trypan blue dye exclusion, clonogenesis, and culturing, respectively, after exposure in suspension to therapeutic levels of ultrasound (1 MHz; continuous wave; spatial peak intensity 0.9, 1.7, and 2.6 W/cm2; 5 min at 37 degrees C) . Acoustic emission from the cavitating cell suspensions was recorded as the rms value of the half-harmonic and noise components combined . Cell viability and survival appeared better correlated with acoustic emission than with spatial peak intensity, implying that acoustic emission may provide a more direct measure of insult to the cells in a cavitating field than the incident intensity . Biological assay results of growth were well correlated with spatial peak intensity but not with acoustic emission levels, which seems to imply that for this end point incident intensity is a more directly interacting parameter than cavitational activity, provided however that the latter is present . Further refinement of the technique for measuring acoustic emission from cell suspensions should permit separate measurements of the half-harmonic and noise components . When thus refined, it may provide the means to demonstrate cavitational action without resorting to additional experiments to suppress cavitation.

Cryobiology, 1986 Apr, 23(2), 103 - 15
Vitrification of human monocytes; Takahashi T et al.; Human monocytes purified from peripheral blood by counterflow centrifugal elutriation were cryopreserved in a vitreous state at 1 atm pressure . The vitrification solution was Hanks' balanced salt solution (HBSS) containing (w/v) 20.5% Me2SO, 15.5% acetamide, 10% propylene glycol, and 6% polyethylene glycol . Fifteen milliliters of this solution was added dropwise to 1 ml of a concentrated monocyte suspension at 0 degrees C . Of this, 0.8 ml was drawn into silicone tubing and rapidly cooled to liquid nitrogen temperature, stored for various periods, and rapidly warmed in an ice bath . The vitrification solution was removed by slow addition of HBSS containing 20% fetal calf serum . The numerical cell recovery was about 92% and most of these retained normal phagocytic and chemotactic ability . Differential scanning calorimeter records of the solution show a glass transition at -115 degrees C during cooling and warming, but no evidence of ice formation during cooling . Devitrification occurs at about -70 degrees C during warming at rates as rapid as 80 degrees C/min . The amount of devitrification is dependent upon the warming rate . Freeze-fracture freeze-etch electron microscope observations revealed no ice either intra- or extracellularly in samples rapidly cooled to liquid nitrogen temperatures except for small amounts in some cellular organelles . However, if these cell suspensions were warmed rapidly to -70 degrees C and then held for 5 min, allowing devitrification to occur, the preparation contained significant amounts of both intra- and extracellular ice . Biological data showed that this devitrification was associated with severe loss of cell function.

Biull Eksp Biol Med, 1986 Apr, 101(4), 468 - 71
{Clonogenic stromal cell count in the bone marrow of mice}; Latsinik NV et al.; The number of fibroblast colonies in bone marrow cultures depends on FCFC concentration in explanted cells and FCFC cloning efficiency . For mouse bone marrow the efficiency of fibroblast colony formation increases in the presence of the feeder (irradiated bone marrow of spleen cells) . Colony-stimulating feeder activity does not depend on the presence of phagocytic and stromal cells in the feeder cell population . Trypsinization of the bone marrow leads to the release of additional FCFC and the increase of their concentration in bone marrow cell suspensions.

Scand J Clin Lab Invest, 1986 Apr, 46(2), 143 - 9
Endotoxin-induced human and porcine leucocyte reactions in vitro; Andersen OK et al.; Tube migration of a fixed number of PMN cells in plasma (5 X 10(9) cells/l) was approximately reduced by 50% when 1 ml of cell suspension was exposed to 20 micrograms E . coli endotoxin in 100 microliter NaCl for 2 hours . Procoagulant activity in monocytes increased approximately eight-fold when 1 ml of whole blood was exposed to 2 ng endotoxin in 10 microliters NaCl for 2 hours . Chemiluminescence in both PMN cells (5 X 10(9) cells/l) and mononuclear cells (2 X 10(9) cells/l) in plasma was markedly increased when 100 microliters cell suspension was added to 100 microliters Luminol, exposed to 20 micrograms endotoxin in 100 microliters NaCl and tested immediately . Decreased lysosomal enzyme levels in PMN and mononuclear cells were demonstrated when 1 ml cells in plasma (the same cell numbers as aforementioned) were incubated for 4 hours at 37 degrees C with 200 micrograms endotoxin in 100 microliters NaCl . Similar results were obtained in human and porcine leucocytes, making the pig a suitable animal for studies of humoral and cellular reactions to infections complications following trauma and major surgery.

Curr Eye Res, 1986 Apr, 5(4), 257 - 62
Experimental ocular malignant melanoma in Sinclair swine; Burns RP et al.; An animal model of malignant melanoma of the eye was established by transplanting a cell suspension from cutaneous melanomas into the anterior chamber of the eye in Sinclair Farm miniature swine . The frequency of tumor takes in the eye was increased from 8.9% to 22% by treating the animals simultaneously with subconjunctival triamcinolone acetonide . As an animal model for hematoporphyrin derivative--photoradiation treatment of human malignant melanoma of the eye, this does not appear to be a good research tool because of the sporadic incidence of tumor takes, the rapid growth of tumor within the eye causing glaucoma, and the dark iris pigmentation of successful tumor takes, which hides extensive underlying ciliary body tumor.

J Natl Cancer Inst, 1986 Apr, 76(4), 703 - 9
Spontaneous immortalization rate of cultured Chinese hamster cells; Kraemer PM et al.; Chinese hamster cell cultures derived from either fetal cell suspensions or adult ear clippings invariably became permanent cell lines during conventional subcultivation . The immortal cell cultures arose from rare spontaneous cellular events during the in vitro cultivation of cells with limited proliferative capacity . Immortality was not related to rare, precommitted cells from the animals . The expansion of clones of cells with limited life-span to form permanent cell lines was routinely successful only when the initial, unsubdivided culture achieved a total number in excess of 10(6) cells . On the basis of this observation, a serial clonogenicity assay was developed for determining the life-span of the cells with limited proliferative capacity and for determining whether a cell population is immortal . In addition, the technique of clonal expansion was used for a fluctuation analysis to determine the rate of immortalization . This analysis yielded a rate of 1.9 X 10(6) per cell per generation.

Mem Inst Oswaldo Cruz, 1986 Apr-Jun, 81(2), 225 - 32
RS virus diagnosis: comparison of isolation, immunofluorescence and enzyme immunoassay; Siqueira MM et al.; Two techniques for rapid diagnosis, immunofluorescence (IFAT) and enzyme immunoassay (EIA), have been compared with virus isolation in tissue culture for the detection of respiratory syncytial virus (RSV) in specimens of nasopharyngeal secretions . The specimens were obtained from children under five years of age suffering from acute respiratory illness, during a period of six months from January to June 1982 . Of 471 specimens examined 54 (11.5%) were positive by virus isolation and 180 (38.2%) were positive by immunofluorescence . The bacterial contamination of inoculated tissue cultures unfortunately prevented the isolation of virus from many samples . Specimens from 216 children were tested to compare enzyme immunoassay and immunofluorescence . Of these 60 (27%) were positive by EIA and 121 (56%) were positive by IFAT . Our results suggest that the EIA technique although highly specific is rather insensitive . This may be because by the time these tests were done the original nasopharyngeal secretions were considerably diluted and contained more mucus fragments than the cell suspension used for IFAT . Of the three techniques, IFAT gives the best results although EIA may be useful where IFAT is not possible.

J Immunogenet, 1986 Apr-Jun, 13(2-3), 219 - 27
Differential expression of HLA class I and II antigens in primary and metastatic melanomas; Lopez Nevot MA et al.; Class I and II histocompatibility antigen expression was studied in cryostat sections of biopsy tissues from 15 patients diagnosed as suffering from malignant melanoma, using monoclonal antibodies against HLA class I and II monomorphic determinants and an indirect immunofluorescence technique . Class I antigens were detected in three of the four primary melanomas and in five of the eleven metastatic melanomas . Class II antigens were expressed only in metastatic melanomas, in three out of eleven cases . Some tumour cell suspensions were obtained and short-term cultures were established . Radiobinding and immunoprecipitation studies were carried out in two cases, named M6 and M8 . The results were comparable to those obtained with direct immunofluorescence . We modulated the expression of class I and II HLA antigens with interferon in M6 when adapted to tissue culture . This melanoma was class I and II negative; after IFN gamma treatment it became strongly positive for class I and II antigens . In addition we have demonstrated, using Southern blot analysis with the restriction enzymes PvuII and EcoRI, that the M6 melanoma does not have any detectable alterations in its class II beta genes.

Jpn J Pharmacol, 1986 Apr, 40(4), 591 - 3
Functional compensation by transplantation of cell suspensions of embryonic mesencephalon into the striatum of rats with 6-hydroxydopamine lesions; Watanabe H et al.; Neuronal cell suspensions were implanted into the striatum of female rats that showed apomorphine-induced rotation and a reduction in striatal dopamine after intranigral treatment with 6-hydroxydopamine . The apomorphine-induced rotation was significantly suppressed by the transplantation in 12 out of 64 rats . DOPA accumulation and dopamine level (6.3 and 3.4%, respectively, compared with those of uncompensated rats) in the striatum following treatment with an amino acid decarboxylase inhibitor were slightly restored in compensated rats.

J Reprod Immunol, 1986 Apr, 9(1), 33 - 47
Immunosuppressive properties of human placenta: study of supernatants from short-term syncytiotrophoblast cultures; Culouscou JM et al.; Using collagenase and mechanical treatment to attempt to eliminate cellular contamination such as macrophages and decidual cells, trophoblast enriched cell suspensions were isolated from the human placenta . With a view to assessing the role of trophoblast in impairing maternal rejection of the fetus, supernatants (SPl4) were prepared from these placental cells after short-term culture (4 h) . The immunosuppressive activity of these supernatants was studied following application to mitogen-stimulated human lymphocyte cultures and mixed lymphocyte cultures . In both cases a reproducible inhibition was observed . The ability of these substances to induce a non-specific inhibitory effect was ascertained by observing mouse lymphocyte responses to mitogens or alloantigens . To gain further insight into in vivo fetal protection against anti-paternal cells, we also examined the effects of SPl4 on CTL generation . It was found not only that CTL generation was markedly depressed but also that SPl4 drastically impaired cell-mediated lympholysis at the effector level . To characterize the factors involved in our observations, SPl4 was subjected to dialysis and to chromatography . In the first case, it was found that these factors were not amenable to dialysis . In the second case, we obtained on an Ultrogel AcA 44 column two fractions with immunosuppressive activity . Following our previous work on human syncytiotrophoblast, we analyzed only the low molecular weight inhibitory fraction, which was chromatographed again on Ultrogel AcA 202 . The molecular weight of the immunosuppressive factor(s) was estimated to be around 3.5 kDa . We postulate that human trophoblast releases soluble factors around the fetus which may act to protect it against maternal immunological rejection.

Magn Reson Med, 1986 Apr, 3(2), 312 - 6
Effects of antimalarial drugs on oxygen consumption by erythrocytes infected with Plasmodium berghei: an ESR study; Butler KW et al.; Oxygen consumption in mouse erythrocytes infected with Plasmodium berghei has been followed by electron spin resonance (ESR) spectroscopy of nitroxide radical spin probes . The parasitized red cell suspension is mixed with the spin probe CTPO (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) in a closed chamber . Oxygen consumption is monitored by the increasing resolution of the superhyperfine splittings of the spin label . The antimalarial drugs quinacrine, primaquine, and quinine are shown to decrease the rate of oxygen consumption of the parasitized erythrocyte suspensions . The spin-label method offers advantages over conventional polarographic and spectrophotometric assays for highly parasitized cell populations where cells are fragile and contain oxidized hemoglobin as well as hemoglobin-derived pigments.

J Leukoc Biol, 1986 Apr, 39(4), 371 - 83
Fractionation of rat alveolar macrophages by isopycnic centrifugation: morphological, cytochemical, biochemical, and functional properties; Chandler DB et al.; Studies on alveolar macrophages have usually been performed on a single cell suspension obtained by lung lavage . However, recent evidence on the diversity of functions of the alveolar macrophage suggests that the macrophage is not a single population, but one composed of several subpopulations of macrophages . One approach toward determining if alveolar macrophages are heterogeneous would be to separate subpopulations based on density . To accomplish this, we developed a continuous gradient of iso-osmotic colloidal silica (Percoll) that separated alveolar macrophages from Fischer 344 rats into 18 density-defined subpopulations . The density-defined alveolar macrophage subpopulations were then characterized and were shown to be significantly different based on morphological, cytochemical, biochemical, and functional analysis . The results of this study suggest that alveolar macrophages are heterogeneous and that a continuous iso-osmotic gradient of colloidal silica is an efficient and reproducible method for separating subpopulations.

In Vitro Cell Dev Biol, 1986 Apr, 22(4), 201 - 11
Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations; Kreamer BL et al.; A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability . The method has been applied to cells isolated by a variety of collagenase digestion techniques . This procedure involves the low-speed centrifugation (50 X g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells . The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo{a}pyrene or 2-acetylaminofluorene.

J Invest Dermatol, 1986 Apr, 86(4), 359 - 62
Monoclonal anti-interleukin 2 (15-2) antibody binding to granular layer keratinocytes of human skin; Dreno B et al.; Among several monoclonal antibodies (moABs) directed against human interleukin 2 (IL-2), the 15-2 moAB raised in our laboratory against unglycosylated recombinant IL-2 (produced in Escherichia coli) cross-reacted with a human skin epitope . This moAB gave a strong staining on the cell-surface membranes of keratinocytes from the granular layer of the epidermis . In addition, the 15-2 moAB stained 15% of epidermal cell suspensions obtained from suction blisters and reacted with cells from the spinous layer in parakeratosis and psoriasis, as well as with spinous epithelioma cells . Preincubation of the 15-2 moAB with pure human recombinant IL-2 abrogated skin binding, whereas a polyclonal antikeratin antiserum did not block 15-2 skin binding . Two other anti-IL-2 moABs, one directed against unglycosylated recombinant IL-2 (17-2 moAB) and one against glycosylated natural IL-2 (9B11 IE5 moAB), were unreactive on skin . Taken together, the data suggest that the 15-2 moAB binds to an epitope cross-reacting with, but different from, IL-2 which is located in the cell-surface membranes of granular layer cells . This cross-reactive epitope may provide a useful probe for the study of human epidermal cell differentiation.

J Invest Dermatol, 1986 Apr, 86(4), 355 - 8
Enhanced angiogenic capability of monocyte-enriched mononuclear cell suspensions from patients with systemic scleroderma; Marczak M et al.; Different subsets of peripheral blood mononuclear cells (MNC) from 15 patients with systemic scleroderma were tested for their ability to evoke angiogenesis in a xenogenic system . The angiogenic capability of total MNC from patients with systemic scleroderma was lower than that of normal human cells, irrespective of the form of the disease . However, the capability of a monocyte-enriched subset of MNC from patients with scleroderma was found to be increased, as compared with their total MNC and with that of the corresponding subset from healthy individuals . This might be due to the activation of monocytes in the disease.

Immunol Lett, 1986 Apr, 12(4), 231 - 5
Subclustering of CD1 monoclonal antibodies based on the reactivity on human Langerhans cells; Schmitt D et al.; Among the differentiation antigens expressed by lymphoid cells, CD1 monoclonal antibodies (MCA) distinguish a group of antigens expressed by cortical thymocytes . Some of them (OKT6 and BL6) have been shown to react with normal human skin Langerhans cells (LC) . Using indirect immunofluorescence on skin sections and LC-enriched epidermal cell suspensions obtained by skin trypsinisation, we have studied the reactivity of eight new CD1 MCA . Our immunocytochemical observations indicate that four groups of CD1 MCA can be distinguished on the basis of their reactivity on LC . NA.1.84, SK9/Leu6, 10 D.12.2 and I19 identify LC in skin and in suspensions, but 4A.76 and NUT2 were negative in both . M.241 became negative after trypsinisation of epidermal cells and D47 reacted with an antigen expressed on LC only after enzymatic treatment . These results demonstrate the heterogeneity of the antigens recognized by CD1 MCA . Some of them do not react with normal human LC . Some antigens appear to be masked in the epidermis and are expressed only after trypsin treatment . Our data confirm the heterogeneity of CD1 molecules recently documented on the basis of biochemical analysis.

Arthritis Rheum, 1986 Apr, 29(4), 471 - 9
Stimulation of neovascularization by human rheumatoid synovial tissue macrophages; Koch AE et al.; Synovial tissue from patients with rheumatoid arthritis was enzymatically dissociated, and single cell suspensions were fractionated into subpopulations by centrifugation on continuous Percoll gradients . Five fractions (F1-F5) with densities of 0.991-0.998 gm/ml, 0.998-1.042 gm/ml, 1.042-1.062 gm/ml, 1.062-1.082 gm/ml, and 1.082-1.180 gm/ml, respectively, were prepared . F3 consistently contained the highest number of macrophages, while F2 and F4 contained substantially fewer macrophages . Macrophages present in F2, F3, and F4 were enriched by differential adherence to fibronectin-coated collagen gels . These macrophage-enriched cell preparations were found to be Fc and C3 positive, esterase positive, and peroxidase negative, to stain positively with anti-HLA-DR, anti-Leu-M3, OKM1, and OKM5 monoclonal antibodies, and to show characteristic features of macrophages by electron microscopy . Macrophages from F3 consistently induced neovascularization in rat corneas, while equal numbers of macrophages from F2 and F4 did not . Fibroblastic synovial cells and cells that did not adhere to fibronectin-coated collagen gels did not induce neovascularization . Within the rheumatoid synovium, there appears to be a major subpopulation of macrophages capable of inducing neovascularization, a process vital to the development of the rheumatoid synovial pannus.

Toxicol Appl Pharmacol, 1986 Mar 15, 82(3), 474 - 80
Macrophage uptake of a lipoprotein-sequestered toxicant: a potential route of immunotoxicity; Kaminski NE et al.; An experimental system was chosen to investigate the bioactivity of a lipoprotein-sequestered toxicant at the cellular level based on recent studies demonstrating receptor-mediated uptake of lipoproteins by macrophages . Rat peritoneal exudate cell suspensions (PEC) were exposed to DDT and lipoprotein-sequestered DDT, followed by measurement of DDT uptake, metabolism, and cellular toxicity . In vitro uptake assays demonstrated that PEC suspensions treated for 10, 20, and 30 min with 2.5 microM lipoprotein-sequestered DDT had approximately a twofold increase over the amount of DDT associated with PEC treated with 2.5 microM free DDT . PEC were assayed for DDT metabolites to serve as a measure of the cellular internalization of the toxicant after treatment in vitro for 18 hr with either 1.5 microM DDT or lipoprotein-sequestered DDT . Evidence of DDT metabolism was only observed with PEC which had been treated with lipoprotein-sequestered DDT . These cells contained significantly higher amounts of DDT metabolites as compared to cells treated with unsequestered DDT (over an eightfold difference) . Assays measuring macrophage phagocytic activity indicated that macrophages treated for 4.5 hr in vitro with 2.5 microM lipoprotein-sequestered DDT showed significant inhibition in their ability to phagocytize yeast particles . These results suggest that serum lipoproteins may facilitate the cellular uptake of lipoprotein-sequestered toxicants leading to altered cellular function (phagocytosis).

J Immunol, 1986 Mar 15, 136(6), 2305 - 10
Monoclonal antibody 45-2D9 conjugated to the A chain of ricin is specifically toxic to c-Ha-ras-transfected NIH 3T3 cells expressing gp74; Roth JA et al.; Monoclonal antibody 45-2D9 recognizes a 74K Mr glycoprotein determinant on a c-Ha-ras oncogene-transfected cell line (45-342) . An immunotoxin was made by conjugating this antibody to the A chain of ricin toxin (RTA) . The immunotoxin could mediate essentially complete inhibition of leucine and thymidine incorporation by 45-342 cells prepared as single cell suspensions from tumors grown in vivo . Addition of ammonium chloride to the culture medium potentiated this inhibition, but the magnitude of this effect was dependent on incubation time and cell concentration . The immunotoxin effects were noted at concentrations 100-fold lower than similar effects caused by unconjugated RTA, and the 45-2D9 antibody had no effect in the assay system . Immunotoxins directed against antigens not expressed by 45-342 were not effective, and the 45-2D9 immunotoxin was not specifically toxic to other transfected cells not expressing the gp74 antigen . After a 72-hr incubation, lysis of 80% of the 45-342 cells was demonstrated by trypan blue exclusion . Complete inhibition of 45-342 colony formation was achieved at 10 days with a 10(-9) M concentration of the specific immunotoxin . These results indicate that an immunotoxin with specific reactivity towards an oncogene-transformed cell can be made, and that such cells derived from fresh tumors are susceptible to immunoconjugate-mediated toxicity.

J Immunol, 1986 Mar 15, 136(6), 2005 - 10
Evidence of extensive lymphocyte death in sheep Peyer's patches . I . A comparison of lymphocyte production and export; Reynolds JD; The metaphase arrest technique was used to determine the rate at which cells divide in the Peyer's patches (PP) and the thymus of 5 to 8 wk old lambs . The metaphase indices of these tissues were determined by analyzing cell suspensions of tissues taken before and 1, 2, 3, and 4 hr after metaphase arrest was initiated with i.v . vincristine . The metaphase indices increased in both tissues at a linear rate, which provided an estimate of the rate at which cells entered mitosis and of the lymphocyte birth rate . The ileal PP had the highest lymphocyte birth rate, 2.8% of the lymphocytes entered mitosis each hour; the rate was lower in jejunal PP (1.0%/hr) and thymus (0.5%/hr) . With these values and estimates of the lymphocyte content in all PP (1.45 X 10(11)) and in the thymus (1.71 X 10(11)), it was calculated that the hourly lymphocyte production by PP in a lamb was 3.61 X 10(9) cells, which is four to five times greater than for the thymus (0.82 X 10(9)) . Lymphocyte production in PP could then be compared with the number of lymphocytes that emigrated from the small intestine . Newly produced cells leave PP via the intestinal lymph, which could be collected from the entire small intestine after removal of the mesenteric lymph nodes . Cells entered the lymph at a rate of 0.8 X 10(9)/hr, but the output fell rapidly during chronic lymphatic drainage, a procedure known to deplete long-lived recirculating cells . It was concluded that most of the cells in intestinal lymph were recirculating cells, and newly formed lymphocytes produced in PP probably account for less than 25% of the total or 0.2 X 10(9)/hr . It seems unlikely that emigration could occur at a rate comparable with the rate of production in the PP . At most, only 5% of the PP cells seemed destined to leave their site of production, and it is proposed that most die within the PP follicles . The high mortality rate associated with the production of large numbers of B lymphocytes in lamb PP seems likely to have a significant impact on the nature of the contribution that these tissues make to the immune system.

Biochem J, 1986 Mar 15, 234(3), 605 - 10
Transamination of glutamate to tricarboxylic acid-cycle intermediates in cultured neurons correlates with the ability of oxo acids to support neuronal survival in vitro; Facci L et al.; Cultures of central-nervous-system neurons at low densities require for their survival exogenous pyruvate, alpha-oxoglutarate or oxaloacetate, even in the presence of high glucose concentrations . Most other alpha-oxo acids support cell survival only in the presence of alpha-amino acids which transaminate to alpha-oxoglutarate, oxaloacetate or pyruvate . The alpha-oxo acids therefore operate as acceptors of amino groups from appropriate donors to generate tricarboxylic acid-cycle-relevant substrates, and these alpha-oxo acids provide for neuronal support only insofar as they make it possible for exogenously supplied alpha-amino acid precursors to generate intracellularly one of the three critical metabolites . To examine more closely the relationship between transamination activity and neuronal survival, we measured 14CO2 production from {14C}glutamate in the presence of appropriate alpha-oxo acid partners by using 8-day-embryonic chick forebrain, dorsal-root-ganglion and ciliary-ganglion neurons . Neuronal survival was measured concurrently in monolayer neuronal cultures maintained with the corresponding amino acid/oxo acid pairs . Forebrain and ganglionic cell suspensions both produced 14CO2 from {14C}glutamate, which accurately correlated with 24 h neuronal survival . Concentrations of glutamate or alpha-oxo acid which provide for maximal neuronal survival also produced maximal amounts of 14CO2 . The same ability to generate CO2 from glutamate (in the presence of the appropriate alpha-oxo acids) can ensure neuronal survival in 24 h cultures and therefore must meet energy or other metabolic needs of those neurons which glucose itself is unable to satisfy.

Eur J Biochem, 1986 Mar 3, 155(2), 311 - 8
Enzymic synthesis of isoflavones; Kochs G et al.; The NADPH and oxygen-dependent conversion of (2S)-naringenin to genistein catalyzed by a microsomal preparation from elicitor-treated soybean cell suspension cultures has been resolved into two steps . In the first step (2S)-naringenin is converted to a product (P-2) which yields genistein in a second step . The chemical behaviour of P-2 and its ultraviolet and mass spectral data are consistent with a 2-hydroxyisoflavanone structure . The conversion of (2S)-naringenin to P-2 requires NADPH, oxygen and cytochrome P-450 . The participation of cytochrome P-450 was demonstrated by CO inhibition of the reaction and its partial reversal by light, and by inhibition with typical cytochrome P-450 inhibitors . On a Percoll gradient the membrane fraction which catalyzes P-2 formation coincides with marker enzymes for the endoplasmic reticulum and with the position of cytochrome P-450 . Enzymatic activity for conversion of P-2 to genistein is mainly present in the supernatant of the 160 000 X g fraction . This reaction, formally a dehydration, does not require NADPH or oxygen.

Life Sci, 1986 Mar 3, 38(9), 779 - 87
A thymus factor influences the in vitro testosterone secretion of Leydig cells in the rat; Pedernera E et al.; Thymus extracts obtained from 15-day-old rats were fractionated through molecular sieve chromatography, and the fractions assayed in vitro by changes produced in the testosterone secretion of Leydig cells obtained from adult rat testes . Fractions corresponding to 27-28000 mol wt of the thymus extract diminish the testosterone secretion of Leydig cells stimulated with hCG . No changes in the basal testosterone secretion were produced by the presence of the thymus fractions . The inhibitory effect is dose related and persists during 180 min of incubation . Fractions of the same mol wt obtained from liver, heart and spleen do not modify the testosterone secretion of Leydig cells . The inhibitory activity of the thymus factor disappears after heat or trypsin treatment . Further fractioning in preparative flat bed electrofocusing makes manifest that the inhibitory activity is focused at pH 4.7 . The data demonstrate the existence in rat thymus of a factor, probably of protein nature, which modifies the in vitro hCG response of a testis cell suspension.

Ultrasound Med Biol, 1986 Mar, 12(3), 209 - 16
Lack of effect of pulsed ultrasound on ABO antigens of human erythrocytes in vitro; Miller DL et al.; The efficacy of pulsed ultrasound in removing ABO blood group antigens from human erythrocytes was investigated in vitro . Cell suspensions were exposed to 5.25 MHz focused ultrasound with 1.23 microseconds pulses, at 0, 20, and 37 degrees C using spatial peak, pulse average intensities of 11, 126, and 1000 W/cm2 and pulse spacings of 10, 100, and 1000 microseconds . A second experiment involved application of 7.5 MHz pulses of 0.77 microseconds duration and 8 W/cm2 SPPA intensity which were spaced 1.25 ms apart . Exposed cells were tested for agglutination by antibody to determine changes in antigen expression . In addition, supernates from exposed cells were tested for the presence of soluble antigen . A sensitive capillary tube agglutination technique was developed for these experiments . No detectable antigen removal occurred as a result of any of the pulsed ultrasound exposures as compared to sham exposures . A positive control, which employed antigenic material prepared from cells disrupted by ultrasonic cavitation, indicated that the assay could detect the soluble antigen equivalent of about one cell in 10,000.

Brain Res, 1986 Mar, 390(2), 239 - 47
The developmental appearance of alpha-bungarotoxin binding sites on rodent spinal cord neurons in cell culture; Schaffner AE et al.; {125I}alpha-Bungarotoxin specifically binds to a subpopulation of rodent spinal cord neurons in vitro . Binding first becomes apparent between 1 and 2 weeks in culture and then increases dramatically after 3 weeks . Similarly, cell suspensions from freshly dissociated embryonic spinal cords do not bind toxin whereas cell suspensions from 1 week old neonates demonstrate specific binding of {125I}alpha-bungarotoxin . In vitro, binding is inhibited more effectively in the presence of nicotinic rather than muscarinic agents . Autoradiography of {125I}alpha-bungarotoxin binding to 4-week-old cultures revealed a uniform labeling pattern over cell somas and processes . Although the relation of toxin binding to functional acetylcholine receptors is not known, the appearance of toxin binding sites may have some developmental significance for the maturation of cholinergic transmission or the maintenance of synaptic connections.

In Vitro Cell Dev Biol, 1986 Mar, 22(3 Pt 1), 141 - 9
Growth and differentiation of primary rat keratinocytes on synthetic membranes; Vaughan FL et al.; The attachment, proliferation, and differentiation of primary cultures of keratinocytes isolated from murine epidermis were monitored after purified cell suspensions were seeded and incubated in vitro on various synthetic membranes . Concomitant studies of the effects of attachment factors added to synthetic membranes before use as substrata for keratinocytes were also done . The study demonstrated that a synthetic membrane composed of nylon was superior to other membranes and to plastic control culture vessels in supporting the growth of murine keratinocytes . Although laminin enhanced initial attachment and proliferation of cells on nylon membranes, the untreated substratum was more effective for extended incubation . Stratification and differentiation of these keratinocytes on the nylon substratum was enhanced by raising confluent cultures (7 d) to the air-medium interface so that they were in contact with medium only from the bottom . Cultures raised for 14 d produced many morphologic markers of the epidermis and closely resembled the architecture of this tissue in situ.

Invest Ophthalmol Vis Sci, 1986 Mar, 27(3), 285 - 95
Rod photoreceptor cells dissociated from mature mice retinas; Lolley RN et al.; Intact rod photoreceptors were dissociated from pronase-treated whole retinas of adult mice by repeated passage through a plastic pipette tip . Hemocytometer counts of the cell suspensions indicate that, during a series of ten dissociation steps, a total of about 1-2 million intact photoreceptor cells are dissociated from one adult mouse retina, with less than 5% contamination from Muller cells and neurons of the inner retina . Visual cells with rod outer segments (ROS) and synaptic terminals are released in each step, but they occur in the greatest number during the sixth to ninth steps; detached ROS are released most frequently in the early steps, and neurons of the inner retinal layers appear in the later steps of dissociation . Nuclei are found in each step . Cell intactness was estimated by Trypan blue and Erythrosin B exclusion and by microscopic analysis using differential interference optics or scanning electron microscopy . The cells bind lectins (concanavalin A, Ricinis communis, and wheat germ agglutinin but not peanut agglutinin), displaying surface topography like that observed in situ . The metabolic capacity of dissociated cells was assessed by measuring the utilization of 32P inorganic phosphate for the synthesis of phospholipids and for the light-dependent phosphorylation of rhodopsin . Mature photoreceptor cells were estimated to contain, on average, 6.4 X 10(-12) g DNA, 2.3 X 10(-12) g RNA and 42-64 X 10(-12) g protein . The dissociation procedure provides a population of photoreceptor cells that appears suitable for microscopic, electrophysiological, and biochemical analysis.

Cancer Res, 1986 Mar, 46(3), 1539 - 43
Feasibility of a concanavalin A-peroxidase labeling method to detect cancerous and precancerous lesions of the uterine cervix; Davina JH et al.; For the detection of cancerous and precancerous lesions in cervical cytopathology, the feasibility of a concanavalin A-peroxidase labeling procedure was tested and compared with the Papanicolaou method . To this end, the percentage of labeled flattened epithelial cells with a morphologically normal appearance present in cervical cell suspensions was determined . It was found that the mean labeling percentage of the control group was 71% (SD, 11%) . The means for mild, moderate, and severe dysplasia groups were, respectively, 54% (SD, 19%), 48% (SD, 13%), and 44% (SD, 16%) . The mean for the carcinoma in situ group was 32% (SD,11%), and for the squamous cell carcinoma group 16% (SD, 5%) . It appeared that the labeling percentage gradually decreases with increasing atypia of the epithelium as confirmed by histological observation . A complete dissimilarity was found between healthy individuals and cancer patients . In a follow-up study it was found that the mean labeling percentage did not alter in cases of an unchanged stage of disease . A reestablishment of the normal concanavalin A-peroxidase labeling percentage often appeared once the cancerous or precancerous lesion was treated . In conclusion, the concanavalin A-peroxidase labeling method can be considered as a supplementary technique to the Papanicolaou method for the early detection of cervical cancer . It reduces the effect of sampling and screening errors of the Papanicolaou method, and it allows a more objective cytological diagnosis . In addition, it may possess prognostic significance.

Andrologia, 1986 Mar-Apr, 18(2), 224 - 9
{Change in sodium and potassium concentration in human seminal plasma infected experimentally with Ureaplasma urealyticum}; Mehl M et al.; The concentration of sodium and potassium ions was measured by flame photometry in human seminal plasma and human sperm cells as well as in a sperm cell suspension infected experimentally with Ureaplasma urealyticum during incubation as long as 22 hours . The sodium concentration is 86.2 +/- 21.97 mmol/l cells in sperm cells and 120.8 +/- 15.87 mM in seminal plasma . The potassium concentration is 33.2 +/- 16.35 mmol/l cells in sperm cells and 26.7 +/- 4.92 mM in seminal plasma . The sodium concentration decreased considerably in the suspension medium of sperm cells during the first hour of incubation and increased again subsequently . The potassium concentration in the suspension medium changed only little . The sodium concentration in the infected sperm cell suspension was insignificantly increased compared with the mycoplasma-free controls . The potassium concentration was slightly higher in the infected specimens than in the controls . There was no mycoplasma-induced effect on the concentration of sodium and potassium in the sperm suspension after experimental infection.

J Reprod Fertil, 1986 Mar, 76(2), 587 - 96
Flow cytometry and sorting of meiotic prophase cells of female rabbits; Larsen JK et al.; We present a new, flow cytometric method by which cells in various stages of the meiotic prophase can be quantitated and sorted in partly enriched fractions . Ovarian cells of 3-16-day-old rabbits were mechanically dispersed and fixed in ethanol and aldehydes . The cell suspension was stained with the DNA fluorochrome mithramycin and analysed and sorted in a FACS IV cell sorter according to the fluorescence and forward light scatter distribution . Cells sorted onto slides were stained with haematoxylin and eosin and differentially counted in the microscope . In the diploid fraction, preleptotene cells were more fluorescent than somatic cells . Leptotene cells were found throughout the S fraction and the tetraploid fraction . Zygotene and pachytene cells caused a major peak in the tetraploid region with 10-25% more fluorescence than somatic cells . Cells in diplotene had 5-15% more fluorescence than somatic cells . Mitotic cells were 20-40% more fluorescent than somatic cells and scattered the light more intensely than did meiotic cells with the same fluorescence.

Am J Clin Pathol, 1986 Mar, 85(3), 297 - 304
Detection of lymphocyte antigens in tissues placed in transport medium . Comparison with cryostat fresh-frozen section technic . An immunologic study of 56 cases; Sheibani K et al.; It has been suggested recently that use of a transport medium (Michel's medium) satisfactorily preserves the antigenicity of surface markers on lymphocytes for immunologic evaluation . Transport medium has been reported to be especially useful when immediate preparation of a specimen for immunologic study is not possible . Because adverse conditions such as changes in temperature during the transport of fresh-frozen tissue may markedly alter immunologic markers, it is desirable to transport fresh tissue in a medium that does not adversely affect the immunoreactivity of the tissue antigens . Therefore, we undertook a comparative study in which we compared cryostat-cut, fresh-frozen sections and tissues fixed in transport medium . The specimens were obtained from 56 consecutive patients who had various malignant lymphomas and benign lymphoid disorders . The results of our study indicate that the morphologic and immunologic findings obtained from the frozen sections that had been maintained in the transport medium (Michel's) may be confusing and may be interpreted inaccurately . This study also confirms our previous observation that the inconclusive or spurious results occasionally obtained with cell suspension technics can be avoided if the immunohistochemical technics are applied to cryostat-cut, fresh-frozen sections . We conclude that immunohistochemical study of cryostat-cut fresh-frozen sections remains the technic of choice for the identification and evaluation of both the morphologic and immunologic characteristics of tissues involved by lymphoproliferative diseases.

