Curr Eye Res, 1987 Nov, 6(11), 1309 - 17 Calcium does not act as a second messenger for adrenergic and cholinergic agonists in corneal epithelial cells; Cork RJ et al.; The role of changes in intracellular {Ca2+}i as a second messenger in response to either adrenergic or cholinergic agonists was determined in isolated bovine corneal epithelial cells . {Ca2+}i was measured in suspensions of cells loaded with either of the fluorescent indicators quin2 or indo-1, as well as in single cells loaded with fura-2 . Fluorescence from the cell suspensions was measured in a spectrofluorometer while single cell fluorescence was measured using a modified fluorescence microscope with a photon counting photometer . Cells were loaded with these dyes by incubation in Ringer's (pH 8.1) containing 2-50 microM of the acetoxymethyl ester of the indicator . Fluorescence was measured before and after exposure to either, one of the adrenergic agonists isoproterenol, phenylephrine or epinephrine, or the cholinergic agonist carbachol . The resting {Ca2+}i level from the quin2 experiments was 115 nM +/- 41 nM (SEM) (n = 23) whereas with fura-2 it was 71 +/- 10 nM (n = 30) . In no case did we see any change in {Ca2+}i within 15 min after addition of any agonist but we were able to observe increased calcium when 0.5 microM ionomycin was added to either the same or untreated cells . The disparity in the resting levels determined by the two methods may result from various calibration problems . Our results indicate that changes in {Ca2+}i have no second messenger role in response to these agonists. Br J Cancer, 1987 Nov, 56(5), 561 - 9 Analysis of tumour cell composition in tumours composed of paired mixtures of mammary tumour cell lines; Miller BE et al.; In order to quantitate the effects of tumour subpopulation interactions, we have devised a method to determine the subpopulation composition of tumours by using paired tumour cell lines able to grow in different selective media . Line 4T07 forms colonies in thioguanine but not in HAT and line 168 forms colonies in HAT but not in thioguanine . An independent technique of determining tumour cell content was used to validate this method: line 168 and 4T07 cells are distinguishable by flow cytometry after staining with propidium iodide for DNA content . Mixtures of cell suspensions prepared from each unmixed tumour, as well as from tumours arising from mixtures of these lines, were analysed by both the colony formation assay and by the DNA content assay . The colony formation assay yielded values in good agreement with the DNA content assay, but was considerably more sensitive in that it was able to quantitate minority subpopulations that constituted less than 10% of the tumour . Both methods revealed that in tumours arising from mixtures, the tumour cells were almost entirely line 4T07, even when the inoculum had contained a high proportion of 168 cells . Since line 168 cells are very tumorigenic per se, these results suggest that line 4T07 cells are capable of interfering with 168 proliferation in mixed tumours, either directly or through a host-mediated mechanism. J Pathol, 1987 Nov, 153(3), 281 - 8 Immunocytochemical evidence that endometrial stromal granulocytes are granulated lymphocytes; Bulmer JN et al.; Endometrial stromal granulocytes (EGs) are prominent in late luteal phase human endometrium and in early pregnancy decidua . They have been believed to develop from endometrial stromal cells and to secrete relaxin . Recent immunohistochemical studies have suggested that EGs are derived from bone marrow but this has been difficult to prove, mainly because the characteristic cytoplasmic granules are not preserved in frozen tissues . Two separate approaches have now been employed to investigate the cellular lineage of EGs . Formalin-fixed paraffin-embedded sections of first trimester decidua were labelled by an immunoperoxidase method with four monoclonal antibodies (mAbs) reactive with routinely fixed and processed tissues . In addition, acetone-fixed smears of decidual cell suspensions were labelled with a panel of mAbs . Sections and smears were counterstained to demonstrate the characteristic cytoplasmic granules of EGs . Endometrial granulocytes were LCA+, CD2+, MT1+, and UCHL1+, which provides evidence that they are leucocytes . EGs are probably members of the large granular lymphocytes series and may have an essential role in normal implantation and placentation. Acta Cytol, 1987 Nov-Dec, 31(6), 825 - 33 Immunocytochemistry of cerebrospinal fluid; Yam LT et al.; In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined . Immunocytochemical studies could be performed on either cell suspensions or smears . The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears . Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity . The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique . The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining . Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears . In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method . These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses. Am J Clin Pathol, 1987 Nov, 88(5), 560 - 9 Synaptophysin identified in metastases of neuroendocrine tumors by immunocytochemistry and immunoblotting; Wiedenmann B et al.; Synaptophysin, an Mr 38,000 integral membrane glycoprotein of neurotransmitter vesicles, has been identified in diverse primary neuroendocrine (NE) tumors of both neural and epithelial origin (Wiedenmann and co-workers, Proc Natl Acad Sci USA 1986; 83: 3500-3504) . In the present study, metastases of several types of NE tumors, including medullary thyroid carcinoma, gastrinoma, insulinoma, small (oat) cell carcinoma of the lung, gastrointestinal carcinoid, and neuroblastoma, were examined for the presence of synaptophysin by immunocytochemistry, with the use of tissue sections as well as centrifuged cell suspensions and by immunoblotting of tumor proteins . The results show that expression of synaptophysin can be maintained during formation of metastases . Therefore, the authors propose that synaptophysin antibodies be used for the positive identification of metastatic NE tumors, notably in differential diagnosis . The possible implications of these findings for tumor diagnosis are discussed. Z Naturforsch {C}, 1987 Nov-Dec, 42(11-12), 1207 - 14 4-(2'-Carboxyphenyl)-4-oxobutyryl coenzyme A ester, an intermediate in vitamin K2 (menaquinone) biosynthesis; Kolkmann R et al.; Enzyme preparations from Mycobacterium phlei, Escherichia coli and Galium mollugo cell suspension cultures were incubated in the presence of 4-(2'-carboxyphenyl)-4-oxobutyrate (i.e . o-succinylbenzoic acid, OSB, 1), ATP, coenzyme A and Mg2+ . The main product isolated from the incubation mixture was 4-(2'-carboxyphenyl)-4-oxobutyryl coenzyme A ester (2) as determined by comparison with synthetic coenzyme A esters . Synthetic and enzymically formed 4-(2'-carboxyphenyl)-4-oxobutyryl coenzyme A ester (2) was shown to be enzymically converted to an intermediate in vitamin K2 biosynthesis viz . 1,4-dihydroxy-2-naphthoic acid (5) . The enzymic formation of 2-(3'-Carboxypropionyl)benzoyl coenzyme A ester (3) and 4-(2'-carboxyphenyl)-4-oxobutyryl-di-coenzyme A ester (4) was also observed . They appeared in minor amounts, however . These esters were not convertible to 1,4-dihydroxy-2-naphthoic acid (5). Br J Haematol, 1987 Nov, 67(3), 267 - 71 Myeloid progenitors from the bone marrow of patients with vitamin D resistant rickets (type II) fail to respond to 1,25(OH)2D3; Nagler A et al.; The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been shown to enhance the growth of human granulocyte/macrophage haemopoietic progenitors in vitro and to induce these cells to differentiate along the monocyte/macrophage pathway . In order to evaluate the relationship between specific receptors for 1,25(OH)2D3 and the role of 1,25(OH2D3 in the regulation of haemopoietic cell differentiation, we examined the effect of haemopoietic cell differentiation, we examined the effect of 1,25(OH)2D3 on the in vitro growth and differentiation patterns of marrow myeloid progenitor cells from two patients with 1,25(OH)2D3 resistant rickets, resulting from defective receptors to vitamin D . A significant rise in the frequency of myeloid colonies in control marrow cell cultures was induced by 2 X 10(-9) to 2 X 10(-7)M 1,25(OH)2D3 . This rise reached a plateau at 2 X 10(-9)_2 X 10(-8) M 1,25(OH)2D3, resulting in a maximal 54 +/- 9% increase in colony numbers . In contrast, no stimulatory effect could be detected when 1,25(OH)2D3 was added to cultured marrow cells from the patients with 1,25(OH)2D3 resistance . Analysis of colony composition revealed that 2 X 10(-8) and 2 X 10(-7) M, 1,25(OH)2D3 induced a 50 +/- 26% increase in the frequency of colonies composed only of monocytes/macrophages in control, but not in the patients' marrow cell cultures . The effect of 2 X 10(-8) and 2 X 10(-7) M 1,25(OH)2D3 on progenitor cell differentiation towards monocytes/macrophages was also observed in marrow cell suspension cultures . Whereas 1,25(OH)2D3 induced a 81-136% increase in the frequency of monocytes in control marrow cells, no effect could be detected on the generation of mature monocytes in marrow cells of the 1,25(OH)2D3 resistant patients . Our results show that marrow granulocyte/macrophage progenitor cells from patients with 1,25(OH)2D3 resistance fail to respond to 1,25(OH)2D3 . We thus demonstrate that the effect of 1,25(OH)2D3 on the proliferation and differentiation of haemopoietic progenitor cells is mediated through its binding to specific cytoplasmic receptors. In Vivo, 1987 Nov-Dec, 1(6), 327 - 34 Multiparameter analysis of four human renal cell carcinoma xenografts in nude mice; Karthaus HF et al.; Four human renal cell carcinoma xenografts (RC2, RC14, RC43, NC65), maintained in nude mice for several years, were investigated in a multi - disciplinary study, using (immuno) histochemical, biochemical and ultrastructural techniques . Histological, cellular, nuclear and biological characteristics were investigated . All tumors showed histologically recognizable features of human renal cell carcinomas, although marked differences between the four tumors were seen, both at the histological and ultrastructural level . Flowcytometric analysis of tumor cell suspensions allowed DNA quantification as well as the detection of subpopulations . Immunohistochemical staining procedures using tissue specific antibodies against intermediate filament proteins revealed two populations of tumor cells . Most tumor cells in three of the xenografts coexpressed cytokeratins and vimentin, while in RC43 most of the tumor cells expressed only vimentin . Northern blot analysis showed a higher expression of vimentin mRNA in all tumors as compared to normal kidney tissue . RC43 showed a three-fold higher level of vimentin mRNA than the other xenografts . Growth potential in the human tumor cloning system was evaluated by temporal growth pattern analysis . These experiments showed that the xenografts resemble human primary renal cell tumors in different ways, and reflect different characteristics that can be present in human renal cell carcinoma. J Immunol, 1987 Nov 1, 139(9), 3107 - 11 Natural killer cell inhibition of young spherules and endospores of Coccidioides immitis; Petkus AF et al.; The recent development of a method for culturing the parasitic form of Coccidioides immitis by using conditions compatible with the growth of lymphoid cells has enabled us to investigate the role of natural killer (NK) cells in defense against this pathogenic fungus . Pure cultures of spherules and endospores were grown in RPMI 1640 which contained 10% calf serum . Single cell suspensions of young spherules and endospores were incubated in the presence of freshly isolated human peripheral blood lymphocytes (PBL) . After a 4-hr incubation, the colony-forming ability of the fungus was significantly reduced . Leu-11 is a monoclonal antibody that binds to the Fc receptor of NK cells . When PBL were incubated in the presence of this monoclonal antibody and complement, the colony-forming ability of C . immitis was not reduced, indicating that the effector cell involved in reduction of colony-forming units is also recognized by the Leu-11 monoclonal antibody . Classical NK activity can be enhanced by preincubation with interferon; the inhibitory activity of the PBL which are responsible for the reduction in colony-forming units of C . immitis is similarly enhanced by pretreatment with interferon . When PBL are incubated in the presence of young spherules and endospores for 24 hr, the cellfree supernatants will kill U937 target cells . In addition to stimulating the release of NK cytotoxic factor, C . immitis is susceptible to inactivation when incubated in the presence of factors released by PBL which have been incubated in the presence of either K562 or C . immitis . Other evidence reported by this laboratory demonstrates that C-reactive protein is present on the surface of NK cells and that antibody to this molecule blocks NK-mediated killing of standard tumor cell targets . Pretreatment with anti-C-reactive protein also blocks the ability of PBL to inhibit the colony-forming capacity of this fungus . These data suggest that the cell that inhibits the in vitro growth of the pathogenic fungus, C . immitis, is an NK cell. J Immunol, 1987 Nov 1, 139(9), 3062 - 9 Purification and characterization of human skin mast cells . Evidence for human mast cell heterogeneity; Lawrence ID et al.; Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species . Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells . In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity . Comparative studies of mast cells from these several sources have uncovered several major differences among them . Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively . Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C . Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects . First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond . Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells . These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses. J Immunol, 1987 Oct 15, 139(8), 2551 - 5 Enhanced antigen-presenting capacity of cultured Langerhans' cells is associated with markedly increased expression of Ia antigen; Shimada S et al.; Recent studies indicate that when epidermal Langerhans' cells (LC) are cultured for 2 to 3 days they, in comparison to freshly prepared LC, exhibit markedly enhanced ability to stimulate T cell proliferative responses in oxidative mitogenesis and in the mixed epidermal-leukocyte reaction . In this study, we determined whether cultured LC enhance antigen-specific T cell responses, and whether such enhanced stimulatory capacity correlates with the level of Ia antigen expressed on LC . We used C3H/He (Iak) epidermal cells as stimulators and, as responder cells, both the trinitrophenyl-specific clones D8 and SE4, which were assayed for {3H}dThd incorporation, and the pigeon cytochrome c specific hybridoma 2C2, which was assayed for interleukin 2 production . Cultured LC induced 10 to 100 times greater proliferation or interleukin 2 production by responder cells than did freshly prepared LC . The intensity of I-Ak and I-Ek, expressed on cultured LC as assessed by immunofluorescence and flow cytometry, was found to be 10 to 36 times greater on a per cell basis than that on freshly prepared LC . Depletion of LC from fresh epidermal cell suspensions by anti-Iak and complement or treatment with 50 mJ/cm2 medium range ultraviolet light or cycloheximide before culture abrogated both the increase in Ia expression and antigen-specific clonal proliferation . The results suggest that when LC are removed from their usual epidermal milieu, they express increased amounts of Ia and become more potent stimulators of T cell responses. Cancer Res, 1987 Oct 15, 47(20), 5316 - 22 Importance of extended growth potential and growth factor independence on in vivo neoplastic potential of primary rat mammary carcinoma cells; Ethier SP et al.; Recently developed culture systems that allow extended growth of normal rat mammary epithelial (RME) cells were used to directly compare the proliferative potentials and growth factor requirements of primary normal and primary neoplastic rat mammary cells . RME cells were obtained from 45- to 55-day-old inbred female Lew rats and rat mammary tumor (RMT) cells from 7,12-dimethylbenz(a)anthracene- or N-nitroso-methylurea-induced mammary carcinomas . To compare the proliferative lifespan of RME and RMT cells, colony forming efficiencies were determined after consecutive passages over a 70-day culture period . Whereas the proliferative potential of RME cells declined with time in culture, RMT cells from five separate mammary carcinomas had colony forming efficiencies that increased with serial passage . By the end of the 70-day culture period, colony forming efficiencies for RMT cells were 100- to 1000-fold higher than those for RME cells . To compare the growth factor requirements for RME and RMT cells, a serum-free culture medium that supports RME cell growth was used and the influence of specific growth factors was examined by deletion experiments . Cells from 5 of 18 primary mammary carcinomas exhibited requirements for insulin, epidermal growth factor, and cholera toxin identical to those of RME cells . In contrast, cells from 9 of 18 tumors expressed independence of one, two, or all three of these factors for growth in serum-free culture . To examine the in vivo growth potential of primary RMT cells, samples of the cell suspensions used to initiate primary cultures were transplanted into the interscapular white fat pads of syngeneic female recipients . Transplantation of cells from growth factor dependent tumors yielded nonneoplastic mammary outgrowths . In contrast, transplantation of growth factor independent tumor cells yielded grossly visible tumors in 100% of the recipients within 4 weeks of transplantation . Thus, our results indicate that cells from all primary mammary carcinomas have dramatically enhanced growth potential in long-term culture relative to RME cells . Furthermore, a subset of these tumors are also independent of growth factors required by RME cells, and expression of growth factor independence is associated with high neoplastic potential in vivo. Neuroscience, 1987 Oct, 23(1), 73 - 86 Gabaergic neurons of the hippocampus: development in homotopic grafts and in dissociated cell cultures; Robain O et al.; The hippocampus taken from E18-E19 rat embryos was dissociated into a cell suspension and was either grafted into the hippocampus of adult rats or cultured . The growth of GABAergic neurons was examined using a GABA directed antiserum . The implanted tissue was capable of survival and growth without exhibiting a laminar organization . Most of the various morphological neuronal types could be observed, establishing different types of synapses; however, granule neurons were rarely encountered . A substantial proportion of GABA-positive neurons was detected within the graft with profuse labelling of the neuropil . In cultures issued from the same cell suspension, GABA-immunoreactive neurons were numerous and had different morphologies . Altogether these data suggest that GABA neurons express a high potential for growth and sprouting in vitro and in vivo. Radiat Res, 1987 Oct, 112(1), 105 - 15 The radiation response of a human colon adenocarcinoma grown in monolayer, as spheroids, and in nude mice; West CM et al.; A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice . The growth and radiation responses of the cells under these different growth conditions were compared . The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively . The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively . The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar . Values for D0 were 1.5 Gy and for n between 3 and 5 . Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6 . Oxygen enhancement ratios were approximately 3.3 . Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively . The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids . Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors. Cryobiology, 1987 Oct, 24(5), 412 - 9 Seasonal changes in recovery of cryopreserved murine lymphocytes resemble endogenous rhythms of unfrozen cells; Brock MA; Seasonal changes in the resistance of C57BL/6 mouse splenocytes to cryopreservation stress were expressed in both the recovery of viable cells and the levels of responses of T and B lymphocytes to mitogens in vitro . Single cell suspensions in 10% Me2SO were cooled at 1 degree C/min, the optimum velocity which was determined by using a range of cooling rates during January and May, the months of minimum and maximum recoveries of viable cells, respectively . After rapid thawing and washing, ethidium bromide-fluorescein diacetate staining delineated viable and nonviable cells . Cultures containing 0.5 X 10(6) viable cells were stimulated with the T lymphocyte mitogens, phytohemagglutinin and concanavalin A, and the B lymphocyte mitogen, lipopolysaccharide . Tritiated thymidine was added to each culture for the last 18 hr of the incubation period, and its incorporation by activated dividing cells was determined . Recoveries of viable cells were high from March through July and then declined to minimum levels in January and February . During the seasons of low recoveries, greater numbers of cells lysed in response to the freeze-thaw cycle . Activation of both T and B lymphocytes by mitogens was maximal in the spring and summer and then declined to only 40% of unfrozen control levels in October . The patterns of activation resembled those of the previously documented endogenous seasonal rhythms in levels of blastogenesis of unfrozen cells . These seasonal differences in cryopreservation properties of lymphocytes from inbred mice living under constant conditions reinforce the previously reported endogenous annual rhythmicity in cellular functions. Circ Res, 1987 Oct, 61(4), 548 - 54 Release of endothelium-derived relaxing factor from freshly harvested porcine endothelial cells; Hartmann A et al.; Vascular relaxation in rabbit aortic preparations induced by acetylcholine is endothelium-dependent . The nature of the endothelium-derived relaxing factor (EDRF) has not been ascertained because it is very labile (reported half-life 6-50 seconds) . To obtain a stable source of EDRF, a system was developed in which the relaxing factor was continuously produced by freshly harvested porcine endothelial cells . Endothelial cells were collected from aortas by exposing the endothelial lining to collagenase 0.1% . Cells were washed and concentrated by repeated centrifugation to obtain a high cell count (7.2 X 10(6) cells/ml) . Endothelium-deprived aortic strips from rabbits were incubated in these cells suspended in tissue culture medium and fetal calf serum . The strips were precontracted with histamine . Acetylcholine was added to induce EDRF release . Significant relaxation of endothelium-deprived aortic strips was observed . Superoxide dismutase, an enzyme known to protect EDRF against inactivation, caused further relaxation, which was inhibited by the addition of hemoglobin, an agent known to inhibit the relaxing action of EDRF . Even without the addition of acetylcholine, hemoglobin caused contraction of the denuded aortic strips in suspension of porcine endothelial cells, demonstrating spontaneous EDRF release . Hemoglobin had no effect in cell-free medium . Endothelial-cell-dependent relaxation occurred without attachment of endothelial cells to the endothelium-deprived aortic strips: when the cell suspension was replaced by cell-free medium, relaxation did not occur after acetylcholine . Scanning electron microscopy showed no attachment of endothelial cells to the subendothelial layer . It can be concluded that freshly harvested endothelial cells produce endothelium-derived relaxing factor with an without stimulation by acetylcholine. Appl Biochem Biotechnol, 1987 Oct, 15(3), 191 - 200 Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system; Suzuki M et al.; A novel tumor-detection system consisting of complement-mediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells . The present system consists of a CCD image sensor, image memory board, personal computer, and microscope . When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred . Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy . The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory . After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction . L-10 cells in mixed cell suspension were determined specifically by the system . Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min . Since this method does not require a cell washing process, automation of the whole system is possible. Bone Marrow Transplant, 1987 Oct, 2(3), 271 - 8 Effects of low dose rate irradiation on human marrow hematopoietic and microenvironmental cells: sparing effect upon survival of stromal and leukemic cells; Laver J et al.; The effects of different dose rates of in vitro irradiation on the proliferative capacity of marrow stromal, hematopoietic and leukemic colony-forming cells (CFC) are described . Marrow cell suspensions, HL-60 cells and trypsin-dispersed fibroblasts were irradiated at 5 or 45 cGy/min and then assayed for CFC . Irradiation at low (5 cGy/min) compared to high (45 cGy/min) dose rate showed a significant difference in survival of stromal and of HL-60 cells, but not of hematopoietic progenitors: the respective D0 values were 170 and 120 (p = 0.003) for marrow fibroblastic progenitors (CFU-F); 145 and 110 (p = 0.005) for passaged marrow fibroblasts (CFU-F); 170 and 140 (p = 0.045) for HL-60 cells; 85 and 85 for multipotential CFC (CFU-mix); 125 and 120 for erythroid progenitors (BFU-E); and 115 and 120 for granulomonopoietic progenitors (CFU-GM) (p = 0.5 for hematopoietic clonogenic cells) . Marrow suspensions did not establish confluent stromal layers in long-term marrow cultures following irradiation with 600 cGy at 45 cGy/min, whereas after 840 cGy at 5 cGy/min confluent stromal layers were obtained . This indicates that low dose rate-sparing effect applies to all stromal cell progenitors . Confluent stromal layers derived from progenitors surviving irradiation sustained hematopoiesis as well as controls when co-cultured with fresh hematopoietic cells . Adherent layers in long-term marrow cultures irradiated after establishment with doses less than or equal to 1500 cGy at 5 or 45 cGy/min also showed normal hematopoietic supportive function when co-cultured with freshly isolated hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS) Clin Exp Immunol, 1987 Oct, 70(1), 143 - 51 Adoptively transferred reactivity to