Cell suspensions

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 induce IgM synthesis in human B cells were found to be present in the 15-20-kDa fraction of the supernatant of mononuclear cells activated with concanavalin A (Con A) and phorbol ester . Previously, it has been shown (Sauerwein, R . W . et al., Eur . J . Immunol . 1985 . 15: 611) that interleukin 2 (IL2) in this material is able to induce T cell-dependent IgM secretion in normal B cells . Evidence was obtained for the presence of another factor distinct from IL2 that could replace T cells in the induction of B cell differentiation . We have analyzed this factor with the use of a neoplastic B cell population of prolymphocytic origin that was functionally nonresponsive to IL2 . T cell-replacing factor (TRF)-like activity and IL2 could be separated by ion-exchange chromatography, although a small amount of IL2 was recovered in the TRF fractions . This small amount of IL2 was found to be crucial for the observed TRF activity . Moreover, a substantial amount of monomeric Con A was detected in the TRF preparation . Our studies show that Con A in the presence of IL2 can act as a potent inducer of helper function in lower numbers of T cells for normal and neoplastic B cells . Functional assays for T cell contamination in B cell suspensions are therefore of limited value because they are determined by the efficiency of the stimulating signal . Particularly in those B cell factor preparations, obtained from mitogen-activated T cells with an obligatory or unidentified role of IL2, the possible effect of a contaminating mitogen must be considered.

Bone Marrow Transplant, 1987 Aug, 2(2), 155 - 63
T lymphocyte depletion of human peripheral blood and bone marrow using monoclonal antibodies and magnetic microspheres; Gee AP et al.; It has previously been demonstrated that graft-versus-host disease can be overcome in patients receiving HLA-mismatched bone marrow transplants by prior in vitro depletion of T lymphocytes from the marrow . In this report we describe the use of monoclonal antibodies and magnetic microspheres for the depletion of T cells from peripheral blood and bone marrow . The target cells are sensitized with antibodies directed against the CD2, CD3, CD4 and/or CD8 cell surface antigens, captured by magnetic beads coated with sheep anti-mouse IgG antibody and collected by placing the cell suspension in a magnetic field . This simple, rapid procedure results in the efficient removal of T cells from peripheral blood and from bone marrow without affecting the colony-forming potential of normal hematopoietic stem cells . The procedure is capable of being scaled up for the treatment of larger volumes of marrow that are required for clinical transplantation.

Aust Vet J, 1987 Aug, 64(8), 239 - 40
Detection of K88 and K99 fimbrial antigens on Escherichia coli by coagglutination; Murray CJ; Coagglutination was used to detect K88 and K99 fimbrial antigens on Escherichia coli, and results were compared to an enzyme immuno assay (EIA) . When pili suspensions were tested by both methods, 28 of 66 cultures were shown to have K88 and 11 of 31 cultures had K99 antigens . No pili suspensions were positive by coagglutination that were not positive by EIA . Testing of cell suspensions gave equivalent results to pili suspensions for K99 when tested by coagglutination . Two cell suspensions reacted with the K88 coagglutination which could not be confirmed by testing of pili suspensions, while a further 20 out of 43 cultures gave equivalent results with both cell and pili suspensions for K88 when tested by coagglutination.

Neuroscience, 1987 Aug, 22(2), 481 - 97
Neural grafting in a rat model of Huntington's disease: striosomal-like organization of striatal grafts as revealed by acetylcholinesterase histochemistry, immunocytochemistry and receptor autoradiography; Isacson O et al.; Grafts of fetal striatum were implanted in the form of a cell suspension into the brains of rats with prior ibotenic acid lesions of the caudate-putamen . The grafts were placed in three different sites: the lesioned caudate-putamen, or the denervated (but otherwise undamaged) globus pallidus and substantia nigra . After 3-6 months survival the grafts were investigated by means of immunohistochemistry and receptor autoradiography in combination with routine histology and acetylcholinesterase histochemistry . The grafts placed within the lesioned caudate-putamen were at least 10-fold larger larger than those placed in the substantia nigra region, with the grafts placed in the globus pallidus being of intermediate size . In all locations the acetylcholinesterase staining had an uneven, patchy distribution, which was most pronounced in the grafts located within the caudate-putamen . These patches did not bear any obvious relationship to variations in density of the neuronal perikarya within the grafted tissue . Many of the neuropeptide-immunoreactive neuron types present in the normal striatum, such as those containing substance P, {Met}enkephalin, somatostatin, cholecystokinin and neuropeptide Y were also detected in the grafted striatum along with acetylcholinesterase-positive staining . Acetylcholinesterase-positive, {Met}enkephalin-positive, substance P-positive and tyrosine hydroxylase-positive markers all showed uneven, patchy distributions in the grafts . This was also the case for the distribution of dopamine D2 and opiate receptors (as revealed by {3H}spiroperidol and {3H}diprenorphine autoradiography, respectively), whereas muscarinic receptor binding was even throughout the grafts . As is the case in the so-called striosomal patches (neurochemically defined compartments) in the immature intact striatum during the early postnatal period, patches of high acetylcholinesterase staining in the grafts showed partial correspondence with patches of high {Met}enkephalin fibre staining, and dopamine receptor density, and (although to a lesser degree) also with patches of high opiate receptor density and high substance P-immunoreactivity . This correspondence of patches also occurred between tyrosine hydroxylase fibre staining and acetylcholinesterase staining as revealed by grafts placed into the substantia nigra . These results suggest that the fetal striatal cell suspension grafts will give rise to a fairly normal range of striatal neuron and receptor types and that they develop at least some of the striosomal features characteristic for the normal striatum.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Haematol, 1987 Aug, 39(2), 148 - 53
Comparative study of eosinophil purification on Nycodenz, Metrizamide and Percoll density gradients; Brattig NW et al.; Studies on the metabolism of human eosinophils and their interaction with helminthic parasites have been hampered by the difficulty in obtaining these granulocytes in sufficient purity and quantity . In this report, we describe for the first time the application of the density medium Nycodenz for the purification of eosinophils from peripheral blood . Nycodenz density gradient centrifugation is compared with Percoll and metrizamide gradient centrifugation with special emphasis on cell yield, purity of cell suspensions, and functional integrity of separated cells . Percoll gradients provide purer preparations (mean value 94 +/- 15%) of eosinophils from blood with moderate eosinophilia than Nycodenz or Metrizamide (58 +/- 29 and 88 +/- 13%, respectively) . The recovery rates of eosinophil-enriched fractions were low after Nycodenz centrifugation (29 +/- 19%) in comparison to Metrizamide (60 +/- 21%) or Percoll (70 +/- 16%) . Eosinophils (as neutrophils), prepared by the 3 techniques, function metabolically (superoxide anion generation) and in assays measuring phagocytosis (S . aureus, zymosan particles) . Percoll gradient isolated eosinophils (as neutrophils) adhere to and immobilize larvae of Onchocerca volvulus.

Biochem Biophys Res Commun, 1987 Jul 31, 146(2), 510 - 6
Paclobutrazol inhibition of sterol biosynthesis in a cell suspension culture and evidence of an essential role for 24-ethylsterol in plant cell division; Haughan PA et al.; Growth of a celery (Apium gravidens) cell suspension culture was inhibited by the synthetic plant growth regulator paclobutrazol . Paclobutrazol caused a reduction in the incorporation of {2-14C}acetate into the 4-demethyl sterols (campesterol, sitosterol, stigmasterol) but radioactivity accumulated in the 4 alpha-methylsterols . The accumulating 4 alpha-methylsterols were identified as obtusifoliol and cycloeucalenol indicating that paclobutrazol was inhibiting sterol biosynthesis by blocking 14 alpha-demethylation . The inhibition of celery cell growth by paclobutrazol could be partially overcome by addition of cholesterol to the culture medium . However, addition of stigmasterol restored growth to the control value suggesting an essential role for a 24-ethylsterol to support plant cell division.

Biochemistry, 1987 Jul 28, 26(15), 4757 - 63
Photoaffinity analogues of methotrexate as folate antagonist binding probes . 2 . Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells; Price EM et al.; A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate . The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Kt of 506 +/- 79 nM and a Vmax of 17.9 +/- 4.2 pmol min-1 (mg of total cellular protein)-1 . Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate (I50 = 1.0 microM) . The parent compounds of the iodinated photoprobe inhibit {3H}methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a Ki of 66 +/- 21 nM . UV irradiation, at 4 degrees C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein . This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound . Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate . In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated . Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr 38K and 21K) derived from the cell homogenate supernatant.

Biochim Biophys Acta, 1987 Jul 23, 901(2), 265 - 72
Role of Na+/H+ antiport in intracellular pH regulation by rabbit enterocytes; Shimada T et al.; The steady-state intracellular pH (pHi) of isolated rabbit enterocytes was determined using 9-aminoacridine, a fluorescent weak base, and the null-point method with digitonin . When cells are incubated in a Na+-containing solution, the estimated value of pHi was in the range of 7.10-7.20, whereas it was 6.60-6.70 when cells were incubated in a Na+-free solution, indicating an important role of external Na+ in maintaining pHi at a slightly alkaline level . Pulse injection of Na+ into a Na+-free cell suspension induced a slowly developing alkalinization of pHi . The time course of the alkalinization was found to be dependent on the Na+ concentration . Li+ had the same effect as Na+, while K+ had a slight effect . Amiloride inhibited the effects of Na+ dose-dependently . These results indicate that the Na+/H+ antiport plays an important role in maintaining the pHi at a neutral or slightly alkaline level in the intact enterocytes.

Biochim Biophys Acta, 1987 Jul 10, 901(1), 138 - 46
Parallel investigation of exocytosis kinetics and membrane fluidity changes in human platelets with the fluorescent probe, trimethylammonio-diphenylhexatriene; Kubina M et al.; A simple, flexible and sensitive fluorescence method is described, which, from the same experiment, provides coupled quantitative informations on membrane fluidity changes and exocytosis, and reliable kinetic analyses of these effects, in intact cell suspensions . The method is based on the features peculiar to trimethylammonio-diphenylhexatriene (TMA-DPH), a fluorescent hydrophobic probe, which, in intact cells, is incorporated specifically into the plasma membranes, according to an instantaneous partition equilibrium . The method was tested on human platelets upon stimulation with various agents, such as human alpha-thrombin, adenosine diphosphate (ADP), adrenaline and ionomycin, which act through different types of mechanism . The experimental conditions were chosen to allow platelet shape change and exocytosis, but no aggregation . The kinetics and the dose-dependence of the changes in TMA-DPH fluorescence intensity and anisotropy were compared to the simultaneous physiological responses of platelets to the same stimuli, under the same conditions . Quantitative correlations were established between serotonin secretion and the increase in fluorescence intensity, whereas fluorescence anisotropy, which monitors membrane fluidity changes was associated with platelet shape change . The specificity of the effects was confirmed with appropriate antagonistic or modulating agents.

J Biochem (Tokyo), 1987 Jul, 102(1), 1 - 4
Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope; Kato K et al.; Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation . Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm . On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.

Jpn J Pharmacol, 1987 Jul, 44(3), 293 - 302
Effect of several d-morphinans on ascites tumors in mice; Kigoshi S et al.; Dextromethorphan and its analogues (DM 16, DM 34, DM 72, DM 75 and DM 96) were examined for their effect on Ehrlich ascites carcinoma or ascites sarcoma-180 in female mice of the ddY strain . The suspension of Ehrlich carcinoma cells or sarcoma-180 cells was prepared from mice at 10 days after i.p . inoculation of the cells, using Hanks' balanced salt solution, and the cell suspension was inoculated i.p . into mice (2 X 10(6) viable cells/mouse) . The chemicals dissolved in physiological saline containing 5% HCO-60 were then injected i.p . into the mice once daily for 5 successive days (5-40 mg/kg/day) . In addition, mice given the tumor cells were treated with the saline containing 5% HCO-60 alone for 5 days (untreated mice) . In groups of mice bearing Ehrlich ascites carcinoma or ascites sarcoma-180, the mean survival time of mice treated with 20-40 mg/kg/day of DM 96 was more than twice that of the corresponding untreated mice . The mean survival time of mice treated with 20 mg/kg/day of DM 96 was also longer than that of mice treated with 40 mg/kg/day of the other chemicals, irrespective of the ascites tumors . Concerning these survival times, the LD50 (i.p.) of DM 96 in mice differed slightly from that of other chemicals (88 mg/kg and 77-106 mg/kg) . These results indicate that DM 96 is more active than the other chemicals against the ascites tumors in mice.