J Microsc, 1986 Mar, 141 ( Pt 3), 263 - 76
Low temperature light microscopy and its application to study freezing in aqueous solutions and biological cell suspensions; Korber C et al.; The freezing of biological cell suspensions can be understood in terms of ice formation in the external suspension medium and the cellular reactions to the changing environment . Cryomicroscopy allows a quantitative analysis of both categories of phenomena . Besides freezing stages of appropriate thermal design, the components used for that purpose include a microcomputer (PSI 80) based control system, an image analysis system (Intellect 100) and a spectrophotometer (MPV compact) . The investigation of extracellular ice formation is focused on the following effects: The redistribution of solutes in the residual liquid and the resulting concentration profiles are determined photometrically or densitometrically . The transitions between various morphologies of the ice-liquid phase boundary (planar-cellular-dendritic) can be related to interface instability theories . With respect to solute segregation, the studies also involve the formation of bubbles from supersaturated gaseous solutes and freezing potentials resulting from the differential incorporation of cations and anions into the solid phase . The interaction between particles or cells and the advancing ice front is determined from critical interface velocities marking the transition between repulsion and entrapment . The effects of freezing on biological cells are studied mainly with blood cells, especially lymphocytes . The water efflux due to osmotical gradients across the membrane yields volume shrinkage curves which are recorded and analysed from video images for various cooling rates . Beyond a certain threshold cooling rate, intracellular ice starts to form, and different crystallization morphologies can be detected . The intracellular crystallization temperatures depend on cooling and warming rates as well as on the presence of penetrating cryoadditives . A fluorescence viability is used to determine the percentage of damaged cells immediately after thawing.

Am J Physiol, 1986 Mar, 250(3 Pt 2), F386 - 95
Indirect immunoselection of late distal cell populations from rabbit kidney cortex; Vandewalle A et al.; This study describes a method for the separation of distal cell populations based on the sequestration of proximal cells on immunoadsorbent columns (CNBr-activated Sepharose 6MB) bound with three brush-border monoclonal antibodies (S6-Mab) . A high yield of isolated cell suspension from rabbit kidney cortex was prepared by mechanical dissociation after perfusion and incubation of the kidneys with 10(-3) M EDTA . The sequestration of the proximal cells was achieved in two sequential chromatographic steps . About 92% of the applied cells were first retained on an S6-Mab column after a 60-min stationary stage and the unbound cells were submitted by direct flow to a second S6-Mab column . In such conditions, 8 X 10(6) cells were recovered when starting with 331 X 10(6) cortical cells . The efficiency of the proximal cell depletion process was confirmed by an 80% decrease in brush-border enzymes, a very low phosphoenolpyruvate carboxykinase activity, and absence of cells bearing long microvilli, as ascertained by electron microscopy . This immunodepleted cell population presented the enzymatical characteristics of cells from the more distal segments . As compared with the initial cell suspension, these cells exhibited higher hexokinase (2.3 times), succinate dehydrogenase (1.5 times), and Na+-K+-ATPase (2.6 times) activities . In addition, adenylate cyclase activities remained sensitive to parathormone, arginine vasopressin, and isoproterenol . The functional capacity of these immunodepleted cells was assessed by an almost complete exclusion of eosin dye, a low Na+ and high K+ intracellular content, and a high respiratory rate of oxygen consumption . In conclusion, this immunoselective process makes it possible to obtain subpopulations of renal cortical cells possessing the main characteristics of the distal, connecting, and collecting cells for physiological and metabolic studies.

J Natl Cancer Inst, 1986 Mar, 76(3), 541 - 8
Accessory cell activity of murine tumor-associated macrophages; Dougherty GJ et al.; The accessory cell activity of macrophages associated with the murine 3-methylcholanthrene-induced fibrosarcoma FSa was investigated . On the basis of Fc receptor expression and phagocytic activity, 20-25% of cells present within enzymatically disaggregated tumor cell suspensions could be classified as macrophages . These cells were approximately 50% I-Ak positive but did not express the Mac-1 antigen . T-cells played an important role in regulating I-Ak expression, and macrophages obtained from tumors grown in nu/nu mice were I-Ak negative . Tumor-associated macrophages were shown to possess potent accessory cell activity and were fully capable of reconstituting the primary anti-calf red blood cell plaque-forming cell (PFC) response of Sephadex G-10-passed spleen cells . This function required the presence of the I-Ak-positive subpopulation, and macrophages treated with anti-Ia serum and complement or obtained from tumors grown in nu/nu hosts lacked accessory cell activity . Tumor-associated macrophages were also able to provide the essential accessory cell function required for cooperation between tumor-specific TH cells and normal B-cells in the generation of an anti-trinitrophenyl (TNP) PFC response in the presence of TNP-coupled FSa antigen . These results suggest that progressive growth of the FSa tumor in vivo cannot be readily attributed to a defect in the accessory cell function of tumor-associated macrophages.

J Histochem Cytochem, 1986 Mar, 34(3), 307 - 15
Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis; Virtanen I et al.; The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins . In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers . Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained . Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers . On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required . All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI . Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies . This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions . Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins . A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells . This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also . The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties . The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.

J Immunol, 1986 Mar 1, 136(5), 1688 - 92
In vitro production of interleukin 1 by normal and malignant human B lymphocytes; Pistoia V et al.; In this study, the capacity of normal and neoplastic B lymphocytes to release interleukin 1 (IL 1) has been investigated . Peripheral blood B cells from normal donors were isolated by depletion of E rosetting cells and by positive selection of cells expressing surface immunoglobulin (sIg) or the B1 marker . Peripheral blood B cells from patients with B cell chronic lymphocytic leukemia (B-CLL) were purified by removal of E rosetting cells followed by complement-mediated cytotoxicity with selected monoclonal antibodies . All of the normal B cell suspensions and the large majority of the B-CLL cells produced in culture high amounts of IL 1 in the absence of any apparent stimulus . Control experiments ruled out that small numbers of monocytes in the B cell suspensions could represent the source of IL 1 . These data support the contention that B cells participate to the immune response as accessory cells for T cell activation not only by physically presenting antigen, but also by releasing IL 1.

Blood, 1986 Mar, 67(3), 777 - 83
Short-term culture of acute myeloid leukemia blasts: analysis of acquired susceptibility to activated natural killer cells; Moingeon P et al.; Following a cryopreservation step, short-term cultures of circulating leukemic blasts from a patient with acute myeloid leukemia (AML) were performed . Because cultured tumor cells became susceptible to natural killer (NK) activity, in vitro alteration of the blasts was studied . Immediately after thawing, cell suspensions consisted of a relatively homogeneous population of undifferentiated blasts . In culture, tritiated thymidine uptake by the leukemic cells was low during the first 24 hours and then increased (X20) to a peak on day 7 . The cell concentration started to increase on day 4 . On day 8, less than 10% of the cultured cells still appeared as undifferentiated blasts, whereas up to 60% were granular and 30% to 40% had a monocytoid morphology . Prior to being cultured, the blasts were resistant to resting and IL2-activated natural killing . When the kinetics of in vitro acquired susceptibility were studied, it was found that maximum cytotoxicity against these leukemic cells was reached within 24 hours . Thus, the blasts had become NK-sensitive prior to increase in DNA synthesis, proliferation, and differentiation based on morphological and cytochemical criteria . In contrast, there was a positive correlation between acquired susceptibility and surface expression of an activation antigen, termed TNKtar . To dissect further the mechanisms of acquired susceptibility, a series of six NK clones representing four distinct phenotypes of NK active lymphocytes were tested against the leukemic cells . Immediately after thawing, blasts were essentially resistant to all clones, whereas they were strongly killed by 5 of 6 clones when cultured for 24 hours . Cold target inhibition assays indicated that resistance of fresh blasts was likely to be due to a binding defect . These results suggested that tumor cells became susceptible because they surface-expressed NK target structure(s) in the early phase of an activation process leading to their proliferation and/or differentiation . This hypothesis was substantiated for one clone, termed JT9, because the anti-TNKtar antibody blocked cytotoxicity of JT9 cells against the cultured blasts.

Cancer Res, 1986 Mar, 46(3), 1176 - 81
Synergistic activity of doxorubicin and the bisdioxopiperazine (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane (ICRF 187) against the murine sarcoma S180 cell line; Wadler S et al.; The bisdioxopiperazine (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)-propane (ICRF 187) abrogates doxorubicin cardiotoxicity in every mammalian species tested, but its effect on doxorubicin antitumor activity remains poorly understood . In order to better define the anthracycline-bisdioxopiperazine interaction, the ability of murine sarcoma S180 cells to form colonies in soft agar and their capability to proliferate in microtiter wells were assayed after exposure to drug at varying doses and schedules . Incubation of cell suspensions for 1 h with doxorubicin, 0.1 microgram/ml, with or without (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane, 80 micrograms/ml, produces additive cytotoxicity for the combination . Prolonged incubation (24 h) with the same drugs produces synergistic cytotoxic and antiproliferative effects at 1- and 2-log order reductions in dose . These studies indicate that the antineoplastic activity of the single agents doxorubicin and (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane is enhanced when the drugs are used in combination, and that this phenomenon is highly dose and schedule dependent.

Acta Radiol Oncol, 1986 Mar-Apr, 25(2), 127 - 30
Early stage carcinoma of the uterine cervix . Effects of intracavitary radium treatment on lymphoid cells in blood and pelvic lymph nodes; Onsrud M et al.; Sixteen patients with early stage carcinoma of the uterine cervix treated with primary radical hysterectomy and pelvic lymphadenectomy were compared with 17 patients who four to six weeks before the operation received intracavitary treatment with radium . The calculated radiation dose to the pelvic wall was approximately 10 Gy . The distribution of lymphoid cells in blood and pelvic lymph nodes was studied by an indirect immunofluorescence technique using monoclonal antibodies . The radium treated group showed a significant reduction of circulating OKT4+ (T helper) and OKT8+ (T suppressor/cytotoxic) lymphocytes . The number of Leu7+ (natural killer) cells and 1D5+ cells (monocytes) was not changed, but the ratio between monocytes and T cells was increased after radium therapy . In cell suspensions obtained from the pelvic lymph nodes, the radium treatment induced a significant reduction of the OKT4+ cell fraction . It is concluded that this low dose rate regimen of intracavitary treatment induces changes in the immune system which are of the same type as those seen after external field irradiation.

Acta Endocrinol (Copenh), 1986 Mar, 111(3), 411 - 8
Isolation, purification and culture of Sertoli cells from immature piglet testes; Renier G et al.; Several studies suggest a role of Sertoli cells in the control of Leydig cell steroidogenesis . In order to verify this hypothesis, we have developed a system for the purification of pig Sertoli cells . These cells were then characterized by their morphological appearance in light and electron microscopy, their ability to bind {125I}follicle stimulating hormone (FSH) and their functional capacity as evaluated by adenosine 3',5' monophosphate (cAMP) accumulation and lactate production when in primary culture under basal and FSH-stimulated conditions . Crude Sertoli cell suspensions from immature porcine testes were fractionated on discontinuous Percoll gradients (densities 1.025, 1.039, 1.055, 1.080 g/ml) . Highly purified Sertoli cells were contained in the second band (d: 1.039) generated on the gradient . These cells demonstrated morphological and functional integrity as evidenced by binding specifically {125I} FSH and by responding to FSH stimulation (by an increased production of cAMP and lactate after 3 days in primary culture), but not to human chorionic gonadotrophin (hCG) . This preparation represents a useful model for the study of Sertoli cell functions and their interation with Leydig cells in the regulation of testicular steroidogenesis.

Blood, 1986 Mar, 67(3), 682 - 8
CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa; Jenkins RB et al.; Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen . We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa . Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies . On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media . Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study . After the radioactive pulse, the cell suspension was solubilized with nonionic detergent . To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose . The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1-1D-Sepharose) or on a control monoclonal antibody resin . Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography . Autoradiograms of diethylamine eluates from HP1-1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions . Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1-1D-Sepharose . Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material . This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.

Clin Immunol Immunopathol, 1986 Mar, 38(3), 319 - 26
Loss of allogeneic T-cell activating ability and Langerhans cell markers in human epidermal cell cultures; Demidem A et al.; Human Langerhans cells are the only epidermal cells that express the T6 and HLA-DR antigens and are responsible for the in vitro allogeneic T-cell proliferative responses in the mixed skin cell lymphocyte reaction (MSLR) . To investigate the presence of Langerhans cells in normal human epidermal cell cultures, epidermal cell suspensions obtained from normal human skin specimens and from the subsequent epidermal cell cultures were analyzed by indirect immunofluorescence for the presence of T6 and HLA-DR determinants . In parallel, MSLRs were conducted with suspensions of cultured epidermal cells as stimulatory cells . These studies present evidence that when human epidermal cells are grown in culture, they loose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR and T6 antigens . The T6 antigens were lost during the first 2 weeks of culture, while HLA-DR determinants were still expressed by a small number of cells and were progressively lost through duration of cultures . The loss of HLA-DR antigens closely paralleled the progressive inability of human epidermal cells in culture to stimulate allogenic T cells in MSLR.

Am J Physiol, 1986 Mar, 250(3 Pt 1), C506 - 16
Characterization of monoclonal antibodies to rabbit renal cortical cells; Ronco P et al.; The immunological heterogeneity of the rabbit nephron was investigated using monoclonal antibodies . Seventeen antibodies have been produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with unfractionated rabbit renal cortical cell preparations . Sixteen antibodies reacted with proximal tubular cells: 11 with the brush border and 5 with basolateral membrane or intracytoplasmic components . Only one of the latter was specific for constituents of the proximal tubule . One antibody reacted with the cortical collecting tubule . Eight of the anti-brush-border antibodies were further characterized by immunoprecipitation of detergent-solubilized radiolabeled brush-border membrane vesicles . Seven proteins with subunits ranging in molecular weight from 90,000 to greater than 340,000 were identified . Systematic survey showed that one of these proteins with a subunit molecular weight of 115,000 exhibited leucine aminopeptidase activity . Selected monoclonal antibodies bound to Sepharose 4B immunoadsorbents were used to deplete solubilized brush-border membrane vesicles of a given antigen and to identify leucine aminopeptidase . Furthermore, the obtention of specific antibodies directed against the proximal tubule allowed us to set up a simple method for renal cell separation: isolated renal cortical cells could be depleted by 80% in proximal cells by passage over columns of Sepharose 6MB covalently linked with three different monoclonal anti-brush-border antibodies, thus leading to cell suspensions considerably enriched in tubule cells originating from the more distal segments of the nephron.

J Natl Cancer Inst, 1986 Mar, 76(3), 485 - 92
Effects of D-glucosamine and 6-azauridine on nucleotide contents, 5-fluorouridine uptake, and cytotoxicity in TA3 mammary tumor cells; Holstege A et al.; In TA3 mammary carcinoma cells in suspension culture, D-glucosamine X HCl (GlcN) induced a diversion of uridylate from UTP into UDP-N-acetylhexosamines, reducing the intracellular pool of UTP and eliciting an increased rate of de novo uridylate synthesis . This rise in de novo synthesis was completely suppressed by addition of 6-azauridine (6-AzaUrd) to the cell suspension in vitro or in the solid TA3 mammary tumor in NMRI mice in vivo . A synergistic depletion of UTP pools to less than 6% of the UTP in controls was observed in TA3 cell suspensions exposed to GlcN and 6-AzaUrd . In solid TA3 tumors in vivo, UTP was reduced by this combination to 19% of the control value . A high sensitivity of the solid tumor to inhibition of pyrimidine synthesis de novo was indicated by the reduction of the UTP content after administration of 6-AzaUrd alone . UTP deficiency in TA3 tumor cells was accompanied by CTP deficiency . In addition, 6-AzaUrd caused a lowering of GTP in the neoplastic tissue . Host liver was resistant to 6-AzaUrd but responded to treatment with GlcN with a decrease in UTP to 67% . Uridine-cytidine kinase was less inhibited in the presence of lowered UTP and CTP, which are potent feedback inhibitors of the enzyme, and enabled an enhanced formation of phosphorylated derivatives of 5-fluorouridine (FUrd) . Aside from the formation of 5-fluoro-UTP, we have identified 5-fluoro-UDP-N-acetylhexosamines (FUDPHexNAc), which accumulated when FUrd and GlcN were sequentially administered . Treatment of TA3 cells with FUrd after a pretreatment with 6-AzaUrd and GlcN resulted in a 2.5-fold increase in {14C}FUrd uptake and a duplication of 5-fluorouridylate incorporation into the RNA . The proportion of FUDPHexNAc increased to 58% of the phosphorylated FUrd metabolites, as compared to 6% in TA3 cells exposed exclusively to FUrd . In vivo chemotherapy of mice bearing TA3 ascites tumors was most effective with respect to tumor growth inhibition and animal survival when GlcN and FUrd were combined.

Cytometry, 1986 Mar, 7(2), 157 - 62
Isolation of viable type II alveolar epithelial cells by flow cytometry; Wilson JS et al.; We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO) . Fischer-344 rat lungs were dispersed into single-cell suspensions by a technique that yields a high number of cells (4-5 X 10(8) cells/lung, congruent to 85% viable), congruent to 11% of which are type II cells . Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0-0.02 micrograms/ml AO and analyzed by flow cytometry . Parameters analyzed included axial light loss (ALL) and red fluorescence (RF) . Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity . Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at greater than or equal to 85% purity . The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.

Brain Res, 1986 Feb 26, 366(1-2), 346 - 9
The rotating 6-hydroxydopamine-lesioned mouse as a model for assessing functional effects of neuronal grafting; Brundin P et al.; Mice were first administered intrastriatal injections of 6-hydroxydopamine and subsequently a sub-group was given neural cell suspension grafts prepared from 14-day-old fetal ventral mesencephalic mouse tissue . Six and 8 weeks after transplantation the mice in the grafted group exhibited a significant reduction in amphetamine-induced turning behaviour towards the lesioned side compared to non-grafted lesioned controls . Six of the 7 mice that had surviving grafts containing histofluorescent dopamine neurons eventually showed a reversed motor side bias with more amphetamine-induced turning in a direction away from the transplant.

Cancer, 1986 Feb 15, 57(4), 808 - 11
Primary breast cancer . Flow cytometric DNA pattern in relation to clinical and histopathologic characteristics; Thorud E et al.; Cell suspensions of 59 primary operable mammary carcinomas in women were subjected to flow cytometric DNA analyses . The results were correlated to histopathological grading, clinical staging, menopausal status and estrogen receptor status . Thirty-two tumors showed a DNA peak above +25% of murine lymphocyte level and were named distinct aneuploid (AN) . Twenty-seven tumors did not show such a peak and were named near diploid (ND) . Low histopathological malignancy grade was associated with a high frequency of ND tumors (P less than 0.05) . Small tumors (T1) were predominantly ND (14/19), while larger tumors (T2-3) showed a high frequency of AN tumors (27/40) (P less than 0.01) . Tumors occurring in post-menopausal women were often AN, while those arising in premenopausal women were frequently ND (P less than 0.06) . No clear correlation was found between the DNA ploidy pattern and estrogen receptor status . There was a tendency that patients with AN tumors had a higher frequency of relapses during the first 4-5 years of follow-up than those with ND breast carcinomas.

J Immunol, 1986 Feb 15, 136(4), 1510 - 5
The antimetastatic function of concomitant antitumor immunity . II . Evidence that the generation of Ly-1+2+ effector T cells temporarily causes the destruction of already disseminated tumor cells; Dye ES; Progressive growth of the P815 mastocytoma in an immunocompetent host evokes the generation of an antitumor immune response that can be measured in terms of the production of cytolytic Ly-1+2+ T cells in the draining lymph node and spleen . This immunity, designated concomitant immunity, is present on day 6 of tumor growth, peaks on day 9, and decays progressively thereafter . It fails to develop in mice made T cell deficient by thymectomy and lethal whole-body gamma-radiation, and reconstituted with syngeneic bone marrow cells (TXB mice) . Employment of a mouse survival assay, capable of enumerating metastatic P815 cells in cell suspensions, showed that the P815 tumor metastasizes to the draining lymph node and spleen at the same rate in normal and TXB mice for the first 6 days of growth of an intradermal P815 tumor . By day 6 of tumor growth there were approximately 10(3) P815 cells in the draining lymph node in both types of mice . However, during the generation of concomitant immunity between days 6 and 9, the number of metastatic P815 cells in the draining lymph nodes and spleens of normal tumor-bearing mice declined by nearly 90% . After day 12, however, the number of tumor cells in the nodes and spleens increased concordantly with the decay of concomitant immunity . These findings, together with the demonstration that T cell-deficient mice failed to restrain the number of metastatic P815 cells in the draining lymph node and spleen, suggest that concomitant immunity is an important defense mechanism against the development of systemic disease . Additional evidence consistent with this interpretation was provided by studies which showed that adoptive immunization with spleen cells from concomitant immune donors significantly prolonged the median survival time of TXB tumor-bearing mice by destroying a substantial proportion of P815 tumor cells already seeded in the draining lymph node . Adoptive immunization also delayed the appearance of metastatic tumor cells in the spleen.

J Immunol, 1986 Feb 15, 136(4), 1504 - 9
The antimetastatic function of concomitant antitumor immunity . I . Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay; Dye SE; A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor . The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p . into mice and their mean survival time . It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells . This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p . into 800-rad irradiated test recipients . The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.

Int J Cancer, 1986 Feb 15, 37(2), 201 - 7
Tissue-specific markers in flow cytometry of urological cancers . II . Cytokeratin and vimentin in renal-cell tumors; Feitz WF et al.; Nine primary human renal-cell tumors (RCT), one lymph-node metastasis, 4 human xenografts of a RCT in nude mice and a rat RCT line were analyzed by flow cytometry (FCM) using propidium iodide for DNA analysis and antibodies to cytokeratin and vimentin in the indirect immunofluorescence technique for labelling of specific tumor-cell populations . By means of 2-dimensional FCM analysis, vimentin- and cytokeratin-positive (tumor) cells were compared and their DNA content and proliferative fraction analyzed separately from those of cytokeratin-negative stromal and inflammatory cells . In primary human RCT, 2 subpopulations of cells were detected and analyzed separately . Small numbers of tumor cells with an abnormal DNA stemline were also detected . In addition, co-expression of intermediate filament proteins of both the cytokeratin and the vimentin types was detected in the aneuploid cell population . Comparison of 2 model systems of RCT with primary human RCT revealed a similar pattern of tumor-cell subfractions within these tumors . The 2-parameter FCM analysis permits the detection of subpopulations in complex cell suspensions and the quantification of these fractions, as well as analysis of their cellular DNA content.

Eur J Biochem, 1986 Feb 3, 154(3), 559 - 62
Leukotriene C4 metabolism by hepatoma cells deficient in the uptake of cysteinyl leukotrienes; Weckbecker G et al.; Uptake and metabolism of the cysteinyl leukotrienes C4 and E4 (LTC4 and LTE4) were studied in AS-30D hepatoma cell suspensions and compared with rat hepatocytes . The hepatoma cells were deficient in the uptake of {3H}LTC4 and {3H}LTE4 but took up, in control experiments, L-{14C}glutamine and {14C}adenosine in a time-dependent manner . By contrast, isolated hepatocyte suspensions incubated under the same conditions took up {3H}LTC4 and {3H}LTE4 as well as L-{14C}glutamine and {14C}adenosine . The hepatoma cells deficient in the uptake of cysteinyl leukotrienes metabolized extracellular {3H}LTC4 to {3H}LTD4 and to {3H}LTE4 . Addition of acivicin, an inhibitor of gamma-glutamyltransferase, largely prevented metabolism of {3H}LTC4 by the hepatoma cells . Sonication of the cells did not enhance the formation of {3H}LTD4 and {3H}LTE4 from {3H}LTC4 . We conclude that ectoenzymes of AS-30D hepatoma cells catalyze the conversion of LTC4 to LTE4 via LTD4 . As compared to hepatocytes, these neoplastic cells have lost the uptake system for cysteinyl leukotrienes and may serve in studies on leukotriene metabolism by cell-surface enzymes.

FEBS Lett, 1986 Feb 3, 196(1), 44 - 8
Mechanism of o-aminophenol metabolism in human erythrocytes; Tomoda A et al.; o-Aminophenol was found to be rapidly metabolized to a brown compound in the presence of purified human oxy- and methemoglobin, coupled with the oxidation and reduction of these hemoglobins by o-aminophenol . The final product of o-aminophenol was identified as 2-aminophenoxazine-3-one, by using spectrophotometry and HPLC . The metabolism of o-aminophenol was also observed in human erythrocytes . The production rates of 2-aminophenoxazine-3-one in the cells were very fast, but these were strongly decreased by bubbling carbon monoxide into the cell suspension when intracellular hemoglobin was in the ferrous state . The production of 2-aminophenoxazine-3-one from o-aminophenol in the cells was completely suppressed by cyanide and azide when intracellular hemoglobin was in the ferric state . These results suggest that oxy- and methemoglobin are involved in metabolism of o-aminophenol to 2-aminophenoxazine-3-one in human erythrocytes.

Biol Reprod, 1986 Feb, 34(1), 195 - 206
Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes; Bucci LR et al.; A method to obtain several highly enriched populations of testis cell types from rats of a single age is described . Single cell suspensions from immature rat testes were prepared after enzymatic removal of interstitial cells . Cells were separated on the basis of size into four fractions (bulk preparations) or eight fractions (analytical preparations) by centrifugal elutriation . These elutriator fractions were further separated by equilibrium density centrifugation in Percoll gradients . In this manner, populations of 2 X 10(7) type A spermatogonia (51% purity), 3 X 10(7) type B spermatogonia (76% purity), 5 X 10(7) zygotene/early pachytene spermatocytes (56% purity), 3 X 10(7) midpachytene spermatocytes (70% purity), and 4 X 10(7) Sertoli cells (89% purity) could be obtained from 50 immature rats within 6 h after killing . Purities, determined by examination of cytologic smears, were verified by Coulter volume and flow cytometric DNA determinations . These separation methods were used to obtain cell populations for characterization of levels and synthesis of high mobility group proteins in the early stages of spermatogenesis.

Exp Cell Res, 1986 Feb, 162(2), 566 - 73
Neurons in cell culture survive freezing . Effect on electric membrane properties; Scott B et al.; A preliminary technique is described for freezing and thawing of intact neural cell cultures and neural cell suspensions of adult mouse dorsal root ganglia which permitted the neurons to retain to a remarkable degree their usual morphological and electrophysiological characteristics . Determination of ten electrical membrane properties (EMP) indicated significant quantitative alterations including increased specific membrane resistance and duration of the action potential and decreased resting membrane potential . The pattern of altered EMP was discussed in terms of the possible ionic site of freezing damage.

Eur J Cancer Clin Oncol, 1986 Feb, 22(2), 219 - 23
Sinus histiocytes in axillary lymph nodes from patients with breast cancer: macrophage characteristics and activation level; Steele RJ et al.; Cell suspensions from 16 tumour-free axillary lymph nodes from breast cancer patients were prepared, using collagenase digestion to free the sinus histiocytes from the fibrous stroma of the nodes . The histiocytic cells so obtained were then characterized using four surface markers: Fc(IgG) receptors, C3 receptors, DR antigen and a macrophage-associated antigen (defined by the monoclonal antibody VEP-7) . In addition phagocytosis was assessed using IgG-coated red cells, and both lysozyme and alpha-1-antitrypsin were localized by means of immunoperoxidase staining . The results demonstrated that the majority of sinus histiocytes carried surface macrophage markers, but that a minority displayed phagocytosis and the presence of lysozyme or alpha-1-antitrypsin.

Am J Obstet Gynecol, 1986 Feb, 154(2), 408 - 11
Improving the yield of direct chorionic villus slide preparations; Bhatia B et al.; Low cytogenetic yields in the processing of small chorionic villus sampling have in some instances, limited its applicability . We have modified "standard" techniques to increase the number of interpretable metaphases by (1) using a gravity method for cell suspension spreading and (2) using a special high-quality slide glass . Both modifications reduce cell damage and increase interpretable mitotic figures and may allow a cytogenetic diagnosis in some instances in which a diagnosis might not otherwise be possible with standard methods.

Proc Natl Acad Sci U S A, 1986 Feb, 83(4), 1135 - 9
Growth and differentiation of cerebellar suspensions transplanted into the adult cerebellum of mice with heredodegenerative ataxia; Sotelo C et al.; Cell suspensions from cerebellar primordia of 12-day mouse embryos were grafted into the cerebellum of 4-month-old Purkinje cell degeneration (pcd) mutant mice and examined 2-3 months later . In contrast to those of nontreated mutants, all of the grafted cerebella exhibited Purkinje cells that had migrated into the molecular layer, where they were clustered over its superficial two-thirds . These Purkinje cells develop flattened dendritic trees perpendicular to bundles of parallel fibers . Ultrastructural examination of their synaptic inputs and outputs disclosed that (i) as in normal cerebella, climbing fibers and axons from basket and stellate cells synapse on thick dendrites, whereas parallel fibers almost exclusively contact the distal spiny branchlets, and (ii) a substantial number of Purkinje cell axons reach their appropriate targets in the deep cerebellar nuclei, where they establish synaptic connections on large and small neurons . These results indicate that embryonic Purkinje cells grafted into the cerebellum of adult mice with heredodegenerative ataxia integrate themselves very specifically into the cerebellar circuitry of the recipient mouse, where they can replace the missing Purkinje cells . They also provide a morphological basis favoring the notion of functional restorative capabilities of neural grafts in systems in which neurons are connected in an almost point-to-point manner.

Cell Immunol, 1986 Feb, 97(2), 238 - 47
Pertussigen enhances antigen-driven interferon-gamma production by sensitized lymphoid cells; Sewell WA et al.; We have studied the effects on interferon-gamma (IFN-gamma) production of pertussigen, a protein toxin from Bordetella pertussis that augments and prolongs delayed-type hypersensitivity (DTH) reactions . Lymphoid cell suspensions from immunized mice were incubated with antigen or mitogen, and the culture supernatants were assayed for IFN-gamma . The production of IFN-gamma on exposure to specific antigen or concanavalin A was greatly enhanced if mice were given pertussigen at the time of immunization . There was no detectable IFN-gamma production when cells were exposed to saline or to an irrelevant antigen . The effect of pertussigen on antigen-driven IFN-gamma production correlated with its effect on the capacity of the same cell populations to transfer DTH . The enhanced IFN-gamma production by cells from mice given pertussigen could not be attributed to an increased antigen-presenting capacity of this cell population . Production of IFN-gamma was abolished if the cells were pretreated with emetine, but not with mitomycin C, and the release of IFN-gamma was not detected in the first 8 hr of culture . After immunization with pertussigen, IFN-gamma was produced by lymph node and spleen cells from 7 days onward and both cell types produced IFN-gamma until at least 30 days after immunization . It is suggested that the augmentation of antigen-specific IFN-gamma production may contribute to the prolonged DTH reactions induced by pertussigen in vivo.

Am J Physiol, 1986 Feb, 250(2 Pt 2), R260 - 6
Studies on avian erythrocyte metabolism . XIV . Effect of CO2 and pH on P50 in the chicken; Isaacks R et al.; The O2 affinity (P50) of erythrocyte suspensions from 18-day chick embryos, from 2-, 5-, 8-, and 14-day chicks, and from mature chickens decreased with increasing concentrations of either CO2 or H+, particularly at a pH near 7.4 and at 37 degrees C . A greater effect on delta P50's was observed from increasing H+ concentration from pH 8.0 to 6.8 in cell suspensions from 18-day embryos (28.8 Torr at 0% CO2) and adult chickens (55.1 Torr at 0% CO2) than from increases in CO2 concentration at any given pH . The Bohr effect (delta log P50/delta pH) in the absence of CO2 was -0.508 and -0.479 for cell suspensions from 18-day chick embryos and adult birds, respectively . The specific effect of CO2 on the Bohr effect, regardless of the CO2 concentration, indicates that the delta P50/0.1 pH is approximately 1.35 and 2.45 Torr for the embryo and adult chicken blood . These results indicate that increasing H+ and CO2 concentrations markedly affect the P50 of chicken blood and that even subtle changes in either could play a significant role physiologically in regulating blood P50 in birds.

Nippon Yakurigaku Zasshi, 1986 Feb, 87(2), 99 - 104
{Stimulation of pituitary-adrenocortical system by cepharanthine}; Yoshikawa N et al.; We observed that cepharanthine might exert its anti-allergic action by stimulating the secretion of corticosterone . The present experiments were carried out to investigate stimulation of the pituitary-adrenocortical system by cepharanthine . Administration of cepharanthine to rats produced increases in plasma and adrenal corticosterone levels . Administration of cepharanthine to propranolol pretreated rats also produced increases in plasma and adrenal corticosterone levels and plasma ACTH level . The elevation of corticosterone level induced by cepharanthine was considered to be the specific effect of cepharanthine . Cepharanthine did not increase plasma corticosterone level in rats in the state of dexamethasone suppression of the pituitary-adrenocortical system, in which the level was lowered . Administration of cepharanthine to Bordetella pertussis vaccine induced beta-adrenergic blocked rats also produced increases in plasma and adrenal corticosterone levels . The production and release of corticosterone from an adrenal cell suspension were not influenced by cepharanthine in vitro . These results suggest that cepharanthine stimulates the pituitary-adrenotropic function.

Mol Cell Endocrinol, 1986 Feb, 44(2), 147 - 58
Characterization of luteinizing hormone-releasing hormone receptor binding in rat pituitary cell monolayer cultures; influence of intercellular communication; Andries M et al.; Receptors for the luteinizing hormone-releasing hormone (LHRH) were characterized in rat pituitary cells cultured for 3 days as monolayers on coverslips using 125I-{D-Ala6-Pro9-LHRH-NEt} as the labeled ligand . The monolayers were left intact during the binding assay . Specific binding displayed the various characteristics of binding to the physiological LHRH receptor . Various kinetic data corresponded to those reported previously . However, in these cultured cells, in which binding was tested in a physiological medium, the dose response of competition for binding by LHRH agonists ranged over a smaller concentration range (less than 2 orders of magnitude) than that by LHRH antagonists . In a cation-free buffer competition curves of agonists and antagonists were parallel but the apparent dissociation constant was lower than in the physiological medium . In cultures of pituitary cell populations separated by unit gravity sedimentation, the specific binding increased with the proportional number of gonadotrophs in the various populations . However, when the gonadotroph-richest population (approximately equal to 70% gonadotrophs) was cultured after recombination with gonadotroph-poor populations, binding capacity significantly increased . Microscopic examinations suggested that this phenomenon was the consequence of disrupting cellular contacts among gonadotrophs . It is concluded that certain characteristics of LHRH receptors tested on cells in a tissue-like organization and in a physiological environment are different from those reported previously in disrupted cells or monodispersed cell suspensions and that intercellular communication is an important factor controlling LHRH receptors.

Mol Immunol, 1986 Feb, 23(2), 193 - 200
Human melanoma-associated antigens: analysis of antigenic heterogeneity by molecular, serologic and flow-cytometric approaches; Morgan AC Jr et al.; The relationship of antigenic heterogeneity to the epitope recognized by an antibody was examined with monoclonal antibodies to human melanoma-associated antigens . Expression of the human melanoma-associated antigens, 250-Kd glycoprotein/proteoglycan and p97, was examined quantitatively by flow cytometry on fresh cell suspensions of human melanoma . Percent positive cells and mean fluorescence intensity were consistently higher with antibody 9.2.27 to the 250-Kd glycoprotein/proteoglycan than with antibody to p97 . In addition, assessment of percent positive cells in multiple skin lesions biopsied from individual patients indicated that in 26 of 30 lesions, greater than 90% of the cells stained positively with 9.2.27 . This relative lack of antigenic heterogeneity with antibody 9.2.27 contrasted with previous reports which showed considerable antigenic heterogeneity with other antibodies to the 250-Kd glycoprotein/proteoglycan . The explanation for this distinction was sought by quantitative flow cytometric and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques . Comparison by flow cytometry and immunoperoxidase of three antibodies, which recognized distinct epitopes of the 250-Kd glycoprotein/proteoglycan, indicated that 9.2.27 reacted more intensely with cultured cells and tissue sections than other antibodies to the same antigen . Examination by SDS-PAGE indicated that 9.2.27 could immunoprecipitate a larger proportion of 250-Kd glycoprotein molecules than other antibodies . In addition, immunodepletion experiments in gels indicated that the 9.2.27 determinant was present on a higher proportion of 250-Kd glycoprotein molecules than PG-2 antibody to a separate determinant . It is likely that 9.2.27 antibody displays less antigenic heterogeneity because its epitope is represented on a higher proportion of the antigen molecules . Thus, not only the nature of the antigen but also the epitope recognized by an antibody influences the degree of antigenic heterogeneity.