M . leprae in nude mice infected with M . leprae; Shannon EJ et al.; Reversal reactions are manifestations of delayed hypersensitivity to M . leprae and are thought to be usually accompanied by manifestations of effective cell-mediated immunity (CMI) as measured by bacterial clearing . These experiments were designed to study the induction of reversal reactions in M . leprae-infected, congenitally athymic nude mice using adoptive transfer of CMI . Splenic cell suspensions derived from unimmunized heterozygous nu/+ mice, and those vaccinated with heat-killed M . leprae, viable BCG and a mixture of the two antigens were diluted to contain 10(4), 10(5), 10(6), 10(7) lymphocytes/0.1 ml and infused intravenously into multibacillary nude mice . The production of reversal reactions in leprous nude mice in response to adoptively transferred CMI was studied in a quantitative fashion . Dose responsive induction of reversal reactions, apparent by footpad inflammation and swelling, decreased morphological indices (MI) of the bacteria and mononuclear cell infiltrations, histopathologically, were observed . For nude mice receiving cells primed with 3.9 X 10(5) living BCG alone, the effective dose 50% (ED50) was 1.0 x 10(6) lymphocytes to induce reversal reactions . For those receiving cells primed with 10(7) M . leprae the ED50 was 3.7 x 10(5) lymphocytes . For nude mice receiving cells primed with a mixture consisting of 1/2 the above dose of BCG + 1/2 the above dose of M . leprae, the ED50 was 6.8 x 10(4) lymphocytes. Urology, 1987 Oct, 30(4), 333 - 6 Flow cytometric analysis of relative mean DNA content of urogenital cancer cells in fresh and paraffin-embedded materials; Nakamura K et al.; The relative mean DNA content calculation was performed by flow cytometry on single cell suspensions prepared from fresh and paraffin-embedded specimens of 10 patients with surgically resected urogenital cancer . Samples were processed by a modified method of Hedley et al . including two hours of pepsinizing time, ribonuclease digestion, and propidium iodide staining . The mean DNA content which is a quantitative description of flow cytometric characteristics was significantly correlated between the fresh and paraffin-embedded materials (n = 10, r = 0.869, p less than 0.01) . This method allows for the objective, retrospective analysis of DNA content in relation to diagnosis and prognosis of urogenital cancer. Infect Immun, 1987 Oct, 55(10), 2502 - 8 Adoptive transfer of immunity to Treponema pallidum Nichols infection in inbred strain 2 and C4D guinea pigs; Wicher V et al.; T lymphocytes purified from lymph nodes and spleens of chancre-immune, inbred strain 2 guinea pigs, when infused into syngeneic guinea pigs, conferred protection against challenge with Treponema pallidum subsp . pallidum Nichols . No protection was conferred by similar injections of cell suspensions from normal guinea pigs or guinea pigs immunized with T . phagedenis biotype Reiter or T . pallidum-free testis supernatants from infected rabbits . Similar results were obtained with homozygous C4D guinea pigs . After several months of infection, 2 of 11 strain 2 and 1 of 8 strain C4D recipients of T . pallidum-immune cells developed an erythematous reaction of short duration at the injection site; 2 of these recipients were positive for T . pallidum . Throughout the experimental period the humoral response to treponemal antigens was substantially lower in the adoptively immune guinea pigs than in various unprotected control groups . Passive immunity to infection with T . pallidum, however, seems to be dose related, since asymptomatic infection persisted for as long as 3 months after challenge in strain 2 guinea pigs transfused with 10(8) T . pallidum-immune lymphocytes, but not in C4D recipients of twice as many immune cells. Blood, 1987 Oct, 70(4), 1063 - 8 A two-color flow cytometry assay for detection of hairy cells using monoclonal antibodies; Kristensen JS et al.; We have developed a simple two-color immunofluorescence assay equally suited for microscopy and flow cytometry detecting hairy cells (HCs) in single cell suspensions, based on the concomitant reactivities with the B cell-specific monoclonal antibody B1 (CD20) and the monocyte/HC-associated antibody SHCL-3 (CD11c) . Thus, HCs can be demonstrated in peripheral blood, bone marrow, and spleen specimens from hairy cell leukemia (HCL) patients even when they constitute less than 1% of the cell suspension . Likewise, admixture experiments with normal mononuclear cells and the MOLT-4 T-acute lymphocytic leukemia (ALL) cell line demonstrated that HCs could be detected in amounts as low as 1% . The validity of this assay has been ascertained by the lack of double marker positivity in cell suspensions from B-chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML) patients that only expressed B1 or SHCL-3, respectively . Furthermore, other malignant blood diseases, including malignant lymphomas, acute leukemias, and chronic leukemias disclosed no double marker positive cells . In a clinical setting, this assay was used for purifying HCs (by flow cytometry) from the peripheral blood from patients with no apparent morphological evidence of circulating HC infiltration and for monitoring the effect of interferon therapy . In conclusion, this assay should be of value for both diagnosis and monitoring patients with HCL. Am J Surg Pathol, 1987 Oct, 11(10), 779 - 87 Distinction between undifferentiated (small noncleaved) and lymphoblastic lymphoma . An immunohistologic study on paraffin-embedded, fixed tissue sections; Brownell MD et al.; The morphologic differentiation between malignant lymphoma of the small noncleaved cell (SNC) type and lymphoblastic lymphoma (LBL) is at times difficult, particularly when fresh tissue is not available for immunologic studies . We have examined the reactivities of a panel of monoclonal and polyclonal antibodies, including LN-1, LN-2, and antibodies to immunoglobulin light chains, leukocyte common antigen (LCA), Leu-M1, vimentin, S-100 protein, lysozyme, and alpha-1-antitrypsin, in paraffin-embedded, B5- and formalin-fixed tissue involved by SNC or LBL . The immunophenotypes in all of the cases included in this study had been characterized previously in fresh-frozen sections or cell suspensions . Among 21 samples of B5-fixed SNC, LN-1 was reactive in 17 and LN-2 in 18 cases . Among 13 B5-fixed LBL, LN-1 was reactive in two cases and LN-2 was reactive in two cases . Each of 20 B5-fixed samples of SNC was reactive with at least one of the antibodies, whereas 10 of the 13 B5-fixed samples of LBL were negative for both antibodies . Lesser reactivity was evident in formalin-fixed tissues, with only nine of 27 SNC specimens positive for LN-1 and 16 of 27 positive for LN-2 . Most or all of the SNC and LBL samples were negative for immunoglobulin light chains, Leu-M1, vimentin, S-100 protein, lysozyme, and alpha-1-antitrypsin . The majority of both SNC and LBL were positive for LCA . We conclude that LN-1, preferably in combination with LN-2, can be used for distinguishing between SNC and LBL in paraffin-embedded, B5-fixed tissue when fresh tissue is not available. Vet Med (Praha), 1987 Oct, 32(10), 633 - 40 {The growth of attenuated strains of canine parvovirus, mink enteritis virus, feline panleukopenia virus, and rabies virus on various types of cell cultures}; Zuffa T; The growth characteristics were studied in the attenuated strains of canine parvovirus CPVA-BN 80/82, mink enteritis virus MEVA-BN 63/82 and feline panleucopenia virus FPVA-BN 110/83 on the stable feline kidney cell line FE, and in the attenuated canine distemper virus CDV-F-BN 10/83 on chicken embryo cell cultures (KEB) and cultures of the stable cell line VERO . When the FE cultures were infected with different parvoviruses in cell suspension at MOI 2-4 TKID50 per cell, the first multiplication of the intracellular virus was recorded 20 hours p . i . In the canine parvovirus, the content of intracellular and extracellular virus continued increasing parallelly until the fourth day; then, from the fourth to the sixth day, the content of extracellular virus still increased whereas that of intracellular virus fell rapidly . In the case of the mink enteritis virus the release of the virus into the culture medium continued parallelly with the production of the cellular virus until the sixth day . In the case of the feline panleucopenia virus the values concerning free virus and virus bound to cells were lower, starting from the second day p . i . When KEB or VERO cultures were infected in cell suspension with the canine distemper virus at MOI about 0.004 per 1 cell, the replicated intracellular virus was first recorded in the KEB cultures five hours after infection but in the VERO cultures only 20 hours after infection, with a timely release of the virus into the culture medium in both kinds of tissue . In the KEB and VERO cultures the highest values of infection titres were recorded on the fourth day p . i., the course of virus multiplication on the cells being parallel with its release into the culture medium. J Endocrinol, 1987 Oct, 115(1), 71 - 6 ACTH and adrenal aerobic glycolysis . II: Effects of aminoterminal peptide fragments on lactic acid and steroid production by mouse adrenocortical cells; Hinson J et al.; The effects of shortening the ACTH molecule from either end of the peptide chain on adrenal glycolysis and steroidogenesis were examined in mouse adrenal cell suspensions . Shortening the (1-24) sequence to (1-17), (1-16) and (1-14), thereby interfering with the basic tetrapeptide (15-18) assigned to the address message, progressively reduced both glycolytic and steroidogenic potencies by four, six and ten orders of magnitude respectively, without impairing the capacity for maximal excitation . The glycolytic potency of the (1-18) sequence, which was amidated at the C-terminal, equalled that of ACTH (1-24), but the steroidogenic potency was reduced by an order of magnitude . The (1-13) sequence of alpha-MSH, which contains substitutions at both terminals, had glycolytic and steroidogenic potencies intermediate between those of ACTH(1-16) and ACTH(1-17) . Deletion of Ser1,Tyr2 from ACTH(1-18)-NH2 reduced both potencies by an order of magnitude . ACTH(11-24) and (7-38) were inactive or inhibitory . The capacity for excitation was further examined by comparing responses to peptide fragments (1-4), (1-10), (1-13), (4-10), (4-11), (5-10), (5-14), (7-13) and (11-24) at a concentration of 1 mmol/l . All fragments, excepting (1-4), (5-10) and (11-24) were active . The activities of fragments (5-14) and (7-13), as opposed to (5-10), suggest that the requirements for methionine in position 4 may be replaced by the (11-13) tripeptide.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1987 Oct, 258(1), 226 - 32 Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L.) cell suspension cultures; Kuhnl T et al.; Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate . trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate . trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates . The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor . NADH and ascorbic acid cannot substitute for NADPH . Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase . Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsibl for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate . The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light . We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters . Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway. J Immunol, 1987 Sep 15, 139(6), 1861 - 6 The in vivo effects of interleukin 1 . I . Bone marrow cells are induced to cycle after administration of interleukin 1; Neta R et al.; We have previously reported that interleukin 1 (IL-1) administration 20 hr before irradiation protects mice from lethal effects of radiation . The recovery of total nucleated bone marrow cells and of hematopoietic progenitor cells was enhanced in IL-1 treated, as compared to untreated, irradiated mice . This suggested that IL-1 administration may affect the cells in the bone marrow of normal mice . Intraperitoneal administration of recombinant IL-1 resulted in bone marrow cell enlargement and increased cycling of these enlarged cells . In addition, the capacity of bone marrow cells from IL-1 treated mice to proliferate in response to granulocyte macrophage-colony-stimulating factor (GM-CSF) in cell suspension cultures was enhanced . The above effects were not genetically restricted as C57BL/6, B6D2F1, C3H/HeN, and C3H/HeJ mice showed similar responses . A comparative study showed that 100 ng of IL-1 was much more effective in stimulating bone marrow cells by the above criteria than 5 micrograms GM-CSF . Since IL-1, unlike CSF, can not be demonstrated to have a direct in vitro stimulatory effect on bone marrow cells, the aforementioned in vivo effects of IL-1 are presumably mediated by other hematopoietic growth factors . We have previously shown that IL-1 induces the appearance of high titers of CSF in the serum . Consequently hematopoietic growth factors that are generated at local sites following IL-1 administration may mediate the observed cell cycling effect. J Biol Chem, 1987 Sep 5, 262(25), 12085 - 91 An expanded two-state allosteric model for interactions of human hemoglobin A with nonsaturating concentrations of 2,3-diphosphoglycerate; Kister J et al.; Oxygen binding curves (OEC) for red cell suspensions have a biphasic shape and reduced n50 values when the concentration of 2,3-diphosphoglycerate (DPG) is lowered by aging or experimental procedures . The mechanism for the abnormal shape of the OEC has been related to variations in the activity of free DPG . DPG binds to tetrameric Hb at a single site, and in red cells its normal concentration is equivalent to that of tetrameric Hb . This equivalence renders the oxygen affinity of Hb and the shape of the OEC very sensitive to small changes in the activity of DPG . The OEC for stripped Hb solutions in the presence of nonsaturating concentrations of DPG also exhibit a biphasic shape but with much larger changes in the n values than observed for red cells . Upon addition of chloride, a known competitor of DPG binding to Hb, the shape of the OEC becomes similar to that of red cell suspensions with the same DPG/Hb ratio . Studies on Hb solutions in the presence of varying concentrations of DPG, but without chloride, have revealed that the cofactor shifts the entire OEC to the right, including both its upper and lower asymptotes . This finding indicates that DPG lowers the intrinsic oxygen affinity for both the T and R states . Theoretical considerations leading to a successful modeling of OEC obtained under varying conditions of DPG and chloride require an expanded two-state allosteric model in which allowance is made for DPG-dependent variations in the dissociation constants of oxygen for both the T and R conformations. Biomaterials, 1987 Sep, 8(5), 360 - 6 Microencapsulation of CHO cells in a hydroxyethyl methacrylate-methyl methacrylate copolymer; Dawson RM et al.; Chinese hamster ovary fibroblasts, as model cells, have been microencapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer (HEMA-MMA) by interfacial precipitation . The polymer containing approximately equal to 75 mol% HEMA, dissolved in polyethylene glycol 200 (PEG 200) was coextruded with the cell suspension (4-6 X 10(5) cells/ml in the alpha-MEM with 10% foetal calf serum +/- Ficoll 400/PBS) through a concentric needle assembly . Polymer solution droplets, containing cells, were blown off the end of the needle assembly by a coaxial filtered air stream into a nonsolvent bath containing phosphate buffered saline (PBS) with 5 ppm Pluronic L101, overlaid with hexadecane . The nascent capsules hang at the hexadecane/PBS interface while the solvent is extracted into the aqueous nonsolvent, to precipitate the polymer around the cells . The resultant capsules were 500 microns-1 mm in diam . with a microporous sponge-like interior, and also very tough and flexible . The cells survived encapsulation based on subculture ability, retention of some fluorescein diacetate (FDA) activity over 5 d and direct light microscopic evidence of cell growth over 10 d after histological sectioning and staining . However, cell growth was not uniformly observed (especially in the FDA assay) and this was attributed to space limitations for growth within the microporous interior . Continued development of this process and adaptation to cells such as pancreatic islets is expected to lead to hybrid artificial organs which are capable of ameliorating metabolic disorders such as diabetes. Microvasc Res, 1987 Sep, 34(2), 200 - 10 Oxygen transport studies of normal and sickle red cell suspensions in artificial capillaries; Stathopoulos NA et al.; Oxygen transport from normal and sickle red cells was studied under known and carefully controlled conditions simulating the microcirculation . Oxygenated red cell suspensions became deoxygenated as they traversed silicone rubber artificial capillaries of 27 microns diameter . Oxygen saturation values of the flowing red cell suspensions were measured at several axial positions along the artificial capillary by use of a microspectrophotometric technique . Oxygen saturation decreased with increasing distance from the entrance of the artificial capillary and was influenced strongly by the flow rate . Under the same hematocrit and flow conditions, the rate of oxygen saturation decrease was significantly higher for the sickle red cells than that for the normal red cells . Similar results were obtained by use of a mathematical simulation of oxygen transport in the microcirculation for both normal and sickle red cells . Sickle red cells would be expected to have a higher diffusional resistance to oxygen transport than would normal red cells . However, the higher diffusional resistance is more than offset by the lower oxygen affinity of the sickle cells . The difference in oxygen affinity appears to account for the difference in oxygen transport rates between normal and sickle red cells. J Reprod Fertil, 1987 Sep, 81(1), 59 - 64 Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro; Mitchell BS et al.; Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro . During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells . Albumin was also detected in GMG cells at the same stages . The IgG and albumin were not detected during the next 4 days in culture . When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells . When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively . Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated . The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo. J Acoust Soc Am, 1987 Sep, 82(3), 794 - 9 Inversion of ultrasonic scattering data for red blood cell suspensions under different flow conditions; Lucas RJ et al.; Recent results for low-frequency scattering by correlated random distributions of nonspherical particles averaged over orientation are applied to invert ultrasonic data for red blood cell suspensions under different flow conditions . The inversion procedure isolates a correlation parameter (c) representing a process in which the volume fraction (w) of particles increases linearly, and also a cell population parameter P . Reduced data records of scattering versus hematocrit are compared with S(c;w)P, where the generalized fluctuation function S is proportional to the variance in particle number, and P is proportional to the backscattering cross section of an isolated particle . The peak scattering for the different flow processes occurs at values of w ranging from about 0.15 for the most uniform to 0.25 for the least, corresponding to c values of about 2.1 to 0.4, as compared with w approximately equal to 0.13 and c = 3 for hard (repulsive at contact) spheres or aligned ellipsoids . The lower values of c suggest weaker repulsion between the deformable cells and effective interparticle attraction (aggregative trends), and c approximately equal to 2 may also involve flow alignment of the discoids. Surgery, 1987 Sep, 102(3), 485 - 92 Acceleration of B16 melanoma growth in mice after blood transfusion; Francis DM et al.; Evidence suggests that blood transfusions depress immunologic reactivity; as some tumors are influenced by the immune status of their host, it is possible that transfusions could promote tumor growth by impairing host immunity . The influence of blood transfusion on the growth of a transplantable B16 melanoma was examined in nude (athymic) CBA mice and immunocompetent C57 BL/6J mice . Recipients were given infusions of saline solution or syngeneic or H-2-incompatible allogeneic blood transfusions on two occasions 3 days apart . Infusions were begun 10 days before inoculation of a single cell suspension of B16 melanoma . Growth was determined by measurements of primary tumor volume and tumor weight after excision . There was no statistically significant difference in tumor size or weight between the three recipient groups of athymic mice . However, immunocompetent mice given H-2-incompatible allogeneic blood had higher rates of tumor engraftment--saline solution recipients versus allogeneic recipients: chi 2 = 13.2, df = 1, p less than 0.001; syngeneic recipients versus allogeneic recipients: chi 2 = 2.97, df = 1, p greater than 0.05 . In the allogeneic group significantly larger and heavier tumors developed than in mice given syngeneic blood or saline solution . The study indicates that H-2-incompatible allogeneic blood transfusions can influence the growth of a transplantable murine tumor by a mechanism that involves a cell-mediated immune response. J Clin Endocrinol Metab, 1987 Sep, 65(3), 575 - 80 Identification of human growth hormone messenger ribonucleic acid in pituitary adenoma cells by in situ hybridization; Pixley S et al.; The technique of in situ hybridization was used to examine human GH gene expression in human GH-secreting pituitary adenoma cells . Five somatotroph adenoma specimens obtained at surgery were dispersed into single cell suspensions . Hybridization experiments were performed on cells immediately after dispersal or on cells cultured for 48-72 h in a defined serum-free medium . Tritiated GH cDNA was used to probe fixed cells, and hybridization was determined by liquid autoradiography . Of the freshly dispersed adenoma cells probed with GH cDNA, more than 70% contained GH mRNA, as determined by counting silver grains per cell . Significant cellular grain counts were obtained for GH cDNA-probed cells from all five tumors, while negative controls showed negligible silver grain counts . In cultured cells derived from three of five tumors, an average of 40% contained detectable GH mRNA . Therefore, quantitative in situ hybridization is a useful technique to demonstrate the expression of GH mRNA in human pituitary adenoma cells. Invest Ophthalmol Vis Sci, 1987 Sep, 28(9), 1599 - 604 Ocular metastasis of in vivo and in vitro derived syngeneic murine melanoma; Harning R et al.; We examine ocular metastasis of syngeneic murine melanoma in C57Bl/6J mice and compare the metastatic capability of B16F10 tumor cells maintained in vivo with those maintained in culture . We demonstrate that as long as the tumor cells are derived from an in vivo source, intraocular tumor readily metastasizes to the lungs . When in vivo derived tumor is introduced as an intracameral (ic) cell suspension, or as a solid fragment implanted on the iris, 100% of the animals die with extensive pulmonary metastasis 5-6 weeks later . In contrast, when B16F10 cells are passaged five times in culture and inoculated ic, a marked decrease in the frequency and extent of metastasis, and an increase in survival is seen . These studies demonstrate an alteration in the ability of cultured B16F10 cells to metastasize from the eye . When metastasis of in vivo derived tumor from the eye was compared with metastasis from an extraocular location, the extent and frequency of pulmonary metastasis and survival of hosts was the same . The effect of enucleation on the metastasis of B16F10 from the eye has only previously been examined using cultured cells . In this paper, we demonstrate that the efficacy of enucleation depends upon whether B16F10 melanoma cells have been passaged in vivo or in vitro. Exp Neurol, 1987 Sep, 97(3), 564 - 76 Susceptibility to pentylenetetrazol-induced and audiogenic seizures in rats with selective fimbria-fornix lesions and intrahippocampal septal grafts; Cassel JC et al.; At the age of 31 days, Long-Evans female rats received electrolytic lesions either of the medial fimbria (N = 24), of the dorsal subcallosal fornix (N = 24), or of both structures (N = 24) . Ten rats were used as sham-operated controls . Ten days later, half the rats of each lesion group received intrahippocampal grafts of acetylcholine-rich fetal basal forebrain cell suspensions . Twelve months after grafting, all surviving rats, except six grafted rats which became very difficult to handle (because they developed convulsive behavior) were tested for reactivity to pentylenetetrazol (30 mg/kg, i.p.) and to sound (120 dB, 90 s) . Grafted rats were found to be more reactive to the drug and less reactive to sound than their nongrafted counterparts with similar lesions . Reactivity to pentylenetetrazol of grafted rats was correlated with body weight and extent of graft-induced hippocampal damage, but not with graft size . Reactivity to sound, which was apparently not dependent on hippocampal damage or graft size, might be related to enhanced graft-derived cholinergic activity in the hippocampus . Our results show that intrahippocampal septal grafts interfere in opposite directions with the seizure-inducing treatments used, so that it can be assumed that the physiologic mechanisms by which grafts do so and by which these treatments induce seizures are likely to be different. Biull Eksp Biol Med, 1987 Sep, 104(9), 325 - 7 {Effect of antiglobulin serum on the expression of M-cholinoreceptors of spleen lymphocytes of intact and immunized mice}; Ado AD et al.; Using a labelled blocker of M-cholinoreceptors (M-CR)--3H-Quinuclidinyl benzilate--the number of the receptors on spleen lymphocytes has been determined before and after immunization of CBA and BALB/c mice with antiglobulin serum . The incubation of non-separated spleen cell suspension with antiglobulin serum decreased the number of M-CR by 14%, while the incubation of the enriched B-lymphocyte suspension decreased it by 32.5% . The immunization of animals with ovalbumin or bovine red blood cells increased the serum effect on M-CR expression in non-separated lymphocyte suspension and had practically no influence on the serum effect in B-lymphocyte suspension . Thus, the effect on immunoglobulin receptors of B lymphocytes has a pronounced influence on M-CR expression, which may