J Exp Zool, 1987 Jul, 243(1), 137 - 51
On the diversity of sperm basic proteins in the vertebrates: V . Cytochemical and amino acid analysis in Squamata, Testudines, and Crocodylia; Kasinsky HE et al.; The variability of sperm basic proteins in representatives of three reptilian orders, Squamata, Testudines, and Crocodylia, has been examined by cytochemistry, acid-urea polyacrylamide gel electrophoresis, and amino acid analysis of amidoblack-stained bands . Snakes contain type 3B intermediate sperm basic proteins by cytochemical criteria . Electrophoresis of basic proteins from epididymis chromatin as well as from testis and ductus deferens cell suspensions shows two fast-moving bands in the vicinity of herring protamine . These proteins are triprotamines containing about 27 mol % arginine, along with lysine and histidine . Lizards have type 1 protamines in their sperm nuclei cytochemically and also show a two-banded electrophoretic pattern similar to that of snakes . However, these proteins are triprotamines, similar to those in snakes with 25 mol % arginine . It may be that these are testis-specific proteins of the spermatid stage in lizards since a cytochemical transition can be observed from type 3A intermediate proteins in spermatids of testis to type 1 protamine in mature sperm of ductus deferens . Turtles contain type 3A intermediate sperm basic proteins cytochemically and basic proteins from epididymis chromatin display both a prominent band and a minor band close to, but slightly slower than, the two bands for snakes and lizards . Amino acid analysis of these bands shows that these basic proteins are also triprotamines but with a higher level of arginine, about 48 mol %, than that in snake and lizard sperm proteins . Basic proteins from epididymis chromatin of a single Mississippi alligator show three main bands moving close to the bands of snakes, lizards, and turtles . These proteins have amino acid compositions typical for triprotamines, with 28-39 mol % arginine . The data indicate that the sperm basic proteins of representatives of 25 species in three reptilian orders are very similar, in contrast to the diversity of sperm protein types found in frogs (Kasinsky, Huang, Kwauk, Mann, Sweeney, and Yee: J . Exp . Zool., 203:109-126, '78; Kasinsky, Huang, Mann, Roca, and Subirana: J . Exp . Zool., 234:33-46, '85a) . This appears to be part of a macroevolutionary trend from diversity of sperm basic proteins in frogs to relative constancy in reptiles (Kasinsky, Mann, Pickerill, Gutovich, and Byrd, Jr.:J . Cell Biol., 91:1879, '81; Kasinsky, Mann, Lemke, and Huang: In: Chromosomal Proteins and Gene Expression, Plenum Press, New York, pp . 333-352, '85b) . We present the hypothesis that one factor for such a trend resides in the fact that fertilization is internal in reptiles but external in anurans.

Am J Physiol, 1987 Jul, 253(1 Pt 2), H147 - 53
Reflectance measurements of hematocrit and oxyhemoglobin saturation; Steinke JM et al.; Fiberoptic oximeters measure oxyhemoglobin saturation from the optical reflectance of whole blood, but the calibration of such oximeters is hematocrit dependent . Therefore, using photon-diffusion theory and an empirical approach, we have developed a new reflectance method that determines hematocrit and correspondingly corrects the oxyhemoglobin-saturation measurement . Our method employs four fiber-optic light guides, a photodetector, and three inexpensive light-emitting diodes (one with emissions at 660 nm and two at 813 nm) . Hematocrit is determined from the ratio of reflectances from the differently spaced emitting fibers at 813 nm and is used to correct the 813-660 nm measurement of oxyhemoglobin saturation . In red cell suspensions, the mean difference between reflectance measurements of hematocrit and conventional determinations was only 2.09% (r = 0.99), and when compared with conventional gasometric measurements of oxyhemoglobin saturation, the reflectance method yielded the same calibration curve for different hematocrits and gave a mean difference of only 2.67% . Although the technique is demonstrated with a cuvette appropriate for an extracorporeal circulation in animal experiments, it could possibly be further developed for fiber-optic catheter oximeters.

J Neurosurg, 1987 Jul, 67(1), 106 - 9
A reproducible model of metastatic brain and ocular tumor by hematogenous inoculation of the VX2 tumor in rabbits; Frank JA et al.; A metastatic brain-tumor model has been developed in rabbits by infusing the VX2 carcinoma into the internal carotid artery to simulate hematogenous dissemination of tumor . In a series of 25 New Zealand White rabbits, multiple metastases arose in the hemisphere of 24 (96%) and in the eye of 22 (92%); in all instances ocular metastases were ipsilateral to the site of infusion . Ocular metastases were visible in the anterior chamber in 80% of animals 3 to 12 days after the infusion of VX2 tumor cell suspension . All rabbits deteriorated neurologically or died by Day 15 after the inoculation . Multiple metastases were demonstrated by magnetic resonance imaging as early as 5 to 7 days after infusion of the tumor cells and were confirmed at autopsy . This technique models hematogenous metastases to the brain and eye and is useful in evaluating the response of metastases to chemotherapy and radiation therapy directed to the brain and eye.

Invest Ophthalmol Vis Sci, 1987 Jul, 28(7), 1131 - 7
Transplantation of cultured rabbit retinal epithelium to rabbit retina using a closed-eye method; Lopez R et al.; We have developed a closed-eye technique for transplanting cultured rabbit retinal epithelial cells to Bruch's membrane of the rabbit . A glass micropipette containing a suspension of 3H-thymidine-labeled, cultured retinal pigment epithelial (RPE) cells is inserted through a pars plana incision and positioned adjacent to the neural retina . A jet stream from the pipette is used to make a small retinal hole and bleb detachment . Patches of host retinal epithelium lift off with the neural retina, creating areas of bare Bruch's membrane . The cell suspension is injected into the subretinal space, and labeled cells can be seen attached to Bruch's membrane as early as 1 hr later . The neural retina spontaneously reattaches within 24 to 48 hr, bringing photoreceptor outer segments in direct contact with the transplanted cells . Phagocytosis of outer segment material by transplanted cells can be seen as early as 24 hr after surgery . This closed-eye technique offers an advantage over the open-sky method used previously in that it allows for reattachment of the neural retina and at least a partial return of function in the transplanted retinal epithelium.

Cancer Lett, 1987 Jul, 36(1), 71 - 81
In vitro proliferation of primary human head and neck squamous cell carcinomas evaluated by flow cytometry; Bijman JT et al.; In vitro proliferation of primary human head and neck squamous cell carcinomas was investigated using single cell suspensions and tissue explants of primary specimens and xenografts from 20 tumor specimens . The evaluations of the cells emerging in culture were performed with flow cytometry . Epithelial-like cells proliferated in serum-free medium, while no fibroblast-like cells were observed in culture . The epithelial-like cells could be subcultured several passages before senescence occurred . Conditioned medium or serum supplementation was necessary for a sustained outgrowth of malignant squamous cells as documented by flow cytometry . From a tumor line established in nude mice slowly proliferating tumor cells emerged . After 4-5 months in culture tumor cells seemed to be adapted to the culture conditions used . This resulted in a more consistent tumor cell proliferation . Early passage cultures from primary human head and neck squamous cell carcinomas are clearly difficult to obtain either from primary human specimens or from tumor lines established in nude mice.

Ann Inst Pasteur Immunol, 1987 Jul-Aug, 138(4), 549 - 60
Basal and lipopolysaccharide-inducible membrane alkaline phosphatase of lymphoid cells from mice with immune system dysfunctions; Mosbach-Ozmen L et al.; The expression of membrane alkaline phosphatase (mAlPase) activity is an enzymatic marker of activated but not resting B cells which can be used on unseparated lymphoid cell suspensions . It is higher in lymphoid cell suspensions from mice with higher proportions of B cells (athymic mice) or with more activated B cells (autoimmune mice) than in those of control mice.

Eur J Immunol, 1987 Jul, 17(7), 921 - 8
B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens; Kroese FG et al.; Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced . The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs . HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes . HIS22 and HIS24 detected B lineage-associated antigens expressed by major subpopulations of B cells . HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone . In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively) . In bone marrow the vast majority of cytomplasmic mu+ pre-B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis . The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells . As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22, or HIS24 . In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells . HIS22 did not bind to Ig- peripheral lymphocytes . Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches . As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen . In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen . This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.

Transplantation, 1987 Jul, 44(1), 106 - 12
Prolonged survival of cultured keratinocyte allografts in the nonimmunosuppressed mouse; Hammond EJ et al.; The effect of in vitro culture on the survival of allografts of epidermal keratinocytes has been examined using a mouse model . Female BALB/c tail epidermal cells were cultured from single cell suspensions to form confluent sheets that were grafted onto male CBA recipients using a transplantation technique that ensured separation of donor graft from host skin . Animals were killed at defined intervals, and the status of the grafts determined histologically . Full thickness skin allografts rejected at 13-15 days . Allografts of epidermis (obtained by enzymatic cleavage at the dermoepidermal junction) rejected at 19-20 days . Cultured keratinocyte allografts were not rejected at least within 70 days and had a histological appearance identical to syngeneic controls . The expression of MHC class I and class II determinants and the leukocyte common (Ly5) surface marker on the donor cells before and after culture were examined using indirect immunofluorescence and monoclonal antibodies . These and other cytochemical studies showed that freshly dissociated tail epidermal cells contained 0.3% of cells that expressed membrane-bound ATP-ase activity, Ia antigens, and the Ly5 surface antigen . These are the Langerhans' cells of the epidermis . In culture, these cells decrease so that by day 8 of culture, no such cells can be detected . At confluence, there are no Ia positive cells, but all cells express MHC class I antigens and stain with an antikeratin antibody . The loss of Ia expression on culture correlates with a decreased stimulation of the class II H-2d-restricted T cell clone D7.1 by cultured keratinocytes compared with freshly dispersed epidermal cells . Furthermore, cultured keratinocytes fail to prime allogeneic mice as determined by the survival of whole thickness skin grafts, whereas freshly dispersed cells induce an accelerated rejection . The results suggest that the survival of cultured keratinocyte allografts is due to the elimination of cells expressing Ia antigens and supports the passenger leukocyte theory of graft rejection.

Exp Hematol, 1987 Jul, 15(6), 715 - 8
Detection of residual murine LPC-1 myeloma cells from bone marrow cell mixture after purging by 4-hydroperoxycyclophosphamide; Frondoza CG et al.; Mixtures of BALB/c bone marrow (5 X 10(6) and LPC-1 myeloma (2 X 10(6) cells have been purged in vitro with 100 microM 4-hydroperoxycyclophosphamide (4HC) for 30 min at 37 degrees C . Elimination of tumor cells was assessed by monitoring newly synthesized tumor-specific IgG 2a kappa in vitro and by reinjecting the cells subcutaneously into the hindlegs of mice . Drug-treated LPC-1 cells had no detectable tumor production and did not reproduce tumors . Untreated cells regrew as solid tumors and killed the host within 3-4 weeks . Bone marrow cell suspensions purged of tumor cells were then used to reconstitute lethally pretreated mice injected 24 h earlier with 300 mg/kg cyclophosphamide (CY) . Mice injected with CY alone died within 10 days . Those reconstituted with bone marrow cells or with purged bone marrow-tumor cell mixture lived longer than 7 months . Mice reconstituted with untreated bone marrow-tumor cell suspension grew tumors, had detectable tumor-specific IgG 2a kappa in their serum, and died by day 44 . These studies demonstrate that the success of myeloma cell purging can be determined by monitoring newly secreted tumor protein and that 4HC successfully eliminates malignant plasma cells in vitro without impairment of normal bone marrow stem cell functions.