Exp Hematol, 1986 Feb, 14(2), 101 - 7
Selective removal of clonogenic neoplastic B cells from human bone marrow using anti-HLA-DQ antibodies and complement; Falkenburg JH et al.; Polymorphic HLA-DQ (DC/MB) determinants appeared to be not expressed on human hematopoietic progenitor cells (HPC), using several murine monoclonal and human polyclonal antibodies in a complement-dependent cytotoxicity (CDC) assay . Since mature HLA-DR-positive malignant lymphoma cells prove to be HLA-DQ positive, an attempt was made to remove clonogenic neoplastic DQwl-positive B cells selectively from DQwl-positive marrow samples without affecting hematopoietic progenitor cells . Using a combination of a clonogenic tumor cell assay, an HPC culture assay, and a mixed-tumor-cell-HPC culture assay, selective elimination of more than 98% of clonogenic neoplastic cells from tumor-cell-contaminated bone marrow suspensions was achieved with monoclonal anti-DQ antibodies and complement without depletion of HPC . These results indicate that anti-HLA-DQ antibodies can be used in autologous bone marrow transplantation to deplete the bone marrow cell suspension of DQ-positive malignant cells.

Biochem Biophys Res Commun, 1986 Jan 29, 134(2), 711 - 5
Recovery of transferrin receptors on hepatocytes membrane after collagenase perfusion; Kishimoto T et al.; Hepatocyte cell suspensions obtained by collagenase perfusion method did not have transferrin (TF) receptors . However, after incubation at 37 degrees, they appeared to gain TF receptors, the number of which was the function of incubation time at 37 degrees . It is suggested that hepatocyte TF receptors are collagenase-sensitive . This study can explain previous observations that hepatocyte isolated with collagenase treatment of the tissue do not bind TF at 4 degrees but take up TF at 37 degrees.

J Immunol Methods, 1986 Jan 22, 86(1), 39 - 44
Turbidimetric microassay for macrophage-mediated antibody-dependent cellular cytotoxicity; Rummage JA et al.; An improved microassay for quantitation of murine macrophage-mediated antibody-dependent cellular cytotoxicity (ADCC) has been developed . The method is based on the turbidimetric measurement of sheep erythrocyte or nucleated (L1210) target cell suspensions at 630 nm with an automatic microtiter plate densitometer . The novel method was applied to demonstrate dose-related increases in murine macrophage mediated ADCC with varying antibody concentration, effector:target ratio, and incubation time . Advantages of the turbidimetric method were shown over the 51Cr-labeled target cell method by direct comparisons in that the new method was 2-4 times more sensitive and allowed repeated readings of the same plate after various incubation time intervals . The method provides further advantages of (1) elimination of the need for expensive and hazardous radioactive materials, (2) relative ease and rapidity in which experiments may be performed and quantitated, (3) sensitivity and reproducibility, and (4) versatility of the assay for measuring cytotoxicity of either erythrocyte or nucleated target cells.

Biochem J, 1986 Jan 15, 233(2), 571 - 6
Is the availability of substrate for the tricarboxylic acid cycle a limiting factor for uncoupled respiration in sycamore (Acer pseudoplatanus) cells?
Journet EP, Bligny R, Douce R.
Protoplasts obtained from sycamore (Acer pseudoplatanus) cell suspensions were found to be highly intact and to retain a high rate of O2 consumption . If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured, leaving the fragile amyloplasts largely intact . Distribution of enzymes of glycolysis in plastids and soluble phase of sycamore protoplasts indicated that the absolute maximum activity for each glycolytic enzyme under optimum conditions exceeded the estimates of the maximal rate at which sycamore cells oxidize triose phosphate . Passage of protoplasts through the fine nylon mesh produced a 3-5-fold decrease in O2 consumption . However, addition of saturating amounts of respiratory substrates and ADP restored an O2 consumption equal to that observed with uncoupled intact protoplasts . Taken together, these results demonstrated that neither the overall capacity of the glycolytic enzymes in sycamore cells nor the availability of respiratory substrates for the mitochondria is ultimately responsible for determining the rate of uncoupled respiration in sycamore cells.

Biochem Pharmacol, 1986 Jan 15, 35(2), 325 - 9
Formation and transport of xenobiotic glutathione-S-conjugates in red cells; Eckert KG et al.; In vitro studies with freshly drawn human erythrocytes showed 4-dimethylaminophenol, a cyanide antidote, to be rapidly metabolized with the formation of a transient S,S-(2-dimethylamino-5-hydroxy-1,3-phenylene)bis-glutathione conjugate and a stable S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)tris-glutathione conjugate . The stable tri-glutathionyl derivative was actively transported across the red cell membrane with an apparent Vmax = 1 nmol/min/ml red cell suspension (15 g hemoglobin/100 ml) and Km = 0.5 mM . The transport system was strictly unidirectional, inhibited completely by sodium fluoride and reduced to one-fifth by lowering the temperature from 37 to 22 degrees . Similarly S-(2,4-dinitrophenyl)-glutathione, the glutathione-S-transferase mediated glutathione-S-conjugate with 1-chloro-2,4-dinitrobenzene, was unidirectionally transported, a process which was inhibited by sodium fluoride . Kinetic analysis revealed two different transport processes: Vmax = 0.9 nmol/min/ml, Km = 1.4 microM and Vmax = 4.5 nmol/min/ml, Km = 700 microM . Mutual inhibition of the low affinity transport system was found for both glutathione-S-conjugates . The apparent energies of activation for all these transport processes and for GSSG were identical (70 kJ/mol) suggesting at least one common carrier for the excretion of the three glutathione-S-conjugates.

Brain Res, 1986 Jan 8, 362(2), 344 - 9
In vivo measurement of spontaneous release and metabolism of dopamine from intrastriatal nigral grafts using intracerebral dialysis; Zetterstrom T et al.; Spontaneous release and metabolism of dopamine (DA) from intrastriatal grafts of fetal mesencephalic DA neurons was measured by intracerebral dialysis . Mesencephalic DA cell suspensions were implanted into the head of the caudate-putamen in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the mesostriatal DA pathway . Four months later, when tests for amphetamine-induced turning behaviour showed that the grafts had become functional, loops of dialysis tubing were implanted into the striatum on the grafted side and the contralateral non-lesioned side of the grafted rats, and in a similar position in the denervated caudate-putamen of 6-OHDA lesioned control rats . Dialysis perfusates collected from the 6-OHDA lesioned striata showed a reduction of about 95-98% in DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) . In the grafted animals these levels had recovered to about 40% of control for DA and to 12-16% of control for HVA and DOPAC . In addition, the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was increased in the grafted striata compared to both the lesioned and non-lesioned controls . Amphetamine had little or no effect on DA release in the 6-OHDA lesioned rats, but caused a marked increase in DA release in the grafted rats, this response being proportional to that seen in intact striata . Since the subsequent histochemical analysis showed that the dialysis probe had been located in the transplant-reinnervated part of the caudate-putamen, the results provide additional evidence that the grafted DA neurons exert their functional effects through a continuous active transmitter release from their newly-established terminals in the reinnervated host target.

Neuroscience, 1986, 17(1), 89 - 98
Enhanced graft survival in the hippocampus following selective denervation; Gage FH et al.; The trophic effects of denervation on the survival of fetal cholinergic neuronal cell suspensions grafted to the hippocampal formation of the rat were assessed in the present study . Young adult female rats were injected with cell suspensions of neurons obtained from the fetal basal forebrain region into the hippocampal formation simultaneously with (or without) a fimbria-fornix transection, which removes the hippocampal cholinergic afferents . Four to six months later, one group of grafted animals was evaluated histochemically for: transplant volume; number of acetylcholinesterase-positive cells, and size of acetylcholinesterase-positive cells in the graft . A parallel study was conducted to determine the total number and size of the acetylcholinesterase-positive cells in the septal-diagonal band-substantia innominata complex of the adult rat, to match with the cell survival and growth in the grafts . A second group of grafted rats was taken in parallel for biochemical analysis of choline acetyltransferase activity in the grafted hippocampus . The transplant volume in the rats with fimbria-fornix transection was greater than twice the volume seen in animals without fimbria-fornix lesion . In addition, the number of acetylcholinesterase-positive cells in the transplant was twice as great in the denervated animals as in the non-denervated ones . However, the number of acetylcholinesterase-positive cells per mm3 of graft volume did not differ between the two groups, suggesting that the trophic effect of the denervation was not specific for the cholinergic neurons, but affected the entire grafted tissue . The hippocampal choline acetyltransferase activity of the animals that received the fimbria-fornix lesion simultaneously with transplantation was about three times higher than that of the rats that received grafts but no simultaneous fimbria-fornix transection . A control experiment with animals that received an aspirative lesion of the retrosplenial cortex, transecting the perforant path input, revealed no enhancing effect of hippocampal choline acetyltransferase activity over non-lesioned grafted animals . Thus, the denervation-enhancing effects of the fimbria-fornix lesion appear to be selective and not the result of a general wound-induced mechanism . These results strongly support the contention that neurotrophic factors are released as a result of denervation in the adult hippocampal formation, and that these neurotrophic factors can support survival and growth of central cholinergic neurons . However, the factors involved do not appear to be specific for the cholinergic neurons, but rather have their trophic effects on many types of cells.

J Microsc, 1986 Jan, 141 ( Pt 1), 119 - 26
Microscopic observation and video recording of cellular response to sudden changes in environment; Eskelinen S et al.; A method to record responses of single cells to rapid changes in their environment is described . A rectangular glass microcapillary is filled with the cell suspension under investigation and placed under a light microscope . With the aid of a micromanipulator a smaller microcapillary with a narrowed tip, which was still over 10 times wider than the cell diameter, is driven into the larger, cell containing, microcapillary . The environment of the cells is changed by injecting a new medium via the smaller capillary and the cell responses are recorded using a video recording system . The usefulness of the system has been examined by studying the time course of the osmotic swelling of erythrocytes after water injection and the shape transformations caused by lysophosphatidylcholine.

Cytogenet Cell Genet, 1986, 41(3), 181 - 4
An improved fixation method for chromosome preparation of Chinese hamster, Chinese hamster-human hybrid, and mouse cell lines; Korthof G; Lowering the proportion of acetic acid in the standard 1:3 acetic acid:methanol chromosome fixative used both during initial fixation and subsequent washing produced up to a 20-fold increase in the yield of intact metaphases from cultures of several permanent cell lines . Although this inhibited chromosome spreading, addition of various acetic acid-methanol mixtures immediately after the cell suspension was dropped onto slides increased the degree of spreading and resulted in well-spread, cytoplasm-free metaphases.

Leuk Res, 1986, 10(3), 303 - 12
Identification of L3 leukemia and Burkitt's lymphoma cells by flow cytometric quantitation of nuclear and cellular RNA and DNA content; Walle AJ; Separate quantitations of nucleic acids of isolated nuclei and cells of L3 leukemia/Burkitt's lymphoma cell populations of peripheral blood (PB), bone marrow (BM) and lymph node (LN) cell suspensions of 17 patients were performed by acridine orange (AO) flow cytometry (FCM) . The cell populations were analysed with respect to cell cycle characteristics and DNA/RNA distribution histograms of cells in various compartments of the cell cycle using mean value, coefficient of variation (CV), third moment about the mean as a measure of skewness (MOM3) . The correlations between numbers of L3 blasts detected by microscopy and aneuploid cells quantified by FCM in 10 patients with aneuploid disease were r = 0.75 and r = 0.85 for BM and PB cell populations, respectively . Content of both nuclear and cellular RNA was 3-4 times higher in L3 cells than in normal donor lymphocytes . Percentages of cells in SG2M phase of L3 populations were significantly different from control populations in BM (p less than 0.01) and PB (p less than 0.0001) . All samples with L3 blasts had abnormal CV of the DNA, and abnormal CV and MOM3 of the RNA peaks of G0/1 cell histograms . All samples with no blasts by morphology had normal DNA and RNA patterns . AO FCM correctly classified all specimens as involved or not involved with disease in accordance with the cytomorphological diagnoses . Combining AO FCM with prior cell separation increased the sensitivity of the method to detect abnormal cells to 0.02%, or 0.5 L3 cells per microliter of blood.

Int J Radiat Oncol Biol Phys, 1986 Jan, 12(1), 89 - 93
Differential kinetics of thermal resistance (thermotolerance) between murine normal and tumor tissues; Urano M et al.; The kinetics of thermotolerance were studied in mouse normal and tumor tissues . Early generation isotransplants of a spontaneous fibrosarcoma, FSa-II, were used . Tumor cell suspension was transplanted into the C3Hf/Sed mouse foot . Hyperthermia was given by immersing animal feet into a water bath . The TG (tumor growth) time, the time required for half the treated tumors to reach 1000 mm3 from the first treatment day, was the end point . Foot reaction was studied as a normal tissue response . The RD50, the treatment time that induces a loss of one toe or greater reaction in half the treated animals, was analyzed . Thermotolerance developed rapidly and extensively in normal and tumor tissues . A significant difference was observed in the decay of thermotolerance between these tissues . The decay of thermal resistance was incomplete in the murine foot tissue, even at 14 days after the initial heat treatment . Although some experimental difficulties were involved in the tumor study, present results suggest a complete decay of thermotolerance in the FSa-II tumor in 8 days after a 7.5 min treatment of 45.5 degrees C . Thermal resistance again developed in both tissues following the second heat treatment, which was given 7 or 8 days after the first heat treatment . The development, maximum magnitude and decay of the second thermal resistance was comparable to those of the first thermal resistance in each tissue.

Blood, 1986 Jan, 67(1), 110 - 8
Influence of oxygen tension on the viscoelastic behavior of red blood cells in sickle cell disease; Nash GB et al.; Although the rheological behavior of sickle cell suspensions and of hemoglobin S solutions is known to be strongly dependent on oxygen tension (PO2), little data exist concerning the influence of PO2 on the viscoelasticity of individual HbSS RBC . We have used micropipette aspiration techniques to test the deformation response of both HbSS and control HbAA RBC over a wide range of PO2 at 23 degrees C . Sickled, spiculed HbSS cells were present for PO2 approximately less than 35 mm Hg; for a number of these cells, the deformation response was essentially elastic and an effective membrane rigidity (EMR) was calculated . EMR increased with decreasing PO2 and was approximately 5 to 50 times higher than the equivalent rigidity of oxygenated HbSS RBC . In addition, the rate of membrane deformation was very slow for sickled cells; the half-time for the deformation process increased as PO2 was lowered and was about two orders of magnitude longer than the equivalent time for normal RBC . Other sickled cells exhibited plastic deformation when subjected to comparable deforming forces and experienced irreversible membrane deformation and budding . At all PO2 levels tested, some HbSS RBC remained as discocytes; these cells had normal membrane elasticity and membrane viscosity . Furthermore, changes in PO2 did not affect the membrane properties of HbAA RBC . Thus, gross abnormalities in the deformation response of HbSS RBC were only detected after morphological sickling had occurred . These abnormalities most likely arose from changes in the cytoplasmic HbS viscoelasticity and, if present in vivo, would be expected to impair the flow of HbSS cells in the microcirculation.

Cancer Res, 1986 Jan, 46(1), 99 - 105
Simultaneous measurements of macrophage-induced cytostasis and cytotoxicity of EMT6 cells by flow cytometry; Stevenson AP et al.; Cytostasis and cytotoxicity as well as survival and growth of EMT6 cells following exposure to tumoricidal macrophages have been examined using multiparameter flow cytometry . Macrophages were resolved from tumor cells, and the number of surviving EMT6 cells at different interaction times was determined by adding, to each cell suspension, fluorescent latex particles the concentration of which was known and counting them along with the cells . The percentages of EMT6 cells in S phase were determined by analysis of DNA histograms and compared to the percentages as determined by autoradiography . The progression of cells through the cell cycle was also examined during exposure to macrophages by dual parameter analysis of DNA content and bromodeoxyuridine incorporated into DNA prior to exposure to macrophages . The results showed that, by 4 h of interaction with tumoricidal macrophages, EMT6 cells stopped progressing; tumor cell progression was inhibited in all phases of the cell cycle . By 24 h, there was an absolute decrease in survival . By 48 h, however, surviving tumor cells which escaped the tumoricidal activity of macrophages exhibited a population doubling rate similar to control cells.

Cancer Res, 1986 Jan, 46(1), 198 - 202
Lack of development of thermotolerance in early progenitors of murine bone marrow cells; Mivechi NF et al.; We have studied the sensitivities of four hematopoietic stem cell types to heat stress as well as their abilities to develop thermotolerance . Granulocyte-macrophage colony forming units were the most heat resistant bone marrow progenitors tested . Of the erythroid progenitors tested, erythrocyte colony forming units were more resistant than the two more primitive erythrocyte burst forming units . To determine their ability to develop thermotolerance, hematopoietic precursors were heated in vivo at 43 degrees C for 30 min . At various times thereafter the hematopoietic stem cells were flushed from female C3Hf/Sed mouse preheated tibia . The bone marrow cell suspensions were then heated in vitro and plated for colony formation . The four stem cell precursors differed markedly in their abilities to develop thermotolerance . The thermotolerance induced in granulocyte-macrophage colony forming units reached a maximum at 3-6 h after heating and disappeared by 24-48 h . The thermotolerance in erythrocyte colony forming units (0.5 units erythropoietin/ml media) reached a maximum at 3-6 h and disappeared by 48-72 h . The maximum level of thermotolerance reached by granulocyte-macrophage colony forming units and erythrocyte colony forming units was approximately the same . On the contrary, the two more primitive erythrocyte precursors which were grown by the addition of 2.5 and 5 units erythropoietin/ml of media do not develop thermotolerance.

Neurochirurgie, 1986, 32(6), 514 - 8
{Transplantation of serotonin neurons in the olfactory bulb of the adult rat}; Mansour H et al.; Serotonergic neurons have their perikaraya concentrated in the raphe nuclei . In the present experiment, pieces of raphe nuclei containing serotonergic neuroblasts were dissected from 14 days rat embryos, mechanically dissociated, and 1 microliter of a cell suspension containing 30,000 cells was injected slowly under stereotaxic control in the olfactory bulb of adult rats . Three weeks after transplantation, the brain of the rats were processed for immunocytochemical detection of 5-HT . In all of the grafted animals, 5-HT neurons were found in the olfactory bulb . They exhibited the morphology of adult neurons, with well developed axon and dendrites, extending far from the injection site . Ultrastructural immunocytochemistry disclosed afferent and efferent synapses to the grafted neurons . Three preliminary results indicates that the transplanted neurons have been integrated in the host's neuronal circuitry . Ongoing electrophysiological studies are now directed at investigating the functional status of these grafted neurons.

Dev Comp Immunol, 1986 Fall, 10(4), 571 - 84
Comparative phenotypic and ultrastructural characteristics of OKT6-positive cells in normal peripheral blood (adult and infant), in cord blood and in epidermis; Dezutter-Dambuyant C et al.; Simple and rapid procedures have been developed to isolate T6-positive cells in epidermal cell suspensions, in peripheral blood of adult, and in cord blood . The isolated T6-positive cells were tested by double immunogold labelling at ultrastructural level to analyze their phenotype (T6/HLA-DR antigens) and their morphological features in order to understand their origin, their fate, and their relationship to the early precursors of dendritic cells, immature neonatal lymphoid subsets, and immature macrophagic leukocytes.

Eur Biophys J, 1986, 14(2), 67 - 81
Flicker spectroscopy of erythrocytes . A sensitive method to study subtle changes of membrane bending stiffness; Fricke K et al.; Frequency analysis of thermally excited surface undulations of erythrocytes leading to the flicker phenomenon is applied to determine biochemically and physically induced modulations of the membrane curvature elasticity . Flicker spectra of individual cells fixed to the window of a flow chamber by polylysine are taken by phase contrast microscopy, enabling investigations of the reversibility of the structural modifications . The spectra may be approximated by Lorentzian lines in most cases . By measuring the amplitude (at zero frequency) and the line width, effects of the structural changes on the curvature elastic constant, Kc, and the wavelength distribution of the undulations may be studied separately . Effect of physically induced modifications: The temperature dependence of the flicker spectra are taken from 10 degrees C to 37 degrees C . Above 20 degrees C, Kc decreases with increasing temperature whereas the reverse holds below this limit . The latter anomalous behaviour is explained in terms of a conformational change associated with protein and lipid lateral phase separation . The bending stiffness increases when the cells swell osmotically, owing to surface tension effects . The dependence of the flicker spectra on the viscosity of the suspension medium agrees with the theoretical prediction . Biochemically and drug induced modifications: 5 vol% of ethanol leads to a pronounced and reversible suppression of the long wavelength undulations without altering the discoid cell shape and without affecting the bending stiffness appreciably . Adsorption of dextran to the glycocalix increases Kc by a factor of 1.6 at saturation . The bending stiffness is increased by a factor of 1.3 after cross-linking the proteins with the SH-oxidizing agent diamid . Injection of Ca++ into the cell via ionophores evokes (within 10 min) the formation of fine--probably spectrin free--spicules . This leads to an increase in Kc by a factor of 1.3 which is explained in terms of a lateral condensation of the spectrin/actin network . The spicule formation and Kc change is completely reversible (within 2 min) after perfusion with Ca++-free buffer . Cholesterol depletion leads first to a continuous increase in Kc without change of the cell shape whereas a sudden discocyte- to echinocyte transformation sets in below a critical steroid content . The latter transition is also observed in cell suspensions and is reminiscent of a phase transition . The anti-tumor drug actinomycin D evokes an increase in the bending stiffness Kc by a factor of two, suggesting that its effect is at least partially due to a modulation of the membrane structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Neoplasma, 1986, 33(6), 707 - 11
Comparative chromosome examination of AKR mouse lymphoma after its i.p . transplantation with thymus- or spleen-derived lymphoma cells into hybrid mice; Selypes A et al.; A spontaneous AKR female mouse lymphoma was transplanted with its thymus cell suspension (10(6) cells i.p.) into AKR female mice . Lymph node cell suspension derived from one of the leukemic mice was injected (10(6) cells i.p.) into AKR females . The lymphoma cells "homing" to the thymus of one of the AKR females were suspended in Parker solution and 5 X 10(6) cells were given i.p . 5 (C3H X AKR) F3 hybrid mice (group 1) . The lymphoma cells "homing" to the spleen of the same AKR female were also suspended, and 5 X 10(6) cells were injected i.p . into 5 (C3H X AKR) F3 hybrids (group 2) . Chromosomes were prepared from the thymus and the spleen of two mice in both groups . The karyotypes derived from the hybrids of group 1 were compared to that of the group 2 . It was found that the lymphoma cells "homing" to the thymus could be characterized by the trisomy of chromosome 15, while the lymphoma cells "homing" to the spleen had primarily the trisomy of chromosome 18 . The results indicate that the thymus manifestation of the spontaneous AKR lymphoma is heterogeneous, and it contains at least two major subpopulations of the lymphoma cells.

Blood Cells, 1986, 12(1), 249 - 70
Cellular and rheological factors contributing to sickle cell microvascular occlusion; Kurantsin-Mills J et al.; Sickle (HbSS) erythrocytes contain subpopulations that are heterogeneous in shape, size, and density and exhibit abnormal microcirculatory behavior . Their phthalate esters density distributions quantitatively distinguish subpopulations of HbSS cells from density profiles of normal (HbAA) erythrocytes . Filtration of HbSS cell suspensions, devoid of leukocytes, through 5-microns Nucleopore filters at constant flow rate (29.5 microliters/s) yields pressure-time curves that demonstrate deformability of the sickle cells to be several-fold less than equivalent suspensions of normal (HbAA) cells . For a cell flux of 6.43 X 10(5) cells/s, the rate of the rise of the pressure (Pi/t) following 1-2 s of the initial pressure reading indicates occlusion of the filter pores by the dense cell fraction . Rats exchange-transfused with human sickle (HbSS), normal (HbAA), or autologous rat erythrocytes were used to investigate the flow dynamics of these cells in the mesenteric microcirculation by intravital videomicroscopy . Time-averaged velocities of the autologous rat red cells in 16-30 microns (i.d.) arterioles ranged from 1.10 to 1.25 mm/s with varying flux and wall shear rates . Time-averaged velocities of the HbAA cells in single 15-35-microns arterioles ranged from 1.16 to 1.24 mm/s with wall shear rates similar to the estimates for the autologous cells . In contrast, sickle cells exhibited time-averaged velocities of 0.38-0.45 mm/s with lower wall shear rates in 10-35 microns single unbranched arterioles with three times less volumetric flux . In some arterioles, sickle RBCs with a high axial ratio of 3-4 and low deformability showed apparent adhesion to endothelial surfaces and occluded precapillary junctions or entry points for several seconds until dislodged by the higher flow velocity . Within single unbranched vessels or at microvascular bifurcations, sickle elliptocytes and sickle echinocytes with low deformability and axial ratios of 3-4 obstructed flow and exhibited residence times of 6-75 s at the sites of occlusion, thereby causing stasis and increasing the local apparent viscosity . Thus, both the in vitro and in vivo data demonstrate the rheological disequilibrium state induced by HbSS cells as they traverse artificial micropores or course through successive segments of the microcirculation . The specific tendency of dense cells with high axial ratio (ISCs) to manifest precapillary junctional blockade and prolonged residence times implicates this cell fraction in the initiation of microvascular occlusion.

Arch Dermatol Res, 1986, 278(6), 454 - 9
Defective monocyte and polymorphonuclear leukocyte chemotaxis and clinical characteristics in atopic dermatitis; Ternowitz T et al.; Using highly purified cell suspensions, monocyte (MO) and polymorphonuclear leukocyte (PMN) chemotaxis was measured by the 51Cr-labeled cells technique in 30 adult patients with atopic dermatitis (AD) . MO chemotaxis was depressed in 60% of the patients; in one-third both MO and PMN chemotaxis was impaired . All patients with normal MO chemotaxis had normal PMN chemotaxis . The defective chemotaxis was related to the presence of cutaneous infection and to the activity of the disease . Cutaneous infection was observed in 70% of the patients with low MO and PMN chemotaxis . We found no relation between the chemotaxis defects and serum IgE levels . Presence of asthma in addition to AD did not influence the results . Preincubation of normal leukocytes with AD plasma did not alter the chemotactic responses . Plasma from atopics had a lower capacity for inducing migration than normal plasma using leukocytes from healthy subjects as test cells.

J Ultrastruct Mol Struct Res, 1986 Jan, 94(1), 30 - 6
Improved transmission electron microscopy (TEM) of cultured cells through a "floating sheet" method; Arnold JR et al.; Cultured cells provide isolated systems for both biochemical and morphological studies . Previous methods of processing cell culture specimens for electron microscopy (EM) have been limited to sectioning either a monolayer or centrifuged cell suspensions which are not morphologically intact . In our improved method, N-butylglycidyl ether is added to cell cultures (2-5 min with agitation) following in situ fixation (3.0% glutaraldehyde in 0.1 M Pipes, pH 7.2, for 20 min, osmium tetraoxide 4% for 20 min) . A thin pliable "sheet" of cells floats free from the plastic culture device and can be manipulated (centrifuged or folded) to obtain a vast number of morphologically intact cells for examination . We have examined several cell types (vascular smooth muscle, lung, liver, and endothelial cells) grown in two types of plastic culture flasks (Nunc and Falcon) . This new method provides excellent EM morphology, maximizes the number of cells examined, and allows determination of cell orientation since a remnant of the dissolved flask remains loosely bound to the bottom of the cells.

Eur J Nucl Med, 1986, 12(4), 163 - 7
In vitro function of radiocolloid-labelled monocytes; Ensing GJ et al.; Human monocytes in a mononuclear cell suspension were labelled specifically using 111In-Fe-colloid, with the ultimate goal of using them for monocyte kinetic studies in haematological malignancies . In order to examine the function of the labelled monocytes, in vitro function tests were performed on cell suspensions containing labelled or non-labelled monocytes . These tests were: phagocytosis of opsonized zymosan particles and ability of monocytes to mature into macrophages when cultured in glass tissue chambers . There were no significant differences in in vitro function between labelled and non-labelled monocytes . Cell viability was always greater than 90% . During culture, a rapid loss of monocyte-bound radioactivity took place . We conclude that the labelling procedures do not interfere with subsequent in vitro cell function . However, because of the rapid loss of cell bound radioactivity in vitro, monocytes labelled by this method do not seem suited for long-term studies in vivo.

Invasion Metastasis, 1986, 6(4), 197 - 208
Use of drug resistance markers to recover clonogenic tumor cells from occult metastases in host tissues; Miller FR et al.; A variant of metastatic mouse mammary tumor line 410.4 was produced which is resistant to both 60 microM thioguanine and 3 mM ouabain . Occult tumor cells which result from the metastatic spread of this subline are detected by plating cell suspensions from host tissues in selective media containing thioguanine and ouabain (TO) . Only the tumor cells survive and form colonies . Tumor cell colonies were recovered when as few as 6 tumor cells mixed with 1 X 10(6) lymph node cells or normal lung cells were plated in TO . Thus, the method potentially will detect the presence of clonogenic tumor cells when the host tissue contains less than 0.0006% tumor cells . Results suggest that, within 15 min of intravenous injection, less than 10% of the injected cells are clonogenic . In addition, clonogenic tumor cells can be detected in draining lymph nodes prior to the appearance of a palpable tumor implant in the subcutis.

Eur J Clin Pharmacol, 1986, 30(5), 591 - 6
Protein binding of tolfenamic acid in the plasma from patients with renal and hepatic disease; Laznicek M et al.; The protein binding of tolfenamic acid in plasma from patients with renal and hepatic disorders was studied by equilibrium dialysis . Drug binding to the cellular components of whole blood and blood cell suspensions was also measured . Salicylic acid was used as the reference drug in all experiments . Renal and hepatic diseases increased the unbound fraction of tolfenamic acid . Free drug fractions were significantly correlated with changes in creatinine, urea, and total bilirubin, but not with those in albumin or total protein in plasma . Comparison of the theoretical binding parameters in control plasma and similar changes in protein binding in all the plasma samples studied revealed that tolfenamic acid and salicylic acid probably share a common primary binding site . The significance of the correlation permits use of salicylic acid as a model drug for predicting changes in the protein binding of tolfenamic acid . The measurements of binding properties in whole blood and blood cell--buffer suspension showed that tolfenamic acid interacts with the lipid membrane structures of blood cells, while salicylic acid is distributed into the aqueous cell space.

Bioelectromagnetics, 1986, 7(3), 259 - 69
Use of the loss-tangent function in dielectric spectroscopy; Surowiec A et al.; A new method for finding the dielectric parameters of biological substances is presented . The method makes use of the loss-tangent as a function of frequency to identify the dominating relaxation process . The method was tested for a few cell suspensions (blood and lymphocytes) and two tissues (liver and spleen) . The obtained parameters agree well with those calculated from Maxwell-Wagner theory (beta dispersion).

Thymus, 1986, 8(3), 141 - 50
Rat thymus macrophages: an immunohistochemical study on fetal, neonatal and adult thymus; Sminia T et al.; The pre- and postnatal development of the macrophage population of rat thymus is investigated applying enzyme-histochemical and immunohistochemical techniques, both on tissue sections and cell suspensions . A set of three monoclonal antibodies (ED1, ED2 and ED3), each of which recognizes cells of the monocyte-macrophage lineage in the rat, enabled us to distinguish between macrophages in the various compartments of the thymus . The medulla is characterized by ED1-positive dendritic cells, the corticomedullary region comprises numerous monocyte-like ED1-positive macrophages and the cortex contains a particular subpopulation of branched ED2-positive macrophages . Both the medullary dendritic cells and the cortical branched cells show Ia-membrane staining . ED3-positive cells are only occasionally present . During fetal life ED1-positive monocyte-like macrophages and dendritic cells are present . Just after birth ED2-positive cortical macrophages start to develop . Their number increases strongly during the first week after birth . The role of the various subpopulations of thymic macrophages is discussed.

Blood Cells, 1986, 11(3), 421 - 8
A new method of preparing monocyte suspensions from human whole blood; Stoll HP et al.; A method of isolating monocytes from human whole blood is described . The technique is primarily based on simple centrifugation steps that follow Tylose-sedimentation as well as on the use of the new density gradient medium Nycodens . Counterflow centrifugation is not involved . The final monocyte suspension is free of platelets . The contaminating cells are predominantly lymphocytes . As a whole, the method is a modification of the Nycodens technique published by Boyum in 1983, which leads to a total elimination of platelet contamination in the final cell suspension.

Biorheology, 1986, 23(1), 75 - 87
Shear viscoelasticity of suspensions of biological cells with viscoelastic membrane II; Abe K et al.; To discuss the relaxation phenomena of biological cell suspensions, we calculate the complex intrinsic viscosity of dispersions of spherical cells with viscoelastic membrane as a function of the frequency taking account of interfacial tension at both the interfaces of the membrane . The Maxwell model and two kinds of the three-parameter models are used to describe the viscoelasticity of the cell membrane . The results are computed mainly for the Maxwell model similarly in case of the Voigt Model (Abe, K., Takano, Y . and Sakanishi, A . Biorheology 21 405-414, 1984) . The computed results of the four models, the Voigt, the Maxwell and the two kinds of the three-parameter viscoelastic models, are compared with the experimental data.

J Toxicol Environ Health, 1986, 18(3), 491 - 7
Mechanisms of ozone toxicity in cultured cells . I . Reduced clonogenic ability of polyunsaturated fatty acid-supplemented fibroblasts . Effect of vitamin E; Konings AW; The direct action of ozone on viability and survival of normal and modified mouse lung fibroblasts has been studied . By cell manipulation of fibroblasts in culture, the content of polyunsaturated fatty acids (PUFA) in the phospholipids was increased from about 6% to about 40% . The cellular content of alpha-tocopherol (alpha-T) (vitamin E) could be drastically enhanced . Vitamin E supplementation to the cell did not influence the PUFA manipulation . Normal, PUFA, and PUFA(alpha-T) fibroblasts were exposed to ozone by bubbling 10 ppm through the cell suspensions for different periods of time (0-6 h) . No significant effects of the ozone exposure could be established when normal fibroblasts were used . The PUFA fibroblasts, however, were very vulnerable to ozone toxicity, both in terms of dye uptake (Trypan blue) and cell death (clonogenic ability) . When alpha-tocopherol was present in the cell (200 ng/10(6) cells), a clear protection against ozone toxicity was found . It is concluded that ozone toxicity might be higher under conditions of a relative high amount of polyunsaturated fatty acids in the membrane phospholipids of the cell and a low cellular antioxidant capacity . Cellular membranes are probably an important target for ozone-induced cell death.

J Steroid Biochem, 1986 Jan, 24(1), 395 - 9
Antiestrogen binding within different pituitary cell populations . Comparison with androgen and estrogen receptors; Thieulant ML et al.; Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation . They contained 4.8 +/- 0.7% of the cells, 51 +/- 10% of the LH and less than 3% of the PRL (n = 4) . Gonadotrope-depleted fractions were also obtained that contained most of PRL cells . Specific antiestrogen binding sites (AEBS) were quantitated in these populations after destruction of estrogen receptor . Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions . However, AEBS are not specific of a pituitary cell type . Thus, AEBS appear different from estrogen receptors in pituitary gland: by the thermal stability of AEBS, by the localization of AEBS in particulate material, by the uniform distribution of AEBS in different populations which differ markedly for E2 binding sites . Whereas the ratio of binding AE/E2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes . The dissociation constants for AEBS were in the same range (1.16 - 2.27 X 10(-9) M) for the different populations.