be one of the mechanisms of nervous and immune systems interaction. Am Rev Respir Dis, 1987 Sep, 136(3), 710 - 7 Metachromatic cell progenitors and specific growth and differentiation factors in human nasal mucosa and polyps; Otsuka H et al.; We previously demonstrated that fluctuations in circulating metachromatic cell progenitors were inversely related to nasal metachromatic cell (NMC) counts and nasal symptoms in allergic rhinitis . Now, we have quantitated NMC progenitors and lineage-specific growth factors using a hemopoietic colony assay . Cell suspensions from excised collagenase-treated nasal polyps (n = 7) contained 3.8 +/- 1.1 granulocyte colony-forming cells per 10(6) cells plated, compared to less than or equal to 0.5 in human tonsil suspensions, less than or equal to 0.5 in nasal mucosal epithelial scrapings, and 33 +/- 8 in peripheral blood of patients with ragweed allergic rhinitis (p less than 0.01) . The percentage of metachromatic cells in nasal-polyp-derived colonies was 47 +/- 10 compared with 3.0 +/- 0.7 in peripheral blood colonies (p less than 0.005) . Highly potent metachromatic cell colony-stimulating activity (CSA) was detected in supernatants from cultured human nasal epithelial scrapings from both polyps and atopic nasal mucosa, but not from nonatopic nasal mucosa . Supernatants from polyp mononuclear cells stimulated with phytohemagglutinin also contained metachromatic cell-CSA, which had an approximate molecular size of 25 to 70,000 daltons on column chromatography . An IL-3-like activity was also detected in these supernatants . These observations provide further evidence for in situ hemopoietic mechanisms in human nasal mucosa, involving epithelium-derived stimulation of local metachromatic cell progenitor growth and differentiation in allergic rhinitis. Cell Tissue Kinet, 1987 Sep, 20(5), 473 - 83 Cell cycle responses of heterogeneous human colon adenocarcinoma subpopulations to X-irradiation; Bliven SF et al.; The cell cycle responses of two exponentially growing subpopulations of cells (clones A and D), originally obtained from a human colon adenocarcinoma to X-irradiation, were studied using centrifugal elutriation . Cell suspensions were separated by changing counter-current flow rate while keeping the rotor speed constant (1600 rpm) and the composition of eluted fractions was determined using flow cytometry . The X-ray sensitivity of unseparated clone D cells was somewhat greater than that of clone A cells (e.g . 10% greater at the 10% level of survival) . This difference appeared to be due to a greater value of the alpha parameter (one-hit cell killing), using the linear-quadratic equation in which the relative survival S/S0 = exp - (alpha D + beta D2) with dose (D) in Gy . This finding was confirmed in the cell cycle studies where the alpha parameter was always greater for the clone D cells than for the clone A cells . The beta parameter was essentially the same for both cell lines through the cell cycle. Acta Cytol, 1987 Sep-Oct, 31(5), 537 - 56 Applications of monoclonal antibodies in clinical cytology as exemplified by studies with monoclonal antibody B72.3 . The George N . Papanicolaou award lecture; Johnston WW; The monoclonal antibody (MAb) B72.3, reactive with a high-molecular-weight, glycoprotein, tumor-associated antigen, designated TAG-72, has been previously shown to be reactive with formalin-fixed, paraffin-embedded tissue sections of adenocarcinomas of the ovary, colon and breast, but not a variety of normal adult tissues . It has demonstrated utility as an immunocytochemical adjunct for the diagnosis of carcinoma in cell blocks and cytocentrifuge preparations of human serous effusions, with selective reactivity for tumor cells (particularly adenocarcinoma) over reactive mesothelium . Using the avidin-biotin complex (ABC) method of immunoperoxidase staining and formalin-fixed, paraffin-embedded cell suspensions, MAb B72.3 detected tumor cells in effusions from all of 21 patients with adenocarcinoma of the breast . No reactivity was demonstrated in any cell type in benign effusions from 41 patients . In contrast, MAb B72.3 showed no reactivity to leukemic or lymphomatous effusions, or to mesothelial cells from malignant effusions . MAb B72.3 also detected adenocarcinoma cells in effusion specimens from 12 of 12 patients with adenocarcinoma of the lung and 16 of 16 patients with adenocarcinoma of the ovary . MAb B72.3 has recently been used with fine needle aspiration (FNA) biopsy specimens and the corresponding surgically excised tumors to determine cellular reactivity . Using the ABC immunoperoxidase method, fine needle aspirates and corresponding surgically excised tumors were analyzed for TAG-72 expression . Positive staining with MAb B72.3 was observed in needle aspirates of 27 of 27 adenocarcinomas and adenosquamous carcinomas of the lung, 17 of 21 adenocarcinomas of the breast, 6 of 6 adenocarcinomas of the colon and in carcinomas from other body sites . In contrast, 21 small-cell carcinomas of the lung, 13 malignant melanomas, 2 lymphomas and 2 sarcomas did not stain with the antibody . Benign lesions from the breast, lung, pancreas, parotid and thyroid also showed no staining . In many patients, tumor-bearing tissue had also been resected and was available for comparative examination with MAb B72.3 . In more than 90% of these patients, the staining patterns of the tumor cells in the aspirates were found to be predictive of the patterns of antibody reactivity in the comparable surgically resected tumors . From these studies, it is concluded that MAb B72.3 defines a tumor-associated antigen that is expressed in neoplastic cells versus benign cells, that is most selectively expressed in carcinomas and that may be used as a novel adjunct for the diagnosis of neoplasms in effusions and in fine needle aspiration biopsies. Blood, 1987 Sep, 70(3), 860 - 8 Neutrophil elastase produces 52-kD and 30-kD glucocorticoid receptor fragments in the cytosol of human leukemia cells; Distelhorst CW et al.; Characterization of glucocorticoid receptors in leukemia cells is important to understand mechanisms of glucocorticoid resistance but has been impeded by receptor fragmentation in cytosol extracts . We recently found that formation of 52- and 30-kilodalton (kD) glucocorticoid receptor fragments in cytosol of leukemia cells is due to proteolysis and is blocked by diisopropylfluorophosphate (DFP) . In the present study, we identify a 28-kD serine protease in cytosol of leukemia cells that binds {3H}DFP and correlates with the formation of 52- and 30-kD receptor fragments . This protease is immunoprecipitated by antiserum to neutrophil elastase . Limited digestion of {3H}dexamethasone-21-mesylate-labeled receptors by purified neutrophil elastase produces 52- and 30-kD receptor fragments . Receptor fragmentation in the cytosol of leukemia cells in inhibited by methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethylketone, a highly specific inhibitor of neutrophil elastase . The addition of as few as 5% neutrophils to a lymphoid cell suspension provides sufficient elastase to produce receptor fragmentation . Our findings indicate that neutrophil elastase is responsible for receptor fragmentation in the cytosol of leukemia cells . The neutrophil elastase may be endogenous to the leukemia cells or may come from neutrophils that contaminate leukemia cell suspensions. J Physiol, 1987 Sep, 390, 453 - 67 Ryanodine releases calcium from sarcoplasmic reticulum in calcium-tolerant rat cardiac myocytes; Hansford RG et al.; 1 . The hypothesis tested in this study is that ryanodine depletes sarcoplasmic reticulum (s.r.) Ca2+ loading in suspensions of single adult rat cardiac myocytes by effecting Ca2+ release into the myoplasm resulting in an increase in myoplasmic free {Ca2+} ({Ca2+}i) . The latter was monitored by the fluorescent dye, quin2 . 2 . The competency of the technique to detect s.r . Ca2+ release was tested by using caffeine to induce Ca2+ release . The addition of 5-10 mM-caffeine to myocytes loaded with quin2 and incubated in a medium containing 1 mM-Ca2+ gives a large, transient increase in fluorescence, which is interpreted as indicating an increase in {Ca2+}i . If the chelating agent EGTA is added to the cell suspension 1-5 min prior to the caffeine, to a concentration sufficient to decrease extracellular Ca2+ to 0.1-0.15 microM, then caffeine again gives a large, transient increase in fluorescence, indicative of the fact that sarcolemmal Ca2+ transport is not necessary for this response . The ionophore ionomycin also raises {Ca2+}i in a transient manner when added after EGTA . The addition of caffeine prior to ionomycin largely diminishes the response to the latter; however, addition of ionomycin prior to caffeine totally abolishes its effect to increase {Ca2+}i . This is taken to indicate that the intracellular store which is releasable by caffeine--and which presumably reflects the s.r.--is also releasable by ionomycin: ionomycin, however, also gives access to another, minor intracellular pool . 3 . The plant alkaloid, ryanodine, at concentrations of 10(-8) to 10(-6) M, consistently causes a slow and prolonged increase in {Ca2+}i when added to cell suspensions incubated with 1 mM-extracellular Ca2+ . Under conditions precluding net entry of Ca2+ into the cell, viz . 0.1 microM-extracellular Ca2+, ryanodine causes a more limited, partially reversible, increase in {Ca2+}i . 4 . When added prior to EGTA, ryanodine attenuates, or prevents, the subsequent response to caffeine: efficacy depends upon the time of pre-incubation (1-10 min) and the concentration of ryanodine (10(-8) to 10(-6) M) . When the response to caffeine is largely prevented by ryanodine, the response to ionomycin is also severely attenuated, i.e . there is no evidence that ryanodine causes sequestration of Ca2+ within an ionomycin-sensitive pool.(ABSTRACT TRUNCATED AT 400 WORDS) Histochem J, 1987 Sep, 19(9), 497 - 503 Single-cell immuno-beta-galactosidase staining of heterogeneous populations . Practical application on limited cell numbers; Leenen PJ et al.; In this paper we describe a sensitive immunocytochemical staining method, particularly useful for the study of subpopulations of cells in complex mixtures such as bone marrow cell suspensions . E . coli beta-galactosidase is used as a label, which has the advantage that no endogenous activity is observed under the present experimental conditions . Direct sedimentation of cells on to poly-L-lysine-pretreated multi-well slides followed by gentle fixation prevents cell loss during preparation and subsequent incubation steps . Furthermore, analysis of only a few hundred cells per sample is possible . We examined the sensitivity of this method by comparing the percentages of positive cells in a spleen cell suspension after staining with a panel of monoclonal antibodies followed by analysis with the present immuno-beta-galactosidase method or standard flow cytometry . For almost all antibodies used, the percentages of positive spleen cells obtained with the immuno-beta-galactosidase method at least equalled those obtained with flow cytometry . Several fixatives, used to permanently adhere the cells to the slide's surface, were tested for the preservation of both morphological and antigenic structure . Glutaraldehyde and formol acetone proved to be the best choices in this respect . The present method combines high sensitivity with good morphology and is especially useful for immunophenotyping low cell numbers of heterogeneous populations. Arch Biochem Biophys, 1987 Sep, 257(2), 416 - 23 Effects of Ca2+ on phytoalexin induction by fungal elicitor in soybean cells; Stab MR et al.; A glucan elicitor from the cell walls of the fungus Phytophthora megasperma f.sp . glycinea caused increases in the activities of the phytoalexin biosynthetic enzymes, phenylalanine ammonia-lyase and chalcone synthase, and induced the production of the phytoalexin, glyceollin, in soybean (Glycine max) cell suspension cultures when tested in culture medium containing 1.2 mmol/liter Ca2+ . Removal of extracellular Ca2+ by treatment with ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid followed by washing the cells with Ca2+-free culture medium abolished the elicitor-mediated phytoalexin response . This suppression was largely reversed on readdition of Ca2+ . Elicitor-mediated enhancement of biosynthetic enzyme activities and accumulation of glyceollin was strongly inhibited by La3+; effective concentrations for 50% inhibition were (mumol/liter) 40 for phenylalanine ammonia-lyase, 100 for chalcone synthase, and 30 for glyceollin . Verapamil caused similar effects only at concentrations higher than 0.1 mmol/liter, whereas trifluoperazine and 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate did not affect enzyme induction by the elicitor in the concentration range tested . Uptake of alpha-amino isobutyric acid into soybean cells, which was rapidly inhibited in the presence of the glucan elicitor, was not affected by La3+ nor was uptake inhibition by the elicitor relieved by La3+ . The Ca2+ ionophore, A23187, enhanced phytoalexin biosynthetic enzyme activities and glyceollin accumulation in a dose-dependent manner, with 50% stimulation (relative to the elicitor) occurring at about 5 mumol/liter . The results suggest that the glucan elicitor causes changes in metabolite fluxes across the plasma membrane of soybean cells, among which changes in Ca2+ fluxes appear to be important for the stimulation of the phytoalexin response. Microvasc Res, 1987 Sep, 34(2), 223 - 30 Determination of volumetric flow in capillary tubes using an optical Doppler velocimeter; Davis MJ; The purpose of this study was to use the optical Doppler velocimeter of Borders and Granger {(1984), Microvasc . Res . 