Microvasc Res, 1987 Jul, 34(1), 1 - 12
The time course of filtration test as a model for microvascular plugging by white cells and hardened red cells; Reinhart WH et al.; The propensity of white blood cells and rigidified red blood cells to plug narrow channels was studied in an in vitro model . Blood cell suspensions (20 ml) with a hematocrit level of 10% were pumped through Nuclepore filters with a nominal pore diameter of 5 micron at constant flow rates (0.82-6.1 ml/min), and the pressure-time curves were recorded . An initial fast rise (K1) and a later slow rise (K2) of the pressure-time curve were observed; according to earlier studies (Skalak, R., Impelluso, T., Schmalzer, E.A., and Chien, S . (1983), Biorheology 20, 41), K1 reflects the dynamic plugging-unplugging process and K2 the permanent plugging of pores by rigid cells . Changes of the flow rate had no influence on K1 (greater than or equal to 1.6 ml/min) or K2 . The addition of small concentrations of mononuclear leukocytes to the red cell suspensions resulted in a dose-dependent increase in K1, but did not affect K2 . Admixture of partially hardened red cells (0.03% glutaraldehyde for 30 min) with normal red cells at a constant total hematocrit caused increases of both K1 and K2 . From these results we conclude that both white cells and hardened red cells tend to plug narrow channels . White cells, however, may cause less permanent plugging than rigidfied red cells . These results may help to understand microcirculatory disorders seen in sickle cell crisis or leukocytosis and leukemia.

Cytometry, 1987 Jul, 8(4), 372 - 6
An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry; Schutte B et al.; A new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei . Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and decreased background binding . This technique is applicable to cell suspensions, including cultured cells and bone marrow cells . Furthermore, pepsin digestion of ethanol fixed tissue fragments resulted in a high recovery of nuclei in which incorporated BrdUrd could be detected . This possibility, together with the high sensitivity, make this method especially suitable for cell kinetic studies of human solid tumors in vivo.

J Exp Med, 1987 Jul 1, 166(1), 129 - 41
Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts; Owen WF Jr et al.; Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients . After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived . The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF . In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d . In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts . Culture with fibroblasts alone did not support eosinophil survival . The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts . Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition . In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S . mansoni larvae than the freshly isolated, normodense cells from the same donor . Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes . In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.

Cell Immunol, 1987 Jul, 107(2), 348 - 57
Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells; Pietrangeli CE et al.; The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis . Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by {3H}thymidine labeling and radioautography . In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains . The turnover of small lymphocytes also increased . These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC . In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects . The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production . Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.

Proc Natl Acad Sci U S A, 1987 Jul, 84(13), 4586 - 90
Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain; Stingl G et al.; The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2- . Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes . Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain . Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions . The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood.

J Reprod Fertil, 1987 Jul, 80(2), 537 - 44
Comparison of subpopulations of luteal cells obtained from cyclic and superovulated ewes; Hild-Petito S et al.; Peripheral blood samples were collected daily (Days 1-10 after ovulation) and analysed for progesterone content . Luteal tissue was collected on Day 10 after the LH surge, or Day 10 after hCG injection from cyclic and superovulated ewes, respectively . The tissue was enzymically dispersed and an aliquant was utilized for measurement of cell diameters, and staining for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase activity (3 beta-HSD) . The remaining cell preparation was separated into small (10-22 micron) and large (greater than 22 micron) cell fractions by elutriation . Small and large cell suspensions were incubated (37 degrees C, 2 h) in the presence or absence or ovine LH (100 ng/ml) or dbcAMP (2 mM) and progesterone content of the medium was measured . Superovulation did not affect circulating progesterone concentrations, when expressed per mg luteal tissue recorded; basal progesterone production by small or large luteal cells; the unresponsiveness of large luteal cells to ovine LH or dbcAMP; the ratio of small:large cells recovered by dissociation the mean diameter of total cells; or the mean diameter of large cells . However, the mean cell diameter and LH stimulation of progesterone production by small cells were greater (P less than 0.05) in luteal tissue collected from superovulated than in that from cyclic ewes . These differences appear to be an amplification of basic function . Therefore, we conclude that corpora lutea obtained from superovulated ewes can be used to study functional aspects of small and large cells.

Neuroscience, 1987 Jul, 22(1), 169 - 78
Autoregulation of dopamine release and metabolism by intrastriatal nigral grafts as revealed by intracerebral dialysis; Strecker RE et al.; The autoregulation of dopamine release and metabolism by intrastriatal grafts of mesencephalic dopamine neurons was examined in vivo using an intracerebral dialysis technique . Dopamine-rich cell suspension grafts were implanted into the head of the caudate putamen in rats with complete unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine pathway . Six months later behavioural tests indicated that the grafts had reversed the lesion-induced rotational behaviour . Extracellular levels of striatal dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were monitored bilaterally in the halothane-anaesthetized grafted rat, both under basal conditions, and also following low (0.05 mg/kg) and high (0.5 mg/kg) doses of the dopamine receptor agonist apomorphine . The perfusate from the grafted striatum showed levels of dopamine which were not statistically different from those of the intact contralateral striatum, indicating that the baseline release of dopamine from the graft was close to normal . Similarly, 3-4-dihydroxyphenylacetic acid and homovanillic acid levels were well recovered on the grafted side (67% and 52%, respectively, of control values) . Consistent with previous observations, levels of the serotonin metabolite 5-hydroxyindoleacetic acid measured in perfusate collected from the grafted side was elevated significantly above normal . Subsequent histological analysis revealed large grafts, rich in dopamine-containing neurons (mean +/- SEM number equalled 3138 +/- 630), giving rise to an approximately normal density of dopamine-containing fibres in the area of the host caudate putamen surrounding the probe . Treatment with 0.05 mg/kg (subcutaneous) apomorphine did not affect extracellular dopamine recovered from the grafted striatum, while extracellular DA decreased by a maximum of 30% on the intact side . However, a subsequent injection of 0.5 mg/kg apomorphine produced a large decrease of the dopamine recovered from both the grafted (maximum 40% decrease) and intact striata (maximum 80% decrease) . Both the low and the high dose of apomorphine reduced extracellular dopamine metabolite levels, a response which was essentially similar for both the intact and grafted sides . Finally, the dopamine reuptake blocker nomifensine (10(-5) M) added to the perfusion medium produced similar large increases in dopamine in perfusates collected from both grafted and intact striata, while 3,4-dihydroxyphenylacetic acid and homovanillic acid did not change.(ABSTRACT TRUNCATED AT 400 WORDS)

Scand J Immunol, 1987 Jul, 26(1), 1 - 6
Amplification of T-cell response to PPD by epidermal cell suspensions containing HLA-DR-expressing keratinocytes; Tjernlund U et al.; The biological importance of the presence of class II transplantation antigens on highly differentiated epithelial cells such as keratinocytes in certain conditions, is still unknown . We have therefore investigated the antigen-presenting capacity of separated human epidermal cells obtained from tuberculin-reactive skin 6 days after intradermal injection of purified protein derivative (PPD) . Earlier studies have shown a high percentage of HLA-DR-expressing keratinocytes at this time . Peripheral adherent blood cells were used as control stimulator cells and highly purified peripheral blood T lymphocytes as responder cells . The T-cell proliferation in response to PPD in the presence of autologous epidermal cells from normal and tuberculin-reactive skin was measured by {3H}thymidine incorporation on day 6 . The latter cell population, 76-86% of which consisted of HLA-DR-expressing cells as judged by immunocytochemistry, induced a greater T-cell response to PPD than do normal epidermal cells . This discrepancy in the T-cell proliferation could not be explained by a difference in the numbers of anti-Leu 6 or anti-HLA-DQ-reactive Langerhans cells . The present data indicate that epidermal cell suspensions containing HLA-DR-expressing keratinocytes induce a greater T-cell response to PPD than do normal epidermal cells.

Life Sci, 1987 Jun 22, 40(25), 2415 - 20
In vivo and in vitro effect of naloxone on prolactin response to TRH in rat; Velkeniers B et al.; In adult male Wistar rats submitted to a standardized noise stress, intravenous TRH induced a prolactin (PRL) secretory response . Prior IV naloxone administration not only lowered plasma PRL levels in those stressed rats but abolished also the stimulatory action of TRH . This effect was further studied by superfusion experiments on enriched PRL cell suspensions (70% lactotrophs) from female adult Wistar rats . Naloxone kept unaffected the basal PRL secretion but lowered significantly that induced by TRH . These experiments suggest a dual effect of naloxone on rat PRL secretion, one exerted on central opioid receptors lowering stress-related increased basal PRL levels, the other inhibiting the TRH-dependent PRL secretion exerted at the lactotroph level itself.

Life Sci, 1987 Jun 22, 40(25), 2421 - 8
Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions; Horiuchi T et al.; Effect of adrenergic activity on the adrenal steroidogenesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions . Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release . Both prazosin (alpha 1 antagonist) and yohimbine (alpha 2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively . Both atenolol (beta 1 antagonist) and ICI 118-551 (beta 2 antagonist) also blocked (-)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively . Neither (-)-isoproterenol nor (+/-)-norepinephrine at concentrations of 10(-6) M potentiated aldosterone release stimulated by angiotensin II or ACTH . These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.

J Immunol, 1987 Jun 15, 138(12), 4329 - 35
Forssman glycolipid, an antigenic marker for a major subpopulation of macrophages from murine spleen and peripheral lymph nodes; Bethke U et al.; Three hybridoma clones were isolated after hybridization of a mouse myeloma line with splenocytes from rats immunized with Forssman glycosphingolipid (Fo) . Two of these clones produced Fo-specific monoclonal antibodies (MAB) of the IgM class, one MAB of the IgG2c class . In complement-dependent depletion experiments and immunofluorescence studies on the nature of Fo-positive leukocytes in CBA/J mice the following results were obtained: whereas blood monocytes, polymorphonuclear leukocytes, and lymphocytes were Fo negative, 5 to 10% of suspended spleen cells were positive . The majority of these were macrophage-like, glass- and nylon-adherent, nonspecific esterase-positive phagocytizing cells carrying Ia and globoside markers . These cells participated as accessory cells in the mixed lymphocyte culture reaction . In cell suspensions from axillary and inguinal lymph nodes, 2% were Fo positive . They were enriched up to 70% in the glass-adherent, esterase-positive population from this source . In contrast, no Fo-positive cells were detected in mesenteric lymph nodes, and less than 0.1% of the resident peritoneal macrophages bore this marker . The percentage of Fo-positive cells increased to 1% in thioglycollate-elicited peritoneal cells . Immunostaining of cryosections of lung and liver tissue showed alveolar macrophages and Kupffer cells, respectively, to be Fo negative.

J Biol Chem, 1987 Jun 5, 262(16), 7825 - 30
Endogenous lectin from cultured soybean cells . Chemical characterization of the lectin of SB-1 cells; Malek-Hedayat S et al.; A lectin has been identified in the cell line, SB-1, originally derived from the roots of Glycine max . This lectin, which we shall refer to as SB-1 lectin, was isolated on the basis of its carbohydrate-binding activity (affinity chromatography on Sepharose column derivatized with N-caproyl-galactosamine) and its immunological cross-reactivity (immunoblotting with rabbit antibodies directed against seed soybean agglutinin (SBA} . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of SB-1 lectin revealed a major polypeptide (Mr approximately equal to 30,000) which co-migrated with seed SBA . This form of the lectin was observed in fractions purified from culture medium of SB-1 cells or supernatant fraction of SB-1 cell suspension after enzymatic removal of cell wall . Extracts of SB-1 cells under some other conditions yielded a major band (Mr approximately equal to 60,000) as revealed by SDS-PAGE and immunoblotting with rabbit anti-seed SBA; prolonged incubation of these samples in the presence of SDS resulted in the appearance of the 30-kDa polypeptide . It appears that the 60-kDa band represented a highly stable, even under SDS-PAGE conditions, dimeric form of the 30-kDa subunit . The SB-1 lectin derived from the culture medium was compared with seed SBA by gel filtration and by peptide mapping after limited proteolysis; no difference between the lectins from the two sources was found . Extracts of soybean roots fractionated on N-caproyl-galactosamine-Sepharose affinity columns yielded, upon elution with galactose, polypeptides of Mr 30,000 and 60,000 . These results suggest that soybean roots contain a lectin whose polypeptide composition corresponds to that of seed SBA and SB-1 lectin.