Microbiol Immunol, 1986, 30(12), 1281 - 90
A study on location of synthetic site which mainly synthesizes and delivers fifth component of complement system in vivo; Geng L et al.; Tissue sites for synthesis of the fifth component of complement (C5) in vivo have been investigated by using allogeneic bone marrow chimeras and bone marrow chimeras which were transplanted in addition with hepatocytes . Our prior studies have demonstrated that bone marrow chimeras which had been prepared by transplanting marrow cells from C5-sufficient donor mice into irradiated C5-deficient recipients lacked detectable levels of C5 in the sera . However, when such potentially C5-deficient {C5(+)---C5(-)} chimeras were introduced into their spleens by means of injections of fully dispersed single cell suspensions of hepatocytes isolated from the C5-sufficient donor strain, they accepted the transplantation of hepatocytes for prolonged periods and developed a measurable amount of C5 in the sera . These results indicate that C5 protein in sera is not synthesized in significant amount by cells that are descendants of bone marrow cells but rather that this complement component is synthesized and delivered to the blood in vivo by somatic cells including liver cells that are not derivatives of the bone marrow.

Mech Ageing Dev, 1986-87 Jan, 37(3), 197 - 210
Temporal order vs . variability in activation of lymphocytes from aging mice; Brock MA; Changes in patterns of circannual rhythms in the in vitro activation of splenic lymphocytes from aging C57BL/6 mice may account both for reports of conflicting results in tests of immune function and for the assumed increase in variability in data obtained using cells from older mice . Single cell suspensions from 4-6, 15 and 24-28-month-old mice living in presumably constant environments were cultured in vitro with mitogens, and the incorporation of tritiated thymidine by dividing cells was determined . Free-running periods of T and B lymphocyte rhythms of blastogenic responses increased in cells from both older groups; amplitudes of the T but not B cell rhythms were reduced in senescent mice . This results in continuously changing phase relationships of both T and B cell rhythms, without consistently higher levels of activation of cells from young animals, thus obscuring at intervals the age-related declines in function . Because data were collected for several years, it was clear from the patterns of the rhythms and also the combined data that the greatest ranges in levels of activation ("variability") characterized T and B cells from the youngest mice rather than older animals.

Ann Pathol, 1986, 6(3), 183 - 91
{Immunoperoxidase study of 18 malignant lymphoblastic lymphomas (LMLbl) in children}; Caillaud JM et al.; Eighteen cases of malignant lymphoblastic lymphomas in children were studied by immunoperoxidase technique on frozen tissue sections and/or cytological samples . Different monoclonal or polyclonal antibodies were used: OKT8, OKT11, OKB2, OKIal, anti immunoglobulins (mu, kappa, lambda chains) . The results were compared with those of classical histology: three histologically unclassified malignant lymphoblastic lymphomas were linked to a Bor T line (one pre-B lymphoma and two T lymphomas) . One diagnosis of T lymphoma could not be confirmed by immunoperoxidase technique . The technique did not reveal any surface immunoglobulins in two cases of malignant lymphomas of Burkitt type . Every one of the remaining 12 cases gave concordant results between morphological and immunological studies . In 12 cases it was possible to compare the results of immunological typing on tissue sections and cell suspension . The results were concordant in 11/12 cases . An advantage of this immunochemical technique on frozen sections and/or cytological samples is that it gives good visualisation of the cells and their reactivity with the antibodies.

Padiatr Padol, 1986, 21(3), 275 - 82
{Treatment of stage IV neuroblastoma with high-dose melphalan and autologous bone marrow transplantation following in vitro preliminary treatment of the bone marrow with the active cyclophosphamide derivative Asta Z-7654}; Urban C et al.; The case of a 4 year 8 months old boy with neuroblastoma of unknown primary, metastatic to the bone and to the bone marrow is presented . After achieving a partial remission with six cycles of conventional chemotherapy, the patient was given supraconventional chemotherapy (melphalan 220 mg/m2 bolus i.v.) in an effort to eliminate residual disease . Prior to the administration of the drug, 560 cc of autologous bone marrow, morphologically free of tumor was harvested (total 110 X 10(8) nucleated cells) and concentrated to a mononuclear cell fraction with a total of 10 X 10(8) cells . After in vitro purging with the stable metabolite of 4-hydroperoxycyclophosphamide ASTA Z 7654 (40 micrograms/2 X 10(7) mononuclear cells/ml), the mononuclear cell suspension was retransfused 10 hours following the application of high dose melphalan . Hemopoietic reconstitution was delayed with a platelet count reaching 70,000/microliter only after seven months . At the time of this writing (20 months after diagnosis and 16 months after autologous bone marrow transplantation) there is no evidence for active disease according to the bone scan and multiple bone marrow biopsies . In view of the dismal prognosis of patients with neuroblastoma, stage IV it is recommended that further patients should be treated with a slightly modified protocol of the cooperative austrian neuroblastoma study.

Cancer Immunol Immunother, 1986, 23(1), 51 - 5
Isolation and dissociation of immune complexes from pleural effusions of lymphoma patients; Patel GV et al.; Immune complexes (IC) isolated from pleural effusions of lymphomas with favorable and unfavorable prognoses were of IgG type . These IC were further dissociated by ion exchange chromatography using 8 M urea . The antibody was found to be a high molecular weight protein (1.5 X 10(5) daltons) and reacted with antihuman IgG immunologically while a second peak obtained on ion exchange chromatography may be an antigen moiety with a molecular weight of 3.2 X 10(4) daltons as it reacted immunologically with the antibody . Strong cytoplasmic fluorescence was observed with various cell suspensions of lymphomas when reacted with the antibody preparations . The antisera raised against two different antigen fractions prepared from two lymphomas--nHL and LL showed positive fluorescence with both nHL and LL suspensions . The absorption of these rabbit antibodies with individual cell extracts or with antigen preparations also entirely blocked the cytoplasmic staining . The antigen moiety (PK-II) may have a common origin in the disease process . Pleural effusions from patients with unfavorable and favorable prognoses showed identical patterns of separation of IC components.

Acta Diabetol Lat, 1986 Jan-Mar, 23(1), 43 - 9
A technique for the isolation of highly viable pancreatic B-cells from ob/ob mice; Norlund LK; A method has been developed to prepare free islet cells in suspension from adult ob/ob-mice . About 200 collagenase-isolated pancreatic islets were pooled in 4 ml of calcium-free Krebs-Ringer-HEPES buffer supplemented with 1 mM EGTA and 10 micrograms/ml DNAase . The islets were gently shaken in a water-bath for 10 min at 30 degrees C . Then, the cell suspension was filtered through a nylon screen and centrifuged through ice-cold, dense albumin . The isolated cells, of which more than 99% were B-cells, appeared well preserved both in light- and electron-microscopy . Out of the isolated cells, 7.1 +/- 0.5% took up Evans Blue and were thus considered non-viable.

Histochemistry, 1986, 84(2), 131 - 8
Long-term culture of renin containing tissue; Minuth WW et al.; Thin cortical tissue explants from kidneys of hydronephrotic mice were excised and incubated in different culture media containing growth and proliferation factors . Over a period of several months the content of renin in the explants and in the culture medium was repeatedly measured, to define the conditions necessary for the maintenance of renin production in a long-term culture . The best results were obtained when culturing the renal tissue in Dulbecco's medium (DMEM) with 10% fetal calf serum, 6 units/100 ml platelet-derived growth factor and 200 ng/ml glycylhistidyllysine . Renin was still present within the cells and in the culture medium after more than six months . Prevention of dedifferentiation, as evidenced in this case by the maintenance of renin production, seemed to be dependent on specific extracellular matrix proteins of renal origin . If the explants were dissociated from their matrix components by collagenase, a gradual loss of renin production was observed within 5 days . Complementation of the collagenase-digested cell suspension with different nonrenal extracellular matrix materials did not afford the stabilizing effect of the original pericellular matrix.

Ontogenez, 1986 Jan-Feb, 17(1), 27 - 36
{The stromal colony-forming cell (CFUf) count in the bone marrow of mice and the clonal nature of the fibroblast colonies they form}; Latsinik NV et al.; The clonal nature of FCFC-derived stromal colonies was tested by chromosomal analysis in mixed cultures of CBA and CBAT6T6 bone marrow cells depleted of macrophages and myeloid cells . Inoculation of the bone marrow cell suspensions in flasks coated with poly-l-lysine has revealed practically no stromal aggregates among the explanted cells . The coincidence of karyotypes within the stromal colonies in the mixed cultures proved that the FCFC-derived colonies were cell clones . It was shown by indirect immunofluorescence with antibodies to type 1 collagen that the mouse bone marrow FCFC-derived colonies consisted of stromal fibroblasts . The cloning efficiency of the bone marrow FCFS depends on the explantation density of cells; a stable colony-forming efficiency could be reached only in the presence of feeder cells (irradiated bone marrow) . In the bone marrow cells suspensions obtained by trypsinization the amount of FCFC is markedly higher than in the suspensions of mechanically disaggregated bone marrow cells.

Appl Environ Microbiol, 1986 Jan, 51(1), 91 - 4
Evaluation of carriers used in the test methods of the Association of Official Analytical Chemists; Ascenzi JM et al.; Revision of the official test method for the determination of the tuberculocidal activity of disinfectants is being undertaken . The current procedure lacks precision and accuracy and is not quantitative . Variability associated with carriers and the lack of temperature control were evaluated in this paper . The use of porcelain versus stainless steel carriers was also evaluated . When carriers of either type were contaminated with Mycobacterium bovis BCG, the number of organisms on the carriers varied by as much as 1.0 on the log10 scale . The average number of organisms attached to each porcelain carried was 1.10 x 10(5) CFU (range, 2.7 x 10(4) to 2.7 x 10(5) CFU), whereas the average number of organisms attached to each stainless steel carrier was 1.38 x 10(5) CFU (range, 2.9 x 10(4) to 4.0 x 10(5) CFU) . The average number of cells attached to the carrier was directly proportional to the number of cells in the contaminating cell suspension . Variations in drying time did not alter the number of cells attached to the carrier . When porcelain carriers were placed in a test solution, the average number of organisms washed from the carriers was 55% of the total, with a range of 19 to 80%, whereas for stainless steel carriers, the average number was 82% of the total, with a range of 52 to 96% . Data for B . subtilis spores were similar to those for M . bovis BCG, suggesting that there may be similar problems with the Association of Official Analytical Chemists sporicidal test, which uses carriers . It was also found that the lack of an exacting temperature control could influence the outcome of the test . Changes in temperature as little as 1 degree C could influence the rate of killing of M . bovis BCG.

Int Arch Allergy Appl Immunol, 1986, 79(3), 296 - 304
Effects of cyclosporin A, antilymphocyte serum and donor-specific transfusions on murine delayed-type hypersensitivity and skin graft survival; Mottram PL et al.; The effect of immunosuppressive reagents cyclosporin A (CsA) and rabbit anti-mouse antilymphocyte serum (ALS) on the response to alloantigens was studied in inbred mouse strains . Alloantigen was given either as a cell suspension which induced a delayed-type hypersensitivity reaction (DTH), or as a full-thickness skin graft . Dose-response studies showed that DTH reactions in CBA mice sensitised to BALB/c cells were reduced to background levels when recipient mice were treated with 100 mg/kg CsA on days 0, 4 and 6 after primary alloantigenic challenge . The response to a second challenge was significantly decreased by CsA treatment during primary or secondary exposure to alloantigen and CsA was as effective as ALS in abrogating both primary and secondary DTH reactions . Survival of full-thickness grafts of BALB/c skin on CBA mice was increased from 9 to 23 days by ALS treatment on days -1 and +2, with grafts given on day 0 . Long-term treatment with CsA, from day -14 to +12, also prolonged graft survival from 9 to 18 days but donor-specific transfusions, with or without concomitant ALS or CsA treatment, decreased graft survival and often sensitised the recipients . This occurred with transfusions administered from -63 to -7 days and on the day of grafting . Thus, in H-2 mismatched mice, both CsA and ALS treatments produced a state of tolerance when administered during short-term exposure to alloantigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Biochem, 1986, 18(2), 109 - 13
Effect of extracellularly generated free radicals on the plasma membrane permeability of isolated pancreatic B-cells; Grankvist K et al.; Previous experiments on alloxan diabetogenicity suggest that alloxan increases the permeability of B-cell plasma membranes by generation of noxious free radicals . Whether the radicals are generated intra- or extracellularly has however been disputed . To test if extracellularly generated free radicals could decrease trypan blue exclusion of dispersed islet cells, a radical-generating solution of xanthine oxidase/hypoxanthine was employed . The solution increased dye uptake by cells in the cell suspension . Superoxide dismutase and catalase but not scavengers of hydroxyl radicals protected against the increase in dye uptake . Both L- and D-glucose protected the cells from injury . It is concluded that extracellularly generated free radicals induce damage to the plasma membrane of islet cells . The result strengthens the hypothesis of plasma membrane damage by extracellularly generated free radicals as the primary event in alloxan diabetogenicity and may provide a link for explanation of damage caused by islet inflammation in juvenile diabetes.

Cytometry, 1986 Jan, 7(1), 8 - 17
Cytopress: automated slide preparation of cytologic material from suspension; Oud PS et al.; This paper describes a new automated system to prepare slides of cytological material from suspension . The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure-fixation . Using cervical cells as a model, results show that a well-defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate . Contamination is very small . Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown . In general, more than one slide can be made from one sample . Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way . This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.

Acta Cytol, 1986 Jan-Feb, 30(1), 70 - 4
Use of the cytocentrifuge for electron microscopy investigations; Lorenzetti E et al.; A simple method of cytocentrifugally processing cell suspensions for conventional and histochemical investigations at the ultrastructural level is described . Fixed sediments from cell-poor suspensions are resuspended in an albumin-buffered solution . A few drops of the albumin cell solution are cytocentrifuged, leaving a cell disc on a plastic support . A brief dipping in a paraformaldehyde-glutaraldehyde mixture transforms the cell disc into a compact thin fragment attached to the plastic support . Cytocentrifugation of cell-rich suspensions, on the other hand, produces a thicker cell disc, which can be easily detached from the plastic slide . In both cases, the postfixation, dehydration and infiltration are directly carried out on the cell disc . The present method is particularly useful for the ultrastructural study of cell-poor suspensions and can also be performed on cell suspensions previously stained with several histochemical procedures.

J Biochem Biophys Methods, 1986 Jan, 12(1-2), 81 - 8
Preparation of suspensions of pancreatic islet cells: a comparison of methods; Kohnert KD et al.; A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase . Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method . The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature . With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92% . Cell viability (dye exclusion) was more than 90% . Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods . Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment . Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.

J Immunopharmacol, 1986, 8(3), 289 - 97
Angiotensin II suppression of human mononuclear cell reactivity is associated with enhanced OKT8+ lymphocyte thymidine incorporation; Simon MR et al.; Recent evidence suggests that angiotensin II may participate in the regulation of inflammation . We previously reported that angiotensin II inhibits human peripheral blood mononuclear cell reactivity and acts directly on lymphocytes . These observations are again confirmed . In addition, purified OKT8+ but not OKT4+ T cell suspensions stimulated with phytohemagglutinin revealed increased thymidine incorporation when simultaneously cultured for 48 hours with angiotensin II . These findings suggest that angiotensin II may inhibit mononuclear cell thymidine uptake through stimulation of suppressor lymphocytes contained within the OKT8+ subpopulation.

Cell Immunol, 1986 Jan, 97(1), 51 - 8
Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice . II . B-cell subsets: enteric antigen exposure is associated with immunoglobulin isotype switching by Peyer's patch B cells; Carlson JR et al.; The aim of this study was to quantify the response of Peyer's patch B cells, surface IgA-bearing (sIgA) B cells, and surface IgM-bearing (sIgM) B cells to Giardia muris infection . Following infection of a cohort of immunocompetent BALB/c mice with G . muris cysts, Peyer's patch cell suspensions were prepared at serial time points during the infection, incubated with fluorescein-conjugated monoclonal antibodies directed against murine leukocytes, B cells, sIgA B cells, sIgM B cells, or T cells, and analyzed by flow cytometry . Of total Peyer's patch leukocytes, the percentages of B cells, sIgA B cells, and sIgM B cells in uninfected BALB/c mice were 64.7 +/- 2.0% (mean +/- SEM), 30.3 +/- 1.5%, and 52.5 +/- 2.4%, respectively . The total number of Peyer's patch leukocytes increased significantly (1.8 X) during G . muris infection, and returned to control levels as the infection was cleared . The percentages of Peyer's patch T and total B cells did not change significantly during Giardia infection . However, sequential changes were observed in the percentages and numbers of sIgM and sIgA B cells during the infection . Peyer's patch sIgM B cells rapidly increased in percentage and number, reaching maximum levels 1 week after cyst inoculation . After remaining constant the first week, the number of Peyer's patch sIgA B cells increased during the second week of G . muris infection, reaching a maximum level 11-14 days after cyst inoculation . The data support the hypothesis that immunoglobulin isotype switching in Peyer's patches is induced by antigen exposure.

Cancer Immunol Immunother, 1986, 22(1), 31 - 6
Treatment of adenocarcinoma in the peritoneum of mice: chemoimmunotherapy with IL-2-stimulated cytotoxic lymphocytes as a model for treatment of minimal residual disease; Salup RR et al.; We have used a transplantable murine adenocarcinoma of renal origin (Renca) introduced to the abdomen by i.p . injection of a tumor cell suspension, to study the therapeutic potential of adoptive immunotherapy and/or biological response modifiers (BRMs) . This tumor model is therapeutically challenging since the tumor grows progressively resulting in extensive peritoneal carcinomatosis, with hemorrhagic ascites, metastases to abdominal lymph nodes, liver, most serous membranes, spleen, and in some animals, pulmonary metastases . Without therapy, death occurs invariably in 36 +/- 3 days . In vitro, the tumor is lysed by lymphocytes obtained from the peritoneal cavity of mice treated with human recombinant interleukin-2 (rIL-2) and by cytotoxic lymphocytes stimulated by in vitro culture with human rIL-2 . Treatment of i.p . Renca with a single i.p . injection of the chemotherapeutic agent doxorubicin hydrochloride (DOX), or adoptive transfer of in vitro stimulated cytotoxic lymphocytes together with rIL-2 cured 50% and 20% of the tumor-bearing mice, respectively . In contrast, combined therapy with DOX and adoptive transfer of in vitro stimulated cytotoxic lymphocytes and rIL-2 cured the majority (90%) of tumor-bearing mice . These results suggest that administration of immunotherapy with in vitro activated cytotoxic cells together with human rIL-2 substantially enhances the effectiveness of chemotherapy.

Agents Actions, 1986 Jan, 17(3-4), 390 - 1
Augmentation of thromboxane production in vitro by polymorphonuclears and macrophages exposed to IL1/LP; Conti P et al.; Human peripheral blood polymorphonuclear leukocytes (PMNs) and murine peritoneal macrophages (M phi) were stimulated to generate thromboxane upon treatment with highly purified human interleukin 1/leukocytic pyrogen (IL1/LP) at various concentrations . Thromboxane B2 was measured by radioimmunoassay in the cell-free supernatants of cell suspensions after 1 hour incubation at 37 degrees C . Thromboxane B2 amounts increased in a way which depended on the dose of IL1 added to the cell cultures.

Int Arch Allergy Appl Immunol, 1986, 79(2), 164 - 8
Cyclosporin A-induced suppression of ongoing IgE antibody formation in the mouse; Okudaira H et al.; Persistent anti-ovalbumin (OA) IgE antibody formation in the mouse was suppressed by oral administration of cyclosporin A (Cy A) . Spontaneous anti-OA IgE antibody formation in vitro was also suppressed by Cy A added to the culture . Anti-OA IgG antibody responses in vivo and in vitro were less affected by Cy A . Cy A-induced immunosuppression was T cell-dependent since removal of T cells from the immune spleen cell suspension abolished the Cy A-induced suppression of antibody formation . Supplementing normal spleen T cells resulted in recovery of Cy A-induced suppression of spontaneous antibody formation in vitro . Cy A-induced suppressor T cells carried both Lyt 1 and Lyt 2 surface markers.

Ann Intern Med, 1986 Jan, 104(1), 44 - 7
T-cell intestinal lymphoma associated with celiac sprue; Loughran TP Jr et al.; A patient with a long history of celiac sprue developed a pleomorphic intestinal lymphoma . Cell suspensions of tumor cells and immunoperoxidase labeling of cell surface antigens in frozen tissue sections clearly showed the lymphoma to be of T-cell origin, despite the presence of other nontumor cells bearing enzyme markers of histiocytes . These findings may have important implications on the cellular origin of lymphomas developing in patients with celiac sprue.

Ann N Y Acad Sci, 1986, 465, 427 - 34
Radionuclides in detecting active granuloma formation . Gallium-67 scintigraphy and histopathology with autoradiographic findings; van Maarsseveen A et al.; Granuloma formation studies were performed on lungs of guinea pigs sensitized with FCA over 2 to 17 months . Prolonged time of sensitization revealed more granulomatous pulmonary tissue . An intravenous booster of FCA in the animals that had been sensitized for 3 months yielded enhanced granuloma formation within 5 days . The histopathology of these lungs was comparable with that seen in lungs of animals after 17 months of sensitization without booster . Enhanced local proliferation of macrophages, measured by {3H}thymidine incorporation and autoradiography, was seen in the lungs of the animals that had received boosters . Moreover, 67Ga scintigraphy was strongly positive in these animals . Scintigraphy of cell suspensions of pulmonary tissue from these animals showed that 67Ga was predominantly taken up (quantitatively as well as qualitatively) by the alveolar macrophages . Cell suspensions of sarcoidosis patients, prepared in the same way, showed only a low level of 67Ga uptake, one comparable to that of the pulmonary cell suspensions of the sensitized animals that had not received boosters . It is suggested that a negative scintigraphy in patients with chronic pulmonary granulomatous disorders could be (partly) explained by the absence of activated macrophages.

J Natl Cancer Inst, 1986 Jan, 76(1), 151 - 8
Clonal origin of tumors induced by ultraviolet radiation; Burnham DK et al.; Skin tumors were induced in (C3H/HeN X C3H/HeN-PGK-1a)F1 female mice, heterozygous at the X-linked phosphoglycerate kinase-1 (PGK-1) locus, by exposure to the carcinogenic influence of ultraviolet radiation (UVR), 3-methylcholanthrene {(MCA) CAS: 56-49-5}, or benz{a}pyrene {(BP) CAS: 50-32-8} . An assessment of the clonal origin of these tumors was accomplished through an analysis of the PGK-1 enzyme phenotype expressed by the transformed cells . In vitro culture was employed as a means of depleting nontransformed cells of host origin from the induced tumors . Cultured lines derived from tumors induced by each of the above agents were found to express only one of the two enzyme forms encoded by the host genotype, consistent with the probability that all the UVR-, MCA-, and BP-induced tumors examined in this study were monoclonal in origin . For further substantiation of the monoclonality of UVR-induced tumors, 2 UVR-induced tumors were enzymatically dissociated immediately following excision from the primary hosts, and the resulting cell suspensions were cloned in soft agar . Upon analysis, each set of clones selected in soft agar expressed only a single PGK-1 enzyme form . To rule out the possibility that the apparent monoclonality of culture-adapted tumor lines was due to in vitro selection, tumors that arose in UVR-treated PGK-1a/b female heterozygote mice were transplanted into PGK-1a and PGK-1b homozygous recipients . These transplanted tumors expressed a single PGK-1 allozyme following growth in recipients that were genetically homozygous for the major PGK-1 enzyme form expressed by the tumor prior to transplantation . These data strongly support the concept that most, if not all, UVR-induced tumors are derived from the progeny of a single transformed cell . This observation is important to the understanding of the nature of tumor-specific transplantation antigens expressed by individual UVR-induced tumors and indicates that such antigens also are clone specific . In addition, the results of this study indicate that polyclonality does not play a significant role in the generation of cellular and phenotypic heterogeneity known to be present within individual UVR-induced tumors.

Dev Genet, 1986, 7(2), 99 - 108
Regulation of late gene expression in a temperature-sensitive cohesion-defective mutant of Dictyostelium discoideum; Saxe CL 3rd et al.; We have analyzed the expression of a series of developmentally regulated genes in the Dictyostelium discoideum strain JC-5 . This strain has been previously described as a temperature-sensitive, cohesion-defective derivative of FR17, itself a temporally deranged mutant of wild-type NC-4 . At restrictive temperature (27 degrees C), JC-5 initially acquires EDTA-resistant cell contacts but at the time of tip formation (12 hr) loses the ability to make specific cell-cell associations and regresses to an amorphous mound of cells . WE have found that genes preferentially expressed in either prespore or prestalk cells are expressed prior to the appearance of the cohesion defect in JC-5; the specific cell contact system defective in this strain is necessary for neither the proper initiation nor maintenance of expression of either prespore of prestalk genes . We have also found, by use of an in vitro cell suspension system, that JC-5 is temperature-sensitive with respect to gene expression several hours before the defect in cell cohesion is observable . Our data suggest that the defect in JC-5 is due to a specific lesion not in the late cohesion system but rather in a more general component that is required earlier in the developmental process.

J Neurosci Res, 1986, 16(2), 387 - 96
Glial culture on artificial capillaries: electron microscopic comparisons of C6 rat glioma cells and rat astroglia; Tiffany-Castiglioni E et al.; The functional association of astroglial footplates with blood vessels is important because astrocytes may provide a channel between the blood and neurons deeper in the brain parenchyma for the passage of ions and metabolites . This hypothesized function is very difficult to study in vivo or in monolayer cultures . We have produced a three-dimensional cell culture model of perivascular astroglia by means of an artificial capillary system . Conventional primary cultures of astroglia were first prepared from neonatal rat cerebral hemispheres in 75-cm2 tissue culture flasks . After 25 days, the cells were seeded in Amicon Vitafiber hollow fiber culture vessels . Direct seeding of brain cell suspensions was not successful . A culture unit consists of a bundle of hollow, semi-permeable polysulfone fibers encased in a plastic shell . The fibers were coated with fibronectin and bovine serum albumin, and astroglia were seeded on their outer surfaces . Warmed medium was pumped through the lumina of the fibers . After 13 days the cells were fixed with paraformaldehyde and examined . Scanning electron microscopy revealed the tubes to be uniformly covered with astroglia with short processes that contacted nearby cells . Transmission electron microscopy showed glial filaments and gap junctions . Astrocyte cultures were compared morphologically to C6 rat glioma cells in hollow fiber culture . The astrocytes formed a monolayer, whereas C6 cells formed a stratified culture . Furthermore, C6 cells did not form gap junctions . Astrocytes have been hypothesized to take up K+ discharged to the extracellular space by depolarizing neurons and move it to areas of low concentration, i.e., to act as a K+ spatial buffer . Our culture system should permit direct testing of this hypothesis.

Brain Res, 1986 Jan, 389(1-2), 77 - 84
Intrastriatal grafting of dopamine-containing neuronal cell suspensions: effects of mixing with target or non-target cells; Brundin P et al.; The effects of target and non-target cells on the growth and function of intrastriatal grafts of mesencephalic dopamine neurons have been studied in rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal dopamine pathway . Cell suspensions of ventral mesencephalon from 14-15-day-old rat fetuses (rich in developing dopamine neurons) were either grafted alone or grafted after mixing with equivalent numbers of cells obtained from the striatum (a major dopamine target area) or spinal cord (a non-target area for the mesencephalic dopamine neurons) . The combined mesencephalic and striatal grafts gave rise to a greater area of dense innervation in the host caudate-putamen than grafts of mesencephalic cells alone or grafts of mesencephalic cells mixed with spinal cord cells . The number of surviving catecholamine-containing neurons did not differ significantly in the different types of grafts . In addition, there was an altered outgrowth pattern in the combined mesencephalic-striatal grafts consisting of small round islands of intensely fluorescent catecholamine-containing fibres, often in close association with the grafted dopamine neurons . In a subsequent biochemical study it was found that combined mesencephalic-striatal grafts exhibited dopamine levels and turnover that did not differ from grafts containing mesencephalic cells only . The mesencephalic-striatal cografts showed a trend toward enhanced behavioural effect, in terms of greater reduction in amphetamine-induced rotation asymmetry, when compared to other graft groups . It is suggested that the addition of embryonic striatal target cells can exert stimulatory effects on morphological development, and possibly functional parameters, of fetal dopamine cells also in vivo after intrastriatal grafting.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differ, 1986 Jan, 18(1), 17 - 26
Mode of action of erythropoietin and glucocorticoids on the hepatic erythroid precursor cells: role of prostaglandins; Mayeux P et al.; The hypothesis that prostaglandins, and especially PGE2, are the second messengers of erythropoietin (Ep) and that glucocorticoids inhibit Ep action by inhibiting PG synthesis was tested on the erythroid cell line from fetal rat liver . The optimal (10(-9) M) stimulatory concentration of PGE2 did not reproduce, by far, the maximal effect of Ep on the growth of CFUE erythroid colonies . Ep did not increase PGE2 release in liquid culture media of cell suspensions made of the whole erythroid line or enriched (over 85%) in precursor cells . Ep did not modify the turnover rate of arachidonate . Nevertheless, indomethacin partially inhibited Ep effect on CFUE development, and this inhibition was abolished by PGE2 . These results suggest that PGE2 potentiates Ep action but is not its second messenger . Spontaneous PGE2 release in liquid culture media brought about concentrations of the order of 10(-9) M, and 10(-7) M dexamethasone completely inhibited this release . Part of (but not all) the anti-Ep effects of glucocorticoids might thus be mediated this way . Dexamethasone effects required previous protein synthesis.

Differentiation, 1986, 33(2), 111 - 20
Altered cyclic-AMP receptor activity and morphogenesis in a chemosensory mutant of Dictyostelium discoideum; Barclay SL et al.; The functional properties of the cell-surface cyclic-AMP receptor that controls chemotaxis were found to be altered in an aggregation mutant of Dictyostelium discoideum . The mutant aggregated without stream formation and had a tenfold increased cell-density requirement for the initiation of aggregation . After aggregation, mounds formed multiple tips and subsequently subdivided to give multiple fruits that were small and abnormally proportioned . Cyclic-AMP-induced light-scattering changes in cell suspensions indicated that the mutant had a diminished response to external cyclic-AMP signals . Associated with these altered functional responses was a physical change in the cyclic-AMP sensory system . Cyclic-AMP-binding studies showed that the parent had two classes of cyclic-AMP binding sites, i.e., Kd = 32 and 110 nM . In contrast, the mutant had two- to threefold or more high-affinity sites (Kd = 25 nM) and altered low-affinity sites (Kd less than 3 microM) . These results indicate that both affinity classes of binding site are independently mutable . This observation suggests that the two affinity classes can be interconverted by mutation, or the mutation alters a single molecular species and its equilibrium between binding sites with different affinities for cyclic AMP, as postulated in receptor cycling models.

Biomed Biochim Acta, 1986, 45(10), 1227 - 36
Maturation of rabbit reticulocytes: strong decline of the turnover of polyphosphoinositides; Maretzki D et al.; The {32P}phosphate labelling of ATP and polyphosphoinositides (PI) of intact rabbit reticulocytes and mature erythrocytes was compared . Despite the fact that reticulocytes have a 30-fold higher turnover of ATP than mature erythrocytes, the 32P-labelling of the cellular ATP pool was identical . The 32P-Pi entry into the cells is the rate-limiting step for 32P-phosphate isotopic equilibration of the ATP-pool and is independent of maturation . The rates of 32P-phosphate incorporation into PI-4,5-P2 and into PI-4-P, respectively, were 17-fold and 8-fold higher in reticulocyte-rich cell suspensions than in erythrocytes . The specific radioactivity of PI-4,5-P2 labelling reaches 55% and that of PI-4-P 40% of the ATP specific activity . However, a rapid isotopic equilibration of the polyphosphoinositides, was found . These findings indicate the existence of a heterogeneity of polyphosphoinositide pools . Reticulocyte membranes lose about 30% of their polyphosphoinositides during maturation proportional to the total loss of phospholipids.

J Membr Biol, 1986, 93(1), 33 - 42
Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange; Montrose MH et al.; Suspensions of LLC-PK1 cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF . Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal . They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na+ and K+ gradients comparable to those of cells in monolayer culture . The steady-state intracellular pH (7.05 +/- 0.01, n = 5) of cells which have recovered in (pH 7.4) Na+-containing medium is not affected over several minutes by addition of 100 microM amiloride or removal of extracellular Na+ (Na+o less than 1 mM) . In contrast, when the cells recover from an acid load (caused by NH4 preincubation and removal), the recovery is largely Na+ dependent and is sensitive to 100 microM amiloride . These results suggest that with resting pH near neutrality, both Na+o/H+i and Na+i/H+o exchange reactions are functionally inactive (compared to cellular buffering capacity) . In contrast, Na+o/H+i exchange is activated by an increased cellular acid load . This activation may be observed directly either as a stimulation of net H+ efflux or net Na+ influx with decreasing intracellular pH . The extrapolation of this latter data suggests a "set point" of Na+/H+ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.

Int J Tissue React, 1986, 8(3), 199 - 203
Effect of l-carnitine on human neutrophil activity; Schinetti ML et al.; The effect of l-carnitine on human neutrophil oxidative metabolism was investigated, both on superoxide production and luminol amplified chemiluminescence (CL) in phorbol-myristate-acetate (PMA) stimulated cells . L-carnitine either preincubated 10 minutes with the cells, before PMA challenge, or added simultaneously to the stimulator, inhibited superoxide generation . When tested in an O2- -generating system, such as xanthine-xanthine oxidase, l-carnitine did not act as an O2- scavenger . On the PMA induced CL response the drug was ineffective as an inhibitor, if preincubated with the cell suspension before activation . When added together with PMA, l-carnitine significantly inhibited the CL . Taken together the results reveal that the drug might affect the interaction of PMA with its specific receptor in human neutrophils, that is protein kinase C.

Vet Med Nauki, 1986, 23(6), 8 - 11
{Use of a microvirus-neutralizing reaction in diagnosing transmissible gastroenteritis (TGE)}; Belopopska P et al.; A micro virus-neutralization reaction was designed and tested to detect antibodies to the virus of transmissive gastroenteritis . Use was made of a stable cell line, SPEV, and a laboratory strain of the virus that had been adapted to it . The optimal concentration values of the cell suspension and the normal calf serum contained in it were determined . A total of 90 blood serum samples from pigs were comparatively investigated for the presence of virus-neutralising TGE antibodies, the tube test being performed trough the inoculation of the cells in suspension . On the other hand, the micro virus-neutralization test was carried out in two variants: the sera were diluted via Mikrotiter micropipettes and micropipettes of the Takachi apparatus . It was found that the micro virus-neutralization test in its two variants was not inferior in terms of sensitivity to the tube test, was more readily applicable, and was less material consuming.

Leuk Res, 1986, 10(8), 973 - 88
Differences in the plasma membrane glycoproteins of cultured myeloblastoid and promyelocytic human leukemia (HL60) cells; Peyman JA et al.; Since the mechanisms that control synthesis of surface and internal granule membranes are closely-related within the Golgi apparatus, we have compared the plasma membrane proteins and glycolipids of cells of the human promyelocytic line HL-60 with those of its agranular myeloblastoid variant (HL60-D), and of other human myeloid lines (KG-1a, KG-1 and ML-2) . Proteolytic degradation by granule enzymes altered the protein profiles unless multiple inhibitors were included in the cell suspension before lysis and during subsequent handling of the extracts . Polyacrylamide gel electrophoretic profiles of the proteins accessible to lactoperoxidase-catalyzed 125I-labeling or to periodate {3H}-borohydride labeling, as well as those of the glycoproteins bound to and eluted from immobilized concanavalin A, showed distinct patterns . The apparent molecular weights of the two major sialylated glycoproteins were larger in cell lines with a greater content of azurophilic granules . Also, the blastic line incorporated less fucose into glycolipid and contained less complex gangliosides and neutral glycolipids than did the parent . These data demonstrate that, within the limits of this culture model, cells capable of cytoplasmic granule production express a different constellation of surface components.