27, 117-127} to determine the ratio of centerline red cell velocity to mean velocity of blood flowing in small glass tubes . Red blood cell suspensions were perfused at different rates through capillary tubes (15-100 microns, id) while measuring centerline velocity (Vmeas) . Mean red cell velocity (Vmean) was calculated from measurements of volume flow in the tubes . With the effective slit width (sensor size) of the velocimeter set at 9.0 microns, the ratio of Vmeas/Vmean averaged 1.54 +/- 0.03 (mean +/- SEM), and was essentially independent of tube size . When the slit width was increased, the ratio of Vmeas/Vmean was significantly lower and appeared to vary as a function of tube diameter . These results are compared with previous measurements using other velocimeters, and the relationship between relative sensor size and ratio of Vmeas/Vmean is discussed. Arch Biochem Biophys, 1987 Sep, 257(2), 430 - 8 Purification and characterization of tyrosine aminotransferase activities from Anchusa officinalis cell cultures; De-Eknamkul W et al.; Three activities of tyrosine aminotransferase (TAT; EC 2.6.1.5), the enzyme which catalyzes the first step of the tyrosine pathway leading to the formation of rosmarinic acid (alpha-O-caffeoyl-3,4-dihydroxyphenyllactic acid), have been extensively purified from cell suspension cultures of Anchusa officinalis L . and subsequently characterized . TAT-1, TAT-2, and TAT-3 differ slightly in native molecular weights (180,000-220,000) and are composed of subunits (4 X 43,000 for TAT-1 and 4 X 56,000 for TAT-2) . All three enzymes show a pronounced preference for L-tyrosine over other aromatic amino acids, but TAT-2 and TAT-3 can also effectively utilize L-aspartate or L-glutamate as a substrate . For amino acceptor cosubstrates, either oxaloacetate or alpha-ketoglutarate can be utilized equally well by TAT-1, while the former is the most effective alpha-keto acid for TAT-2 and the latter is the best for TAT-3 . All the TAT activities display high pH optima (8.8-9.6), and are inhibited by the tyrosine metabolite 3,4-dihydroxyphenyllactate . TAT-2 and TAT-3 are also inhibited by rosmarinic acid. Biokhimiia, 1987 Sep, 52(9), 1469 - 73 {Changes in cAMP levels in bacterial cells during the cell cycle}; Lazareva AV et al.; The changes in the cAMP level during the cell cycle in the synchronous cultures of E . coli were demonstrated . Two maxima in the cAMP level were revealed during each generation period . In the cell cycle of 40-45 min duration the first increase was observed approximately in the middle of the cycle, i . e., it was coincident with the initiation of DNA synthesis . Under these conditions the cAMP level increased 8-10 times (from 0.5 to 5.0 pmole per ml of cell suspension) . The second, less pronounced increase in the cAMP level was observed immediately before or during the cell division and was probably related to the regulation of the cell wall formation. Proc Natl Acad Sci U S A, 1987 Sep, 84(17), 6055 - 9 Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase; Howard AD et al.; Placental alkaline phosphatase {orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1} is a member of a diverse group of membrane proteins whose attachment to the lipid bilayer is mediated by a phosphatidylinositol-glycan . To investigate structural aspects of the glycolipid anchor, cultured WISH cells were used because we found that they produce the enzyme in abundant quantities . When cell suspensions were incubated with purified phosphatidylinositol-specific phospholipase C, most of the placental alkaline phosphatase was released from membranes in a hydrophilic form . On incubation of the cells with {14C}ethanolamine, {14C}myristic acid, or myo-{3H}inositol, each was incorporated into the phosphatase near the carboxyl terminus, showing that these components, which are found in other phosphatidylinositol membrane-linked proteins, are also present in placental alkaline phosphatase. Tissue Antigens, 1987 Sep, 30(3), 97 - 103 A common epitope between HLA-B27, -B13 and -B37 alloantigens defined by a monoclonal antibody; Bourel D et al.; An anti-HLA-B27 monoclonal antibody produced by the hybridoma technique is described . This BD.7 reagent is a cytotoxic IgM antibody . Its reactivity was studied by lymphocytotoxicity tests, indirect immunofluorescence tests and biochemical analysis against an extensive panel of peripheral blood mononuclear cells . All HLA-B27 positive samples, either from normal subjects or from patients with Ankylosing Spondylitis, were recognized by this reagent . Moreover, a cross-reaction was observed with HLA-B13 cells, and a new unexpected reaction with all HLA-B37 cell suspensions . The interest of such a reagent is discussed. Cytometry, 1987 Sep, 8(5), 529 - 33 Technique and staining optimization leucoconcentration; Pierrez J et al.; In cytometric clinical application, it is important to obtain cell suspensions rapidly with as little cytological alteration as possible . A procedure has been achieved to prepare cell suspensions for flow cytometric analysis . The leucoconcentration technique, first described by Herbeuval for cytologic analysis, has been modified to be applied in cytometry . This technique involves Saponin lysis of red cells of peripheral blood or bone marrow samples that have been previously fixed with picric acid alcohol solution . Cells in suspension are not shifted and tinctorial affinity is not modified . Then cells have been stained with Mithramycin . Each parameter defined by Crissman has been analyzed to define the best staining conditions . The availability of Leucoconcentration with Mithramycin-DNA-staining permits determination of cell cycle with a fine resolution. Cytometry, 1987 Sep, 8(5), 479 - 87 Solid tumor preparation for flow cytometry using a standard murine model; Ensley JF et al.; The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions . Carcinomas containing a high desmosomal content, such as well-differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare . The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3-methylcholanthrene induced murine SCC for the comparative testing of preparative techniques . Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested . Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense . Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points . Ethanol-water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time . A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis. J Immunol Methods, 1987 Aug 24, 102(1), 127 - 41 Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials; Topalian SL et al.; The potential utility of tumor-infiltrating lymphocytes (TIL) in the adoptive immunotherapy of human tumors has been suggested by murine experiments showing these cells to be 50-100 times more powerful than LAK cells in treating advanced metastatic disease . A method for the large-scale expansion of human TIL for the use of these cells in clinical trials is described in this report . TIL were successfully expanded on an experimental scale from 24 of 25 consecutive human tumors, including six melanomas, ten sarcomas, and eight adenocarcinomas . Tumors were digested enzymatically to yield single cell suspensions which were cultured in RPMI 1640 medium with 10% human serum and 1000 U/ml recombinant interleukin-2 . Lymphocytes constituted from 3% to 74% of single cell tumor suspensions, and expanded from 2.9-fold to 9.1 X 10(8)-fold over a culture period ranging from 14 to 100 days . Nine of 24 TIL cultures lysed fresh autologous tumor targets in 4 h chromium release assays . Cell surface phenotyping identified cultured TIL as activated cytotoxic/suppressor T cells . Subsequently, large-scale expansion of TIL was successful in generating more than 10(10) lymphocytes in five of eight consecutive cases . Clinical trials employing the adoptive transfer of expanded TIL to patients with metastatic disease have begun. Biochim Biophys Acta, 1987 Aug 19, 930(1), 1 - 9 Differential effects of stimulus termination on excitation and desensitization of folic acid receptors and guanylate cyclase in Dictyostelium discoideum; de Wit RJ et al.; The response of guanylate cyclase to addition of extracellular stimuli is well documented . Here we report for the first time the response of guanylate cyclase to removal of stimuli . Three methods were employed to terminate rapidly a stimulus of folic acid . (1) Addition of a highly active folate deaminase preparation, or (2) 12-fold dilution of the stimulated cell suspension, or (3) addition of an excess concentration of a non-agonistic derivative of folic acid, i.e., 2-deaminofolic acid, which chases the folate agonist from its cell-surface receptors . Accumulation of cGMP terminated instantaneously upon addition of deaminase, but degradation of the synthesized cGMP was not observed until 10-12 s after stimulation . Also in a cGMP phosphodiesterase-lacking 'streamer' mutant an instantaneous termination of further cGMP accumulation was observed upon stimulus removal . This suggests that the termination of cGMP accumulation is due to inactivation of guanylate cyclase instead of a steady state of cGMP synthesis and degradation . Further accumulation of cGMP was approx . 75% reduced upon dilution of a cell suspension after stimulation with both agonists . Stimulation by 300 nM folic acid or by 30 nM N10-methylfolic acid (a more potent agonist) yielded identical results . However, upon addition of deaminofolic acid the accumulation of cGMP continued normally if the cells had been stimulated with N10-methylfolic acid, but only slightly in the case of a folic acid stimulus . The effect of stimulus duration on desensitization was monitored; it was observed that 50% desensitization was induced by stimulation for 1 s, while 4 s was sufficient for maximal desensitization . Short stimuli were observed to elicit high levels of desensitization without much excitation of guanylate cyclase . A desensitization-like process was observed at the level of the folate-binding chemotactic receptors as well . Relationships between the cGMP response data and folic acid receptor kinetics are discussed. Anal Biochem, 1987 Aug 15, 165(1), 133 - 6 Assay of tryptophan decarboxylase from Catharanthus roseus plant cell cultures by high-performance liquid chromatography; Pennings EJ et al.; An assay is described for the enzyme tryptophan decarboxylase from plant cell suspension cultures . It is based on the fluorometric detection of tryptamine by HPLC on a LiChrosorb RP-8 Select B column . Tryptophan decarboxylase from Catharanthus roseus was induced by transferring 14-day-old cells into an induction medium . Optimum activity was found 2 days after transfer, the increase being 5- to 10-fold . When kept at -15 degrees C the crude enzyme lost half its activity in about 7 days . The rate of the decarboxylation reaction was linear for at least 3 h at 35 degrees C. Arch Biochem Biophys, 1987 Aug 15, 257(1), 177 - 85 Studies on avian erythrocyte metabolism . XVI . Accumulation of 2,3-bisphosphoglycerate with shifts in oxygen affinity of chicken erythrocytes; Isaacks RE et al.; The ability of the chicken erythrocyte to accumulate 2,3-bisphosphoglycerate (2,3-P2-glycerate) and its effect upon the oxygen affinity (P50) of the cell suspensions have been determined . Erythrocytes from chick embryos, which contain 4-6 mM 2,3-P2-glycerate, and from chickens at various ages, which contain 3-4 mM inositol pentakisphosphate but no 2,3-P2-glycerate, were incubated with inosine, pyruvate, and inorganic phosphate . Red blood cells from 20-day chick embryos incubated in Krebs-Ringer, pH 7.45, containing 20 mM inosine and 20 mM pyruvate had an increase in 2,3-P2-glycerate from 4.3 to 11.9 mM after 4 h . Importantly, as 2,3-P2-glycerate concentration increased there was a corresponding increase in P50 of the cell suspension . Further, erythrocytes from 9- and 11-week, and 7-, 14-, 24-, and 28-month-old chickens when incubated similarly with inosine and pyruvate accumulated 2,3-P2-glycerate with corresponding increases in P50 of the cell suspensions . The ability of the red cell to accumulate this compound under the incubation conditions used apparently decreases with age of the bird (e.g., 11.9 mM in the 20-day embryo to 1.1 mM in the 28-month-old chicken after 4 h incubation) . Despite the presence of significant amounts of inositol-P5, the accumulation of 2,3-P2-glycerate markedly decreases oxygen affinity of the cell suspensions . The delta P50/mumol increase in 2,3-P2-glycerate in the red cells of the 20-day chick embryo after 4 h incubation is 1.5 Torr; conversely, the delta P50/mumol decrease in 2,3-P2-glycerate in the red cells of the 17-day embryo after 6 h incubation in the presence of sodium bisulfite is 2.8 Torr . The demonstrated ability of the chicken erythrocyte to accumulate 2,3-P2-glycerate in response to certain substrates suggests that regulation of concentration of this compound could contribute significantly to regulation of blood oxygen affinity in birds. J Biochem Biophys Methods, 1987 Aug, 14(5), 267 - 72 Rapid analysis of cellular lipids without