Diabetologia, 1987 Jun, 30(6), 434 - 6
Rheological properties of white blood cells are changed in diabetic patients with microvascular complications; Vermes I et al.; Erythrocyte and white blood cell suspensions were prepared from 22 Type 1 (insulin-dependent) and 37 Type 2 (non-insulin-dependent) diabetic patients and from 57 control subjects . Cell filterability was studied with the new "St . George's Filtrometer", which can discriminate between cell deformability and filter occlusion . A pronounced increase of filter clogging was found in diabetic patients compared with control subjects . There was no significant difference between Type 1 and Type 2 diabetic patients, but a significantly increased clogging was found in patients with retinopathy compared with retinopathy-free patients . Considering that filter occlusion is mainly due to leucocytes, our results show a reduced filterability of white blood cells in diabetic patients . Altered white blood cell function may act as an additional factor in the impairment of microvascular circulation in diabetic patients.

Histochem J, 1987 Jun-Jul, 19(6-7), 337 - 44
Cellular heterogeneity in normal human urothelium: quantitative studies of lectin binding; Ward GK et al.; A panel of 10 FITC-labelled lectins (MPA, PNA, ConA, DBA, SBA, RCA-120, WGA, UEA, GS-I, GS-II+) was applied to cryosections of seven specimens of normal urothelium . Seven of the lectins (MPA, ConA, RCA, WGA, UEA, GS-I and GS-II) showed a pattern of increasing fluorescence intensity from basal to superficial cells of the urothelium whereas PNA, DBA and SBA showed more uniform binding throughout the urothelium . Urothelial cell suspensions labelled with FITC-lectins were studied by flow cytometry to quantify the variation in binding to different cells types . Three cellular subpopulations were identified in normal urothelium on the basis of their optical properties . Fluorescence intensity due to specific lectin binding was then measured separately for each subpopulation . Although there was some variation among individual cases, a general pattern emerged in this small series . WGA, RCA, and GS-II bind in large quantities to all urothelial cells while PNA, SBA, ConA and DBA show little binding . MPA, RCA, UEA and GS-I showed the most marked increase in fluorescence intensity from basal to superficial cells as observed microscopically and quantified by flow cytometry.

Anal Biochem, 1987 Jun, 163(2), 440 - 5
Disintegration of isolated cells; Loffler BM et al.; A simple device has been developed for the disintegration of isolated cells in isotonic or hypertonic media . It is based on the extrusion of a cell suspension through a small orifice under controlled pressure . The diameter of this interchangeable orifice has to be adapted to the cell type to be disrupted . The conditions fulfill the requirements for an efficient disruption of the plasma membranes and for good preservation of the particularly fragile subcellular components, lysosomes and peroxisomes, of rat hepatocytes and rat Leydig cells.

Neuroscience, 1987 Jun, 21(3), 685 - 98
Intracerebral transplantation of cultured neurons after reaggregation in a plasma clot; Lindsay RM et al.; In order to be able to transplant neural cells which have been either manipulated in vitro or maintained in culture for the purpose of cell-type enrichment, we have developed a novel plasma clot method which permits reaggregation of previously dissociated cells such that they can be implanted as highly localized transplants rather than as dispersion-prone cell suspension grafts . To establish the method, enzymatically dissociated cells prepared from hippocampal primordia of late embryonic rats were immediately reaggregated into plasma clots and transplanted to the hippocampal formation of adult recipients . By using fluorescein-labelled bovine plasma to form the plasma clot grafts of reaggregated cells, the fate of the plasma clot protein matrix was followed at different post-operative survival times . Initially, 4-5 days post-operative, the plasma clot maintained the grafted cells in a loose sponge-like sack at the site of implantation . After 2-3 weeks, the transplanted cells were more compact and fused with the host neuropil, and the plasma clot matrix had largely been degraded . At 1 month or longer survival, there was no distinguishable boundary between transplant and host, and there was little or no evidence of any remaining plasma clot matrix or proteins . The plasma clot method was subsequently applied to the transplantation of cultures enriched in pyramidal cells . Enrichment for pyramidal cells was achieved by eliminating mitotic cells (dentate granule cells and glia) by brief (200 rad) irradiation of hippocampal primordia followed by dissociation and maintenance in monolayer culture for 4-6 days . Fibres from host dentate granule cells grew into the pyramidal cell-enriched transplants and established mossy fibre terminals on the donor cells . In transplants between embryonic and adult rats, donor cells were identified at long survival times by prelabelling donor cells in utero or in vitro with {3H}thymidine prior to transplantation . In transplants between embryonic and adult mice, donor tissue from A Thy-1.1 strain mice was transplanted to congenic A strain (Thy-1.2) mice such that the donor cells bearing the Thy-1.1 cell surface glycoprotein could be later identified by immunocytochemical staining with antibodies specific for the Thy-1.1 antigen . Reaggregation and transplantation of dissociated cells in a plasma clot thus provides a novel method whereby prior manipulation of neural tissue (separation of neurons and glia, enrichment for specific types of neurons, or glia etc.) can be used to great advantage in studying host-transplant connectivity and in assessing those factors which are critical in sustaining the survival of grafted neural tissue.

Can J Physiol Pharmacol, 1987 Jun, 65(6), 1213 - 9
Sodium-dependent D-glucose transport in brush-border membrane vesicles from isolated rat small intestinal villus and crypt epithelial cells; Freeman HJ et al.; Differentiation and maturation of enterocytes occur with migration from the crypt to villus compartments . To investigate the effect of epithelial cell differentiation on sodium-dependent D-glucose transport, brush-border membrane vesicles were prepared from small intestinal epithelial cell suspensions selectively isolated from villus and crypt populations . Enterocytes were isolated with a morphologically monitored sequential cell dissociation method . Thymidine kinase, sucrase, and alkaline phosphatase activities were measured as differentiation markers of specific cell populations . Brush-border membrane vesicles were purified and their kinetic characteristics defined with a rapid filtration method under conditions of a zero-trans, 100 mM cis-NaSCN gradient . Typical "overshoot" phenomena characteristic of sodium D-glucose cotransport were observed for both villus (five- to eight-fold equilibrium values) and crypt brush-border membrane vesicles (two- to four-fold equilibrium values) . Kinetics analyses of the initial D-glucose flux in brush-border membrane vesicles suggested the presence of at least two sodium-dependent D-glucose carriers in the villus and only a single carrier in the crypt compartments . These data indicate that sodium D-glucose cotransport occurs in brush-border membranes of both villus and crypt populations . Moreover, quantitative and qualitative differences between these two membrane populations suggest that epithelial D-glucose transport processes are differentiation dependent and reflect the degree of enterocyte development.

Br J Exp Pathol, 1987 Jun, 68(3), 461 - 74
Tumour cell trapping in rat mesenteric lymph nodes; Cobb RA et al.; We describe a new experimental model of mesenteric lymph node metastasis in the rat, involving afferent mesenteric lymphatic inoculation of tumour cell suspensions via glass microcannulae . This model has been used to perform a series of experiments to investigate whether the rat mesenteric lymph node trapped tumour cells . Afferent mesenteric lymphatic inoculation of suspensions of transplantable sarcoma cells in inbred hooded Lister rats resulted in tumour growth in the inoculated lymph node in 100% of rats, with no tumour growth at any other site . The same procedure performed on rats which had previously undergone mesenteric lymphadenectomy resulted in growth of tumour in the lungs . Using 125Iododeoxyuridine (IDUR) labelled sarcoma cells we have shown that although radioactivity decreased significantly in the mesenteric lymph node up to 24 h following afferent lymphatic inoculation, there was no evidence that tumour cells reached thoracic duct lymph . We conclude that the rat mesenteric lymph node trapped sarcoma cells.

Br J Exp Pathol, 1987 Jun, 68(3), 453 - 60
Morphological and functional characteristics of mouse mammary carcinoma cells separated on Nycodenz columns; Ford TC et al.; Tissue from four mouse mammary carcinomas was enzymatically disaggregated and cells from the resulting cell suspension were fractionated on a discontinuous density gradient column (5-20%) of Nycodenz (Nycomed A.S . Oslo) . The cell fractions separating at the 10-15% and 15-20% interfaces (density 1.082 and 1.110 g/ml respectively) contained a mean of 83.2 +/- 10.8 (s.d.) and 79.9 +/- 17.4 tumour cells . Compared with the original cell suspension these cell bands contained less cell aggregates and cell debris . The cells in the bands also showed an equivalent ability to grow in tissue culture and to form pulmonary tumours on i.v . injection into isogenic mice, when compared with the tumour cells in the original suspension . The relatively pure preparations of carcinoma cells thus separated may be of value in limiting the unwanted effect of normal cell contamination when testing neoplastic cells in vitro for sensitivity to drugs or hormones.

Neurosci Lett, 1987 Jun 1, 77(1), 15 - 9
Rat Schwann cell remyelination of demyelinated cat CNS axons: evidence that injection of cell suspensions of CNS tissue results in Schwann cell remyelination; Blakemore WF et al.; Injection of a suspension of embryonic rat central nervous system (CNS) cells into an area of persistent demyelination, produced in the cat spinal cord by injecting ethidium bromide into an area previously exposed to 40 Grays of x-irradiation, results, initially, in Schwann cell remyelination of the demyelinated axons . However, the Schwann cells are subsequently rejected; a response which confirms that the remyelinating cells are of rat origin.

Diagn Cytopathol, 1987 Jun, 3(2), 91 - 101
A morphologic, immunologic, and cytometric approach to the classification of non-Hodgkin's lymphoma in effusions; Katz RL et al.; The biologic and clinical heterogeneity of the various subtypes of non-Hodgkin's lymphoma is related to differences in morphologic, immunologic, and kinetic properties . Comprehensive studies characterizing these features in lymphomatous effusion have yet to be reported . We recently studied 27 effusion specimens from 26 patients with clinically suspected or confirmed lymphoma . Wright-Giemsa- and Papanicolaou-stained cytologic preparations, acridine orange nucleic acid flow cytometry, and immunoperoxidase staining of cell suspensions using antibodies to a battery of T and B cell markers were evaluated and compared with prior histologic accessions . Specimens were classified by cytologic characteristics according to the International Working Formulation Scheme and by acridine orange nucleic acid flow cytometry using the parameters of DNA, RNA, and proliferative activity . Correlation of the cytometric and morphologic data demonstrated that with increasing cytologic grade of lymphoma, the proliferative activity increased progressively and distinguished between grades (P less than 0.01) . Immunologic studies identified B cell phenotype in 16 specimens, T cell in three, and true histiocytic lymphoma in one; one lymphoma had no cell markers (null cell) . Six effusions proved to be inflammatory and reactive according to surface marker studies . Classification by cytologic characteristics showed good correlation with histologic classification performed previously . Immunologic study of cytologic specimens gave results identical to those achieved by frozen-section immunohistologic examination . Thus, immunologic and cytometric parameters can be readily performed on effusion specimens and aid in the diagnosis and classification of lymphomas.

Cell Immunol, 1987 Jun, 107(1), 188 - 200
Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants; Hale ML et al.; Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation . Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis . From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive . Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive . In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests . These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.