Acta Radiol Oncol, 1986 Jan-Feb, 25(1), 63 - 9
Changes in energy metabolism following roentgen irradiation of in vivo growing Ehrlich ascites tumour cells studied by 31P magnetic resonance spectroscopy; Skog S et al.; The energy metabolism in Ehrlich ascites tumour cells following in vivo irradiation of a dose of 5.0 Gy was studied in vitro in their ascites fluid up to 48 hours using 31P magnetic resonance spectroscopy measuring ATP, ADP and inorganic phosphate (Pi) . The results are also related to radiation induced changes in cell cycle composition . ATP was reduced by more than 50 per cent 20 to 24 hours after irradiation but normalized at 48 hours . ADP was reduced to about half the normal level 24 to 48 hours after irradiation . When the ATP and ADP had reduced levels, the inorganic phosphate increased correspondingly . Addition of glucose to the ascites cell suspension at the time of minimum ATP level immediately raised the ATP:Pi ratio . Since the glucose concentrations in blood and in ascites fluid following irradiation were also reduced, lack of glucose for energy production might have been a major contributing factor for the reduced ATP production.

J Exp Pathol, 1986 Summer, 2(4), 229 - 45
Cellular immunity and lymphocyte populations in developing uremia in the rat; Raskova J et al.; Changes in cellular immunity and in lymphocyte populations have been studied in rats developing chronic renal insufficiency following 5/6 nephrectomy . Animals remain stable for a period of six months (BUN 40-60 mg/dl); then BUN slowly increases for 2-3 months, followed by rapid deterioration and death of the animals . Skin allotransplants showed no change in survival when transplanted fifteen weeks after nephrectomy; when transplanted 22 weeks and later after surgery, their survival was prolonged . The response of splenic cells in the mixed lymphocyte reaction (MLR) was unchanged for 15 weeks after surgery but became significantly reduced after 20 weeks . At the same time we observed an increased suppressor cell activity in splenic cell suspensions and an inhibitory effect of the uremic serum in the MLR . Resistance to tumor induction by syngeneic adenovirus 12-transformed cells was decreased in the late stages of uremia as measured by tumor development in these animals . Induction of cytolytic T cells in vitro was reduced at 24 weeks after operation; at 30 weeks virtually no cytolytic T cell activity was induced . There was also a decrease in natural killer cell activity in the late stages of uremia . These changes in immune response were correlated with the analysis of the lymphocyte sub-populations by staining with monoclonal antibodies and flow cytometry . During the development of uremia no significant changes were found in the lymph nodes . The thymus underwent a severe involution 20 weeks and later after nephrectomy . In the peripheral blood there was a significant decrease in the numbers of helper T cells . The helper T cell subset was also sharply reduced in the spleen of uremic rats at 20 weeks and later after operation.

Arch Dermatol Res, 1986, 278(3), 199 - 205
The presence of IgE molecules on epidermal Langerhans cells in patients with atopic dermatitis; Bruynzeel-Koomen C et al.; Skin sections of clinically involved and clinically normal-looking skin from patients with atopic dermatitis were incubated with anti-human IgE antibodies using the indirect immunoperoxidase technique . Apart from positive dermal anti-IgE staining, positive epidermal anti-IgE staining was also observed . The morphology of the epidermal staining cells suggested the involvement of dendritic cells . This was confirmed by positive immuno-double labelling with OKT6 and anti-IgE . This phenomenon seemed to be specific for atopic dermatitis since skin sections from normal non-atopic controls, patients with allergic asthma, contact dermatitis, and schistosomiasis showed no epidermal anti-IgE staining . To further elucidate the nature of the epidermal anti-IgE staining cells, epidermal cell suspensions were prepared from clinically involved skin from patients with atopic dermatitis . These cell suspensions also showed positive anti-IgE staining cells and positive immuno-double labelling with OKT6 and anti-IgE . Immunogold electron microscopy with anti-IgE on epidermal cell suspensions from patients with atopic dermatitis showed gold particles on the cell membranes of cells containing Birbeck granules, being Langerhans' cells . Epidermal cell suspensions from normal non-atopic controls were negative . The presence of IgE molecules on epidermal Langerhans' cells, which seems to be specific for patients with atopic dermatitis, provides an explanation for the high frequency of positive patch test reactions to inhalant allergens.

Allerg Immunol (Leipz), 1986, 32(2), 111 - 21
Effects of islet cell surface antibodies on isolated neonatal rat islets of Langerhans in vitro; Schroder D et al.; Islet cell surface antibodies (ICSA) were raised by immunization of rabbits with viable neonatal rat pancreatic islet cell suspensions . By absorption with liver powder and spleen cells unspecific cytotoxic components were removable from the sera . Cytotoxic effect of islet cell antiserum against islet cells was indicated by 51Cr-release . In dependence on serum concentration beta-cell damage induced by ICSA-positive serum was detectable by insulin leakage from neonatal rat islets of Langerhans . The immune cytolytic effect of rabbit anti-rat islet cell surface serum was accompanied by morphological alterations of the islet surface structure as revealed by scanning electron microscopy . Pretreatment of pancreatic islets with cytotoxic islet cell antiserum results in an increased insulin leakage even after the removal of the cytotoxic conditions suggesting the persistence of cell membrane lesions . Insulin secretion of islets of Langerhans first exposed to cytotoxic ICSA-positive serum is still stimulated by 15 mmol/1 glucose plus 0.1 mmol/1 3-isobutyl-1-methylxanthine (IBMX) but taking into account the increased insulin leakage the secretory capacity of these islets is significantly diminished.

Ann Inst Pasteur Immunol, 1986 Jan-Feb, 137C(1), 3 - 10
Interleukin induction of non-specific suppressor cells; Duclos H et al.; We analysed the cellular and molecular signals involved in the induction of non-specific T suppressor (Ts) cells generated in mouse spleen cell suspensions after 3-4 days' culture in the absence of conventional antigen . Using an in vitro model of primary antibody response as a suppressor assay, we showed that purified T cells were unable to generate suppressive activity . This suppression was fully restored when T cells were cultured with macrophages . In this case, Ts-cell generation was markedly increased by the addition of indomethacin suggesting a negative effect of prostaglandins in this phenomenon . Macrophages could be replaced by semi-purified IL1 as well as by recombinant IL2 . Thus, in conditions of severe macrophage depletion, IL1 and/or IL2 are fully capable of inducing Ts-cell generation.

Int J Immunopharmacol, 1986, 8(8), 961 - 6
The effect of cyclophosphamide in vivo on the expression of lymphocyte markers, detected by monoclonal antibodies, in the rat; el-Sady E et al.; Surface markers on lymphocytes from the thymus, lymph node and spleen of the rat were examined in single cell suspensions using a panel of monoclonal antibodies . These were used particularly to investigate Pan T, TH (T-helper), TS/C (T-suppressor-cytotoxic) and Ia antigens . The expression of these markers in rats treated with a single dose of cyclophosphamide (100 mg/kg body weight) was followed for 3 weeks after treatment . Maximum changes were detected at 3 days and recovery took up to 3 weeks to near completion . Comparison was made with histological observations of the effect of CY on these organs . At 3 days, the thymus showed maximum weight loss and gross cortical depletion . This associated with significant drop in the expression of the TS/C marker on small and large lymphocytes . Regeneration of the cortex, beginning at 7 days, was associated with the presence of many large pyroninophilic cells . This was accompanied by an increase in the expression of the TS/C marker on both small and large lymphocytes . The mesenteric lymph node showed marked depletion of the B-cell areas at 3 days . There was also a significant drop in TS/C and Ia expression and a marked rise in Pan T and TH . TS/C expression recovered rapidly with a rebound at 7 days . However, the expression of Pan T and TH did not return to normal until 21 days . In the spleen there was a similar decrease in the lymphocytes populating the B-cell areas at 3 and 7 days with an increase in the expression of Pan T and TH, and a decrease in the expression of Ia.(ABSTRACT TRUNCATED AT 250 WORDS)

Int Arch Allergy Appl Immunol, 1986, 81(3), 269 - 75
Enzyme immunoassay with cell suspensions: studies on reactions between serum antibodies and cell-surface antigens; Wasik M et al.; The following reactions of serum antibodies with cell surface antigens were studied by means of an enzyme immunoassay with cell suspensions: Rh antiserum with human red blood cells; H-2 antisera with murine red blood cells; H-2 antisera with murine thymocytes and hepatocytes; Thy-1 antiserum with murine thymocytes; polyvalent and monovalent HLA alloantisera with human leukocytes and human mononuclear cells, and antisera of murine origin with human and marmoset lymphoid cell lines . In all instances, with the exception of monovalent HLA antisera, the assay proved to be a sensitive and highly specific procedure.

Folia Histochem Cytobiol, 1986, 24(1), 21 - 6
Age related decline in functional T cell subsets in the mouse; Boersma WJ et al.; The changes of two functions of regulatory T cells in mice of different ages were determined . Mice were immunized with SRBC . DTH responsiveness of spleen cells and the production of IL-2 after Concanavalin A stimulation of the same cell suspension was measured . Cell populations were analysed for expression of Lyt-2 and Thy-1 surface markers . The relationship between DTH responsiveness and T cell subset distribution in peripheral blood was analysed . A direct relationship between age related changes in DTH responsiveness and T cell subset distributions in peripheral blood was observed in most individual animals.

Cell Tissue Res, 1986, 246(3), 557 - 60
Change of intracellular fluidity during keratinocyte differentiation measured by fluorescence polarization; Hachisuka H et al.; The fluorescence polarization method was applied to measure the intracellular fluidity of fractionated guinea pig keratinocytes . Guinea pig epidermal cell suspension was obtained by treatment with EDTA and trypsin, and was separated into high, intermediate, and low density fractions using Percoll density gradient centrifugation . Morphological observation and cytofluorometric analysis of DNA content in the fractionated epidermal cells showed that the high, intermediate, and low density fractions were basal, spinous, and granular cell-rich fractions, respectively . Intracellular fluorescence polarization of each fraction was determined by a polarization spectrofluorometer (Hitachi MPF-4, prototype) with fluorescein diacetate . The P-values were calculated for high, intermediate, and low density fractions as 0.192 +/- 0.021, 0.172 +/- 0.019, and 0.147 +/- 0.012, respectively . Since low P-values indicate a high degree of fluidity, the results indicate that intracellular fluidity of keratinocytes is lower in basal cells and higher in granular cells . Dye-binding experiments showed that fluorescein-binding proteins were not detected in the soluble fraction of the epidermal cells . The present findings suggest that intracellular fluidity of the guinea pig keratinocyte increases during the process of its differentiation.

Invasion Metastasis, 1986, 6(5), 313 - 20
Lymphocyte-induced angiogenesis: correlation with the metastatic incidence of two murine mammary adenocarcinomas; Miguez M et al.; Two tumor lines exhibiting different metastasizing capacity were studied with respect to the lymphocyte-induced angiogenesis (LIA) . The MM3 line produce lung metastases with a high incidence . Conversely, the metastatic incidence of M3 line is much lower . Tumor cell suspensions were inoculated in BALB/c mice, 24 h later spleens were removed from the animals . Lymphocyte suspensions were made and then injected intradermally in syngeneic recipient mice . Five days after the injection of lymphocytes the vascular reaction was evaluated in the skin of the recipients by measuring the vessel density . The vascular response induced by lymphocytes from MM3-tumor bearing mice was increased when compared to that of M3-tumor-bearing mice . There appeared to be a positive correlation between metastasizing capacity and the lymphocyte-induced vascular response.

Acta Haematol, 1986, 75(4), 199 - 202
Automated cytochemistry in non-Hodgkin's lymphoma: a new method for determination of cells from lymph node biopsy; Bunyaratvej A et al.; Cell suspension prepared from the lymph node biopsy of patients with non-Hodgkin's lymphoma (NHL), metastatic carcinoma (MC) and non-neoplastic lymphadenopathies (NL) were analyzed by the Hemalog D, automated differential counter . The preparation of lymph node cells is described first in this study . The results show that the percentage of large cells (diameter greater than 13.5 micron) stained negatively with peroxidase (LUC, large unstained cells) was remarkably increased in patients with NHL (mean +/- SEM = 18.6 +/- 3.1%) and was particularly increased in the subgroup, diffuse histiocytic type (31.1 +/- 5.3%) . Patients with MC had a raised percentage of nonspecific esterase-positive cells (9.2 +/- 1.4%) compared to patients in the NHL and NL groups . Patients in the NL group had low percentages of both LUC (5.3 +/- 0.7%) and nonspecific esterase-positive cells (1.8 +/- 0.2%) . Quantitation of cells in the lymph node by using the Hemalog D may assist in the diagnosis of lymph node diseases.

Arch Dermatol Res, 1986, 278(4), 283 - 92
Ultrastructural quantitation of desmosome and differentiation-related keratinocyte membrane antigen; Haftek M et al.; The keratinocyte membrane antigen KM 48 was defined by a new monoclonal antibody obtained after mouse immunization with normal human epidermal cell suspension . Specific reactivity of the antibody with desmosomal regions of keratinocyte cell membrane was demonstrated by immunoelectronmicroscopy . Langerhans cells, melanocytes, and indeterminate cells did not express the KM 48 antigen . Immunogold labelling permitted ultrastructural quantitation of KM 48 antibody binding on keratinocytes from various epidermal layers . A gradual increase in desmosome-related KM 48 antigen expression accompanied differentiation of keratinocytes during their transit from basal to granular layer . Distribution of the antigen on individual cells was uneven . The upper pole of a keratinocyte facing overlying more differentiated cells was always found to be laden with an immunogold marker about twice that of the opposite, lower surface of the cell . The results support the previous reports on gradual development of desmosomes during epidermal cell maturation and open up new possibilities for keratinocyte differentiation studies . They also underline the virtues of the immunogold-labelling method used for cell-surface antigen tracing and quantitation.

Acta Histochem, 1986, 78(2), 185 - 8
Double staining technique using a combination of indirect and direct immunofluorescence with monoclonal antibodies; Kupper H et al.; A simple method for double staining by immunofluorescence is described . If for double staining using monoclonal antibodies of the same species only one antibody is conjugated with FITC or TRITC, a combination of indirect and direct immunofluorescence is possible . For cell staining the following incubation steps are carried out: Monoclonal antibody I (unlabelled, mouse), anti-mouse immunoglobulin serum FITC- or TRITC-conjugated, normal mouse serum for blocking of free binding sites of the anti-mouse immunoglobulin, and monoclonal antibody II (mouse) which is conjugated with an alternative fluorochrome . The use of this method is demonstrated for investigation of single cell suspensions (performed as a slide test) and of cryostat sections.

Acta Histochem, 1986, 78(2), 173 - 8
{Detection of thymocyte antigens by monoclonal antibodies of the BL series}; Kupper H et al.; The reactivity of 4 monoclonal pan T cell antibodies and 6 pan leukocyte antibodies was detected with human thymocytes . The study was performed with single cell suspensions and cryostat sections using immunofluorescence and immunoperoxidase techniques . Monoclonal antibodies with T cell specificity react with different percentages of thymocytes . The antibody BL-T1, which is directed against the sheep erythrocyte receptor, reacts with 90% of thymocytes . These cells were found uniform in the thymus cortex and medulla . On the other hand, the antibodies e.g . BL-T2 and T3 react only with 20% of the thymocytes, identified mainly in the medulla . Pan leukocyte antibodies are also suitable for the detection of different thymocyte membrane antigens . In single cell suspensions, it was found a reactivity of the used antibodies with 20, or 40, or 95% of the cells . The immunohistological findings are described.

Histochemistry, 1986, 84(4-6), 549 - 55
Image analysis combined with quantitative cytochemistry . Results and instrumental developments for cancer diagnosis; Ploem JS et al.; This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens . The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described . LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities . A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications . LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23) . Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure . Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection . Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor . This option allows visual interaction after the machine diagnosis has been made . The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion . The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis) . Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Otolaryngol, 1986 Jan-Feb, 101(1-2), 129 - 34
Phenotyping of mononuclear cells from tonsils and corresponding biopsies using a cytofluorimeter . A comparative study; Plum J et al.; A series of B, T, natural killer cell (NK) and monocyte-specific monoclonal antibodies was used to determine the distribution of lymphocyte subpopulations in cell suspensions obtained from whole tonsils as well as their corresponding biopsies . The majority of the mononuclear cells stained with OKB-7 and anti-Dr . A consistent lower percentage of the B lymphocytes stained with OKB-2 . The T lymphocytes were composed of a majority of OKT4+ lymphocytes . Only a minority of the lymphocytes stained with Leu-7, whereas Leu-llb+ lymphocytes were virtually absent . A significant proportion of the mononuclear cells stained with OKM-1 . There was a highly significant correlation between the distribution of the cells stained with the different monoclonal antibodies obtained from the whole tonsil and the corresponding biopsy (r2 = 0.95) . This study shows that biopsies offer a reliable source to study the lymphocyte subsets in tonsils and that cytofluorimetry can be applied to study the lymphocyte distribution in this organ.

Cytometry, 1986 Jan, 7(1), 76 - 81
Cell size, DNA, and cytokeratin analysis of human head and neck tumors by flow cytometry; Bijman JT et al.; Cell subsets have been discriminated in cell suspensions derived from 37 human head and neck tumors by means of light scatter, DNA, and cytokeratin flow cytometry (FCM) . Cell dispersion was performed overnight at 4 degrees C in two different enzyme mixtures, i.e., trypsin/dithioerythritol and collagenase/DNase, under slight agitation of sliced tumor tissue . Cells were examined before and after fractionation on a discontinuous low-density bovine serum albumin (BSA) gradient . Forward and right-angle light scatter FCM of 23 tumor specimens revealed four main subpopulations with different size and structure . Fractionation of primary cell suspensions on a BSA gradient at unit gravity separated debris, small cells and large cells . DNA FCM of the enriched populations demonstrated a relation between large cells and DNA aneuploidy . Epithelial cells, as recognized by cytokeratin antibodies, were also related with large cells . The results demonstrated the usefulness of light scatter, DNA, and cytokeratin analysis of crude and fractionated tumor cell suspensions for assessment of the efficacy of a particular dispersion technique and to obtain information of the cell subsets dispersed.

Cytometry, 1986 Jan, 7(1), 54 - 63
Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde; Larsen JK et al.; Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide . With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei . Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei . There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence . Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter . The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest . In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.

J Neurosci, 1986 Jan, 6(1), 192 - 8
Neurotoxin-sensitive sodium channels in neurons developing in vivo and in vitro; Couraud F et al.; Fetal mouse brain cells were investigated by 22Na+ flux assays with the aim to determine the ontogenetic time course of appearance of functional voltage-sensitive sodium channels . Their pharmacological properties were assessed by measurement of the response to known neurotoxins, acting at site 1, 2, or 3 of the Na+ channel . Brain cell suspensions, prepared at 11-19 d of prenatal development in vivo, and fetal brain neurons in culture were explored . In vivo neurotoxin-sensitive Na+ influx becomes detectable at 12 d of gestation, in concordance with the time of appearance of saturable binding sites for alpha-scorpion toxin (alpha-ScTx) and saxitoxin . Progression in fetal age or in time in vitro is accompanied by an increase in the initial rate and in the amplitude of Na+ uptake stimulated by batrachotoxin or veratridine . The general pharmacological properties of developing Na+ channels are very similar to the known properties of voltage-dependent Na+ channels in adult nerve: Batrachotoxin acts as a full channel agonist and veratridine as a partial agonist . Their respective apparent affinities are increased in presence of alpha-ScTx, in agreement with the known positive cooperativity of toxins acting at sites 2 and 3 of the Na+ channel . alpha-ScTx alone induces a small increase in Na+ permeability; its effect is greatly amplified in the presence of batrachotoxin or veratridine . The apparent affinity of alpha-ScTx is reduced by cell depolarization . Tetrodotoxin and saxitoxin block the increase in Na+ permeability induced by batrachotoxin, veratridine, and alpha-ScTx.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Invest, 1986 Jan, 77(1), 326 - 9
Histiocytosis X . Purified (T6+) cells from bone granuloma produce interleukin 1 and prostaglandin E2 in culture; Arenzana-Seisdedos F et al.; We have investigated the secretory function of cell suspensions from bone eosinophilic granulomas surgically collected in two patients with histiocytosis X . Unseparated cell preparations spontaneously produced interleukin 1 (IL-1) and prostaglandin E2 (PGE2) . In order to ascertain that this secretion was due to the characteristic Langerhans cell-like histiocytosis X cells predominantly found in the bone lesions, we have purified T6+ cells by the use of a fluorescence-activated cell sorter . Such highly purified cell preparations were found to secrete IL-1 and PGE2 spontaneously in culture . Stimulation with endotoxins and treatment with interferon gamma (IFN gamma) revealed an intense IL-1 secretory function of histiocytosis X cells . Since both IL-1 and PGE2 are able to induce bone resorption in vitro, our findings are compatible with the hypothesis that histiocytosis X cells are responsible for the typical osteolytic lesion observed in histiocytosis X through the local secretion of these two mediators.

J Biol Chem, 1985 Dec 25, 260(30), 16224 - 31
Regulation of peptide amidation in cultured pituitary cells; May V et al.; The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2) . As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly . A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product . Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity . In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor) . Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions . Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary . The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension . Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.

FEBS Lett, 1985 Dec 2, 193(2), 141 - 4
Monensin-dependent and -independent mechanisms of cell-matrix adhesion; Niven VM et al.; Attachment and spreading of human FL cells on a subcellular matrix (SCM) preparation made by treating confluent cell monolayers with deoxycholate are insensitive to the presence of monensin . However, if the cell suspension is surface-iodinated prior to adhesion using the LPO/H2O2 system, cell spreading on SCM is inhibited by 1 microM monensin . The suggested interpretation is that cell surface components required for cell spreading on SCM are inactivated by iodination and need replacement from intracellular reserves by a monensin-sensitive pathway . This pathway is not required in the absence of iodination when sufficient surface components (or a monensin-independent pathway of surface expression) are available . Support for this interpretation is obtained by means of double-iodination experiments in which surface-labelled cells adhere and spread, are detached and labelled a second time and then allowed to adhere again to SCM . Cell spreading in the second case is inhibited by approximately 80%, suggesting that both previously expressed and newly recruited receptors are inactivated.

Biophys J, 1985 Dec, 48(6), 931 - 8
Local oxygen gradients near isolated mitochondria; Clark A Jr et al.; The oxygen concentration in tissue can vary on several length scales . The basic scale of variation is determined by capillary spacing . It is this scale that is manifest in the simplest Krogh cylinder model . A second, smaller scale of variation is associated with the consumption of oxygen by mitochondria . This paper gives a theoretical analysis of these smaller-scale oxygen variations near an isolated mitochondrion . To illustrate the effects of shape, we have carried out the calculations for prolate spheroids as well as for spheres . The principal result is that the local drop in oxygen pressure around a consuming mitochondrion is of the order of (gamma/3K) (3V/4 pi)2/3, where gamma is the oxygen consumption rate per unit mitochondrial volume, K is the Krogh oxygen diffusivity of the surrounding tissue, and V is the mitochondrial volume . The theory is applied to skeletal muscle in vivo and to hepatocytes in cell suspension experiments . In both cases, we find that local oxygen variations produced by oxygen consumption are much smaller than the cell-wide variations produced by the collective effect of all the mitochondria . For example, in maximally consuming skeletal muscle, the drop in oxygen pressure around a consuming mitochondrion is only of the order of 0.03 Torr.

J Anim Sci, 1985 Dec, 61(6), 1576 - 86
Isolation and culture of parenchymal and nonparenchymal cells from neonatal swine liver; Caperna TJ et al.; Procedures for the isolation and monolayer culture of hepatocytes and nonparenchymal (Kupffer and endothelial) cells from livers of neonatal pigs (1 to 15 d of age) are described . Cell suspensions were obtained by a modification of the two-step collagenase perfusion technique . Hepatocytes were collected by low-speed centrifugation and nonparenchymal cell populations were purified by centrifugal elutriation . Hepatocytes were readily maintained in arginine-free medium fortified with either fetal calf serum or bovine serum albumin and oleate for periods as long as 6 d . The ability of cultured hepatocytes to incorporate 3H-leucine and 3H-thymidine into protein and DNA, respectively, demonstrated that cells were metabolically active for at least 3 d in culture . The 3H-leucine incorporation into total cell protein was constant regardless of animal age at the time of cell isolation, while incorporation of 3H-thymidine was influenced by animal age . Incorporation of both precursors was dependent upon duration of culture period in vitro and the type of medium (serum-free vs serum-containing) in which the cells were maintained . Morphological observation and analysis of the DNA and protein levels of hepatocyte monolayers suggest that cells did not replicate during the 3-d incubation period . The ability to isolate and culture metabolically active, nonreplicating hepatocytes from neonatal pigs in a serum-free medium affords opportunities for investigation of the influence of specific hormones and specific growth factors on the uptake and metabolism of nutrients by the liver . Similarly, the neonatal pig will serve as a useful model for the characterization of hepatic nonparenchymal cell metabolism during the neonatal period.

Brain Res, 1985 Dec, 355(2), 282 - 6
Morphology of embryonic neostriatal cell suspensions transplanted into adult neostriata; McAllister JP 2nd et al.; Embryonic neostriatal cell suspensions were transplanted into intact or kainic acid-lesioned neostriata of adult host rats . These transplants survived and were sacrificed at 34-78 days posttransplantation . Nissl and Golgi preparations revealed neurons present within the transplants . Neurons with abundant dendritic spines (Spiny type I) were most frequent, but those with fewer spines (Spiny type II) and smooth dendrites (Aspiny II and III) were also present . These results indicate that neostriatal transplants are populated by the major output and internuncial neurons of the neostriatum.

Toxicol Appl Pharmacol, 1985 Dec, 81(3 Pt 1), 469 - 75
Modulation of stromal cell function in DBA/2J and B6C3F1 mice exposed to benzene or phenol; Gaido KW et al.; Inbred B6C3F1 (B6) mice are more resistant to benzene myelotoxicity than are inbred DBA/2J (D2) mice . This difference may be due to increased sensitivity of the hemopoietic microenvironment in D2 mice to benzene or benzene metabolites relative to B6 mice . The objectives of this investigation were to determine whether stromal cells which support hemopoiesis in the marrow were more sensitive to benzene in D2 mice than in B6 mice and to determine whether these strains would continue to express differences in susceptibility to phenol, an oxidative metabolite of benzene . Mice were given benzene (100 mg/kg) or phenol (100 mg/kg) intraperitoneally, twice a day for four consecutive days . On Day 5 marrow cell suspensions were removed from mice given benzene or phenol and from controls, plated in culture and assayed for (1) the relative number of adherent stromal cell (ASC) colonies present, (2) the number of granulocyte/monocyte precursors from benzene or phenol treated mice that could be supported by ASC from normal mice in coculture, and (3) the number of granulocyte/monocyte precursors from normal mice which could be supported by ASC from benzene or phenol treated mice . After benzene administration, only reductions in body weight and marrow cellularity followed the expected pattern and were reduced to a greater extent among D2 mice than B6 mice . Benzene had no significant effect on ASC colonies or on the number of granulocyte/monocyte precursors present . In contrast, the ability of ASC to support hemopoiesis of granulocyte/monocytes from normal donors was reduced to a greater degree among B6 mice than among D2 mice which paradoxically showed an increase in the ability to support hemopoiesis in coculture . Phenol significantly reduced the ability of ASC to support hemopoiesis of granulocyte/monocyte precursors but no preferential effect between strains was evident . These results suggest that benzene, but not phenol, is metabolized differently between the two strains and that bone marrow stromal cells, components of the hemopoietic microenvironment, are sensitive targets for benzene or its oxidative metabolites.

Surgery, 1985 Dec, 98(6), 1024 - 30
DNA histogram of parathyroid tissue in determining extent of parathyroidectomy; Rosen IB et al.; In an attempt to better define hyperplasia from adenoma and for more accurate discernment of grossly normal but histologically abnormal presumed hyperfunctioning gland, parathyroid tissue obtained at operation was subjected to flow cytometric analysis producing DNA histogram . Seventeen abnormal specimens and seven normal specimens obtained from parathyroidectomy cases were placed in RPMI 1640 culture medium, treated to produce a monodispersed cell suspension, and stained with propidium iodide fluorescent dye to provide a measure of DNA content that could be graphically demonstrated . All cell cycles for affected cell populations could be demonstrated on DNA histogram in the G0G1, S, and G2M phases . Proliferative index was arbitrarily derived by combining percentages of G2M plus S phases . It was apparent that the value so derived showed a tendency for higher value for the abnormal parathyroid tissue but the overlap was sufficient so that no specific discriminating value could be placed on DNA histogram . While flow cytometry may not be of significant use in intraoperative identification of abnormal parathyroid tissue, the values obtained may indicate that a spectrum of activity occurs in hyperparathyroidism that cannot be fully appreciated at the present time and that may in the light of incomplete knowledge manifest itself in the clinical state of hyperparathyroidism and hypercalcemia . The findings of our flow cytometry study may indeed lend credence to the view that all hyperparathyroidism represents a four-gland hyperfunction although this does not support as a consequence routine subtotal parathyroidectomy but should stimulate further inquiry into the pathogenesis of primary hyperparathyroidism.

Gastroenterology, 1985 Dec, 89(6), 1360 - 5
Comparison of leukocytes obtained from the intestinal lumen of Giardia-infected immunocompetent mice and nude mice; Heyworth MF et al.; Previous studies have suggested that Giardia infections are cleared immunologically, but have not defined the mechanism of clearance . The aim of the present work was to compare subpopulations of leukocytes harvested from the intestinal lumen of immunocompetent BALB/c mice, which clear Giardia muris infection rapidly, with those of immunodeficient nude mice, which become chronically infected with Giardia muris . Leukocytes were obtained from the intestinal lumen of Giardia-infected mice, and subpopulations of these cells were quantified after immunofluorescent staining with monoclonal antibodies . Identical numbers of leukocytes were harvested from the intestinal lumen of Giardia-infected immunocompetent mice and nude mice, but the number of these leukocytes bearing the T-lymphocyte antigen Thy-1.2 was smaller in nude mice than in immunocompetent mice . In contrast, no striking differences were observed between the numbers of luminal cytotoxic/suppressor T lymphocytes or macrophages in Giardia-infected nude versus immunocompetent mice . The findings suggest that clearance of Giardia muris infection is not mediated by cytotoxic T lymphocytes or macrophages . Subsequently, T lymphocytes and T-lymphocyte subsets were quantified in cell suspensions prepared from Peyer's patches of immunocompetent BALB/c mice and nude mice . It was found that nude mice have a profound deficiency of Peyer's patch helper/inducer T lymphocytes . This deficiency may account for the inability of nude mice to clear Giardia muris infection at a normal rate.

Calcif Tissue Int, 1985 Dec, 37(6), 625 - 9
Effects of prostaglandin E2 and indomethacin on 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in isolated kidney cells of normal and streptozotocin-induced diabetic rats; Kurose H et al.; The effects of prostaglandin E2 (PGE2) (1 microM) and indomethacin (IN) (20 microM) on 1,25 dihydroxyvitamin D3 production were studied in renal cell suspensions isolated from control, streptozotocin-induced diabetic, and insulin-treated diabetic rats . Renal cortex cells were isolated by enzymatic digestion, and preincubated for 30 min at 37 degrees C with the appropriate additive(s) followed by a 1 h incubation with 8 nM 25-hydroxyvitamin D3 in serum-free medium . Radioactivity incorporated into that fraction of the cell suspension extract co-eluting with synthetic 1,25-dihydroxyvitamin D3 on high pressure liquid chromatography was determined . All animals were raised for 5 weeks on a vitamin D-deficient diet . Isolated kidney cells from vitamin D-deficient rats showed dose-dependent response of 1,25 dihydroxyvitamin D3 production to PGE2 . Cells from control animals demonstrated a stimulatory effect of PGE2 (P less than 0.05) and a suppressive effect of IN (P less than 0.01) on 1,25 dihydroxyvitamin D3 production . In contrast, cells from diabetic rat kidneys failed to respond to these agents, alterations which were reversed by insulin treatment . The accumulated data suggest that depressed synthesis of 1,25 dihydroxyvitamin D3 previously observed in the experimental diabetic rat is due, at least in part, to an impaired production and response to PGE2-like prostaglandins.

Jpn J Cancer Res, 1985 Dec, 76(12), 1147 - 53
Oral infection of a common marmoset with human T-cell leukemia virus type-I (HTLV-I) by inoculating fresh human milk of HTLV-I carrier mothers; Kinoshita K et al.; To obtain definitive evidence that milk-borne infection plays a critical role in the endemy or mother-to-child transmission of human T-cell leukemia virus type-I (HTLV-I), we inoculated concentrated fresh human milk cells obtained from HTLV-I carrier mothers into the oral cavity of a common marmoset (Callithrix jacchus) . Twenty-eight milk samples were collected (5-10 ml each) from 17 carrier mothers in the first week after delivery . Cells in the milk were centrifuged down and resuspended in 1/10 vol of the milk fluid . The concentrated cell suspensions were successively inoculated into the oral cavity of a common marmoset . The marmoset was found to be seroconverted by indirect immunofluorescence assay at 2.5 months after the first inoculation of the milk (3.5 X 10(8) cells in total), and was later confirmed to be infected with HTLV-I by the detection of viral antigen expression in short-term cultures of its peripheral blood T-lymphocytes . The results strongly support the working hypothesis that milk-borne infection plays a significant role in the mother-to-child transmission of HTLV-I.

Tsitologiia, 1985 Dec, 27(12), 1353 - 8
{Ultracytochemical detection of nucleoside phosphatase activity in human peripheral blood cells}; Bonartsev PD et al.; The authors elaborated and described the optimum conditions for fixation, incubation and preparation of human blood cell samples in minimum quantities for ultrastructural and ultracytochemical investigations of 5'-nucleotidase and ATPase activities . The best preservation of the blood cell ultrastructure was obtained after fixation with buffered 1% glutaraldehyde solution followed by postfixation in buffered 1% OsO4 solution . The best ultracytochemical demonstration of 5'-nucleotidase and ATPase activities was achieved after fixation in buffered 2% formaldehyde prior to cytochemical incubation . DMSO added to either fixation or incubation media was shown to damage the plasmalemma and glycocalyx structure in cell suspensions . ATPase in 5'-nucleotidase activities were revealed in plasmalemma, cytoplasmic reticulum, Golgi complex, mitochondria and in the nuclei, in particular, in the perinuclear space, nucleolus and chromatin . With respect to the localization and activity of nucleosidephosphatases, lymphocytes proved to be most heterogenic, with the enzyme activity level directly depending on the rate of ultrastructural differentiation in lymphocytes.

Clin Exp Immunol, 1985 Dec, 62(3), 594 - 9
A cell surface monoclonal antibody (H366) helps to discriminate human cytotoxic from suppressor T cells; Al-Sakkaf L et al.; A study has been undertaken to differentiate T cytotoxic (Tc) and T suppressor (Ts) cell subsets using a monoclonal antibody termed H366 (mouse IgG2b) previously reported to phenotype natural killer and killer (NK/K) cells . Mononuclear cell suspensions from 14 normal subjects were depleted of H366+ cells by means of complement dependent cytotoxicity and the remaining cells were phenotyped with CD8 and CD4 monoclonal antibodies . The effects of depletion with H366 plus complement (C1) on the induction and activity of suppressor and cytotoxic T cells was also examined . The results indicate that H366 antibody recognizes in addition to NK/K cells, a population of Tc but not Ts or helper cells . Therefore, H366 antibody can be useful for obtaining Ts enriched lymphocyte subpopulations and this property may also be used for the enumeration of suppressor cells in the peripheral blood in disease states.

Rev Fr Transfus Immunohematol, 1985 Dec, 28(6), 601 - 12
{Activation of human B lymphocytes by a particulate antigen}; Galanaud P et al.; A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA) . The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test) . The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study) . We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins . The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids . Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function . This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.