extraction; Maziere C et al.; A method is described where cell suspension obtained by scraping of monolayers was directly applied on silica gel plates . Extraction and separation of different lipid classes were simultaneously obtained during chromatography . In the range of validity of the method (no more than 80 micrograms of cellular protein tested for neutral lipids and 30 micrograms for phospholipids), this technique allows rapid lipid analysis of small samples of cultured cells, bypassing all the time- and solvent-consuming extraction and evaporation steps . The method appears to be also suitable for measurement of enzymes of lipid metabolism such as acyl coenzyme A-cholesterol-acyltransferase. Teratology, 1987 Aug, 36(1), 87 - 93 Neuronal degeneration caused by ethylenethiourea in neuronal monocell layers in vitro and in fetal rat brain in vivo; Khera KS; The monocell layers, containing a mixture of neuronal and non-neuronal (primarily glial) cells, were obtained by growing cells dissociated from trypsinized fetal brains of 19-day pregnant rats under optimum conditions . Ethylenethiourea (ETU), at greater than or equal to .5 mM concentrations, caused in monocell layers in vitro a necrosis of neuronal cells and a marked depression in the formation of neurites and fascicles without any noticeable change in the non-neuronal cells . In the in vivo study, ETU orally administered as a single 30 or 45 mg/kg dose to rats on day 19 of pregnancy was found to induce necrosis of neuroblasts in the fetal CNS after 18 and 24 hours of dosing and a high incidence of hydrocephalus in postnatal pups at both doses . In an in vivo/in vitro experiment, rat fetal brains from 19-day pregnant dams which had previously been dosed with 80 or 120 mg ETU/kg were trypsinized and then dissociated into a cell suspension in a nutrient medium . The total number of viable cells in the suspension was significantly reduced compared to the number in the control suspension . The test suspension with cell density adjusted to that in the control suspension grew into monocell layers manifesting a marked decreased population of neuronal cells with a concomitantly increased population of non-neuronal cells . It was concluded that the target of ETU action was most likely the neuronal cells rather than the organization of nervous tissue per se. Gut, 1987 Aug, 28(8), 976 - 80 Phagocytes in cell suspensions of human colon mucosa; Beeken W et al.; Because little is known of the phagocytes of the human colon we enumerated these cells in mucosal suspensions and studied their phagocytic activity . Phagocyte rich suspensions were made by EDTA collagenase dissociation followed by elutriation centrifugation . Phagocytosis was evaluated by measuring cellular radioactivity after incubation of phagocytes with 3H-adenine labelled E coli ON2 and checked microscopically . Dissociation of normal mucosa from colorectal neoplasms yielded means of 1.9 X 10(6) eosinophils, 1.4 X 10(6) macrophages and 2 X 10(5) neutrophils per gram of mucosa . Visually normal mucosa of inflammatory states yielded 2.2 X 10(6) eosinophils, 2.3 X 10(6) macrophages and 7 X 10(5) neutrophils per gram of mucosa . Phagocyte rich suspensions of normal mucosa from tumour patients phagocytosed 21.8% of a pool of opsonised tritiated E coli ON2 and by microscopy 100% of mucosal neutrophils ingested bacteria, 83% of eosinophils were phagocytic, and 53% of macrophages contained bacteria . These results suggest that in the human colonic mucosa, the eosinophil is more abundant than the macrophage and the per cent of those cells exhibiting phagocytosis is intermediate between that of the macrophage and the neutrophil . Thus these three types of cells are actively phagocytic and share the potential for a major role in host defence against invasive enteric bacteria. Biol Chem Hoppe Seyler, 1987 Aug, 368(8), 991 - 9 Antigen-gelonin conjugates . Preparation and application in experimental myasthenia gravis; Brust S et al.; The antigen-specific immune suppression by gelonin-antigen conjugates was tested in two different systems: (i) the horseradish-peroxidase-stimulated T-cell proliferation in vitro and (ii) in vivo with experimental autoimmune myasthenia gravis (EAMG) in the rat . For this, the phytotoxin gelonin, a glycoprotein from Gelonium multiflorum, was purified and linked to the respective antigens . For the in-vitro assay a lymph node cell suspension from rats immunized with horseradish peroxidase was cultured in the presence of this protein and proliferation was measured by {3H}thymidine uptake . In-vitro proliferation was significantly inhibited by adding gelonin-horseradish peroxidase conjugates . The therapeutic effects of antigen-gelonin conjugates were tested in the rat model EAMG . For these experiments rats were immunized with purified nicotinic acetylcholine receptor from electric fish in order to develop EAMG . The success of the immunization was monitored by the change in physical performance tests, the change in anti-acetylcholine receptor antibody titer, and by the change in the number of ionic endplate channels using a novel electrophysiological method . The latter method permits a very accurate assay of functional damage of acetylcholine receptor at the endplate and correlates well with the clinical severity of the disease . Rats were conventionally immunized with acetylcholine receptor from electric fish . After the onset of EAMG as measured by physical performance tests and rise in antibody titer a group of the animals was injected with an acetylcholine receptor-gelonin conjugate and this treatment was repeated seven days later . The loss in functional acetylcholine receptor was significantly smaller in the therapy group than in the untreated EAMG group.(ABSTRACT TRUNCATED AT 250 WORDS) J Pharmacol Exp Ther, 1987 Aug, 242(2), 558 - 65 An examination of the ability of d-tubocurarine to evoke contraction and mediator release from superfused trachea and parenchymal strips isolated from the guinea pig; Fishleder RI et al.; The relationship between curare-induced mediator release and contraction in superfused guinea pig trachea and parenchymal strips was examined . In trachea, curare produced histamine release and contraction with peak release occurring in the first 90 sec (collection period 1) after challenge . Peak contraction developed later (collection periods 4-6) . Curare-induced contraction of parenchymal strips was inconsistent and smaller than that found in trachea . No histamine could be detected in parenchymal strip superfusate samples . Curare also was selective in releasing histamine from monodispersed airway cells vs . peripheral lung cells . No leukotriene bioactivity or immunoreactivity could be detected after curare challenge of tissues or cell suspensions . Tracheal contractions, but not histamine release, occurring early (first 5 or 6 collection periods) after challenge were antagonized by mepyramine, 10(-6) M, and phenoxybenzamine, 3 X 10(-5) M . Combination of FPL55712, 10(-5) M, with mepyramine did not further alter tracheal contraction . Contractions occurring later after challenge and total histamine release were enhanced by indomethacin, 5 X 10(-6) M . Indomethacin also increased contractions in the presence of mepyramine . With mepyramine and indomethacin, LY171883, 1 and 3 X 10(-6) M, and nordihydroguaiaretic acid, 3 X 10(-5) M, antagonized tracheal contractions to curare, 3 X 10(-3) M, but not to 1 X 10(-3) M, without altering histamine release . Indomethacin prolonged return to base line of tracheal tension after challenge with exogenous histamine . After addition of LY171883 or nordihydroguaiaretic acid, the return of tracheal tension after histamine was not different from that seen without drug pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS) Anesthesiology, 1987 Aug, 67(2), 185 - 90 Halogenated anesthetics increase oxygen consumption in isolated hepatocytes from phenobarbital-treated rats; Becker GL et al.; Using suspensions of hepatocytes isolated from phenobarbital-treated and untreated rats (+PB cells and -PB cells, respectively), the authors examined the effects of halothane, enflurane, and isoflurane on O2 consumption (VO2) and on extracellular PO2 and energy status at steady states of O2 and energy metabolism . In +PB cells, all three agents produced increases in VO2 which were largest at 1 MAC and progressively smaller at 2 and 3 MAC . At all three doses, VO2 increases were largest with enflurane (48% at 1 MAC), intermediate with halothane (24%), and smallest with isoflurane (11%) . These anesthetic-induced VO2 increases were abolished by prior addition of a cytochrome P450 inhibitor (metyrapone) to the incubations . In -PB cells, all three agents produced slight and comparable decreases in VO2 at 1 MAC, with further decreases at 2 and 3 MAC . In +PB cell suspensions at steady states of O2 and energy metabolism, 1 MAC enflurane or halothane, but not isoflurane, produced significant declines in steady state PO2 (from initial values of 24 mmHg to values less than 10 mmHg) and reductions in adenosine triphosphate/adenosine diphosphate ratio (ATP/ADP) . These changes were absent in -PB cells exposed to the same conditions or in +PB cells not exposed to anesthetic . The authors conclude that clinical doses of enflurane and, to a lesser extent, halothane produce statistically significant increases in O2 consumption, reflecting enhanced cytochrome P450 activity, in liver cells isolated from phenobarbital-treated rats . Such increases in O2 demand represent a mechanism by which anesthetic metabolism could contribute to intrahepatic hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS) Radiology, 1987 Aug, 164(2), 527 - 30 Ocular and cerebral metastases in the VX2 rabbit tumor model: contrast-enhanced MR imaging; Frank JA et al.; Ocular and cerebral metastases developed after the inoculation of a VX2 tumor cell suspension into the internal carotid artery of 15 rabbits . The hematogenous spread of tumor cells resulted in ocular metastases in 13 of 15 animals (86.7%) and cerebral system metastases in 14 of 15 animals (93%) . Magnetic resonance (MR) imaging with Gd-DTPA demonstrated early disruption of the blood-ocular barrier and blood-brain barrier 5-7 days after infusion of tumor cells . Quantitative assessment of contrast enhancement revealed a mean increase in signal intensity of 145% +/- 51% in the anterior chambers, 102% +/- 70% for choroidal metastases, and 51% +/- 29% for central nervous system (CNS) metastases . These results indicate that contrast-enhanced MR imaging can be used to demonstrate a loss of blood-ocular barrier integrity that is similar to the breakdown of the blood-brain barrier associated with metastatic tumors to the CNS and eye. Photodermatol, 1987 Aug, 4(4), 182 - 6 Stereospecific inhibition of human epidermal cell interleukin-1 secretion and HLA-DR expression by cis-urocanic acid; Rasanen L et al.; UV radiation is known to photoisomerize trans-urocanic acid (trans-UCA) into a stable isomer, cis-urocanic acid (cis-UCA) . To study the possible immunomodulatory effects of cis-UCA, the two isomers were added separately to different in vitro assays employing human epidermal cell suspensions or purified human peripheral T lymphocytes, supplemented with epidermal cells . Cis-UCA but not trans-UCA suppressed interleukin-1 (IL-1) production by epidermal cell suspensions in a dose-dependent fashion and diminished the number of HLA-DR positive epidermal cells to 61% of control values . An inhibitory effect on epidermal cell accessory function could be demonstrated with both isomers of UCA, but only if UVB-irradiated epidermis was used as a source for the epidermal cells . Taken together, the findings of our study lend indirect support to the concept of cis-UCA as a possible mediator of UV-radiation-induced immunosuppression. Invest Ophthalmol Vis Sci, 1987 Aug, 28(8), 1397 - 403 T cell subsets involved in the rejection of metastases arising from intraocular melanomas in mice; Niederkorn JY; The role of cell-mediated immunity in the resistance to spontaneous metastasis of intraocular melanoma was studied in C57BL/6 mice harboring syngeneic B16F10 melanomas . Mice were rendered T cell-deficient by thymectomy and lethal whole-body X-irradiation . Adult thymectomized and bone marrow-restored (ATXBM) mice were selectively reconstituted with immune lymph node cell suspensions that were depleted of specific T cell subsets . The selectively reconstituted hosts were used to evaluate the role of specific T cell subsets in controlling the metastatic spread of intraocular melanomas . The results revealed that T cell-deficient ATXBM mice were highly vulnerable to the metastatic spread of intraocular melanomas . However, this susceptibility could be virtually eliminated by the infusion of either normal or specifically sensitized lymphoid cells . Negative selection experiments demonstrated that the effector cells responsible for protection against metastases resided in a population with the surface phenotype characteristic of cytotoxic T lymphocytes: Thy 1+, Lyt 1+ and Lyt 2+. J Steroid Biochem, 1987 Aug, 28(2), 185 - 8 Inhibition of testosterone biosynthesis by ethanol: multiple sites and mechanisms in dispersed Leydig cells; Widenius TV et al.; Isolated rat Leydig cells were incubated for 2 h in sealed polycarbonate tubes under O2/CO2 atmosphere with 10 mIU/ml human chorionic gonadotropin . 