Arch Pathol Lab Med, 1987 Jun, 111(6), 505 - 12
Recent progress in clinical quantitative cytology; Herman CJ et al.; The last several years have seen changes in quantitative cytologic procedures that have significantly expanded the clinical research and diagnostic applications of quantitative cytologic analysis . Intact nuclei can be isolated from formalin-fixed paraffin-embedded tissue, and the DNA distribution of these nuclei can be analyzed either with flow cytometry or with slide-based image-processing techniques . In addition, more informative DNA analysis has been achieved using antibodies to bromodeoxyuridine-modified DNA, proliferation-associated nuclear antigens, or tissue-type-specific cytoplasmic antigens as second stains for two-parameter analysis . Improvements in both staining and instrumentation have also increased the usefulness of flow cytometry in analysis of "rare events," ie, cell types comprising less than 0.1% to 0.5% of a total cell suspension . Commercially available slide-based image-processing systems have been developed that provide sophisticated cell and tissue analysis functions using inexpensive personal-computer-based systems.

Clin Exp Immunol, 1987 Jun, 68(3), 685 - 93
Studies of human bone marrow treated with soybean lectin and sheep erythrocytes: stepwise analysis of cell morphology, phenotype and function; Schiff SE et al.; Morphological, phenotypic and functional analyses were made of cells obtained at each step after successive treatments of 23 separate human bone marrow suspensions with soybean lectin and sheep erythrocytes (SRBC) . The average total number of nucleated cells harvested was 1.9 X 10(10) and the final cell suspensions contained a mean of 1.9 X 10(9) nucleated cells or 9.2 +/- 4.8% of the initial counts . Monoclonal antibody analyses revealed that both T and B lymphocytes were present in every cell fraction in percentages similar to those found initially until after the first SRBC rosette-depletion . Moreover, both soy lectin agglutinated and non-agglutinated cells exhibited vigorous proliferative responses to phytohaemagglutinin and allogeneic cells . Following the SRBC depletions, no cells having T lymphocyte phenotypes or functions could be detected, whereas 5% of the cells reacted with a monoclonal antibody to B lymphocytes . The final fraction was composed predominantly of immature myeloid cells and blasts and was depleted of erythroid elements, lymphocytes and essentially all mature cells . It contained cells reactive with monoclonal antibodies recognizing undifferentiated T cell precursors (3A1), the transferrin receptor (5E9), and a human progenitor cell antigen (My-10) . The final fraction was also enriched 10-100-fold for CFU-C and 5-10-fold for CFU-GEMN colonies . These studies fail to demonstrate selective removal of T lymphocytes from human bone marrow cells by soybean lectin agglutination.

Br J Dermatol, 1987 Jun, 116(6), 773 - 84
Ultrastructural and phenotypic changes in Langerhans cells induced in vitro by contact allergens; Picut CA et al.; Ultrastructural changes of murine Langerhans cells (LC) were examined following exposure of crude epidermal cell suspensions to the contact allergens dinitrochlorobenzene, nickel sulphate and lead nitrate at various concentrations and for various incubation times . An immunogold labelling technique was employed to study changes in surface expression of MHC Class II (Ia) molecules . In all cases, activation of LC was evident after as little as 15 min exposure and was characterized by a marked increase in surface expression of Ia molecules, prominent rough endoplasmic reticulum and numerous ribosomes and lysosomes . Degenerative changes in LC were apparent to varying degrees depending on the allergen, its concentration and the time of incubation . Degenerative changes included swollen mitochondria, membrane disruption or rupture, loss of density of the cytoplasm (cytolysis), loss of dendritic processes and decreased expression of Ia molecules . In the case of dinitrochlorobenzene, degenerative changes were present and usually severe at concentrations greater than 10 micrograms/ml, while exposure to nickel sulphate and lead nitrate was associated with only mild degenerative changes . These observations indicate that contact allergens have a variety of direct effects on LC, including activation and degeneration, which are dose- and time-dependent . Since these alterations of LC were observed in the absence of other immunologically active cells, peripolesis cannot be involved in these events.

Biochimie, 1987 Jun-Jul, 69(6-7), 671 - 5
Level of messenger RNA encoding small subunit ribulose bisphosphate carboxylase is enhanced by cytokinins in tobacco cell suspension cultures; Axelos M et al.; Chloroplast differentiation is induced by cytokinins in suspension cell cultures of Nicotiana tabacum, line 19M which is independent of the hormone supply for growth . Poly(A)RNA from cells cultured in basal medium or in kinetin-supplemented medium were analyzed by Northern blot and dot blot hybridizations to a 'RUBISCO' small subunit-encoding cDNA probe . It was found that the small subunit-encoding mRNA of cytokinin-supplemented cells was synthesized much earlier during the culture and in amounts one order of magnitude larger than in hormone-starved cells.

Behring Inst Mitt, 1987 Jun, (81), 110 - 9
Cellular oncogenes and lymphocyte activation; Schneider-Schaulies J et al.; In normal mouse splenic B and T cells at least two cellular proto-oncogenes are expressed, i.e . c-myc and c-fos . The expression of c-myc depends on the activation of the cells, but not on subsequent growth . C-fos gene expression appears to be induced by the manipulation involved in preparation of single cell suspensions from spleens . In that respect, c-fos gene expression does not qualify as being significantly involved in transition from G O to S phase while expression of c-myc seems to be correlated with some early events of cell activation leading to growth competence . The kinetics and extent of c-myc gene expression vary with the mitogen used and the type of lymphocyte investigated as examplified by T cells and subpopulations thereof . The expression of both proto-oncogenes in normal mouse spleen cells is finely regulated by an interplay of transcriptional and post-transcriptional control mechanisms . These mechanisms operate differently for the two genes and independently from one another . They also change in predominance at various times, again independently from one another . While we have no evidence that c-fos has significance for the activation of lymphocytes, c-myc is a good candidate for being involved . Thus studies by Susan Corey and collaborators on transgenic mice which constitutively express c-myc in cells of the lymphocyte lineage, indicate that this lineage is profoundly affected . Among others, the effects concern the balance between proliferation and maturation and a constitutive high level of Ia expression, normally only observed in activated cells . Constitutive high expression of c-myc in B cells of these transgenic mice also makes them prone to leukemia possibly due to a series of subsequent events . These last findings also provide an explanation for the need for a very finely tuned regulation of c-myc gene expression as it is here described.

J Steroid Biochem, 1987 Jun, 26(6), 641 - 6
Non-steroidal inhibition of granulosa cell aromatase activity in vitro; Wickings EJ et al.; Treatment of cyclic rats with the substituted triazole R151885 (1,1-di (4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol causes delayed ovulation with suppressed blood oestradiol levels . To determine if R151885 can exert a direct action on ovarian oestrogen biosynthesis, we studied its effect on steroidogenesis in granulosa cell cultures from prepubertal rat ovaries . The cells were incubated for 48 h in medium containing 100 ng human FSH/ml and 10(-7) M testosterone to induce steroidogenic enzymes . When R151885 was also present in the culture medium, there was a marked and concentration-dependent reduction in granulosa cell oestradiol production . Inhibition was half-maximal at approx 3 X 10(-7) M and almost complete at 10(-5) M R151885 . Progesterone and 20 alpha-hydroxy-4-pregnen-3-one production were unaffected except by the highest concentration of the substituted triazole (36% inhibition at 10(-5) M) . Direct assessment of aromatase activity in the 48-h cultured monolayers (oestradiol formation during a 3-h incubation with 10(-7) M testosterone) was made to determine if the inhibitory effect of R151885 was due to reduced aromatase induction/activation . This was not the case, since cells cultured in the presence of 10(-6) or 10(-5) M R151885 had levels of aromatase up to 60% higher than those cultured in its absence . To determine acute effects of R151885 on testosterone (10(-7) M) aromatization, 3-h incubations were carried out using granulosa cell suspensions with high extant aromatase activity due to stimulation by ovine FSH (100 micrograms sc, twice daily for 2 days) in vivo . The triazole acted as an apparent competitive aromatase inhibitor (apparent Km for testosterone 2.5 X 10(-8) M in the absence of R151885 rising to 4.4 X 10(-8) M in the presence of 10(-7) M R151885) . Its potency as an aromatase inhibitor was approximately 10 times greater than that of the naturally occurring steroidal aromatase inhibitor 5 alpha-dihydrotestosterone . Various structurally related substances proved to be even more potent aromatase inhibitors than R151885 . The most active were also substituted 4,4'-difluorophenyl derivatives containing an imidazolyl or pyridyl moiety instead of the 1,2,4-triazolyl substituent in R151885 . This study has identified a novel series of nonsteroidal substances which have the characteristics of potent and specific inhibitors of testosterone aromatization by rat granulosa cells in vitro.

J Endocrinol Invest, 1987 Jun, 10(3), 247 - 53
Linear Percoll gradient centrifugation of rat anterior pituitary cells . A simple method for prolactin cell enrichment; Velkeniers B et al.; Prolactin (PRL) cells were purified from nulliparous normal female adult Wistar rat pituitary cell suspensions by linear Percoll density gradient centrifugation, a procedure yielding single cells . Lactotrophs were found in two different layers, the first containing 70% PRL cells in the density range 1.055 to 1.065 g/ml, the second with 28% PRL cells in the range 1.070 to 1.080 g/ml . Both cell fractions contained more than 90% viable cells with an intact ultrastructure . The physiological integrity of the 70% enriched PRL cells was assessed by their basal PRL secretion, their secretory response to TRH and dopamine, and their cAMP production in a basal situation and after incubation with dopamine.

J Urol, 1987 Jun, 137(6), 1295 - 9
Development and characterization of a monoclonal antibody to human embryonal carcinoma; Khazaeli MB et al.; A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components . The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient . An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian, 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells . The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines . The antibody did not react with 22 other cell lines or with hCG . The antibody was labeled with 131I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later . Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization . Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21 . This antibody thus has selective reactivity with the surface of tumor cells from embryonal carcinoma (testicle) and choriocarcinoma both in vitro and in vivo.

Endocrinology, 1987 Jun, 120(6), 2302 - 7
Calcium release from porcine thyroid microsomes by phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate; Nakamura Y et al.; We have demonstrated Ca2+ mobilization induced by inositol compounds from intracellular stores of isolated porcine thyroid cells that were permeabilized with saponin and from fractionated thyroid microsomes that were preloaded with Ca2+ . In the presence of saponin and antimycin A, a mitochondrial inhibitor, the Ca2+ concentration in the cell suspension decreased after the addition of ATP; the addition of inositol 1,4,5-trisphosphate (IP3) elicited transient elevations in Ca2+ . The half-effective concentration (EC50) of IP3 was about 1 microM . TSH, acetylcholine, and norepinephrine had no effect on the Ca2+ movement, but phosphatidylinositol 4,5-bisphosphate (PIP2) induced a similar Ca2+ release (EC50 = 3 microM) . PIP2-induced release decreased as the Mg2+ concentration was raised . After the release induced by a maximal amount of IP3, PIP2 could induce a further Ca2+ release . We also measured Ca2+ efflux from fractionated microsomes preloaded with 45Ca2+ by ATP-dependent transport . IP3 induced a small efflux of 45Ca2+, but PIP2 was much more effective at low Mg2+ concentrations . More than half of the loaded 45Ca2+ was released by 10 microM PIP2 within 30 sec, and the EC50 was 0.7 microM . These results suggest that Ca2+ mobilization from the microsomes mediated by inositol compounds participates in the regulation of thyroid cell functions.

J Biochem Biophys Methods, 1987 Jun, 14(3), 149 - 60
ESR spectroscopy in the study of antigen processing--uptake of spin-labelled antigens by macrophages; Curtain CC et al.; Macrophages were briefly pulsed with a spin-labelled synthetic polypeptide, poly(L-tyrosine:L-glutamic acid) poly DL-alanine:poly L-lysine (n-TGAL) in the presence and absence of anti-TGAL-antibody, and the electron spin resonance (ESR) spectra of the cell suspension compared with the spectrum of free n-TGAL in solution . Spectral analysis indicated two cell-associated n-TGAL pools, one composed of freely rotating label held in an aqueous environment, susceptible to protease digestion and ascorbate reduction, and a second highly concentrated pool, sequestered intracellularly, and held within a highly ordered, polar microenvironment . The ESR analyses were completed within minutes of antigen pulsing, employed very small numbers of live cells, and did not damage the cells being tested . The utility of the technique in screening fatty acid-antigen conjugates for macrophage uptake was demonstrated.