Proc Natl Acad Sci U S A, 1985 Dec, 82(24), 8800 - 4
Differentiation of catecholaminergic cells in cultures of embryonic avian sensory ganglia; Xue ZG et al.; From the results of previous studies in which developing peripheral ganglia from quail embryos were transplanted into younger chicken embryo hosts, we concluded that spinal and cranial sensory ganglia contain dormant precursors with autonomic potentialities . Here we describe the differentiation of these precursors in vitro, from dorsal root and nodose ganglion cell suspensions . Dorsal root ganglia were removed from quail embryos at 9 to 15 days of incubation, dissociated to single cells, and grown in tissue culture . The differentiation of cells with autonomic features was followed by monitoring properties associated with the adrenergic phenotype (absent from quail sensory ganglia during normal embryonic development) . Provided that the medium was supplemented with chicken embryo extract, numerous cells displaying tyrosine hydroxylase immunoreactivity could be detected from day 4 onward . They possessed long, multiple processes but appeared morphologically distinct from primary sensory neurons . The catalytic activity of tyrosine hydroxylase and of other enzymes required for catecholamine production was demonstrated in the cultures by glyoxylic acid-induced histofluorescence and by radiochemical measurement of the conversion of exogenous tyrosine to norepinephrine . A large proportion of tyrosine hydroxylase-positive cells were found to incorporate {3H}thymidine before and after differentiating . In contrast, recognizable sensory neurons never exhibited adrenergic properties and did not divide . Qualitatively similar results were obtained with cultures of dissociated nodose ganglia . These findings lend further weight to the assumption that latent autonomic precursors are included in the non-neuronal compartment of sensory ganglia.

Cell Biophys, 1985 Dec, 7(4), 251 - 66
Zonal unit-gravity elutriation . A new technique for separating large cells and multicellular complexes from cell suspensions; Andrews P et al.; A new and simple technique, zonal unit-gravity elutriation, has been devised for separating very large cells, multicellular complexes, or small organisms from suspensions consisting mainly of small cells . The separation vessel is a conical chamber with an entrance at the lower, narrower part of the cone and an exit at the upper, wider part of the cone via a dome-shaped lid . A baffle at the entrance prevents turbulence from incoming fluid . Chambers of differing widths and wall slopes are chosen depending on the sedimentation rate of the particles to be separated . A small volume of the cell suspension is placed in the chamber on the bench in a cold-room . Medium stabilized by a shallow density gradient is pumped into the base of the chamber and ascends, creating a decreasing velocity gradient . Cells sediment at unit-gravity against this ascending counterstream, and are separated into bands according to sedimentation velocity . By adjusting the flow rate of the medium, different sizes of cells can be separated . Tumor cells can be enriched, and larger blast cells can be separated from small cells in lymphoid cell suspensions . The procedure produces complete separation of thymic nurse cells (epithelial-lymphoid complexes) from free thymocytes in digested thymus suspensions and produces substantial enrichment of thymic rosettes (macrophage-lymphoid complexes) . A very favorable situation for applying this technique is the isolation of Taenia taeniaformis larvae, which can be completely purified from infected liver suspensions, representing a 4 X 10(5)-fold enrichment of the parasites, with high recovery, in a single 30 min operation.

Blood, 1985 Dec, 66(6), 1233 - 40
Establishment and characterization of a new human eosinophilic leukemia cell line; Saito H et al.; A human eosinophilic leukemia cell line, designated as EoL, was established from the peripheral blood of a patient with Philadelphia chromosome-negative eosinophilic leukemia (EL) . The EoL cell line grows in single cell suspension with a doubling time of 48 hours for about one year . The reactivity of these cells was tested with a panel of monoclonal antibodies; they were found to express surface IA antigen, myeloid antigen (IF10, MY9) and membrane receptors for interleukin 2 (IL-2, Tac antigen) . Under standard culture conditions, a small percentage of cells having more typical eosinophilic characteristics was present . These cells had cytoplasmic granules and were positive for Luxol-fast-blue and eosinophil peroxidase . Under culture conditions to induce the maturation of myeloid cells, such as alkaline medium or addition of dimethyl sulfoxide (DMSO), the frequency of cells with typical eosinophilic features increased to about 40% . In addition, cytogenetic studies showed that cultured cells and original leukemic blasts presented similar chromosome abnormalities . EoL seems to be a unique leukemic line committed to the eosinophilic lineage and can provide a useful in vitro model for the study of malignant eosinophilic properties.

J Biol Chem, 1985 Nov 25, 260(27), 14756 - 63
Low density lipoprotein degradation by rat mast cells . Demonstration of extracellular proteolysis caused by mast cell granules; Kokkonen JO et al.; The interaction between rat serosal mast cells and low density lipoproteins (LDL) was studied in vitro . When rat 125I-LDL was incubated with mast cells, it was bound to a binding site on the mast cell surface but was not internalized by the cells . Even though 125I-LDL was not internalized, its protein component, apolipoprotein B, was rapidly degraded . The proteolytic activity responsible for the degradation of apolipoprotein B was present in the extracellular fluid of mast cells . It could be shown that the degradation was caused entirely by specific cell organelles of mast cells, the granules, which were spontaneously released into the extracellular fluid during preparation and incubation of the cells . In contrast to uncontrolled spontaneous degranulation, a controlled specific degranulation of mast cells can be induced by treating the cells with the compound 48/80 . When increasing amounts of 48/80 were added to mast cell suspensions, a dose-dependent release of granules was observed and an increase in the rate of 125I-LDL degradation resulted . The increase in 125I-LDL degradation closely followed the increase in granule release . Thus, a quantitative relationship between the amount of granules present in the extracellular fluid and the amount of degradation of 125I-LDL could be established . The apolipoprotein part of LDL was extensively degraded by isolated mast cell granules . Analysis by polyacrylamide gel electrophoresis showed that upon incubation of LDL with isolated granules, the apolipoprotein B band rapidly disappeared with simultaneous appearance of several low molecular weight bands . The degradation of 125I-LDL by mast cell granules proceeded optimally at neutral pH and at physiological ionic strength . The results show that mast cell granules are able to efficiently degrade LDL in vitro, once released from mast cells into the extracellular fluid.

Biochim Biophys Acta, 1985 Nov 7, 820(2), 305 - 14
Determination of intracellular conductivity from electrical breakdown measurements; Pilwat G et al.; The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements . The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter) . The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data . The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis . The average value of 180 omega X cm for the intracellular resistivity of intact red blood cells is consistent with the literature.

J Immunol Methods, 1985 Nov 7, 83(2), 209 - 15
Detection of eosinophils using an eosinophil peroxidase assay . Its use as an assay for eosinophil differentiation factors; Strath M et al.; A colorimetric assay for peroxidase has been applied to the detection of eosinophils in bone marrow cultures and tissue cell suspensions . The substrate solution consists of 0.1 mM o-phenylenediamine in 0.05 M Tris-HCl buffer pH 8.0 containing 0.1% Triton and 1 mM hydrogen peroxide . The method is shown to be an easy and reproducible method of detecting eosinophils, with insignificant interference from neutrophils.

Eur J Cell Biol, 1985 Nov, 39(1), 70 - 6
Membrane detachment and release of the major membrane glycoprotein of secretory granules in rat pancreatic exocrine cells; Havinga JR et al.; The major glycoprotein of pancreatic zymogen granule membranes (GP-2) was detected in the medium of acinar cell suspensions from rat pancreas . Its release from the cells was studied in pulse-chase metabolic labeling experiments with radioactive methionine . GP-2 (apparent Mr = 80 000) was found to be processed to a form of slightly lower apparent Mr (75 000) after about 4 h chase . At about the same time this smaller form of GP-2 appeared in the medium . These results are in accordance with earlier findings in vivo . At different chase times acinar cells were extracted with Triton X-114 to separate water-soluble proteins from membrane-associated (hydrophobic) proteins . This experiment showed that GP-2 is slowly converted from a membrane-bound glycoprotein to a soluble glycoprotein after its reduction in apparent molecular mass, causing its detachment from the membrane . Further analysis indicated that the detachment process may occur at the zymogen granule membrane as well as the plasma membrane . Immunocytochemistry on ultrathin cryosections of pancreatic tissue showed that GP-2 is localized on zymogen granule membranes, plasma membranes and in the acinar lumen . Although in much smaller quantities, GP-2 is also present in the granule content . Thus, in summary, GP-2 is synthesized as a true membrane glycoprotein which is gradually processed to a soluble species and is found in the secretion.

Br J Cancer, 1985 Nov, 52(5), 739 - 46
Pharmacokinetics, binding and distribution of Hoechst 33342 in spheroids and murine tumours; Olive PL et al.; The fluorescent stain Hoechst 33342, when injected i.v . into mice, has an LD50 of 300 micrograms g-1 . The stain exits rapidly from the blood, with a half-life of 110 sec following an injection of 10 micrograms g-1, but remains bound within target cells, redistributing with a half-life longer than 2 h . This results in a gradient of drug binding outward from capillaries which can be used to estimate regional perfusion via fluorescence microscopy of frozen tissue sections . For tumour tissues that can be dispersed into single cell suspensions, intracellular Hoeschst 33342 can be quantified by flow cytometry, and cell populations can be selected on the basis of their fluorescence (distance from the vasculature) using a fluorescence-activated cell sorter . Our results in tumours and in spheroids indicate that the rate of stain uptake by different cell subpopulations in situ is much more dependent on stain delivery than on selective uptake . Retention of the stain in spheroids is sufficiently stable to allow cell sorting several hours post-injection . Hoechst 33342 thus appears to have considerable potential as an agent for quantifying tissue perfusion, and for allowing selection of tumour cell subpopulations to assess response to radiation and drugs.

Br J Cancer, 1985 Nov, 52(5), 701 - 5
Selection of tumour cell subpopulations occurs during cultivation of human tumours in soft agar . A DNA flow cytometric study; Tveit KM et al.; To examine whether selection of tumour cell subpopulations occurs during cultivation in soft agar, we compared in 23 human tumours of different histological types the DNA content of cells from colonies formed in soft agar (method of Courtenay and Mills, 1978) with that of the original tumour cells . The ploidy as well as the fraction of cells in S phase were determined from DNA histograms after staining of the nuclei with a propidium-iodide procedure and flow cytometric recordings . In 8 of 17 aneuploid tumours analysed, specific aneuploid subpopulations disappeared during cultivation or new aneuploid populations, not demonstrable in the original cell suspensions, appeared in the colonies . In 9 cases identical aneuploid populations were found in the colonies and the tumours . In one of 6 diploid tumours examined, aneuploid cell populations not revealed in the original cell suspension, were found in addition to diploid cells, whereas 5 tumours gave rise to colonies containing a purely diploid population . The results show that in a variety of human malignant tumours cultivation in soft agar may select specific aneuploid tumour cell populations.

Exp Hematol, 1985 Nov, 13(10), 994 - 8
Neuraminidase increases DNA synthesis of spleen cells induced by native and asialylated erythropoietin; Schooley JC; The uptake of 3H-thymidine by a suspension of spleen cells, obtained from mice made anemic by phenylhydrazine injections, is increased above the values obtained with native human or mouse erythropoietin (Ep) if the hormone is enzymatically asialylated . Preparations of human Ep asialylated by mild acid hydrolysis stimulated DNA synthesis to a lesser extent than the native hormone; however, when neuraminidase was added to the culture medium, the stimulation exhibited by the asialylated Ep was not significantly different from the stimulation shown by the native hormone . The DNA synthesis of these cells is increased even more if neuraminidase and the asialylated hormone are added to the cell suspension . The asialylated Eps are not biologically active in vivo . The sialic acid moieties of Ep and of the cells responding to Ep are involved in the in vitro response of spleen cells to the hormone, possibly by making Ep receptors available to interact with the hormone.

Endocrinology, 1985 Nov, 117(5), 1947 - 52
Examination of the effects of epinephrine, norepinephrine, and dopamine on aldosterone production in bovine glomerulosa cells in vitro; Sequeira SJ et al.; This study was undertaken to explore the possibility that the neurogenic amines epinephrine and norepinephrine may influence aldosterone production in vitro and to examine again the previously reported inhibitory effect of dopamine on aldosterone production . This was accomplished using bovine glomerulosa cell suspensions and a highly specific RIA for aldosterone . Epinephrine and norepinephrine (10(-6) - 10(-10) M) had no significant effect on aldosterone production . Both basal and angiotensin II-stimulated aldosterone production were significantly inhibited by dopamine, 10(-4) M, P less than 0.05 . Basal aldosterone production was unaffected by lower concentrations of dopamine whereas angiotensin II-stimulated aldosterone production was inhibited in a dose-dependent manner that was significant to 10(-6) M dopamine (P less than 0.05) . Pretreatment of glomerulosa cells with the dopamine antagonist metoclopramide impaired the inhibitory effect of dopamine on aldosterone production . This study supports the hypothesis that dopamine may be a significant inhibitor of aldosterone production in vivo . The other neurogenic amines studied, epinephrine and norepinephrine, had no significant effect on aldosterone production in vitro.

Endocrinology, 1985 Nov, 117(5), 1839 - 47
Interaction of estradiol and estriol with uterine estrogen receptor in vivo and in excised uteri or cell suspensions at 37 C: noncooperative estradiol binding and absence of estriol inhibition of estradiol-induced receptor activation and transformation; Muller RE et al.; The partial agonist and antagonist properties of estriol (E3) have been related to the brief nuclear retention of receptor-E3 complexes and to the lower affinity of E3 for the receptor compared to estradiol (E2) . More recently, it was proposed that the partial agonist/antagonist activity of E3 may be due to its ability to eliminate positive cooperative binding of {3H}E2 to cytosolic estrogen receptor . In this model, positive cooperativity is related to receptor activation and transformation . We first examined the long term effects of E3 on E2 action in vivo . Mature ovariectomized rats were treated for 16 days with E2, E3, or mixtures of these two substances delivered through Alzet pumps at a constant hourly rate (E2, 0.04 microgram; E3, 0.4 and 0.04 microgram; E2 and E3, 0.04 and 0.4 microgram) . The effects of E3 on uterine growth, induction of progesterone receptor synthesis, and activation (nuclear binding) of estrogen receptor suggest that when given continuously, E3 acts as a full agonist and does not inhibit E2 action . Furthermore, incubation of uteri at 37 C with {3H}E2 in the presence of a 1- to 20-fold molar excess of nonradioactive E3 did not alter the subcellular distribution of receptor-{3H}E2 complexes (80% nuclear and 20% cytosolic), demonstrating that E3 does not inhibit E2-induced receptor activation (i.e . the increased nuclear binding of receptor) . Similarly, 4S to 5S transformation of {3H}E2-labeled estrogen receptor in intact uteri was not inhibited by E3 . Equilibrium binding of {3H}E2 to uterine cell suspensions at physiological temperature (37 C) was noncooperative; nonradioactive E3 did not alter the affinity of the estrogen receptor for {3H}E2; Dixon plot analysis indicates that E3 is a purely competitive inhibitor of {3H}E2 binding . This, in conjunction with the lower affinity of the receptor for E3 than for E2, adequately explains the agonistic-antagonistic properties of E3.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Immunol Immunopathol, 1985 Nov, 37(2), 230 - 5
Inhibition of neutrophil-mediated cytotoxicity by exogenous adenosine 5'-triphosphate; Cameron DJ; Human polymorphonuclear leukocytes (PMNs) obtained from normal donors kill human tumor cells in vitro . However, if adenosine 5'-triphosphate (ATP) is added to the neutrophil tumor cell suspensions in micromolar concentrations (10-100 microM), there is marked inhibition of neutrophil-mediated cytotoxicity . The inhibitory activity resulted from an effect of ATP on both the effector cells as well as the target cells . When either the effector cells or target cells were preincubated with ATP they became resistant to the effects of the cytotoxic neutrophils . In addition, inhibitory activity was specific to ATP; as it was not demonstrated with GTP, UTP, or CTP . However, when the other adenosine compounds (AMP and ADP) were tested, both AMP and ADP had some inhibitory activity . Cytotoxicity was also inhibited when 100 microM of ATP were added to the neutrophil monolayers either at the time of addition of the tumor cells or 15-60 min after addition of the tumor cells whereas no inhibition of cytotoxicity occurred when ATP was added more than 1 hr after the initiation of the cytotoxic reaction.

Cell Immunol, 1985 Nov, 96(1), 137 - 46
Human autologous rosette-forming cells . IV . Functional role in T-cell activation; Nalet V et al.; Human autorosette-forming cells (auto-RFC), which represent a T4/leu 3a+ cell subset, were investigated for their functional role in T-cell activation . Peripheral blood lymphocytes from healthy subjects either remained unfractionated or were separated into recovered or depleted auto-RFC populations . The phytohemagglutinin P stimulation of these three cell suspensions induced normal levels of proliferation, whereas the pokeweed mitogen activation was significantly decreased in the auto-RFC-depleted population compared to the auto-RFC-recovered subset or unfractionated cells . All three cell suspensions produced IL-2 in response to PHA stimulation . However the levels of lymphokine released were significantly lower in the recovered auto-RFC fraction than in the depleted auto-RFC . After activation, using anti-Tac antibodies, the synthesis of IL-2 receptors was evidenced in all the cell fractions regardless of the presence or absence of auto-RFC . In addition, both auto-RFC absorbed IL-2 activity from a reference supernatant . Taken together, these data suggest that auto-RFC can be expanded by T-cell mitogens, and that once activated, they express IL-2 receptors, but represent a minor source of IL-2 production.

Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Nov, 48(5), 773 - 83
Rubidium transport in X-irradiated human erythrocytes; Haskovec C et al.; Outflow of 86Rb, a radioactive analogue of potassium, from human erythrocytes X-irradiated in vitro was studied with the following results . (1) The 86Rb level in the supernatants of irradiated and control cell suspensions reflected mainly 86Rb outflow and much less its active re-uptake . (2) The effect of irradiation on 86Rb outflow was more pronounced at a low temperature (4 degrees C) than at 37 degrees C; the lowest dose of X-radiation exhibiting a significant effect on 86Rb outflow at 4 degrees C was 2.5 Gy . (3) K/Rb exchange did not seem to play an appreciable role in radiation-induced 86Rb outflow . (4) Calcium and its accumulation in irradiated cells was not found to be the cause of the effect of radiation on 86Rb outflow . (5) The effect of radiation on 86Rb outflow was higher in low Na medium but it was not inhibited by bumetanide . Rb/Na counter- or co-transport do not therefore seem to be involved in radiation-induced Rb+ outflow.

Dev Biol, 1985 Nov, 112(1), 100 - 14
Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules; Adler R et al.; The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described . Retinal cell suspensions in serum-free medium containing the "N1" supplement (J . E . Bottenstein, S . D . Skaper, S . Varon, and J . Sato, 1980, Exp . Cell Res . 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested . Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin . Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN . In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN . No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells . Laminin effects on retinal neurons were clearly substratum dependent . When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development . PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule . Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development . Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.

Clin Immunol Immunopathol, 1985 Nov, 37(2), 213 - 9
Immunohistologic analysis of a human pulmonary alveolar macrophage antigen; Kobzik L et al.; PAM1 is a 200-kDa polypeptide antigen present on lavaged human alveolar macrophages but not on monocytes, peritoneal macrophages, breast milk macrophages, or other normal hematopoietic cells studied by flow cytometry . We have characterized the distribution of expression of this antigen by cells in tissues by using immunohistologic techniques . Normal and diseased lung as well as lymph nodes, spleen, kidney, liver, GI tract, and skin were studied . PAM1 was expressed strongly on the surface and weakly in the cytoplasm of most alveolar macrophages in all 15 of the lung specimens . Occasional interstitial macrophages had weak to moderate staining for this antigen but the majority did not stain . The distribution, pattern, and intensity of staining for PAM1 was the same in normal lung specimens and those with interstitial pneumonitis, despite the increase in mononuclear cells in the latter . Dermal histiocytes and Kuppfer cells expressed PAM1 weakly . Sinus histiocytes in lymph nodes were moderately to strongly positive . Although lymphoid cell suspensions (tonsil) were negative by flow cytometry, five of six lymph nodes had positive cells by immunohistology . PAM1 was also detected on endothelial cells of splenic sinusoids in all 6 specimens but not on any other endothelium . Hence, while PAM1 is expressed most strongly on alveolar macrophages, it can also be demonstrated in other locations using sensitive immunohistologic techniques . Since circulating monocytes are antigen negative and some lung interstitial macrophages bear antigen, PAM1 may be a useful marker for studies of the differentiation of mononuclear cells in the lung.

Z Naturforsch {C}, 1985 Nov-Dec, 40(11-12), 891 - 7
Reculturing of cells from primary CFU-C colonies; Hubner GE et al.; This study was aimed at investigating whether cells of CFU-C derived colonies could form secondary colonies . Bone marrow cultures of volumes of agar medium between 25 microliter and 75 microliter contained in glass capillaries were stimulated with mouse lung-conditioned medium (MLCM) containing granulocyte/macrophage colony-stimulating factor (GM-CSF) . Agar gels with colonies of up to greater than or equal to 20 were blown out into identical culture medium, completely dispersed on a whirl-mix to single cell suspensions, and used for establishing secondary agar cultures . In these secondary cultures considerable numbers of secondary granulocytic, mixed granulocytic/macrophage and macrophage colonies as well as numerous clusters arose . In contrast, when single colonies were recultured, only few secondary cell aggregates were formed . When primary cultures containing up to greater than or equal to 20 cell aggregates were used for serial reculture at intermittent intervals of 3 and 4 days, a 2-7-fold increase of colony-forming cells was found in tertiary cultures as was monitored by 7 day colony counts . And by use of different kinds of CSF-containing media, an over 4-fold increase of secondary over primary colonies was obtained with bovine lung-conditioned medium (BLCM) in primary and L-cell-conditioned medium (LCCM) in secondary 7 day cultures . Primary capillary cultures were found to be devoid of CFU-S . Also, setting up bone marrow cultures in petri dishes and stimulating with MLCM, growth of primary as well as secondary colonies was obtained . The results indicate some self-renewal potential of CFU-C in vitro.

Horm Metab Res, 1985 Nov, 17(11), 576 - 9
In-vitro studies of the development of pituitary and testicular functions in diabetes (C57Bl/KsJ-db/db) mutant mice; Kuhn-Velten N et al.; The hypothesis that male diabetes mutant mice (C57Bl/KsJ-db/db) are suffering from impairment of testicular steroidogenic function and pituitary LH release was tested . A smaller postpubertal increase of testicular weight and a reduction of plasma testosterone and androstenedione levels by 65% at 17 weeks of age were most obvious from the comparison to homozygous lean controls . The ability of constant amounts of Leydig cells, either in crude interstitial cell or in purified Leydig cell suspensions, to respond to maximal doses of hCG or cyclic AMP-was reduced by at least 40% in adult diabetes mice . This defect could be attributed to a 40% decrease of steroid-17 alpha-monooxygenase activity as compared to lean mice . No differences occurred, however, if Leydig cells were submaximally stimulated . GnRH-stimulated pituitary LH release was not significantly changed . The impairment of testicular steroidogenic function in diabetes mutant mice may represent a further aspect of infertility of these animals and of diabetes mellitus.

Lab Invest, 1985 Nov, 53(5), 546 - 55
Vascular sarcomas (probably angiosarcomas) transplanted from suspensions of liver cells from diethylnitrosamine-treated rats; Luquette MH et al.; F344 male rats were given 90 ppm diethylnitrosamine in their drinking water ad libitum in two cycles . Livers containing neoplastic nodules, hepatomas, and no sarcomas in the sections sampled were digested in parallel with 0.05% collagenase, 0.1% Pronase, or 0.25% trypsin . Cells were transplanted into 9- to 19-day-old F344 rats . Despite the absence of sarcomas in the sections examined microscopically from each such liver before digestion and the presence of multiple hepatomas in all sections examined, vascular sarcomas, probably angiosarcomas, were observed in a large proportion of animals injected with the suspensions of cells; hepatomas were not observed in these animals . Morphology by light microscopy, immunohistochemical demonstration of factor VIII, histochemical demonstration of alkaline phosphatase, and the presence of Weibel-Palade bodies strongly suggest that these tumors are angiosarcomas . Similar tumors developed from cells obtained in parallel with the aid of Pronase, collagenase, or trypsin . Cell suspensions obtained with Pronase yielded tumors with the shortest latent period between the injection of cells and the death of one-half of the transplant recipients . The procedure that we used provides a consistent method for the production of transplantable sarcomas . The absence of sarcomas in the single sections taken from donor livers and multiple sections of similar livers not used for transplantation suggests that transplantability of these sarcoma cells is acquired very early in this neoplasm.

J Clin Invest, 1985 Nov, 76(5), 1807 - 11
Effect of atrial peptides on aldosterone production; Atarashi K et al.; This study examines the effects of the synthetic atrial peptides (atriopeptin I, II, and III) on aldosterone and corticosterone production by rat adrenal cell suspensions . Furthermore, we studied the effect of atriopeptin II infusion on the plasma aldosterone response to angiotensin II in the rat in vivo . Atriopeptin I, II, and III decreased aldosterone release from zona glomerulosa cells in a dose-dependent fashion . 10 pM atriopeptin II inhibited basal aldosterone release significantly (P less than 0.01), and 10 nM atriopeptin II or III lowered it by 79% . Atriopeptin II decreased the sensitivity of the glomerulosa cells to adrenocorticotropic hormone (ACTH) and angiotensin II . Atriopeptin II had no effect on basal or ACTH-stimulated corticosterone release by fasciculata-medullary cells . In vivo infusions of angiotensin II with or without simultaneous infusions of atriopeptin II showed that atriopeptin II significantly inhibited the aldosterone response to angiotensin II . This inhibition by atriopeptin II was independent of any effect on plasma renin activity, serum potassium, or ACTH . These data raise the possibility that the atrial natriuretic peptides may affect sodium excretion by the kidney, not only directly, but also indirectly through the inhibition of aldosterone production.

J Clin Endocrinol Metab, 1985 Nov, 61(5), 817 - 24
The in vitro regulation of human thyrocyte HLA-DR antigen expression; Weetman AP et al.; The ability of endocrine organs to express human immune response-associated antigens (Ia), such as HLA-DR, is a subject of intense current interest . In this study, the effects of various potential modulators of thyroid follicular cell HLA-DR expression were examined using in vitro cultures . A culture supernatant containing T-cell-derived lymphokines caused DR antigen expression on 13-18% of thyroid cells; more consistent effects were produced by recombinant gamma-interferon, which led to 46-100% of the thyroid cells becoming HLA-DR positive after 3 days in culture . This effect was both time and concentration dependent and occurred in thyroid cells derived from patients with Graves' disease (n = 7) and Hashimoto's thyroiditis (n = 2) as well as from three subjects with no autoimmune thyroid disease . Thyroid cells stained with the monoclonal antibodies 4F2 and 5E9, which recognize cell activation antigens, regardless of whether they were treated with gamma-interferon . The lectin phytohemagglutinin also induced HLA-DR antigen expression (21-91% of cells positive) . This response was dependent on T cell contamination of thyroid cell suspensions, since the effect was inhibited by cyclosporin A . HLA-DQ antigen expression, identified by the Leu-10 monoclonal antibody, was also induced on thyroid cells by gamma-interferon and phytohemagglutinin . In contrast, neither recombinant alpha-interferon nor interleukin-2 induced HLA-DR antigens . Irradiation reduced the response of thyroid cells to gamma-interferon, but two of the known inhibitors of macrophage Ia expression, prostaglandin E2 and (Bu)2cAMP, did not affect gamma-interferon-induced thyroid cell HLA-DR expression . We were unable to detect interleukin-1 production by thyroid cells . These results suggest that 1) under normal circumstances, thyroid cells are 4F2 and 5E9 positive, but are incapable of expressing Ia antigens and, thus, of activating T cells to initiate autoimmune thyroiditis; and 2) once activated, for example by a virus, T cells could release gamma-interferon and induce thyroid cell HLA-DR and -DQ antigen expression; these Ia-positive thyroid cells could then have a role in maintaining or enhancing the autoimmune response.

Transfusion, 1985 Nov-Dec, 25(6), 547 - 50
Ultraviolet irradiation of platelet concentrate abrogates lymphocyte activation without affecting platelet function in vitro; Kahn RA et al.; We studied the effect of ultraviolet (UV) radiation on platelet concentrates . Samples irradiated at 310 mm for 30 minutes at a dose of 1782 J per m2 showed no loss of platelet function in vitro as determined by adenosine diphosphate, collagen, or ristocetin-induced aggregation . Lymphocytes isolated from irradiated units were unable to act as responders or stimulators in a mixed-lymphocyte reaction . These data suggest that UV radiation of platelet concentrates may result in a cell suspension that is unable to evoke an immunological response.

Eur J Immunol, 1985 Nov, 15(11), 1075 - 9
Direct effect of interleukin 2 on the differentiation of human B cells which have not been preactivated in vitro; Le thi Bich-Thuy et al.; Unfractionated as well as small and large human tonsillar B cells were found to differentiate in the presence of recombinant interleukin 2 (IL2) without requiring an in vitro preactivation signal . These cell suspensions did not contain detectable T cells, monocytes or natural killer cells both at the initiation and the termination of cultures, and did not respond to the T cell-dependent polyclonal B cell activator pokeweed mitogen . They contained very few Tac antigen-bearing cells (less than 2.5%) at the beginning and at the termination of the cultures in the absence of IL2 . In the presence of IL2, only a minimal increase in the number of Tac antigen-bearing cells was noted . The observed differentiation was not due to the effects of contaminating T cells, since cultures purposely contaminated with up to 5% of T cells did not contain greater amounts of IgM, IgG or IgA than cultures without T cells added back . Furthermore, when T cells were present at a ratio of 1 T cell per 9 B cells, the production of IgM was decreased . Taken together, these data support the hypothesis that (a) B cell differentiation in response to IL2 may occur in the absence of in vitro preactivation, and in this case cannot be related to the expression of Tac antigen on the B cell surface; (b) it is not due to helper effects of very low numbers of T cells which might contaminate the B cell suspensions; and (c) it most likely results from a direct effect of IL2 on B cells themselves . The data also point out the fact that, in the presence of IL2, T cells may trigger inhibitory effects on IL2-induced B cell differentiation rather than provide helper signals.

J Immunol, 1985 Nov, 135(5), 3039 - 49
Interactions and quantitative analysis of immunoregulatory cells in the chicken thymus; Boyd RL et al.; Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions . The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells . The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM . When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high . When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter . Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus . With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses . Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed . The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL . If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement . The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells . An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.

Br J Pharmacol, 1985 Nov, 86(3), 571 - 9
The isolated stomach preparation of the mouse: a physiological unit for pharmacological analysis; Black JW et al.; Although oxyntic cell secretion can be studied at many organisation levels between isolated cell suspensions and non-invasive techniques in animals, the isolated, lumen-perfused, stomach preparation of the mouse represents a hierarchical level which eliminates extrinsic regulatory influences but retains all the cellular architecture known to be necessary for physiological responses and so can be defined as the physiological unit of acid secretion . The feeding pattern before and the distending pressure during an experiment have been identified as the main determinants of basal secretion: the combination of an intragastric pressure of 12 cmH2O and the fasted state generated a stable basal secretion over 2 h providing a satisfactory basis for bioassays . Basal acid secretion was lowered by treatment with omeprazole and sodium thiocyanate but not with tetrodotoxin, N-methylatropine or tiotidine, suggesting that basal secretion does not involve nervous stimulation or the local release of histamine under these experimental conditions . The improved assay permitted the full characterization of cumulative agonist concentration-effect curves in single stomach preparations to histamine, 5-methylfurmethide, pentagastrin and isobutyl-methylxanthine . Interestingly, pentagastrin produced sustained stimulation of gastric acid secretion under conditions when there was no pharmacological evidence that histamine secretion was taking place . This finding is discussed in relation to the role of histamine in the control of gastric acid secretion.

Am Rev Respir Dis, 1985 Nov, 132(5), 1019 - 26
Characterization of purified dog mastocytoma cells . Autonomic membrane receptors and pharmacologic modulation of histamine release; Phillips MJ et al.; There is conflicting evidence as to which autonomic receptors mast cells possess and whether the receptors are capable of modulating mediator release . We have studied dog mastocytoma cells because they are available in large numbers in a relatively pure form, unlike normal dog mast cells . Mastocytoma nodules from a dog were excised and disaggregated with collagenase to provide a cell suspension of mastocytoma cells of greater than 92% purity . The presence of autonomic receptors was assessed by both radioligand binding assays and by evaluating pharmacologic modulation of mediator release . In the radioligand binding assays, beta-adrenergic receptors were estimated by {3H}dihydroalprenolol binding, alpha-adrenergic receptors by {3H}prazosin binding and cholinergic receptors by {3H}quinuclidinyl benzilate binding . Nonspecific binding was determined in each case by incubation in the presence of the specific antagonists propranolol, phentolamine, and atropine, respectively . The effect of autonomic agonists on immunologic and nonimmunologic histamine release was examined, using the beta-adrenergic agonists isoproterenol and terbutaline, the alpha-adrenergic agonist phenylephrine with and without propranolol, and the cholinergic agonist acetylcholine . Dose-response curves were constructed both for the autonomic agonists and the histamine-releasing agents . Results from the radioligand binding and the pharmacologic studies were concordant . These dog mastocytoma cells had a high density of beta-receptors (21,500 +/- 3,300; mean +/- SE beta-receptors/cell, n=5) of which the predominant subtype appeared to be beta2 . No evidence was found for the presence of alpha-adrenergic or cholinergic receptors either by direct receptor binding or by their actions on histamine release.

Brain Res, 1985 Oct 28, 346(1), 176 - 80
Cross-species embryonic septal transplants: restoration of conditioned learning behavior; Daniloff JK et al.; Embryonic septal and hippocampal tissue from mice was transplanted between species into the brains of adult rat hosts as cell suspensions . Deficits in the ability to learn a conditioned, hippocampally mediated, forced alternation behavior, which were caused by a bilateral transection of the fornix-fimbria pathway, were ameliorated in the septal transplant recipients . The successful performance of the task was correlated to the density index of acetylcholinesterase in the hippocampal section.

Klin Wochenschr, 1985 Oct 15, 63(20), 1075 - 80
3H-Ouabain binding to human mononuclear leucocytes; Ludwig K et al.; Specific binding of cardiac glycosides to intact human blood cells may be a suitable model for physiological or disease-induced changes in cardiac glycoside binding to human heart muscle . Since the erythrocyte contains no nucleus and has relatively few binding sites compared with heart muscle, intact mononuclear leucocytes were investigated in the present study . Using leucocyte suspensions from 34 normal subjects, 133 measurements of 3H-ouabain binding-were obtained . 3H-Ouabain bound to one type of binding site with an affinity (KD) of 2.8 +/- 1.2 X 10(-9) M, similar to that of human heart muscle . Association and dissociation were slow processes (k+1, 3.9 X 10(4) M-1 sec-1; k-1, 8.1 X 10(-5) sec-1, n = 2) . The number of ouabain binding sites/leucocyte varied from 18,000 to 60,000 (mean +/- SD, 34,600 +/- 9,700), with no correlation with the proportion of monocytes present or with the serum K+-level of the donors . Large inter- and intra-individual differences in binding site number were measured which are probably a result of the heterogeneity of the cell suspension used . Thus, the ouabain binding site on human heart muscle and intact mononuclear leucocytes is probably identical . However, the number of binding sites in mixtures of mononuclear leucocytes shows large and inconsistent intraindividual variations, making these studies unsuitable for quantifying drug- or disease-induced changes in ouabain binding site number.

J Immunol Methods, 1985 Oct 10, 82(2), 281 - 93
Fluoresceinated alpha 2-macroglobulin as a probe for studying macrophages; James K et al.; The potential value of fluorescein-conjugated human alpha 2-macroglobulin as a probe for studying macrophages in murine peritoneal exudate and spleen cell suspensions has been investigated using a fluorescence-activated cell sorter . These studies revealed that the number of alpha 2-macroglobulin-positive cells in the mixtures examined correlated closely with their macrophage content as determined morphologically . Furthermore cells separated on the FACS on the basis of their strong alpha 2-macroglobulin binding exhibited macrophage morphology and expressed Fc receptors on their surface . Conversely the alpha 2-macroglobulin-negative population contained few macrophages or Fc rosette-forming cells . Extensive blocking and comparative binding studies with a number of purified human alpha 2-macroglobulin preparations and derivatives thereof, and a variety of purified proteins, confirmed that the binding of fluorescein-conjugated alpha 2-macroglobulin to peritoneal exudate cells was specific . Furthermore the binding occurs in a highly reproducible manner . These observations suggest that fluorescein-conjugated alpha 2-macroglobulin in conjugation with flow cytometry is a sensitive and reliable method for both identifying and isolating subpopulations of macrophages.

