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 tr