20 mmol/l ethanol reduced the concentration of testosterone (16%, P less than 0.025); raised the concentrations of pregnenolone (60%, P less than 0.001), androstenedione (86%, P less than 0.001) and dehydroepiandrosterone (81%, P less than 0.001); but did not change concentrations of progesterone and 17 alpha-hydroxyprogesterone in the incubation medium . Ethanol also raised the lactate/pyruvate ratio in the Leydig cell suspension . 4-Methylpyrazole (0.5 mmol/l) abolished the ethanol-induced changes . The present results suggest that ethanol inhibits testosterone synthesis in isolated rat Leydig cells at the pregnenolone-to-testosterone pathway by inhibiting 3 beta-hydroxy-5-ene-steroid dehydrogenase/5-ene-4-ene-isomerase catalyzed reactions and the conversion of androstenedione to testosterone . These inhibitions are caused by consequences of ethanol metabolism . A likely mechanism for the former inhibition is that the increase in the NADH/NAD+ ratio in Leydig cells leads to inhibition of reactions catalyzed by 3 beta-hydroxy-5-ene-steroid dehydrogenase/5-ene-4-ene isomerase, but the inhibition mechanism operating at the androstenedione-to-testosterone step remains to be characterized. J Anat, 1987 Aug, 153, 217 - 21 On the mode of sperm autoantigen presentation to the regional lymph node of the testis after vasectomy in rats; McDonald SW et al.; The regional testicular lymph nodes of vasectomised rats, and of sham-operated controls, have been examined, in stained smears of cell suspensions, for the presence of spermatozoa, at intervals of 1-10 weeks after operation . Very few, if any, spermatozoa were found in either group . These findings differ from those reported in rams and boars by Ball & Setchell (1983), and indicate species differences in the mode of presentation of sperm autoantigens to the regional nodes after vasectomy. Ecotoxicol Environ Saf, 1987 Aug, 14(1), 64 - 72 Effects of methyl mercury on arrays of microtubules and macromolecular synthesis in Daucus carota cultures; Czuba M et al.; Cell suspension cultures of Daucus carota were exposed to methyl mercury at concentrations between 0 and 6 micrograms/ml for 1, 3, or 24 hr . Microtubule arrays exhibited no detectable disruption (as compared with controls) when treated with 1, 2, and 3 micrograms/ml methyl mercury . Disorganization of microtubules did occur at higher concentrations (4-6 micrograms/ml) in a concentration/time-dependent manner . No recovery of microtubule arrays was evident when cells were placed in methyl mercury-free medium for up to 7 days . Analyses of soluble protein and carbohydrate content, dry weight, and cell viability (reducing capacity) indicate that methyl mercury exposure has inhibitory effects on cell metabolism . The observed disruption of plant cell microtubules, induced by exposure to methyl mercury, may be secondary in response to an initial inhibition of synthetic pathways and membrane perturbations. Transplantation, 1987 Aug, 44(2), 280 - 6 Hyperthermia for treatment of human split-thickness skin . Differential effects on HLA-DR-expressing cells and keratinocytes; Morhenn VB; Human split-thickness skin was hyperthermia-treated and then the tissue was trypsinized to obtain dispersed cell suspensions . Cells consisting mainly of keratinocytes, obtained from the treated epidermis, retained their capacity to grow in vitro . By contrast, HLA-DR-expressing cells, dispersed from the epidermis as well as the dermis, lost their capacity to stimulate allogeneic peripheral blood mononuclear leukocytes after heat treatment (1.5 hr at 45 degrees C) . The heat-treated epidermal cells did not release a factor that inhibited allogeneic lymphocyte proliferation . Even though the cells expressing HLA-DR antigen were no longer capable of stimulating allogeneic lymphocytes after hyperthermia, they still expressed HLA-DR antigen on their surface up to 72 hr after exposure to heat . The inhibition of stimulation of allogeneic lymphocytes by heat treatment was at least partially reversed by the addition of exogenous interleukin-2, but not interleukin-1 . Thus, functions of the various cell types present in split-thickness skin were differentially sensitive to heat, a characteristic of skin cells that may be useful in skin transplantation. Nippon Yakurigaku Zasshi, 1987 Aug, 90(2), 125 - 32 {Functional improvement produced by transplantation of fetal neuronal cell suspensions in the rat striatum with 6-hydroxydopamine lesions}; Hiyama Y et al.; Behavior improvement from apomorphine-induced rotation, morphological features of nerve cells and in vivo release of dopamine (DA) were examined after the implantation of neuronal cell suspensions into the striatum of female rats with 6-hydroxydopamine lesions . Apomorphine-induced rotation was significantly suppressed in 11 out of 26 rats from two through ten weeks after the implantation . In 3 out of 26 rats, the rotation was significantly suppressed for four weeks after the implantation, although the suppression was reversed by the fifth week . In the remaining 12 rats, the rotation was not inhibited after the implantation . Behavior improvement was concurrent with extension of neurites from anti-DA immunopositive cells to the host caudate . Anti-DA cells were very scant in the absence of behavior improvement . In vivo release of DA and the metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, was detected in the perfusates of the striatum of the animals that were functionally improved by the transplant . Methamphetamine caused an increase in DA release and a decrease in the release of the metabolites in those animals . These results suggest that grafted neuronal cells which are sensitive to methamphetamine probably innervate neuronal elements in the host caudate, and the release of DA from these grafted cells functionally affects behavior improvement. Mutat Res, 1987 Aug, 182(4), 223 - 7 Mutagen detection with a mouse line containing 3 distinct mutations conferring sensitivity; Shiomi T et al.; A mouse-cell mutant sensitive to methyl methanesulfonate (MMS), X-rays, ultraviolet light (UV), and crosslinking agents was selected using the replica plating and cell suspension spotting methods . This mutant (XUM1) is a mitomycin C-sensitive derivative of previously reported XU1, a mutant sensitive to MMS, X-rays and UV . Since XU1 is highly susceptible to the lethal effect of 4-nitroquinoline-1-oxide (4NQO), XUM1 is also hypersensitive to 4NQO . Growth inhibition area tests showed that low concentrations of mutagens were detected with the multiple mutagen-sensitive mutant XUM1 . Hence XUM1 cells will be useful in detecting with high sensitivity a wide range of mutagens and carcinogens which mimic X-rays, UV and crosslinking agents. Cryobiology, 1987 Aug, 24(4), 303 - 10 The influence of cryopreservation on the ultrastructural morphology of human thyroid cells; Shiloh H et al.; The purpose of this study was to investigate the effects of the freeze-thaw procedure on the ultrastructural features of human thyroid cells . Four different stages of thyroid cell preparation were compared: (1) fresh surgical tissue, serving as control, (2) cell suspension before freezing, (3) cell suspension after thawing, and (4) monolayer cell culture, obtained from cells after thawing . Electron microscopic examination of cells from each stage showed that the freeze-thaw procedure used caused only minor intracellular alterations restricted to mitochondria . Some of these organelles showed low-amplitude swelling or occasionally appeared condensed . These ultrastructural changes were not paralleled by a decrease in the vitality or sensitivity of the cryopreserved cells to stimulating agents. Am Rev Respir Dis, 1987 Aug, 136(2), 310 - 5 Receptor-mediated O2- release by alveolar macrophages and peripheral blood monocytes from smokers and nonsmokers . Priming and triggering effects of monomeric IgG, concanavalin A, N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, and cytochalasin D; Nakashima H et al.; Receptor-mediated superoxide (O2-) release by alveolar macrophages and peripheral blood monocytes from smokers and nonsmokers was studied in vitro . When the cells were incubated with monomeric IgG or monomeric Fc(IgG) fragment, no cell O2- release was observed . However, when cytochalasin D (Cyto D) was subsequently added to the cell suspension, we observed a markedly enhanced O2- release . Neither Cyto D alone nor the double stimulation of following Cyto D with monomeric IgG induced O2- release . Concanavalin A (Con A) also had a priming effect on O2 release in combination with Cyto D, as did monomeric IgG or monomeric Fc(IgG) fragment . On the other hand, heat-aggregated IgG, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or phorbol myristate acetate (PMA) induced O2- release without the addition of Cyto D . Thus, we observed 2 different mechanisms in the receptor-mediated O2- release by alveolar macrophages and peripheral blood monocytes . Alveolar macrophages from smokers, which had a higher affinity and a larger number of monomeric IgG binding sites per cell than those from nonsmokers, were more reactive to the double stimulation of following monomeric IgG with Cyto D than to that of Con A and Cyto D, FMLP, or PMA, but for peripheral blood monocytes it was the reverse . We conclude that the binding of monomeric IgG to the Fc(IgG) receptor of alveolar macrophages or peripheral blood monocytes results in a priming effect on the cells for O2- release, and that the regulation of receptor-mediated O2- release by alveolar macrophages differs at least in part from that of peripheral blood monocytes. J Biomed Mater Res, 1987 Aug, 21(8), 1013 - 22 Alternative techniques of seeding cultured endothelial cells to ePTFE grafts of different diameters, porosities, and surfaces; Lindblad B et al.; Attachment of 111Indium-oxine labeled cultured canine venous endothelial cells to expanded polytetrafluorethylene (ePTFE) grafts was evaluated in vitro . Three alternative seeding techniques were studied in grafts having different diameters, porosities, and surfaces, including: (I) manual milking of blood containing endothelial cells within the graft; (II) a two-step procedure of incubating grafts initially with blood and then with an endothelial cell suspension; and (III) mechanical spinning of grafts filled with blood containing endothelium . Method II had significantly higher cell attachment to the graft (11.6%) than did Method I (1.5%) or III (4.7%) . A somewhat higher seeding efficiency was noted in 10-mm-I.D . grafts (11.6%) compared to 6-mm-I.D . grafts (6.3%) . Different graft porosity, created by altering internodal distances, did not cause significant changes in cell attachment (10 microns, 13.4%; 30 microns, 6.3%; 90 microns, 16.0%) . Fibronectin-coated surfaces, which should have enhanced cell adhesion, demonstrated a 6.0% cell attachment, a lower efficiency than the 11.6% observed with a blood coating alone . Acetone-soaked surfaces, which should have predictably exhibited less hydrophobicity, produced quite variable attachments (range 3.4 to 59.7%, mean 23.4%) . In the present investigation the best seeding technique was method II, the two-step incubation procedure . Consistent differences were not noted with various ePTFE graft configurations or surfaces. J Periodontol, 1987 Aug, 58(8), 569 - 73 Phenotypic and functional analysis of T cells extracted from chronically inflamed human periodontal tissues; Cole KL et al.; T-cell subsets extracted from chronically inflamed periodontal tissues were identified using monoclonal antibodies, and their functional activity was analysed using the autologous mixed lymphocyte reaction (AMLR) . Tissue was obtained from a total of 33 adult periodontitis (AP) patients and 6 normal/marginal gingivitis (N/MG) patients . All AP patients had received repeated oral hygiene instruction and root planing prior to the surgery, and the majority (30 out of 33) had at least one site with greater than 6 mm loss of attachment from the cementoenamel junction within the surgical field . The N/MG patients had no loss of attachment, and probing depths were less than 3 mm . Single cell suspensions were obtained following collagenase digestion (90 minutes at 37 degrees C) and mechanical disruption of the tissue . T-cell subsets were identified using an indirect immunofluorescence assay on cells obtained from 19 AP patients and the 6 N/MG patients . The mean (+/- standard error) helper:suppressor (T4:T8) ratio for the AP patients was found to be 0.94 +/- 0.48 compared with 1.65 +/- 0.16 for the N/MG group and 1.51 +/- 0.12 for peripheral blood controls . HLA-DR positive macrophages were identified and were found to include both acid phosphatase (AcP) positive and adenosine triphosphatase (ATPase) positive populations . Functional analysis was carried out using cells extracted from the remaining 14 AP patients . Cells from six of these 14 patients were found to be capable of spontaneous proliferation . Co-culture experiments using autologous T and non-T populations revealed that cells from only four patients were able to respond in an AMLR while those from only one of the 14 patients were able to stimulate the AMLR.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Immunol, 1987 Aug, 17(8), 1145 - 50 Analysis of T cell-replacing factor-like activity: potent induction of T helper activity for human B cells by residual concanavalin A and interleukin 2; Sauerwein RW et al.; At least two factors with the capacity to i