J Biol Chem, 1985 Oct 5, 260(22), 12049 - 53
Studies on neutrophil b-type cytochrome in situ by low temperature absorption spectroscopy; Iizuka T et al.; The b-type cytochrome in porcine neutrophils in situ was studied by the low temperature absorption spectroscopy at 77 K . Absolute spectra of the dithionite-reduced cell suspension revealed the existence of a b-type cytochrome with alpha, beta, and Soret absorption maxima at 558, 528, and 426 nm, respectively . The alpha band was unsymmetrical and showed a main peak at 558 nm with a shoulder at around 556 nm . When the cells were anaerobically stimulated either by phorbol myristate acetate or arachidonate followed by reduction by dithionite, the alpha band split clearly into double peaks at 555.5 and 558 nm, suggesting the presence of at least two states or species of the b-type cytochrome(s) in the cell . By monitoring absolute spectra of neutrophils at 77 K, we examined the possibility of CO binding to the b-type cytochrome . The absorption spectra of reduced b-type cytochrome in the presence and absence of CO, however, were not distinguishable under various conditions including equilibration with CO under high pressure or CO treatments in a dark room or at pH 8.5, 7.0, or 5.5 . In contrast, the spectra of the reduced cytochrome disappeared immediately after exposure to O2, whether or not the cells had been treated with CO . The results indicate that the cytochrome does not form a CO complex in situ but reacts with O2, either directly or indirectly.

Immunol Invest, 1985 Oct, 14(5), 389 - 400
The effect of angiotensin II on human mononuclear cell reactivity: suppression of PHA-P-induced thymidine incorporation; Simon MR et al.; Recent evidence suggests that angiotensin II may participate in the regulation of inflammation . We therefore studied the effects of angiotension II on human peripheral blood mononuclear cell reactivity . Peripheral blood mononuclear cell suspensions stimulated with PHA-P revealed decreased thymidine incorporation when simultaneously cultured for 48 or 72 hours with angiotensin II . Experiments were next conducted to determine whether angiotensin II acted directly on lymphocytes . Complete monocyte depletion (greater than 99.5%) resulted in low PHA-P-induced reactivity which was still inhibited by the addition of angiotensin II . Purified (greater than 99.5%) lymphocytes incubated with angiotensin II prior to reconstitution of mixed mononuclear cell suspensions led to reduced PHA-P-stimulated thymidine incorporation . These findings suggest that angiotensin II can effect mononuclear cell uptake possibly through actions on the lymphocyte population.

J Ultrasound Med, 1985 Oct, 4(10), 513 - 8
The effect of extreme acid and alkali changes upon the echogenicity of blood; Ramos JR et al.; The degree of red cell aggregation is dependent upon multiple conditions . The purpose of these experiments was to ultrasonically determine the threshold and reversibility of human red cell aggregation to extreme pH changes at varying shear rates . Real-time B- and A-mode ultrasonography were used to measure echogenicity . Measurements were recorded at original, acidic, and alkaline pH and following return of both acidic and alkaline pH to neutral pH levels . The results showed that 1) neutral red cell suspensions did not become echogenic at any shear rate; 2) acidification produced a shear-related, partly reversible echogenicity; and 3) alkalinization caused a less intense but more shear-resistant and less reversible echogenicity . Alkalinization produced a microscopically discernible greater persistence of intense red cell aggregation . Cell membrane protein loss was detected by electrophoresis.

J Clin Pathol, 1985 Oct, 38(10), 1096 - 9
Comparison of DNA flow cytometry from fresh and paraffin embedded samples of non-Hodgkin's lymphoma; Camplejohn RS et al.; Cell suspensions were prepared from fresh and paraffin embedded samples of lymph nodes from nine patients with non-Hodgkin's lymphoma . DNA flow cytometry was performed on these samples and the results from fresh tissue compared with those from paraffin embedded material . Results were compared in terms of DNA index (as a measure of aneuploidy) and S phase fraction (as an indication of proliferative activity) . Good agreement was found between the results from the samples prepared by the two methods . The quality of DNA flow cytometry from paraffin embedded material was comparable with or better than that from fresh samples.

Urology, 1985 Oct, 26(4), 356 - 61
Heterogeneity index score (HIS) . New computerized method for classification of human bladder carcinomas using flow cytometry; Tachibana M et al.; The rationale for studying nuclear DNA may be its direct relationship to the aggressiveness of cancer . Recent flow cytometric studies (FCM) of cancer cells show the limitation of the current methods for the accurate determination of the degree of aneuploidy or proliferative characteristics of a tumor cell . Here we report a new methodology for a computerized determination which is well correlated with relative mean DNA content in cell populations analyzed by FCM (heterogeneity index, HI) . A total of seventy-six tissue samples were examined . Twenty-two specimens were benign tissue while fifty-four were histologically malignant bladder tumors . Forty tumors were grade (G)I-II, ten G-III, and four carcinoma in situ . The samples were mechanically minced into a single cell suspension and stained with propidium iodide . An Ortho system 50-H multiparameter flow cytometer equipped with an Ortho 2150 computer was used to determine DNA content and cell number . HI was calculated using the following formulas: (formula; see text) The mean HIS of twenty-two normal and benign tissues was 9.805 +/- 5.6 . The forty G-II tumors had a mean HIS of 23.576 +/- 26.519 . Statistical differences were observed between benign tissue and G-I-II tumors (P = 0.0196) . G-III tumors had a marked increase in HIS of 160.965 +/- 63.404 . The limited study of four carcinoma in situ tumors showed a mean HIS of 45.4 +/- 9.5 . Our computer extrapolation of flow cytometric DNA analysis quantifies an objective description of FCM characteristics and histochemical index which may distinguish the degree of tumor malignancy.

Scand J Haematol, 1985 Oct, 35(4), 399 - 407
Surface immunoglobulin restriction in B-non Hodgkin's lymphomas in cell suspension and on frozen tissue sections; Kluin PM et al.; The diagnostic relevance of different tests for detection of surface immunoglobulin on tumour cells of B-type non-Hodgkin's lymphomas (B-NHL) was investigated by comparison of the direct antiglobulin rosetting reaction (DARR) in suspension with two-colour direct immunofluorescence (DIF) on frozen tissue sections . In benign lymph nodes (n = 27) the kappa/lambda ratio by DARR test ranged from 0.9 to 2.8 . Tested by suspension and frozen tissue analysis, light chain restriction was found in 24 and 27 of 31 cases of B-NHL, respectively . Heavy chain restriction was found in half of the cases (14 of 26) studied in suspension and in almost all (28 of 31) tested on sections . In 9 cases DARR tests showed restriction of more than one Ig class on tumour cells, which was infrequent (2 of 28) in frozen section analysis . Although both tests appeared valuable for routine diagnostic purposes, we found the DIF analysis on tissue sections somewhat more discriminative, especially in detection of heavy chain restriction in B-NHL.

Eur J Cancer Clin Oncol, 1985 Oct, 21(10), 1233 - 43
Factors influencing differential growth of rat mammary tumor fragments and cells transplanted in gland-free and gland-containing mammary fat pads; Alston-Mills B et al.; Transplants from eight primary tumors induced by 7,12-dimethylbenz(a)anthracene were placed into gland-free and gland-containing inguinal mammary fat pads of syngeneic female hosts . Samples were transplanted as 1 mm3 fragments or as cell suspensions of enzymatically dissociated tumors averaging 1.5 X 10(5) cells/ml . Similar differential growth patterns resulted from both types of transplants . In samples from two of eight tumors fragments produced more tumors than did dissociated transplants, regardless of fat pad condition . In one case dissociated transplants gave rise to more tumors than did fragments . Samples from two of eight tumors produced no differences in tumor growth from fragments and dissociated transplants . Samples from the remaining two showed low transplantability . Overall, no significant differences in tumor growth could be attributed to transplant treatment or fat pad condition . Gland-containing fat pads inhibited ductal outgrowth following transplantation while gland-free fat pads favored it . From the data, the greater the lability of the primary tumor, the greater the influence of the host microenvironment on the outgrowth potential of the tumor.

Gynecol Oncol, 1985 Oct, 22(2), 135 - 48
Mullerian inhibiting substance reduction of colony growth of human gynecologic cancers in a stem cell assay; Fuller AF Jr et al.; Mullerian Inhibiting Substance (MIS), a fetal testicular product that causes regression of the Mullerian duct in the male mammalian embryo, was evaluated for its antitumor effect on the premise that a substance active against this genital precursor in the fetus might also be active against tumors derived from these tissues . Increasingly pure fractions of biologically active MIS, prepared from newborn calf testes, were tested in the soft agar colony inhibition assay against single cell suspensions of fresh tumors derived in ascitic or solid form from patients with gynecologic malignancies . Twenty-eight tumor specimens placed in soft agar culture have provided sufficient growth to assess an MIS effect . Twenty-five of these 28 tumors showed significant colony inhibition after incubation with MIS . Increased antitumor response correlated with increased purification of MIS when the same tumor was treated with preparations of different purity . Samples obtained from the same patient at different times, from both ascites and solid tumor sources, produced nearly identical responses to MIS . MIS preparations, previously shown to be active in microcytotoxicity and colony inhibition assays against established human ovarian and endometrial carcinoma lines demonstrate consistent antitumor activity against fresh human gynecologic cancers removed at surgery.

Cryobiology, 1985 Oct, 22(5), 438 - 45
Glycerol-induced baroprotection in erythrocyte membranes; Onuchic LF et al.; A system for applying hydrostatic pressures up to 10,000 atm upon cell suspensions for time intervals from a few seconds to several minutes is described . The K+ content of toad red blood cells was used as an indication of the degree of membrane injury induced by the hyperbaric condition . It is practically not affected for pressures up to 2000 atm in experiments lasting 3 or 10 min . falling markedly for pressures of 5000 or 8000 atm . The duration of the applied pressure and its intensity are additive regarding the magnitude of the baroinjury . Glycerol, a cryoprotective agent . at 4.0 M, confers partial but significant baroprotection, which is characterized by a smaller decline of the cell K+ content of the glycerol-treated cells in comparison to the untreated cells, submitted to the same conditions of pressure and time . Baroinjury is compatible with a reversible mechanism . However, irreversible membrane damage occurs for a pressure of 8000 atm applied for 10 min . Baroinjury is discussed in terms of alterations of the lipid leaflet or of membrane proteins, and the mechanism of baroprotection in terms of stabilization of membrane components, under the effect of high pressure, by the association of glycerol with the proteins or the phosphate head groups of phospholipids.

Am J Med, 1985 Oct, 79(4), 445 - 54
Phenotypic analysis by flow cytometry of surface immunoglobulin light chains and B and T cell antigens in lymph nodes involved with non-Hodgkin's lymphoma; Liendo C et al.; The objective of this study was to demonstrate the diagnostic usefulness of flow cytometric analysis of surface membrane immunoglobulin light chain and monoclonal antibody reactivities in B cell non-Hodgkin's lymphoma . For this purpose, lymph node cell suspensions from 80 patients (20 normal lymph nodes, 11 lymph nodes with benign lymphoid hyperplasia, and 47 lymph nodes with B cell non-Hodgkin's lymphoma) were studied to detect the expression of surface B and T cell differentiation antigens recognized by a panel of monoclonal antibodies (anti-Leu-1, anti-Leu-5, anti-HLA-DR, J-5, anti-BL-1, anti-BL-2, and anti-BL-7) . The clonal excess calculation, percent kappa-positive minus percent lambda-positive/percent kappa-positive plus percent lambda-positive cells per discrete level of fluorescence intensity, was used to study the clonality of surface membrane immunoglobulin light chain expression . Among the BL surface antigens, BL-7 proved to be most consistently expressed in B cell non-Hodgkin's lymphoma (79 percent) . It was also present in 57 percent of lymph nodes with benign hyperplasia . No significant relationships were detected between the patterns of reactivity with the anti-BL monoclonal antibodies and histologic subtypes, although the small number of cases tested in each category precludes any definitive conclusions . Immunophenotypic heterogeneity within subgroups was also observed with expression of the other antigens examined . Monoclonal expression of surface membrane immunoglobulin light chain was seen in 43 of 47 (91 percent) of lymph nodes with non-Hodgkin's lymphoma, three of 11 (27 percent) hyperplastic lymph nodes, and one of 22 (4 percent) normal lymph nodes . When the presence of BL-7 and clonal excess was examined as a panel, 83 percent of B cell non-Hodgkin's lymphomas were positively identified, whereas one normal lymph node and no hyperplastic lymph nodes gave positive results . The simultaneous presence of clonal excess and BL-7 can be a useful diagnostic aid in the differentiation of lymphomatous from hyperplastic lymph nodes . Cytofluorimetry provides a rapid, objective, and reproducible technology to confirm the diagnosis of lymph node involvement in B cell non-Hodgkin's lymphoma.

J Bacteriol, 1985 Oct, 164(1), 95 - 101
Sodium ions and an energized membrane required by Methanosarcina barkeri for the oxidation of methanol to the level of formaldehyde; Blaut M et al.; Methanogenesis from methanol by cell suspensions of Methanosarcina barkeri was inhibited by the uncoupler tetrachlorosalicylanilide . This inhibition was reversed by the addition of formaldehyde . 14C labeling experiments revealed that methanol served exclusively as the electron acceptor, whereas formaldehyde was mainly oxidized to CO2 under these conditions . These data support the hypothesis (M . Blaut and G . Gottschalk, Eur . J . Biochem . 141: 217-222, 1984) that the first step in methanol oxidation depends on the proton motive force or a product thereof . Cell extracts of M . barkeri converted methanol and formaldehyde to methane under an H2 atmosphere . Under an N2 atmosphere, however, formaldehyde was disproportionated to CH4 and CO2, whereas methanol was metabolized to a very small extent only, irrespective of the presence of ATP . It was concluded that cell extracts of M . barkeri are not able to oxidize methanol . In further experiments, the sodium dependence of methanogenesis and ATP formation by whole cells was investigated . Methane formation from methanol alone and the corresponding increase in the intracellular ATP content were strictly dependent on Na+ . If, in contrast, methanol was utilized together with H2, methane and ATP were synthesized in the absence of Na+ . The same is true for the disproportionation of formaldehyde to methane and carbon dioxide . From these experiments, it is concluded that in M . barkeri, Na+ is involved not in the process of ATP synthesis but in the first step of methanol oxidation.

Hum Pathol, 1985 Oct, 16(10), 979 - 85
Monocytoid B lymphocytes: their relation to the patterns of the acquired immunodeficiency syndrome (AIDS) and AIDS-related lymphadenopathy; Sohn CC et al.; It was shown recently that monocytoid cells express B-cell-restricted antigens and polyclonal surface immunoglobulins, and the term monocytoid B lymphocytes (MBL) has thus been offered as a more appropriate designation . Although most commonly seen in toxoplasmic lymphadenitis, MBL have been observed in a variety of reactive and neoplastic conditions involving lymph nodes . In the present study MBL were found in 17 of 22 lymph nodes from 20 patients with the acquired immunodeficiency syndrome (AIDS) and AIDS-related lymphadenopathy . In all 17 samples, the MBL were found in lymph nodes with florid reactive follicular hyperplasia, and they were geographically close to the hyperplastic lymphoid follicles . However, MBL were not detected in lymph nodes showing involuted follicles or lymphocyte depletion . The disappearance of MBL apparently parallels the progressive involution of secondary follicles . Leu-3+/Leu-2+ (T-helper/T-suppressor) ratios were studied in 14 lymph node cell suspension samples and ten peripheral blood samples . The lymph node Leu-3+/Leu-2+ ratios were significantly lower in AIDS-related lymphadenopathy than in non-AIDS-related reactive follicular hyperplasia (P less than 0.001); the peripheral blood ratios were decreased in nine of the ten cases . The diminished T-helper status in patients with AIDS and AIDS-related lymphadenopathy may be relevant to the immunopathogenesis of follicular involution and, indirectly, to the disappearance of MBL.

J Invest Dermatol, 1985 Oct, 85(4), 299 - 303
Purification and in vitro growth of human epidermal basal keratinocytes using a monoclonal antibody; Gottlieb AB et al.; We have made a new monoclonal antibody, EL-2, and used it with an immunorosetting procedure combined with Ficoll-Hypaque gradient centrifugation to purify and culture basal keratinocytes . Immunofluorescence of cell suspensions and immunoperoxidase staining of tissue sections demonstrate that EL-2 reacts with malignant cell lines, activated lymphocytes and monocytes, and basal keratinocytes . Sequential immunoprecipitation studies demonstrate that monoclonal antibodies EL-2 and 4F2 detect the same membrane protein . However, we have extended previous studies by making the new observation that both EL-2 and 4F2 react with cultured melanocytes . Basal keratinocytes were purified from single-cell epidermal suspensions by incubation with EL-2 followed by rosetting with rabbit antimouse IgG antibodies covalently linked to bovine red blood cells . Rosetting (basal) keratinocytes were separated from EL-2 negative cells by Ficoll gradient centrifugation . We obtained basal keratinocyte populations of greater than 90% purity as assessed by reactivity with EL-2 and another basal keratinocyte-specific monoclonal antibody, HCl . Langerhans cell, fibroblast, and melanocyte contamination was negligible . Cultures of basal keratinocytes were enriched in EL-2-reactive cells throughout the entire 19 days of culture studied . EL-2 is being used to characterize disorders of keratinocyte proliferation; EL-2 reacted with both squamous and basal cell carcinomas . EL-2 stained only the basal layer of lesional skin from patients with psoriasis, pityriasis rubra pilaris, and Darier's disease . Purification of basal keratinocytes will be important in biochemical and functional studies of normal skin and in establishing long-term keratinocyte lines from normal cells.

Nippon Sanka Fujinka Gakkai Zasshi, 1985 Oct, 37(10), 2107 - 16
{Study on the direct inhibitory action of luteinizing hormone-releasing hormone on the steroidogenesis of cultured human corpus luteum cells}; Okamoto S; To evaluate the direct inhibitory action of luteinizing hormone-releasing hormone (LH-RH) on the steroidogenesis of the human ovary, the primary cultured human corpus luteum cells were investigated . The following were the effects of the addition of LH-RH: Estradiol (E2) and progesterone were produced and secreted in the cultured corpus luteum cells . In the cytoplasm of the cultured corpus luteum cells, E2 and P-antibody complexes were observed as fine granules by the immunohistochemical staining method . The progesterone production of these cells was not inhibited in the cells cultured with LH-RH 10(-8) Mol alone . The progesterone production of the cells was stimulated in the cells, cultured with gonadotropins (LH, HCG and HMG) . The gonadotropin stimulated progesterone production was inhibited by LH-RH administration in the cells . In the short term incubation of the human corpus luteum cell suspension, the cyclic adenosine monophosphate (c-AMP) accumulation of the cells incubated with LH-RH alone did not change, but the gonadotropin-stimulated c-AMP accumulation of the corpus luteum cells was significantly inhibited by LH-RH . Concerning these results, it is concluded that LH-RH inhibits the gonadotropin stimulated progesterone production directly in vitro . It is suggested that the mechanisms of these inhibitory actions of the LH-RH are related to the gonadotropin receptor-adenyl cyclase systems, c-AMP metabolizing enzyme and/or progesterone metabolizing enzyme.

J Invest Dermatol, 1985 Oct, 85(4), 304 - 8
Fibronectin-mediated keratinocyte migration and initiation of fibronectin receptor function in vitro; Takashima A et al.; Cell suspensions of human keratinocytes, freshly isolated from skin specimens, did not express plasma fibronectin (pFN) receptor function in short-term assays for cell attachment and spreading on pFN-coated culture dishes and binding and phagocytosis of pFN-coated latex beads . These activities were expressed, however, by the cells harvested from primary keratinocyte cultures after 2-4 days of culture . Analysis of the cell types arising during primary culture, based on staining with antikeratin antibodies and bullous pemphigoid (BP) serum, revealed that about 90% of the originally isolated cell population consisted of keratinocytes (keratin-positive) and 30% were basal cells (BP antigen-positive) . After 2 days of culture, 95% of the cells were keratinocytes and 70% were basal cells . In vitro initiation of pFN receptor function also was observed in cells harvested from epidermal explants . After 9 days in culture, the cells that migrated out of the explants also were active in short-term cell adhesion assays, while cells remaining in the central region of the explant had much less activity . In related studies, the role of pFN in epidermal cell migration was analyzed, and it was found that anti-pFN antibodies inhibited migration of keratinocytes out of epidermal explants . Addition of preimmune IgG, however, had no effect . It appears, therefore, that pFN is important in all aspects of keratinocyte adhesion, and the expression of pFN receptor function may be a critical activation step necessary for basal cell phagocytosis and migration during wound healing.

Cancer Lett, 1985 Oct, 29(1), 29 - 36
Activities of drug-metabolizing enzymes in isolated gamma-glutamyltranspeptidase positive preneoplastic hepatocytes from carcinogen treated rats; Lebsanft J et al.; Drug metabolizing enzyme activities were determined in isolated putatively preneoplastic, gamma-glutamyltranspeptidase (gamma-GT) positive hepatocytes from male Wistar rats fed 2-acetylaminofluorene (AAF) . The cells were isolated by affinity binding to anti-gamma-GT antibody coated dishes, the resulting suspension contained 60-87% gamma-GT-positive cells . Cytochrome P-450 dependent metabolism of benzo{a}pyrene, aldrin and ethoxyresorufin was 43-54% lower than in the parent cell suspension, glucuronidation of 3-hydroxybenzo{a}pyrene (3-OH-BP) and hydrolysis of styrene oxide were increased 1.5- and 1.4-fold, respectively . The altered pattern of drug metabolizing enzyme activities in isolated gamma-GT-positive hepatocytes is consistent with the increase in resistance of preneoplastic liver cells to hepatotoxins.

Hematol Oncol, 1985 Oct-Dec, 3(4), 271 - 81
Phenotypic characterization of 'non-T, non-B' acute lymphoblastic leukemia by a new panel (BL) of monoclonal antibodies; Al-Katib A et al.; Peripheral blood and/or bone marrow leukemic cell suspensions from 49 patients with 'non-T, non-B' acute lymphoblastic leukemia (ALL) were analysed by flow cytometry using a new panel of four monoclonal antibodies . Anti-BL1 and anti-BL2 originating from NALM-6 and B35M lymphoblastoid cell lines, respectively . These antibodies recognize B-cell differentiation antigens: a heat stable non-immunoprecipitable antigenic determinant, and a 68 000 daltons glycoprotein molecule, respectively . BL5 and BL6 were derived by immunization with the promyelocytic cell line HL-60, recognizing antigens present on early hematopoietic cells: an 85 000 daltons MW glycoprotein (Pro-Im 1) and a heat stable antigen (Pro-Im2), respectively . All ALL patients studied had L1 or L2 morphology by the FAB classification and a blast count exceeding 50 per cent . There were 25 males and 24 females . Median age was 8 years (range 1-67 years) . Thirty-nine cases were studied at initial presentation and 10 at relapse . Cells from 46/49 cases expressed BL2 and/or BL1, but were not reactive with BL5 or BL6 . Three of 49 cases did not express BL1 or BL2 . However, a small percentage of blasts from one case was positive for BL5 (13 per cent) and the other 2 cases were reactive with BL6 (20 per cent and 36 per cent, respectively) . These were one adult and 2 pediatric patients that had other ALL markers and achieved a complete remission with appropriate ALL therapy . One of the BL6+ cases relapsed after 19 months with a change in phenotype to BL1+ BL2+ BL5- BL6- . This analysis shows that the majority of 'non-T, non-B' ALL's do express B-cell associated antigens (BL1/BL2) argumentative of their B-cell origin . A small subgroup does not express such antigens and may arise from a more immature cell, since they expressed antigens on early hematopoietic stem cells.

J Cell Physiol, 1985 Oct, 125(1), 72 - 81
Lymphocyte membrane potential and Ca2+-sensitive potassium channels described by oxonol dye fluorescence measurements; Wilson HA et al.; A method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (psi) by flow cytometric recording of the oxonol dye fluorescence of single cells . Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of psi than may be obtained by bulk cell suspension techniques employing cationic voltage indicators . Mouse spleen and human blood lymphocyte psi was calculated to be -70 mV . T and B lymphocytes maintain a constant psi as extracellular K+ is varied from 2 to 10 mM and the deviation from K+ equilibrium potentials (EK) is shown to result from Na+ permeability . At {K+}o values greater than 10 mM, lymphocytes behave as K+ electrodes . Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca2+ ionophore A23187 occurs only in T cells . This response was identified as activation of a Ca2+-sensitive K+ channel by pharmacologic manipulations . Hence, T cells depolarized by 4-aminopyridine (4-AP, 10 mM) were observed to return to resting psi by A23187-induced elevation of {Ca2+}i . Cells depolarized by quinine (100 microM) were unaffected by A23187 . The Ca2+-activated channel does not contribute to resting psi in T cells since it may be selectively blocked by quinine (20 microM) or modulated by calmodulin antagonists (5 microM trifluperazine) without affecting resting psi.

J Cell Physiol, 1985 Oct, 125(1), 61 - 71
Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components; Wilson HA et al.; The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbocyanines di-O-C5(3) and di-I-C5(3) . Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements) . The contributions of mitochondrial membrane potential (psi m) and plasma membrane potential (psi pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density . Hence, conditions could be chosen that amplified either the psi m or the psi pm component . Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence . At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed . In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes . Certain problems in calibration of psi pm with valinomycin at low dye concentrations and perturbations of psi pm by mitochondrial inhibitors are presented . These findings address the current controversy concerning psi m and psi pm measurement in intact cells by cyanine dye fluorescence . The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting psi m among cells or changes in psi pm with cell activation may reflect variable susceptibility to dye toxicity rather than intrinsic cell properties.

Neurosci Lett, 1985 Sep 30, 60(2), 133 - 7
Rat fetal brain tissue grafts survive and innervate host brain following five day pregraft tissue storage; Gage FH et al.; Freshly dissected fetal basal forebrain tissue rich in cholinergic neurons was either dissociated into a cell suspension and injected into the cholinergically denervated hippocampus of adult rats, or stored in a preservative medium at 4 degrees C . Five days later the stored tissue was dissociated into a cell suspension and injected in the denervated hippocampus in another group of animals . Six weeks later the grafts were evaluated with acetylcholinesterase histochemistry for the assessment of graft survival, graft tissue volume and extent of reinnervation of the denervated hippocampus . All grafts in both the freshly dissociated and the 'stored' group survived . The stored grafts were on the average 1/5 in volume but had an appropriate and extensive laminated reinnervation pattern within the denervated hippocampus . This method of storing cells for extended periods prior to grafting allows for experimental manipulation of fetal tissue as well as long distance transportation prior to intracerebral grafting.

Eur J Pharmacol, 1985 Sep 24, 115(2-3), 259 - 66
Biochemical mechanisms of regulation of mucus secretion by prostaglandin E2 in rat gastric mucosa; Bersimbaev RI et al.; The effects of prostaglandin E2 (PGE2) and inhibitors of RNA and protein synthesis on rat gastric mucosa were investigated in order to study the cellular and biochemical mechanisms involved in the PGE2-stimulated formation and secretion of gastric mucus . It was shown that PGE2 caused significant stimulation of gastric mucus secretion and this effect of PGE2 was inhibited by cycloheximide but not actinomycin D . The influence of PGE2 on the in vitro incorporation of N-acetyl-{3H}glucosamine and 14C-labelled amino acids in to the glycoproteins representing a major mucus component of the isolated gastric mucosa cells was also studied . The stimulatory effect of PGE2 on incorporation of labelled precursors into glycoproteins of gastric cells was also inhibited by cycloheximide . These results suggest that the effect of PGE2 on mucus production requires ongoing protein synthesis . cAMP can fully reproduce the effect of PGE2 on the formation and the secretion of gastric mucus . The binding of {3H}PGE2 to rat gastric non-parietal cell fractions consisting predominantly of mucoid cells correlated with the ability of PGE2 to increase adenylate cyclase activity in these cells . PGE2 had no effect on adenylate cyclase activity in cell suspensions enriched in parietal cells . These data suggest further that the stimulatory effect of PGE2 on mucus secretion may be mediated by cAMP as a messenger.

Andrologia, 1985 Sep-Oct, 17(5), 461 - 5
Stimulation of protein synthesis in pachytene primary spermatocytes from the human testis by pyruvate; Nakamura M et al.; A sequential enzymatic incubation in collagenase and trypsin was carried out to yield a suspension of viable single cells from the seminiferous epithelium of adult human testis . The cell suspension predominantly consisted of pachytene primary spermatocytes (15%), round spermatids (32%), and condensing spermatids and residual bodies (21%) . Human pachytene spermatocytes were isolated by unit gravity sedimentation using the methods originally developed for murine tissue . The spermatocyte-enriched fraction was 79% pure . When the effect of energy sources on protein synthesis by spermatocytes was examined, the highest rate of protein synthesis with pyruvate was found among four kinds of substrates added at a concentration of 10 mM . As shown with murine spermatocytes, the rate of protein synthesis by the human spermatocytes is probably regulated by pyruvate.

Arch Biochem Biophys, 1985 Sep, 241(2), 453 - 60
Elicitor induction of a microsomal 5-O-(4-coumaroyl)shikimate 3'-hydroxylase in parsley cell suspension cultures; Heller W et al.; Microsomal preparations from parsley cell suspension cultures challenged with an elicitor from Phytophthora megasperma f.sp . glycinea (Pmg) catalyze the formation of trans-5-O-caffeoylshikimate from trans-5-O-(4-coumaroyl)shikimate . Neither the cis isomer nor free 4-coumarate, 4-coumaroyl-CoA, or 5-O-(4-coumaroyl)quinate are substrates for this enzyme . The reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen . NADH, ascorbic acid, and 6,7-dimethyl-5,6,7,8-tetrahydropterine cannot substitute for NADPH . However, NADH enhances enzyme activity observed in the presence of NADPH . Cytochrome c and carbon monoxide inhibit the hydroxylation reaction, suggesting a cytochrome P-450-dependent mixed-function monooxygenase.

J Invest Dermatol, 1985 Sep, 85(3), 191 - 3
Isolation and preliminary biochemical characterization of the human epidermal Langerhans cell; Gommans JM et al.; A method is described for the isolation of human epidermal Langerhans cells (LC) . Following the disaggregation of the epidermis by gentle trypsinization, the cell suspension is incubated with the fluorescein-conjugated monoclonal antibody OKT6 . Using a fluorescence-activated cell sorter, a subpopulation is selected on the basis of positive fluorescence and low forward-scatter, the latter parameter reducing contamination by LC-keratinocyte clusters . The LC averaged 1.7% of the original epidermal preparation . The purity averages 83% as judged either by OKT6 binding or by ATPase activity . Biochemical measurements indicated that the activity of lysosomal enzymes was low with the exception of alpha-mannosidase, which was about 12-fold higher than that of the keratinocyte . The activities of 3 enzymes of intermediary metabolism suggest that the utilization of glucose by the LC is considerably greater than that of the keratinocyte.

Cancer Res, 1985 Sep, 45(9), 4360 - 5
Cellular heterogeneity in normal and neoplastic human urothelium; Mackillop WJ et al.; Cell suspensions derived from 2 specimens of normal human urothelium and 13 human transitional cell carcinomas were studied by discontinuous density gradient centrifugation . The histochemical and proliferative properties of the fractions were evaluated . In normal urothelium, DNA synthesis was restricted to a subpopulation of small round cells which could be partly separated from the more numerous pyramidal and giant cells on the basis of their higher physical density . Normal urothelium did not form colonies in agar . In well-differentiated tumors, the majority of cells present were low-density, elongated cells which histochemically resembled the differentiated pyramidal cells of normal tissue, but DNA synthesis and the ability to form colonies in agar were restricted to a subpopulation of high-density small round cells . Colony size analysis showed that intermediate-density bands contain cells capable of a few divisions which do not form large colonies . With increasing tumor grade, there was an apparent shift into the high-density clonogenic compartment.

Cancer Res, 1985 Sep, 45(9), 4162 - 6
Effect of thermochemotherapy (combined cyclophosphamide and hyperthermia) given at various temperatures with or without glucose administration on a murine fibrosarcoma; Urano M et al.; The effect of combined cyclophosphamide (CY) and heat treatments on a murine tumor was studied at various temperatures . FSa-II tumors, the early generation isotransplants of a spontaneous fibrosarcoma in a C3Hf/Sed mouse, were used . A single cell suspension was transplanted into the animal foot . Hyperthermia was given by immersing animal feet into a water bath maintained at a desired temperature +/- 0.1 degrees C . An average diameter of the tumor at the time of treatment was 4 mm . The tumor growth time, the time required for one-half of the treated tumors to reach 1000 mm3, was the end point . Hyperthermia enhanced the effect of CY at test temperatures ranging from 40.5 degrees - 44.5 degrees C . The enhancement was independent of the temperature when CY was administered 30 min before the beginning of hyperthermia . However, the enhancement was most substantial at temperatures of 40.5 degrees -42.5 degrees C when CY was administered immediately before hyperthermia . The most effective timing of the CY administration was immediately before hyperthermia . The glucose administered 60 min before hyperthermia enhanced the effect of combined CY and hyperthermia when CY was given 30 min before heating . This enhancement was lost when CY was given immediately before hyperthermia . The CY dose response curves at elevated temperatures were downward concave, which may indicate the presence of a CY- and heat-resistant cell population in the tumor . Implications of these observations in clinical hyperthermia were discussed.

Exp Hematol, 1985 Sep, 13(8), 768 - 75
Thymus regeneration by bone marrow cell suspensions differing in the potential to form early and late spleen colonies; Mulder AH et al.; We have determined the thymus-repopulating capacity of purified hemopoietic stem cells, bone marrow cells from mice injected four days previously with 5-fluorouracil (5-FUBM), and bone marrow cells cultured in the presence of stem-cell-activating factor (SAF; SAFBM) . SAF is identical to interleukin 3 (IL-3) . Purified stem cells are more enriched in day-12 CFU-S than in day-8 CFU-S . 5-FUBM consists of CFU-S that give rise to late (day-12) spleen colonies . SAFBM contains predominantly CFU-S that give rise to early spleen colonies (days 6-8) . There is also a net increase in the number of spleen colony-forming units (CFU-S) in these cultures . Thymus regeneration after transplantation with either purified stem cells or 5-FUBM was delayed approximately three days as compared with that after transplantation with normal bone marrow cells . This delay can be ascribed to the absence of prothymocytes in these preparations . Thymus regeneration by SAFBM was delayed approximately ten days as compared with that after transplantation with normal bone marrow cells . The most likely explanation of these results is as follows . The prothymocytes in normal bone marrow produce a relatively limited offspring in the thymus soon after transplantation . This is rapidly replaced by the offspring of newly formed prothymocytes, the results of differentiation of the pluripotent stem cells . These stem cells also give rise to late spleen colonies . Stem cells that give rise to early spleen colonies appear to have lost the capacity for differentiation into the T-cell lineage.

Ultrasound Med Biol, 1985 Sep-Oct, 11(5), 751 - 9
Stable cavitation at low ultrasonic intensities induces cell death and inhibits 3H-TdR incorporation by Con-A-stimulated murine lymphocytes in vitro; Vivino AA et al.; Murine spleen cell suspensions stimulated by Concanavalin A (Con-A) were exposed to 1.6-MHz continuous-wave ultrasound at low intensities (spatial-peak values ranging from 16 to 300 mW/cm2) in the presence of a Nuclepore membrane that contained stabilized gas bodies . The ultrasonically activated gas bodies induced cell lysis and reduced the fraction of intact cells that excluded trypan blue . At a spatial-peak intensity of 75 mW/cm2 (spatial-average intensity 15 mW/cm2), Con-A-induced Methyl{3H}thymidine (3H-TdR) incorporation was reduced in cultures exposed at 24 or 48 hr after addition of Con-A but not at 7 or 13 hr . There was no observable effect on cell survival or 3H-TdR incorporation at spatial-peak intensities below 75 mW/cm2.