J Cell Sci, 1987 Jun, 87 ( Pt 5), 723 - 30
Spike-shaped oscillations in the absence of measurable changes in cyclic AMP concentration in a mutant of Dictyostelium discoideum; Wurster B et al.; Periodic activities of Dictyostelium discoideum cells involve two types of oscillations, spike-shaped and sinusoidal . Spike-shaped oscillations are accompanied by the periodic synthesis and release of cyclic AMP, and cyclic AMP-activated cyclic AMP synthesis is believed to control these oscillations . Experiments described here call into question the importance of cyclic AMP in spike-shaped oscillations . Cell suspensions of strain agip43, an aggregation-deficient mutant of D . discoideum, displayed spike-shaped oscillations in light scattering with period lengths about 1.5 times larger than those of the parent strain . These oscillations were not accompanied by measurable oscillations of cyclic AMP and cyclic GMP . Applied cyclic AMP pulses elicited increases of two- to threefold in the cyclic AMP level and increases of seven- to ninefold in the cyclic GMP concentration . Cyclic AMP additions caused phase shifts in the oscillations of agip43 cells, suggesting that cyclic AMP receptors at the cell surface communicate with the oscillator . We interpret these results in terms of an oscillator not based on cyclic AMP . This oscillator should be coupled to the reaction system involving cyclic AMP synthesis and release . The latter can operate in an oscillatory manner in the parent strain Ax2 but not in mutant agip43.

Am J Pathol, 1987 Jun, 127(3), 580 - 91
Adrenocortical cells of the zona reticularis normally express HLA-DR antigenic determinants; Khoury EL et al.; A distinct population of normal human adrenocortical cells from adult glands spontaneously express HLA-DR, and occasionally also HLA-DQ, antigenic determinants in vivo and in vitro, as detected by immunofluorescence techniques using monoclonal antibodies on frozen tissue sections, primary cultures, and viable cell suspensions . In vivo, these antigenic determinants were found to be confined to the compact-type cells in the zona reticularis . In vitro, the adrenocortical identity of these HLA-DR+ cells was conclusively established by the characteristic change in their morphologic features induced by ACTH1-24 and by the simultaneous detection of tissue-specific adrenal autoantigens in double-label immunofluorescence staining . The cell surface expression of HLA-DR determinants by compact cells persisted in culture for several weeks and was not significantly affected by treatment with ACTH1-24 . On the other hand, fetal adrenocortical cells of both transient and definitive zones were invariably negative for the expression of these HLA Class II antigenic determinants . Because normal human adrenocortical cells also express on their surface the molecules which constitute specific autoantigens in autoimmune adrenalitis, these findings argue against the notion that an ectopic HLA-DR expression associated with an otherwise "silent" autoantigen might trigger an organ-specific autoimmune response against endocrine cells . In addition, the expression of HLA-DR determinants by viable normal reticularis cells provides a readily detectable surface marker which may allow the physical separation of this population for studies in vitro.

Kidney Int, 1987 May, 31(5), 1113 - 20
Cytoplasmic pH regulation in canine renal proximal tubule cells; Reid IR et al.; The precise mechanisms by which the mammalian kidney proximal tubule transports H+ and HCO3- and regulates cytosolic pH (pHi) remain in doubt, though both a H+-ATPase pump and Na+/H+ exchange at the luminal membrane are known to function in the export of protons . The mechanisms of HCO3- transport are less clear though recent reports suggest an important role for an electrogenic Na+/HCO3- symport in the basolateral membrane . The importance of chloride-dependent bicarbonate transport is unknown . In the present studies, the pH-sensitive fluorescent dye, bis-(carboxyethyl)-carboxyfluorescein (BCECF) has been used to study pHi changes in suspensions of canine proximal tubule cells following acidification or alkalinization of the cytosol . Cells were acid-loaded to pH 6.5 by exposure to the H+/K+ ionophore, nigericin . Following removal of nigericin, pHi returned to basal levels (pHi = 7.1) when the cells were resuspended in a buffer containing 100 mM Na+ . This recovery was blocked by removal of Na+ or addition of 0.2 mM amiloride to the cell suspension . In the presence of 0.2 mM amiloride and Na+, partial excretion of the acid load occurred if the buffer also contained HCO3-/CO2, but this effect was blocked by the removal of Na+ or the addition of 1 mM 4-acetomido-4'-isothiocyano-2,2'-stilbene disulfonic acid (SITS) . When cell membrane potential was monitored in these experiments using the potential-sensitive fluorescent dye, bis-(1,3-dibutylbarbiturate) trimethine oxonol, the increase in pHi seen in the presence of Na+ was found to be electroneutral, whereas when that occurred in the presence of Na+, amiloride and HCO3-/CO2 was associated with membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Nippon Sanka Fujinka Gakkai Zasshi, 1987 May, 39(5), 792 - 8
{Chemotherapy sensitivity of gynecologic malignancies determined by the human tumor stem cell assay (HTSCA)}; Obi S; HTSCA was used to study the chemotherapy sensitivity of gynecological malignant tumors . 34 specimens were obtained from 32 patients . 11 of the 34 specimens (32%) yielded a sufficient number of colonies for testing . On these, 51 separate drug assays were done, each at the 1/10 peak plasma level . 20 drugs were identified as active among 51 drugs tested . Clinical correlations of sensitivity (S) and resistance (R) were studied in 6 patients treated with combination therapies, including drugs selected on the basis of in vitro sensitivity . (formula; see text) We conclude that HTSCA can aid in identifying active drugs for use in designing treatment protocols . However, because of difficulty in preparing a single cell suspension, and the small proportion of tumors suitable for testing, HTSCA will not be used in routine drug sensitivity testing until these drawbacks are eliminated.

Brain Res, 1987 May, 430(1), 139 - 44
The avian pecten provides a potent substrate for growth and development of dissociated embryonic neural implants; Ehrlich D et al.; A cell suspension of the optic tecta of 3-day-old chick embryos was injected into the vitreal chamber of 2-day-old posthatch chicks . After a 14-21-day survival period, examination of eyeballs showed that all implants survived and, in 50% of cases, were attached to the pecten . The implants had proliferated and showed a laminated pattern of organization, with small cells in the superficial regions and large cells in the deep regions of the implant . The implants also contained a well-developed neuropil with mature synapses . The host retina was not affected by the presence of the implant . We suggest that the avian pecten represents a highly amenable structure for studies involving the response(s) by damaged retinae to neural implants.

Transfusion, 1987 May-Jun, 27(3), 228 - 33
Viscosity of mixtures of sickle and normal red cells at varying hematocrit levels . Implications for transfusion; Schmalzer EA et al.; Viscosity (eta) in a blood suspension is affected by the total hematocrit (HT) as well as by the deformability of the cells . The impact of these combined factors on the rheologic behavior of sickle cell suspensions and on guidelines for transfusion has not been explored fully . Therefore, the eta of mixtures of washed normal (AA) and sickle (SS) red cells was determined in a rotational viscosimeter as a function of the hematocrit level of SS cells (HS), HT, oxygen tension (PO2), and shear rate . The ratio HT:eta can be taken as an index of potential oxygen delivery . The optimal HT (for maximum HT:eta) became progressively higher as the HS or the HS:HT ratio was lowered: at a given HT, HT:eta rose with a decrease in HS, especially at low HS values . These data support the concept that simple transfusion alone is not as beneficial to the patient as exchange transfusion and that substantial benefit can be obtained by bringing the patient to very low HS levels . The finding that eta rose with HT more steeply when the HS:HT ratio rather than HS was held constant suggested that the absolute level of HS may be more useful than the HS:HT ratio as a guide for a transfusion regimen.

Arch Biochem Biophys, 1987 May 1, 254(2), 491 - 7
Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus; Eilert U et al.; Treatment of one cell line (No . 615) of Catharanthus roseus c.v . Little Delicata with an elicitor preparation of autoclaved and homogenized Pythium aphanidermatum culture resulted in rapid accumulation of indole alkaloids . Alkaloid formation was preceded by rapid transient increases in the extractable activities of the enzymes tryptophan decarboxylase and strictosidine synthase . The induction of these two enzyme activities occurred when cells were transferred to alkaloid production medium or treatment with fungal elicitors . Treatment of this cell line with translational or transcriptional inhibitors prevented the Pythium-induced increases of enzyme activity as well as alkaloid accumulation . When cells were transferred to alkaloid production medium the induction of strictosidine synthase activity preceded that of tryptophan decarboxylase by many hours even when cells were also treated with Pythium elicitor . Results suggested that tryptophan decarboxylase induction proceeds only when endogenous tryptamine levels were decreased by two-third . The internal cellular level of tryptamine, therefore, could regulate expression of tryptophan decarboxylase, whereas induction of strictosidine synthase or of another enzyme in the biosynthetic pathway could control channeling of tryptamine into alkaloids . The results demonstrate that fungal elicitors can be used to facilitate studies of the factors which regulate expression of indole alkaloid pathway enzymes and their ultimate pathway products.

Eur J Immunol, 1987 May, 17(5), 713 - 8
Kinetics of cellular oncogene expression in mouse lymphocytes . II . Regulation of c-fos and c-myc gene expression; Schneider-Schaulies J et al.; Newly isolated lymphocytes from mouse spleens express the c-fos oncogene even in the absence of mitogen with maximal mRNA levels 60 min post preparation of single cell suspension, whereas c-myc mRNA levels increase only after mitogenic stimulation with maximal mRNA levels 6 h post stimulation . The half-lives of c-fos mRNA are generally very short; they increase from 14 min (after 30 min of culture) to 70 min (after 2 h of culture) . The half-lives of c-myc mRNA decrease from 50 min (at 2 and 6 h post stimulation with concanavalin A) to 12 min (at 48 h post stimulation) . The c-fos gene transcription is already turned on in time-0 lymphocytes 10 min after disruption of the organ structure of the spleens and is down-regulated after 2 h and later . In nuclear run-on experiments with nonstimulated lymphocytes there is already significant transcription of the first exon of c-myc, but almost no elongation of the transcript to exon 2 and 3 . In concanavalin A-treated lymphocytes elongation is stimulated about 5-fold within 6 h and returns to background levels at 48 h post stimulation . The nuclear run-on analyses of nonactivated lymphocytes showed a signal for RNA complementary to c-myc mRNA detected with a probe specific for the exon 1/intron 1 boundary of c-myc, which disappeared with increasing time of concanavalin A stimulation . This anti-sense transcription may play a role in regulating the elongation of c-myc transcripts.

Am J Hematol, 1987 May, 25(1), 13 - 27
Acute lymphocytic leukemia: correlation of clinical features with immunocytochemical classification; Twu B et al.; Many immunologic studies of acute lymphocytic leukemia (ALL) during the past decade have demonstrated the close correlation of immunologic phenotypes of ALL subclasses with the clinical presenting features and prognosis . However, the clinical application of conventional immunologic techniques had been very limited because of the requirement of a fresh sample to prepare the mononuclear cell suspensions for study . We studied 81 cases of ALL using immunoperoxidase stain for nuclear terminal deoxynucleotidyl transferase (TdT) and immunoalkaline phosphatase stain for surface markers (using monoclonal antibody J5 for common ALL antigen {CALLA}, Leu-1 for pan-T antigen, and B1 for pan-B antigen) on air-dried smears . The cases were classified as common ALL (TdT+, CALLA+, pan-T-, and pan-B-) (41 cases), null-ALL (TdT+, CALLA-, pan-T-, and pan-B-) (19 cases), T-ALL (TdT+, CALLA-, pan-T+, and pan-B-) (nine cases), B-ALL (TdT-, CALLA-, pan-T-, and pan-B+) (six cases), pre-B-ALL (TdT+/-, CALLA+, pan-T-, and pan-B+) (four cases), or pre-T-ALL (TdT+, CALLA+, pan-T+, and pan-B-) (two cases) . This subtyping of ALL correlated well with known clinical presenting features, prognosis, chromosome analysis in 35 cases with an abnormal clone, and conventional immunologic typing in 38 cases . The data suggest that these simple and practical immunocytochemical stains can be used for immunologic subclassification of ALL.