Proc Natl Acad Sci U S A, 1985 Sep, 82(17), 5756 - 60
Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity; Kull FC Jr et al.; Tumor necrosis factor (TNF) is a proteinaceous toxin shed by stimulated myeloid cells . Murine TNF was radioiodinated to a specific activity of 1 mCi/nmol (1 Ci = 37 GBq) of monomer . 125I-labeled TNF (125I-TNF) retained complete cytotoxic activity and it was immunochemically identical to the native toxin in a quantitative immunoprecipitation assay . It could be shown by competition binding that 125I-TNF bound to intact L929 cells with a specificity equal to that of native toxin . The conditions of time, temperature, and concentration involved in equilibrium specific binding to intact cells were studied in detail . When binding was carried out at 4 degrees C for 18 hr, four cell lines sensitive to 125I-TNF cytotoxicity demonstrated high-affinity binding . The binding reached half-maximal level at 3 pM and saturated at 30 pM . These concentrations approximated those required for cell death . Scatchard analysis gave approximately 1000 sites per cell . J774.1 cells, the source of the toxin, demonstrated similar binding but were not sensitive to 125I-TNF cytotoxicity . Other sensitive cell lines and freshly extracted tumor cells showed specific binding at 3 pM . Normal lymphoid organ cell suspensions and two human tumorigenic cell lines were not sensitive and failed to demonstrate specific binding . 125I-TNF, covalently cross-linked to its receptor on sensitive L-M cells with disuccinimidyl suberate, was isolated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography . Two specific bands were identified . The most prominent band had a mobility corresponding to a molecular mass of 95 kDa and the second band had a molecular mass of 75 kDa . The presence of the binding site appears to be necessary but not sufficient to explain the sensitivity of cells to the cytotoxic action of TNF.

J Natl Cancer Inst, 1985 Sep, 75(3), 545 - 59
Ultrastructural and cytochemical study of the early stages of liver colonization by transplanted neoplastic hepatocytes; Bernaert D et al.; Neoplastic liver cell colonies were induced in the livers of isogeneic F344 rats by intraportal injection of a hepatic cell suspension from diethylnitrosamine-treated donor rats . Examination of the livers 12 days after cell implantation revealed well-demarcated groups of liver cells . The colonies showed alterations of the normal hepatocyte phenotype, which were clearly demonstrated by histologic, cytochemical, and electron microscope techniques . The hepatocytes were markedly deficient in glucose-6-phosphatase and bile canalicular ATPase activities, and they contained numerous mitotic figures . Scanning and transmission electron microscopy allowed characterization of hepatocyte interfaces and the shape of sinusoids and the biliary network . The nodular colonies displayed disorganized, thickened trabeculae separated by dilated sinusoids . In these colonies the hepatocytes proliferated intensely and formed, inside the host parenchyma, revascularized, integrated nodular structures . However, these hepatocytes showed ultrastructural anomalies: large nuclei with prominent nucleoli, many free polysomes, and areas of proliferated smooth endoplasmic reticulum in connection with unfolded cisternae of the rough endoplasmic reticulum . All of these features agreed with the hypothesis previously proposed that the colonies may be precursors of the hepatocarcinomas that ultimately develop in animals given injections of treated liver cells . Direct confirmation, however, still is needed.

J Cell Biol, 1985 Sep, 101(3), 778 - 84
Intracellular pH in Dictyostelium discoideum: a 31P nuclear magnetic resonance study; Jentoft JE et al.; We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum . We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils . Cells continued to develop normally in this set-up . Spectra acquired under these conditions typically show two peaks in the inorganic phosphate region corresponding to pH values of 7.16 +/- 0.03 and 6.48 +/- 0.02 . These peaks are believed to represent the mitochondrial and cytosolic compartments respectively, based on a comparison of these values with published data and the collapse of the two compartments upon addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone . Dictyostelium cells show a remarkable degree of intracellular pH homeostasis . Both mitochondrial and cytosolic pH remained unchanged as extracellular pH was varied from 4.3 to 8.1 . There was also no apparent change in the pH of either compartment after up to 13.5 hours' development in suspension.

Vopr Med Khim, 1985 Sep-Oct, 31(5), 75 - 80
{Activity of membrane-bound enzymes, indices of metabolism in the cytoplasm and various physico-chemical properties of erythrocytes with increased cholesterol level}; Taganovich AD et al.; Activity of membrane-bound enzymes, passive penetration of ions in dose-dependent loading with cholesterol, effect of cholesterol high concentrations on the metabolic patterns in cytosol, viscosity of cell suspension were studied in erythrocytes . Passive cotransport of H+ and Cl- ions via erythrocyte membrane was increased with augmentation in viscosity of the cell suspension . After loading with cholesterol activity of acetylcholinesterase was increased while ATPase and glyceraldehyde-3-phosphate dehydrogenase activities were decreased . The alteration in the enzymatic activity occurred on those sides of the membranes, where these enzymes were localized . Activity of lactate dehydrogenase was decreased in cytoplasm of erythrocytes . The alterations detected may be important in development of ischemic syndrome in hyperlipoproteinemia.

Acta Virol, 1985 Sep, 29(5), 393 - 402
Analysis of reactivation of latent pseudorabies virus infection in tonsils and Gasserian ganglia of pigs; Sabo A; Multiplication of pseudorabies virus (PRV) strain TOP and establishment of latency were followed in the tonsils and Gasserian ganglia (GG) of 8-week-old pigs after oral infection . Already within 24 hr, the titre of PRV in tonsils reached 10(2) TCID50/g tissue, its presence in the oropharynx being detectable by swabbing . Virus multiplication in tonsils culminated on days 3-6 p.i . (10(6)-10(6.5) TCID50/g) and continued till day 11 post-infection (p.i.), when its titre was 10(1.5) TCID50/g the swabs being still positive . The presence of the virus in GG was first proved at 48 hr p.i., but no virus could be found there by titration on day 11 . The virus titre in the GG had essentially followed that in tonsils, however, it was substantially lower during the whole experiment . After primoinfection no shedding of PRV was detected by oropharyngeal swabs taken at 3-5 day intervals during the period of 360 days . Nevertheless, explantation of tonsils and GG, performed between days 60-360 p.i., revealed the presence of PRV latency in 41.1 per cent of animals . The total activation rate in GG cultures was 71.5 per cent, while in the cultures of tonsils 28.5 per cent only . Cocultivation of cell suspensions obtained by trypsinization of GG and tonsillar tissue with chick embryo cells (CEC) did not result in detection of the latent virus though latency was confirmed by explantation of the same tissue samples . However, already 24 hr in culture were sufficient for activation of latency as judged by trypsinization of the explanted tonsillar and GG fragments and their inoculation onto CEC . Estimation of the number of cells carrying PRV in the GG showed that activation occurred in one out of 10(4)-10(5) ganglion cells.

Cancer Res, 1985 Sep, 45(9 Suppl), 4665s - 4670s
Immunohistopathology of lymph nodes in HTLV-III infected homosexuals with persistent adenopathy or AIDS; Biberfeld P et al.; Lymph node biopsies from 43 male homosexuals with persistent generalized lymphadenopathy and from ten acquired immunodeficiency syndrome patients, all with serum antibodies to human T-cell leukemia virus III, were studied with regard to histopathology, immunohistology, and T-cell subsets in cell suspensions . All acquired immunodeficiency syndrome biopsies except one with Kaposi's sarcoma had the same histopathological pattern of follicular depletion, whereas the persistent generalized lymphadenopathy nodes showed a spectrum of changes characterized as follicular hyperplasia, involution with follicular fragmentation, or involution with follicular atrophy . Immunohistology showed a temporal and structural relation between follicular involution, disappearance of follicular dendritic reticulum cells, and follicular invasion by T-cells . These observations suggest elimination of dendritic reticulum cells as part of a pathogenic mechanism in follicular involution . Angiogenesis measured by staining of endothelial cells with antibodies to Factor VIII was increased in many biopsies in stages of involution and depletion . Our observations indicate the occurrence of marked changes not only in T-cells but also in the B-cell compartment of patients with persistent generalized lymphadenopathy or acquired immunodeficiency syndrome . The possibility of staging lymph nodes of these patients by combined histopathology and immunohistology is indicated . This might improve the evaluation of prognosis in these patients . A possible importance of angiogenesis for the tumorigenesis of Kaposi's sarcoma is suggested.

Life Sci, 1985 Aug 12, 37(6), 523 - 30
Fluorescence study on the interaction of salicylate with rat small intestinal epithelial cells: possible mechanism for the promoting effects of salicylate on drug absorption in vivo; Kajii H et al.; The water-soluble drug, salicylate, was rapidly taken up by rat small intestinal epithelial cells . Salicylate, known to enhance the absorption of poorly absorbable drugs by rectum and small intestine, caused a significant decrease in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a slight increase in the fluorescence polarization of 8-anilino-1-naphthalene sulfonic acid (ANS) in the isolated rat small intestinal epithelial cell suspension . An increase in the membrane fluidity of epithelial cells may possibly contribute to the enhancement of drug absorption by salicylate.

Biochim Biophys Acta, 1985 Aug 8, 818(1), 61 - 6
Dication and trication which can increase the permeability of Escherichia coli outer membrane; Katsu T et al.; Recent success in the preparation of the monomer, dimer and trimer in compound 48/80 prompted us to investigate the action of these compounds on Escherichia coli cells . It was found that compound 48/80 inhibited growth of E . coli cells, while the monomer, dimer and trimer in 48/80 did not . However, the following experiments showed that the dimer and trimer disrupted the permeability barrier of the outer membrane of E . coli . First, addition of the dimer or trimer in cell suspension stimulated the uptake of tetraphenylphosphonium cation . Second, the synergistic effect of the dimer on the action of gramicidin caused the efflux of K+ . In experiments using isolated cytoplasmic membrane vesicles, addition of gramicidin alone caused the efflux of K+ . Thus, it was speculated that, with whole cells, the dimer formed some defect structure in the outer membrane, through which gramicidin reached the cytoplasmic membrane and increased the K+ permeability . The temperature dependence of efflux K+ showed that the dimer in 48/80 rendered the outer membrane permeable to gramicidin at temperatures above the phase transition of the outer membrane.

J Biol Chem, 1985 Aug 5, 260(16), 9289 - 97
An intracellular (ATP + Mg2+)-dependent calcium pump within the N1E-115 neuronal cell line; Gill DL et al.; An intracellular (ATP + Mg2+)-dependent Ca2+ pumping mechanism has been identified and characterized within the cultured clonal neuroblastoma cell line N1E-115 . Using cell suspensions treated with 0.005% saponin which selectively permeabilizes the plasma membrane in 95-98% of the cells, it was possible to show clearly that the intracellular Ca2+ pump mechanism is of non-plasma membrane origin and therefore can be compared directly with the Ca2+ pump characterized in detail in synaptosomal membrane vesicles (Gill, D . L., Grollman, E . F., and Kohn, L . D . (1981) J . Biol . Chem . 256, 184-192; Gill, D . L., Chueh, S . H., and Whitlow, C . L . (1984) J . Biol . Chem . 259, 10807-10813) which was proven by flux reversal studies to be derived from the neural plasma membrane (Gill, D . L . (1982) J . Biol . Chem . 257, 10986-10990) . The intracellular Ca2+ pump in N1E-115 cells is distinct from mitochondrial Ca2+ accumulation and is increased up to 8-fold higher as cells reach confluency . In similarity to the neural plasma membrane pump, the intracellular Ca2+ pump within N1E-115 cells has high affinity for Ca2+ (Km = 0.28 microM), is dependent on both ATP (Km = 26 microM) and either Mg2+ or Mn2+ which half-maximally activate Ca2+ pumping at 0.35 mM and 0.32 mM, respectively, and shows similar specificity for Sr2+ and Ba2+ which half-maximally inhibit Ca2+ transport at 50 microM and 1.5 mM, respectively . In contrast to the neural plasma membrane pump, the intracellular Ca2+ pump displays approximately 40-fold higher sensitivity to La3+ (IC50 = 5 microM) and an apparent 400-fold lower sensitivity to VO4(3-) (IC50 = 185 microM), although the inhibitory effectiveness of VO4(3-) is increased 37-fold by a 15-min preincubation of the permeabilized cells with VO4(3-) in the absence of ATP (apparent IC50 = 5 microM) . In further contrast to the neural plasma membrane Ca2+ pump, the intracellular pump within N1E-115 cells is stimulated more than 20-fold by oxalate (giving prolonged linear Ca2+ accumulation), is resistant to low saponin concentrations, and is not modified by calmodulin even after extensive treatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and/or calmodulin antagonist drugs . However, calmidazolium is effective in inhibiting the intracellular Ca2+ pump with an IC50 of approximately 2 microM.

J Immunol Methods, 1985 Aug 2, 81(2), 187 - 97
A procedure for isolation and partial purification of guinea pig lung mast cells; Undem BJ et al.; Pulmonary mast cells were obtained from guinea pig lung using a combination of enzymatic digestion of tissue, centrifugal elutriation, and density gradient centrifugation on Percoll . In the initial procedure, lung tissue was enzymatically digested with collagenase and elastase in four 30 min incubations . Typically, monodispersed cell suspensions contained 4% mast cells . Further purification of these lung mast cells using elutriation and Percoll gradients consistently yielded mast cells of 40-78% (mean 51%) purity . These cells were morphologically intact, viable and found to be functional as determined by histamine release evoked by antigen and anti-guinea pig IgG1 antibody.

J Cell Sci, 1985 Aug, 77, 75 - 85
Density-dependent survival of Ehrlich ascites tumour cells in the presence of various substrates for energy metabolism; Zaporowska-Siwiak E et al.; We have found that Ehrlich ascites tumour (EAT) cells, deprived of any carbon source, and suspended at a density of 2 X 10(5) cells/cm3, begin to die only after 12 h of starvation, though it is known that under these conditions they lose over 80% of their ATP within 30 min . Moreover, we have found that the viability of the cells incubated in the absence of any substrate for energy metabolism is strongly dependent on the density of the cell suspension, and can be significantly improved simply by increasing the suspension density . This prompted us to investigate the density dependence of the maintenance of EAT cell viability in the presence of various substrates for energy metabolism and metabolic intermediates . It was found that: Glucose ensures 48 h viability of EAT cells irrespective of suspension density . Fatty acids and pyruvate as sole carbon source do not improve EAT cell survival . In the presence of glutamine as sole carbon source the EAT cell survival shows dependence on cell-suspension density . At densities of 1.6 X 10(6) to 3.2 X 10(6) cells/cm3 the cell viability is maintained at least as well as in the presence of glucose, but at low cell-suspension densities glutamine does not support cell viability . In the presence of glutamine, addition of 1 mM-inosine and 1 mM-uridine ensures high cell survival irrespective of the cell-suspension density . In the presence of inosine or uridine (10 mM) as sole carbon source, the EAT cell survival is the same as in the presence of glucose and does not depend upon cell-suspension density . Guanosine is less effective, whereas adenosine has no effect at all on the maintenance of EAT cell viability for 48 h . There is no correlation at all between EAT cell survival and the rate of lactic acid production . At a cell-suspension density of 1.6 X 10(6) cells/cm3 the cell survival is of the same order in the presence of glutamine as in the presence of glucose, in spite of the fact that in the first case the rate of lactic acid production is more than 20 times lower . There is no correlation between the capacity of particular nucleosides to support EAT cell survival and their effects on glycolysis and oxygen consumption.

Tsitologiia, 1985 Aug, 27(8), 944 - 6
{Scanning electron microscopy of the surface of Chinese hamster fibroblast-like cells following glycerin treatment}; Glushko TA et al.; The effect of glycerol upon the superficial structure of Chinese hamster fibroblast-like cells was studied . The analysis of scanning electron microscopy data made it clear that glycerol-induced changes in the surface structures were of various character depending on the duration of the contact period . A long-term (4 hour long) exposure to glycerol provoked significant structural changes . Nevertheless, the effects were reversible, judging by the fact that the washing-out of cell suspensions led to the repair of cell structures.

Cancer Res, 1985 Aug, 45(8), 3816 - 24
Induction of heat shock protein synthesis in murine tumors during the development of thermotolerance; Li GC et al.; The function of one or more heat shock proteins (HSPs) may be to confer protection of cells against thermal damage . We examined the induction kinetics of thermotolerance and the synthesis of HSPs in murine tumor models . Squamous cell carcinomas (SCC VII/SF) or radiation-induced fibrosarcomas (RIF) were implanted in the flanks of C3H mice . These flank tumors were first exposed to an elevated temperature (41 degrees-45 degrees C) for a fixed duration, for example, 43 degrees C for 15 min . Some of the tumors were excised immediately, and tumor cell suspensions were made . The other mice with tumors were returned to the cages and left undisturbed for various times up to 72 h before being sacrificed . Again, tumors were then removed and tumor cell suspensions were prepared . These tumor cells were either challenged with a second heat treatment at 45 degrees C in vitro or labeled with {35S}methionine at 37 degrees C in vitro . The tumor cell survival after the combined heat treatments was measured using the in vitro cloning assay . The cellular proteins were analyzed by one- or two-dimensional gel electrophoresis . We found that mild heat shock induced thermotolerance in murine tumors, a result consistent with those of others . The kinetics of induction and decay of thermotolerance depended on the temperature and duration of the priming treatment . Mild heat shock also enhanced the rate of synthesis and accumulation of some HSPs during the development of thermotolerance . For example, after an initial treatment at 43 degrees C for 15 min, the rates of synthesis of HSPs with molecular weights 68,000, 70,000, and 88,000 were greatly enhanced in SCC VII/SF tumors when compared to unheated controls . Qualitatively similar results were seen with radiation-induced fibrosarcoma tumors . The rate of synthesis of Mr 68,000 to 70,000 HSPs reached maximum value (300% of control value) 2 to 4 h after heat shock and decreased to the control value 6 to 24 h later . On the other hand, the rate of synthesis of actin, a major structural cellular protein, remained relatively constant throughout the 72 h of experiments . We then determined the relationship between the synthesis and accumulation of these HSPs and the expression of thermotolerance in murine tumors after a priming heat treatment . The data indicate that the levels of Mr 68,000 to 70,000 HSPs correlate well with thermotolerance.(ABSTRACT TRUNCATED AT 400 WORDS)

Am J Pathol, 1985 Aug, 120(2), 230 - 43
Microwave energy fixation for electron microscopy; Login GR et al.; We have demonstrated that microwave energy (MW) can be used in conjunction with chemical cross-linking agents in order to rapidly fix cell suspensions and tissue blocks for electron microscopy in 7-9 seconds . The optimal MW fixation method involved immersing tissues up to 1 cu cm in dilute aldehyde fixation and immediately irradiating the specimens in a conventional microwave oven for 9 seconds to 50 C . Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hours at 25 C . Stereologic analysis showed that tissue blocks fixed by the MW fixation method did not cause organelles such as liver mitochondria and salivary gland granules to shrink or to swell . Potential applications for this new fixation technology include the investigation of rapid intracellular processes (eg, vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (eg, antigens and enzymes).

J Neurosurg, 1985 Aug, 63(2), 260 - 5
Circulatory disturbance of the spinal cord with epidural neoplasm in rats; Kato A et al.; An experimental model of spinal epidural neoplasm was produced in rats by injecting Walker 256 carcinoma cell suspension anterior to the T12-13 vertebral body . With this model, spinal cord blood flow (SCBF) and its response to CO2 inhalation were estimated by the carbon-14-antipyrine autoradiography and the hydrogen clearance methods . In the early stages after tumor implantation, weakness, axonal swelling, and edema of the white matter were observed, while both SCBF and its response to CO2 inhalation remained normal . In the next stage, the tumor invaded the spinal canal and compressed the spinal cord epidurally . The edema of the white matter progressed, while the gray matter was morphologically intact . The SCBF and its response to CO2 inhalation were altered at both the compression area and caudally in the spinal cord . Changes in response to CO2 inhalation appeared earlier than the SCBF decrease . In the last stage, the SCBF decreased rapidly to the critical level, producing irreversible nervous tissue damage . Microangiographic studies revealed extensive obliteration of the spinal epidural venous plexus and patency of the larger nutritional vessels . From the data obtained, the progressive vascular pathophysiology related to spinal epidural neoplasm is as follows: 1) the vertebral venous plexus is compressed and obliterated in the early stages of the disease, and vasogenic edema appears in the spinal cord; 2) as the tumor grows, mechanical compression of the spinal cord is added and the circulatory disturbance increases; and 3) in the last stage, SCBF decreases rapidly to a critical flow level, and the loss of cord function becomes irreversible.

Chest, 1985 Aug, 88(2), 297 - 9
Two cases of pulmonary Waldenström's macroglobulinemia; Kobayashi H et al.; Two cases of Waldenstrom's macroglobulinemia (WMG) with principally pulmonary manifestation are presented . The disease in both cases was discovered from abnormal shadow in chest x-ray films, without classic features of the disease such as anemia, lymphadenopathy, hepatosplenomegaly or visual disturbance . Transbronchial biopsy (case 1) and open lung biopsy (case 2) revealed diffuse infiltration of lymphoplasmacytoid cells . In both cases, the cytoplasm of proliferating cells stained for monoclonal IgM by the peroxidase-antiperoxidase method . Immunologic examination revealed that almost all tumor cells obtained from bronchoalveolar lavage fluid (case 1) and cell suspension of the right lung (case 2) had monoclonal surface immunoglobulin for IgM . These data established the diagnosis of pulmonary WMG . These cases indicate the importance of immunologic studies for establishing a diagnosis of pulmonary WMG, since histologic findings in biopsy specimens and clinical features are often not specific enough for making this diagnosis.

J Leukoc Biol, 1985 Aug, 38(2), 255 - 65
Localization and characterization of macrophages in murine uterus; Hunt JS et al.; Macrophages are known to be present in the murine uterus and are known to be among those cells comprising the uterine decidual response to pregnancy . The extent of macrophage involvement in the decidual response has not been documented, and there are unresolved questions regarding expression of markers normally associated with macrophages on cells within the decidua . Using tissue immunohistology, macrophages were identified in virgin and pregnant murine uteri . A significant increase in macrophage density was noted during all stages of pregnancy . When uteri from virgin and pregnant mice were enzymatically digested, 10% of uterine cells from virgin and 22% from pregnant mice expressed macrophage markers (binding of rabbit antimouse macrophage serum, Fc gamma receptor expression) . Double labeling immunofluorescence demonstrated that the two markers were associated with the same cells . Those results were confirmed in "panning" experiments using a monoclonal antimouse macrophage reagent . In cell suspensions from pregnant murine (C3H/HeN) uteri, 50% of cells exhibiting macrophage markers were I-Ak positive, and macrophages accounted for nearly all I-Ak positive cells in uterine cell suspensions . The results of this study demonstrate that the murine decidual response to pregnancy includes an increase in Fc gamma receptor-bearing macrophages and that a relatively high percentage of those macrophages are Ia positive.

J Allergy Clin Immunol, 1985 Aug, 76(2 Pt 1), 182 - 8
Localization of tryptase to human cutaneous mast cells and keratinocytes by immunofluorescence and immunoperoxidase cytochemistry with monoclonal antitryptase antibody; Schwartz LB et al.; A monoclonal antibody (H4) against tryptase purified from human pulmonary mast cells was prepared and used as an immunoreactive marker for the cellular localization of tryptase in normal human skin and in lesional skin from subjects with systemic mastocytosis . Mast cells had characteristic metachromatic staining of cytoplasmic granules with Giemsa reagent and were detected in small numbers about superficial vessels in the papillary dermis of nonlesional skin and in large numbers about deep as well as superficial vessels of lesional skin . By both direct immunofluorescence with fluorescein isothiocyanate-H4 and indirect immunoperoxidase cytochemistry with H4, all mast cells were selectively stained . The reactivity was confined to the cytoplasm and was granular in character . In addition, keratinocytes in epidermal tissue and in cell suspensions stained diffusely with H4 antibody . A tryptase-like activity that cleaved tosyl-L-arginine methyl ester (0.003 U/10(6) cells) and was not inhibited by soy bean and lima bean trypsin inhibitors was detected in sonicated suspensions of purified epidermal keratinocytes . Monoclonal antitryptase antibody represents an immunologic probe for the presence of tryptase, a preformed mediator of human mast cells, in tissues and cells.

Eur J Biochem, 1985 Aug 1, 150(3), 415 - 21
Cloning and expression in Escherichia coli of cDNA for serine: pyruvate aminotransferase of rat liver; Oda T et al.; Cloned cDNAs for rat liver serine: pyruvate aminotransferase were obtained by screening of a cDNA expression bank of rat liver with an antibody against the enzyme . Nineteen clones were isolated from 33 000 transformants and most of them had common fragments of cDNA on analysis by digestion with some restriction enzymes . These clones were identified as those containing cDNA for serine:pyruvate aminotransferase by the following criteria . (a) At the nucleic acid level, a 500-base-pair fragment of cDNA prepared by digestion of cDNAs with EcoRI and PstI hybridized with the mRNA coding for serine:pyruvate aminotransferase as judged by hybrid-selected and hybrid-arrested translations . (b) Specific proteins were detected in nine bacterial clones, a 40-kDa protein in one clone and a 39-kDa protein in eight clones . Among them only the 40-kDa protein was found to be solubilized from the cell by sonication, and this protein was immunoprecipitated with an antibody against serine:pyruvate aminotransferase of rat liver . (c) High activity of serine:pyruvate aminotransferase was expressed both in whole cell suspension and sonicated extract prepared from the transformant producing the 40-kDa protein, and 99% of the activity was immunoreactive with the antibody . Two types of mRNA for serine:pyruvate aminotransferase were detected on the RNA blot analysis by using cloned cDNA fragment as a probe . The larger mRNA (approximately 1600 nucleotides) was glucagon-inducible while the smaller one (approximately 1500 nucleotides) was not affected by the hormone.

Scand J Haematol, 1985 Aug, 35(2), 137 - 44
Immunologic subsets in B cell lymphomas defined by surface immunoglobulin isotype and complement receptor--their relationship to survival; Kvaloy S et al.; Cell surface marker profiles were studied on cell suspensions from monoclonal B cell lymphomas . Surface immunoglobulin (sIg) was examined in 178 cases, whereas combined data with sIg and receptors for complement (C3) were available in 99/178 cases . The results showed that B cell lymphomas can be divided into distinct immunological subsets according to surface marker expression . Whereas some histologic subgroups (diffuse centroblastic/centrocytic, centrocytic, immunocytoma) (Kiel classification) consisted of few immunological subtypes, others were more heterogeneous (follicular centroblastic/centrocytic, centroblastic, lymphocytic) . By combining immunological and histological subgroups, more than 40 phenotypes could be identified; this diversity most likely reflects, the heterogeneity of the B cell compartment . As part of the same study the prognostic significance of cell marker phenotypes was examined . Survival analysis undertaken on 149/178 patients did not uncover any significant relationship between type of heavy or light chain expression, C3 receptor expression and clinical outcome . Our data do not confirm recent findings that the type of immunological phenotype may be of prognostic significance.

Scand J Immunol, 1985 Aug, 22(2), 207 - 16
Magnetic monosized polymer particles for fast and specific fractionation of human mononuclear cells; Lea T et al.; Magnetic monodisperse polymer particles were developed and the necessary conditions established to use them for both quantification and fractionation of human peripheral blood mononuclear cell populations . The particles consist of a styrene divinylbenzene core into which magnetite has been deposited by an in situ oxidation process . Thereafter the core has been coated with a hydrophilic polymer containing epoxy and hydroxyl groups . The particles have strong nonspecific binding capacity for protein and can be coated with the appropriate antibodies by physical adsorption only . However, the hydroxyl groups on the outer polymer also make covalent coupling possible . After appropriate blocking they can be used in a rosette assay for quantification of mononuclear leukocytes previously sensitized with monoclonal antibodies . Furthermore, a suitable magnet makes it possible to deplete the cell suspension efficiently of the rosette-forming cells . We have thoroughly investigated the functional properties of human mononuclear cells depleted of T lymphocytes by this technique . Our results show that T cells are virtually completely eliminated, as demonstrated by flow cytometry and various functional assay systems.

Br J Exp Pathol, 1985 Aug, 66(4), 475 - 82
Blood leucocyte infiltration after intravenous injection of ferritin in the rat; Laohapand T et al.; Monocytes infiltrate glomeruli during mesangial deposition of ferritin, and during experimental glomerulonephritis . To determine whether this is solely a local phenomenon, leucocyte infiltration in other organs has been studied following intravenous ferritin injection . Lewis rats received an i.v . injection of 150 mg ferritin/100 g body weight . At 24 h there was a peripheral blood leucocytosis (ferritin-treated rats 26.32 +/- 13.7, control rats 8.54 +/- 2.41 X 10(6) cells/ml) due to increase in polymorphs and monocytes . Bone marrow cell counts fell (ferritin-treated rats 49 +/- 7, control 80 +/- 11 X 10(6)/100 g body weight) . Cell counts on cell suspensions of perfused, enzyme-digested lung, liver and spleen, and lung lavage showed major significant increases in total cell counts: lung 250 +/- 36 (89 +/- 16), lung lavage 2.6 +/- 0.8 (1.4 +/- 0.5), liver 140 +/- 37 (60 +/- 11), spleen 306 +/- 38 (200 +/- 27) X 10(6)/100 g body weight (control values in parentheses) . Cytospin preparations of these suspensions, stained for non-specific esterase showed that the increase in cell numbers was due to increases in non-specific esterase-positive cells (monocytes) and polymorphs . These results demonstrate a generalized leucocyte mobilization, sequestration, and tissue infiltration after i.v . ferritin . The renal glomerulus therefore is not the only site of leucocyte accumulation . These findings may have relevance for studies on inflammation mediated by leucocytes in models of experimental immune complex glomerulonephritis.

Aust N Z J Obstet Gynaecol, 1985 Aug, 25(3), 215 - 20
Chemosensitivity testing of ovarian cancer: results of a rapid in vitro biochemical assay; Khoo SK et al.; There is a need for a predictive test which will assist in the selection of an effective cytotoxic drug and thereby, provide a means of avoiding unnecessary drug-induced toxicity and tumour resistance . In the present study, the viable fraction of the tumour cell suspension from ovarian cancer was used as targets for the effect of the drug, cisplatin, in a 3-hour in vitro assay . DNA synthesis was measured by the incorporation of 3H-thymidine as an index of proliferative activity and RNA synthesis by the incorporation of 3H-uridine as an index of protein metabolism . There was a good correlation between cell activity as determined by the uptake of thymidine and uridine . The degree of drug inhibition was variable over the spectrum of tumour histologic types; thymidine uptake was significantly inhibited by cisplatin in 53% of serous cystadenocarcinomas, and uridine uptake in 21% of tumours . A clear difference in drug effect was observed between those patients who showed clinical response and those who did not . These results support the value of this assay as an indicator of drug sensitivity of the tumour.

Antimicrob Agents Chemother, 1985 Aug, 28(2), 167 - 71
Transfer of amphotericin B from gel state vesicles to mycoplasma cells: biphasic action on potassium transport and permeability; Vertut-Croquin A et al.; The action of amphotericin B on the K+ permeability of Mycoplasma mycoides var . capri cells, containing either cholesterol or ergosterol in their membranes, was studied . When the drug, solubilized in dimethyl sulfoxide, was added directly to the cell suspension, a slightly greater sensitivity to permeabilization was observed for ergosterol-containing cells, confirming the data reported in the literature . When amphotericin B bound to gel state phospholipid vesicles was added to the cell suspension, two effects on cholesterol-containing cells were observed . First, the K+ active transport rates increased; membrane permeabilization and K+ leakage were subsequently detected . For ergosterol-containing cells these sequential events were observed only at amphotericin B concentrations below 10(-6) M . At higher concentrations only K+ leakage was observed . The second permeabilization effect varied with the amphotericin B concentration in different ways in the two types of cells . The permeabilization of ergosterol-containing membranes depended on the amphotericin B/phospholipid molar ratio, whereas the permeabilization of cholesterol-containing membranes did not . In general, the latter remained fairly constant when the total amphotericin B concentration in the medium varied.

J Cell Physiol, 1985 Aug, 124(2), 213 - 8
A collagen:glucosyltransferase at the surface of malignant fibroblasts; Bauvois B et al.; 3T12 fibroblasts possess glucosyltransferases that catalyze the transfer of glucose from UDP-Glucose to galactosylhydroxylysyl residues on collagenous acceptors . The presence of the enzyme activity at the cell surface is indicated by the following findings: a) suspensions of intact cells, as well as intact cell monolayers, glucosylate gelatinized collagen b) glucose transfer is not due to UDP-Glucose hydrolysis and subsequent intracellular utilization of the free glucose c) experiments using cell suspensions with known proportions of broken cells indicate that the glucosyltransferase activity is attributable to intact cells and not to contamination by intracellular enzymes from broken cells . The Km value for UDP-Glucose is about 20 microM . The enzyme has a pronounced requirement for manganese, and shows highest activity between 2 and 10 mM . The optimal Mn2+ concentration for the intracellular gelatin:glucosyltransferase activity is more restricted (5 to 10 mM) . Glucosyltransferase activity is strongly inhibited by diamide and N-ethylmaleimide (5 mM), suggesting that intact sulfhydryl residues present in the enzyme are essential.

J Steroid Biochem, 1985 Aug, 23(2), 153 - 7
Effect of ACTH and prolactin on dehydroepiandrosterone, its sulfate ester and cortisol production by normal and tumorous human adrenocortical cells; Feher T et al.; The effect of ACTH and prolactin on the synthesis of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) was studied in cell suspensions of "normal" and tumorous (adenoma) human adrenal cortex . A stimulation of DHEA and no response of DHEAS production by ACTH in "normal" adrenocortical cell suspension was observed . However ACTH stimulated both DHEA and DHEAS synthesis in tumorous adrenocortical cells . Prolactin did not influence either the basal or the ACTH stimulated DHEA and DHEAS production of adrenocortical cells irrespective of their origin . Our results are compatible with the concept that the biosynthesis of DHEA is under ACTH control, while other factor(s) regulate(s) the sulfate pathway of DHEA secretion under normal conditions . In tumorous adrenocortical cells DHEA may be regulated--at least partly--by ACTH . Prolactin seems to have no direct effect on DHEA and DHEAS synthesis . It is postulated that the relationship between serum prolactin and DHEAS (or DHEA) levels observed by several authors might be an extraadrenal effect of prolactin on adrenal androgens.

Gan To Kagaku Ryoho, 1985 Aug, 12(8), 1552 - 9
{Update of human tumor clonogenic assay in carcinoma of the lung}; Kanzawa F; The human tumor clonogenic assay (HTCA) is a double-layer agar technique, which provides an in vitro prediction of the response of an individual patient's tumor to an antitumor agent . This paper briefly provides an outline of HTCA and its potential use in chemotherapy on patients with lung cancer . In our experience with culturing 123 carcinomas of the lung, 105 specimens (85%) could be subject to more than 5 chemosensitivity tests each by modifying the preparation method of single cell suspension in this system . Growth rate was improved in all types of primary human lung cancer with reasonable consistency . Further, metastatic tumors were capable of being successfully grown in a high percentage of cases, which was comparable to the results obtained for other kinds of tumors . There was no correlation of growth or cloning efficiency with histology, source, or previous chemotherapy . Using 50% or more inhibition on to colony formation as the criterion for chemosensitivity, response rates to vindesine or mitomycin C were 19% or 16%, respectively . The in vitro response rates of these or almost all other antitumor drugs seemed to be comparable to the clinical responses reported by various investigators . Correlation between in vitro chemosensitivity in HTCA and clinical response has been evaluated by various investigators, and the pooled data have demonstrated a good association between in vitro drug sensitivity and clinical response or lack of response . In lung cancer, HTCA had a 57% true positive rate and an 85% true negative rate for the prediction of drug sensitivity and resistance, respectively, of cancer patients to specific chemotherapeutic drugs . Although the system still has to undergo modification to resolve a number of theoretical and practical problems, it has potential uses in lung cancer chemotherapy.

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