J Pathol, 1987 May, 152(1), 13 - 21
Use of the monoclonal antibody WR17, identifying the CD37 gp40-45 Kd antigen complex, in the diagnosis of B-lymphoid malignancy; Moore K et al.; The distribution of the gp40-45 Kd antigen bound by the WR17 monoclonal antibody of IgG2 subclass in normal lymphoid tissue was characterized by immunohistochemistry and immunofluorescence staining with flow cytometric analysis . The predominant staining pattern observed was characteristic of an anti-pan-B-lymphocyte reagent . Weak reactions were observed by immunofluorescence staining of viable cell suspensions with all neutrophils and T-lymphocytes in some normal donors . In tissue sections, B-lymphocytes were stained and no cross reactions were observed with T-lymphocytes, although macrophages stained in some sections . A range of T- and B-cell malignancies were stained with WR17 and the reactivity compared to that observed with other monoclonal antibodies in the CD19, CD21 and CD22 clusters . All B-non-Hodgkin's lymphomas, B-chronic lymphocytic, prolymphocytic and hairy cell leukaemia cells examined were stained by WR17 in indirect immunofluorescence assays, whilst the T-cell tumours were negative . The same pattern was observed in cryostat sections of malignant tissue and in addition some tissue macrophages expressed the CD37 antigen cytoplasmically . Intra-tumour heterogeneity of staining was observed with all the monoclonal antibodies tested, although overall WR17 consistently stained B-cell tumours even when expression of the CD19 pan-B-lymphocyte antigen could not be detected with some monoclonals . Monoclonal antibodies, such as WR17, within the CD37 cluster and binding to the gp 40-45 Kd molecule, bind to mature B-lymphocytes and identify the majority of B-cell malignancies.

Ann Neurol, 1987 May, 21(5), 451 - 7
Altered metabolic properties of cultured skin fibroblasts in Alzheimer's disease; Sims NR et al.; Alzheimer's disease is associated with selective neuronal loss, the cause of which is undetermined . Evidence indicating a predisposing genetic factor associated with this disease suggests that important alterations may be expressed in tissues other than the brain . Because abnormal glucose and energy-related metabolism have been identified in both in vivo and in vitro studies of brain, we conducted a study to examine related measures in cultured skin fibroblasts from six patients with Alzheimer's disease and seven age-matched controls . After 60 minutes' incubation, the production of 14CO2 from {U-14C}glucose and lactate production were significantly higher in the cells from the group of patients with Alzheimer's disease . The increase of 14CO2 production, but not the production of lactate, was most evident after a more rapid period of metabolic activity in the first 10 minutes of incubation . By contrast, 14CO2 production from {U-14C}glutamine, which is probably the major substrate of oxidative metabolism in these cells, was significantly reduced in the Alzheimer's disease cells following longer (120-minute) incubations . Oxygen uptake by cell suspensions was also significantly reduced in the group with Alzheimer's disease . These results indicate that complex metabolic differences are expressed in nonneural tissues from some patients with Alzheimer's disease and may provide important clues to the pathogenesis of this disorder.

Am J Anat, 1987 May, 179(1), 70 - 8
Cytoplasmic bar-like structures of alveolar type II cells: an ultrastructural study in freshly isolated cells from rat lungs; Vincent R et al.; Bar-like structures are tubular cytoplasmic inclusions found in situ in pulmonary epithelial type II cells of several animal species . The physiological significance and mode of formation of these inclusions are not fully established . In this paper, we describe bar-like structures as found in freshly isolated type II cells from rat lungs . Pulmonary cells were dissociated from the tissue with elastase and separated on a discontinuous density gradient of Percoll . The complete isolation procedure yielded 17 X 10(6) type II cells per animal (purity = 80%) . Either from the crude cell suspensions or the purified preparations, only a small fraction of the type II cell population harbored the inclusions (less than 5%) . It is shown that the bounding membranes of the bar-like structures can derive from the endoplasmic reticulum, the nuclear membrane, or the Golgi apparatus . Occasional connections with lamellar bodies were observed, and different levels of complexity in the bar-like structures were also found . The apparent rigid conformation and the orientation of the bar-like structures were taken as evidence for a role of the cytoskeleton in their formation . Because the inclusions do not appear to be new organelles or cellular structures performing a specific function, we propose that their formation may be a transient and limited cellular event in normal cells . However, the stabilization and the generation of the osmiophilic structures, as well as their overproduction, may reflect alterations of the normal physiology of the type II cells.

Pflugers Arch, 1987 May, 408(5), 519 - 23
Effect of triiodothyronine on system A amino acid transport in cells of rat submandibular gland; Eng SP et al.; The effect of L-3,5,3'-triiodothyronine (T3) on alpha-aminoisobutyric acid (AIB) transport in isolated cell suspensions of rat submandibular gland was investigated . The uptake of ATB by these cells appeared to require extracellular Na+ and was inhibited by ouabain (10(-3) M) . Cell suspensions from thyroidectomized rats which have been given three successive doses of T3 on alternate days (50 micrograms/100 g BW) showed a significant increase in AIB uptake compared with cells isolated from thyroidectomized rats treated with diluent . Efflux of AIB from the cell suspension was not affected by T3 . There was no significant changes in AIB uptake 12 h after a single injection of T3 (50 micrograms/100 g BW) . However, there was a significant 49% and 65% increase in AIB net uptake at 24 and 48 h, respectively, after T3 treatment . Under similar conditions, the cell suspension showed a 48% increase in NaK-ATPase activity at 12 h and to a peak of 61% at 24 h . Therefore, changes in NaK-ATPase activity preceded the changes in AIB net uptake upon treatment with T3, implying that AIB uptake is probably mediated, at least in part, by the activity of NaK-ATPase.

J Virol, 1987 May, 61(5), 1435 - 41
Owl monkey astrocytoma cells in culture spontaneously produce infectious JC virus which demonstrates altered biological properties; Major EO et al.; A tumor cell suspension of an explanted JC virus (JCV)-induced owl monkey glioblastoma was inoculated intracranially into four recipient juvenile owl monkeys . Twenty-eight months following inoculation one owl monkey developed a glioblastoma, which was explanted into tissue culture . DNA from both the tumor tissue and tumor cells in culture hybridized to a JCV DNA probe by Southern analysis, indicating that free, as well as integrated, viral DNA may be present . At the time of the second culture passage, viral JCV DNA was extracted from these cells and cloned into a plasmid vector . Nucleotide sequencing of the regulatory region of the cloned DNA demonstrated homology with the prototype Mad-1 strain of JCV and revealed a 19-base-pair deletion in the second 98-base-pair tandem repeat that eliminated a second TATA box . This deletion is characteristic of the Mad-4 strain of JCV, which is highly neurooncogenic . By the third culture passage, 100% of the cells were T-antigen positive . Approximately one-third of the cells in culture hybridized to a biotinylated JCV DNA probe when in situ hybridization was used, a technique that only detects high-copy-number of replicating viral sequences . By the culture passage 5 and continuing through culture passage 14, viable JC virions could be recovered . The T protein synthesized by this virus, now termed JCV-586, differed from both the Mad-1 and Mad-4 strains in that it formed a stable complex with the cellular p53 protein in the tumor cells . Also, the JCV-586 T protein reacted to several monoclonal antibodies made to the simian virus 40 T protein that were not recognized by either the Mad-1 or Mad-4 strains.

Cytometry, 1987 May, 8(3), 287 - 95
DNP-phycobiliproteins, fluorescent antigens to study dynamic properties of antigen-IgE-receptor complexes on RBL-2H3 rat mast cells; Seagrave JC et al.; In RBL-2H3 rat mucosal mast cells, the crosslinking of cell-surface IgE-receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation . We have developed a family of dinitrophenol (DNP)-conjugated fluorescent antigens to study dynamic membrane events associated with these responses . Lysyl groups on the phycobiliproteins, B-phycoerythrin and C-phycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of {3H}serotonin from anti-DNP-IgE-primed RBL-2H3 cells . The binding of these antigens to IgE-receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry . Incubation with 1 microgram/ml DNP42-B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells . Under these conditions, approximately 26 X 10(3) molecules of DNP42-B-phycoerythrin are bound per cell at equilibrium . The rate and extent of antigen binding and of antigen-stimulated mediator release decrease in parallel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced . Secretion stops immediately when the nonfluorescent monovalent antigen, DNP-lysine, is added to degranulating cell suspensions . DNP-lysine also displaces surface-bound antigen when added during the first minutes after multivalent antigen . However, the ability of DNP-lysine to displace surface-bound DNP42-B-phycoerythrin from IgE-receptor complexes decreases progressively with time . Treatment with dihydrocytochalasin B and several analogs that prevent antigen-stimulated F-actin assembly enhances secretion and delays the transition of antigen to its DNP-lysine-resistant form . Cytochalasin treatment also permits the long-range movement of antigen into surface caps.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Pathol, 1987 May, 40(5), 490 - 3
Different reactivity of monoclonal antibodies against common acute lymphoblastic leukaemia antigen (CD10); Haralambidou S et al.; The reactivity of five monoclonal antibodies J5, OKB-cALLA, Nu-N1, Nu-N2 and VIL-A1 against the common acute lymphoblastic leukaemia (common-ALL) antigen (glycoprotein 100, CD10); was investigated by indirect immunofluorescence in cell suspensions, and by immunoperoxidase in cytocentrifuge slides of ALL, chronic B cell lymphoproliferative disorders, and plasma cell dyscrasias . The five monoclonal antibodies gave similar positive results with both techniques only in samples of ALL . J5 was positive in variable degrees by immunofluorescence in the majority of B cell disorders examined but this was not confirmed by immunoperoxidase . OKB-cALLA reacted in a similar way to J5 in both techniques, although with a lower percentage of cells by immunofluorescence . Nu-N1, Nu-N2, and VIL-A1 were mainly negative when tested by both immunofluorescence and immunoperoxidase in B cell disorders other than ALL and therefore seemed to be more specific for the diagnosis of common-ALL.

J Immunol, 1987 May 1, 138(9), 2902 - 5
Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease; Nixon-Fulton JL et al.; C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid) . Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts . Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice . Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level . These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.

Acta Otolaryngol, 1987 May-Jun, 103(5-6), 529 - 36
In vitro chemosensitivity testing of squamous cell carcinoma of the head and neck . A preliminary report; Elprana D et al.; This study deals with in vitro chemosensitivity testing of squamous cell carcinoma of the head and neck by measuring the drug-induced inhibition of {3H}thymidine incorporation . Cell suspensions and tumour slices obtained from four human tumour xenografts were used . These were grown in nude mice and tested in this animal for their sensitivity to bleomycin and cis-platinum . No reliable data could be obtained with the use of cell suspensions because of the rapid decrease in cell viability during incubation . The incubation of 400-microns-thick tumour slices using hyperbaric O2 revealed a stable control level of {3H}thymidine incorporation which persisted for more than 24 h . Cytotoxic drugs only demonstrated a significant and reproducible decrease in the incorporation of {3H}thymidine in those tumours which were found to be sensitive to these drugs in nude mice . This technique seems to be very promising but needs further evaluation of its application on tumour specimens obtained directly from patients.

Cancer Res, 1987 Apr 15, 47(8), 2129 - 35
Platelet aggregating activity mediated by thrombin generation in the NCG human neuroblastoma cell line; Esumi N et al.; Platelet aggregating activity of the NCG human neuroblastoma cell line was compared with that of the HL-60 human promyelocytic leukemia cell line . NCG, in intact cell suspensions and ultracentrifuged pellets, induced platelet aggregation most significantly in heparinized platelet rich plasma (PRP) containing 2.5 units/ml of heparin, but not in the presence of higher concentrations of heparin or 5 mM ethylenediamine-tetraacetate or in citrated PRP . NCG induced platelet aggregation was also inhibited by hirudin or (2R,4R)-4-methyl-1-{N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfon yl)-L- arginyl}-2-piperidinecarboxylic acid (MD 805) in the same manner as that of tissue thromboplastin induced platelet aggregation . HL-60 cells did not induce platelet aggregation in our heparinized PRP assay systems; however, after treatment with neuraminidase HL-60 cells became active in aggregating platelets in either heparinized or citrated PRP . NCG demonstrated high procoagulant activity by either intact cell suspensions or ultracentrifuged pellets . The procoagulant activity of NCG was reduced in Factor VII deficient human plasma as it was in the results obtained by tissue thromboplastin . These results suggest that NCG induces platelet aggregation via thrombin generated through procoagulant activity which is shed in association with microvesicles demonstrated in the ultracentrifuged pellets . This type of platelet aggregating activity found in NCG is significantly different from that of HL-60.

J Immunol, 1987 Apr 15, 138(8), 2604 - 10
Mast cells from the human intestinal lamina propria . Isolation, histochemical subtypes, and functional characterization; Befus AD et al.; With the use of a collagenase dispersion technique, cells were isolated from the lamina propria of the human small and large intestine . The cell suspensions contained 8% mast cells, which on average contained 1 to 2 pg of histamine/cell . With the use of histochemical procedures based upon fixative sensitivity and dye binding, which identify functionally distinct mast cell subtypes in the rat, dispersed human intestinal mast cells contained approximately equal proportions of two histochemical subtypes analogous to those in the rat . Whether these are functionally distinct as in the rat remains to be determined . The histochemically mixed mast cell populations from the human intestinal mucosa secreted histamine in a dose- and energy-dependent manner in response to anti-IgE and A23187, but not 48/80 . Theophylline, doxantrazole, quercetin, and salbutamol all significantly inhibited anti-IgE-induced histamine secretion by human intestinal mast cells, but cromolyn sodium and the experimental antisecretory drugs, nedocromil sodium and FPL 52694, did not inhibit histamine secretion by the mast cell mixture to a statistically significant extent . Cromolyn sodium inhibited histamine secretion by 15 to 30%, and whether this reflected inhibition of one of the two histochemical mast cell subtypes to a greater extent than the other or all the cells to a minimal degree remains to be established . Control investigations of the intestinal cell isolation procedure indicated that these qualities did not reflect effects of the cell dispersal procedure . Further characterization and analysis of intestinal mast cells is essential to determine if functionally distinct mast cell subtypes exist in human tissues.

FEBS Lett, 1987 Apr 6, 214(1), 17 - 20
Release of immunoreactive atrial natriuretic factor from the isolated perfused rat lung; Gutkowska J et al.; The secretion of ANF by the rat lung was demonstrated in the present study . Four forms of immunoreactive ANF were detected in rat lung homogenates, including the 126 amino acid prohormone but only a low molecular mass peptide was released during lung perfusion . The released ANF inhibited forskolin-stimulated aldosterone secretion from rat zona glomerulosa cell suspensions, and this biological effect was comparable to that of the synthetic C-terminal part of the prohormone (Arg-101-Tyr-126).

Biochim Biophys Acta, 1987 Apr 2, 928(1), 1 - 7
Diffusion of singlet oxygen into human bronchial epithelial cells; Nye AC et al.; The respiratory epithelium undergoes morphological and functional changes following exposure to single oxygen . However, mechanisms by which singlet oxygen causes cellular injury are unclear . The present experiments were designed to investigate the possibility that singlet oxygen, a highly reactive species, diffuses into respiratory epithelial cells . Of the various methods for detection of singlet oxygen, the electron spin resonance (ESR) spectrometric technique was judged to be most compatible and sensitive for use with cell suspensions . ESR spectrometry was used to monitor the singlet oxygen-mediated conversion of 2-(9,10-dimethoxyanthracenyl)-tert-butylhydroxylamine, (I), to 2-(9,10-dimethoxyanthracenyl)-tert-butylnitroxide, (II), and its corresponding endoperoxide, (III), in human bronchial epithelial cells treated with extracellularly generated singlet oxygen . In a second series of experiments, bronchial epithelial cells labeled with (I) were treated with singlet oxygen in the presence of 1,4-diazabicyclo{2.2.2}octane, a singlet oxygen quenching agent . The addition of this quenching agent eliminated the ESR spectrum corresponding with (II) and (III) . This result is consistent with the quenching of singlet oxygen by 1.4-diazabicyclo{2.2.2}octane . Collectively, our results indicate that extracellularly generated singlet oxygen diffuses into human bronchial epithelial cells and that this process is a potentially important step in the cytotoxic action of singlet oxygen to the respiratory epithelium.

Clin Chem, 1987 Apr, 33(4), 558 - 61
Rapid detection of leukemia cells by use of a complement-mediated cytolytic reaction and an imaging sensor system; Suzuki M et al.; We describe a system for detection of leukemia cells involving complement-mediated cytotoxic reaction and an image processing system, consisting of a charge-coupled-device image sensor, an image memory board, a personal computer, and a phase-contrast microscope . Then added to a cell suspension, monoclonal antibody specific to the fetal thymus antigen-1 of the mouse leukemia GRSL cell produced cytolysis of only GRSL cells . This cytolysis decreased the brightness of the cells observed by phase-contrast microscopy . The remaining brightness was subtracted from that of the phase-contrast image of the cells before cytolysis, which had been converted to a digital signal and stored in computer memory . Measurement time is 2 s . The time course for complete GRSL cytolysis, as measured with this system, is 12 min; overall measurement time, including reaction time, is approximately 15 min . GRSL cells in a suspension of mixed cells were determined specifically by the system.

Cancer Res, 1987 Apr 1, 47(7), 1973 - 7
DNA flow cytometry and histopathological grading of paraffin-embedded prostate biopsy specimens in a survival study; Lundberg S et al.; Methods to disintegrate old paraffin-embedded tissue blocks for the application of DNA flow cytometry open up new possibilities for retrospective studies on the correlation between tumor cell nuclear DNA pattern and prognosis of the neoplastic disease . In the present work we used such a method to study the relationship between DNA ploidy, histopathological grade, and survival for 50 patients with prostate carcinomas diagnosed 1958-1974 . Plugs of histologically identified tissue from benign and tumor areas were sampled from paraffin blocks of prostate biopsy specimens by using a 4-mm skin biopsy punch . Thirty-micron sections were cut from each plug for dewaxing and disintegration . The cell suspensions obtained were stained with 4',6-diamidino-2-phenylindole dihydrochloride and analyzed by flow cytometry . In about one-half of the cases where two or more plugs were analyzed we found a heterogeneous tumor cell nuclear DNA pattern . No apparent correlation was found between the histopathological grade and the DNA ploidy . Using Cox's multiple regression analysis, we found a significant correlation between DNA ploidy and survival of these patients (P = 0.043) when we controlled for histopathological grade (Dhom grade), acid phosphatase level, occurrence of metastases, age, year of diagnosis, and type of biopsy . The correlation between DNA ploidy and survival was just above the level of significance (P = 0.059) when Gleason grade was substituted for Dhom grade in the regression model.

Agents Actions, 1987 Apr, 20(3-4), 223 - 5
On the isolation of mast cells from human adenoids and tonsils; Grosman N et al.; Human adenoids and tonsils were disintegrated mechanically and the cells dispersed by passage through a stainless-steel screen in EDTA-containing buffer . Collagenase digestion did not increase the yield of adenoidal cells . The mast cell content of the cell suspensions was in the range of 1-10 mast cells/10(4) cells with an estimated mean of 1-2 mast cells/10(4) cells, a value considerably below previous reports on adenoidal cell suspensions . The mast cell content was determined by staining with toluidine blue at low pH (to prevent interference by phagocytes) . The mast cell count as assessed by alcian blue staining and by fluorescence microscopy after FITC-anti-human IgE binding was similar . Various attempts to enrich the cell suspension (i.e . by differential centrifugation, by gradient centrifugation on Ficoll or Ficoll-Hypaque and by velocity sedimentation at unit gravity) all gave negative results.

Naunyn Schmiedebergs Arch Pharmacol, 1987 Apr, 335(4), 445 - 8
The effect of denbufylline on the viscosity of rat whole blood and on the deformability (filterability) of rat blood cell suspensions; Jukna JJ et al.; The novel alkylxanthine, denbufylline {1,3-di-n-butyl-7-(2-oxopropyl)-xanthine} has been examined, in vitro, for effects on the viscosity of rat whole blood and on the filterability of rat blood cell suspensions . For comparison, pentoxifylline was also examined for rheological activity . Denbufylline reduced the viscosity of whole blood at all shear rates utilised, up to 128.5 s-1 . The effect was, however, more pronounced at a low (0.7 s-1) than at a high (94.5 s-1) shear rate indicating that the compound reduces blood cell aggregation and increases blood cell deformability . Denbufylline also increased the filterability of blood cell suspensions . This is further evidence that the compound increases the deformability of blood cells . Denbufylline elevated the filterability of both pure erythrocyte and mixed erythrocyte/leucocyte suspensions, the effect being greatest with the latter . This suggests that denbufylline may influence the deformability of both red and white blood cells . However, the effect on white blood cells, under the experimental conditions employed, is apparently more marked . Pentoxifylline also reduced the viscosity of rat whole blood and increased the filterability of rat blood cell suspensions . However, denbufylline was 10-100-fold more potent in these tests than pentoxifylline.

J Biomed Mater Res, 1987 Apr, 21(4), 419 - 28
The effect of injection of powdered biomaterials on mouse peritoneal cell populations; Pizzoferrato A et al.; The authors suggest an experimental model for the assaying, before implantation, of the tissue reaction that wear particles from artificial joints can cause in the human body . Mice were intraperitoneally injected with biomaterial powders in suspension in saline . The materials used were chromium, chromium oxide, nickel, nickel oxide, aluminum, alumina, alumina-titania, cobalt, molybdenum, titanium, silver, zirconium oxide, iron oxide, and stainless steel . Peritoneal lavage was performed a week after injection . A thorough morphological and quantitative analysis of the cell suspension thus obtained was made both immediately after collection and after a 24 h culture.

Br J Cancer, 1987 Apr, 55(4), 449 - 54
Tumour aneuploidy, prognostic parameters and survival in primary breast cancer; Owainati AA et al.; Cellular DNA content of primary tumours from 280 patients with operable breast cancer was determined by flow cytometry using nuclei from paraffin sections stained with DAPI, and 199 of these patients were followed for 8-13 years after surgery . Tumours from 67 patients have also been analyzed for their DNA content using single cell suspensions from fresh tumour tissue stained with mithramycin and ethidium bromide, and the results compared with those obtained from paraffin blocks of the same tumours . Overall 60% of the tumours contained cells with abnormal DNA content (DNA-aneuploid populations) . Survival and disease free interval were not significantly different in patients with DNA-diploid and DNA-aneuploid tumours when analysed by Mantel's life table method . There was however, an early advantage for patients with DNA-diploid tumours: during the first 30 months after surgery DNA-aneuploidy was associated with higher rate of recurrence and shorter survival . DNA-aneuploidy was strongly related to histological grade . Thus 11/49 (22%) grade I, 60/102 (59%) grade II, and 96/129 (74%) grade III tumours were DNA-aneuploid . Although there was no significant difference in survival of patients with DNA-diploid and DNA-aneuploid tumours overall, there appears to be an unexpected association between DNA-aneuploidy and better survival in grade II patients (P less than 0.01); a similar trend was observed for grade I patients . Although the proportion of DNA-aneuploid tumours was similar in oestrogen receptor positive and negative tumours, DNA-aneuploidy was associated with lower levels of oestrogen receptors in comparison to DNA-diploid tumours . Comparison between the modal DNA values of fresh and paraffin embedded samples showed high rate of comparability (64/67, P less than 0.0001).

In Vitro Cell Dev Biol, 1987 Apr, 23(4), 261 - 6
Isolation, long-term culture, and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells; Nag AC et al.; A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established . The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart . The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk . Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period . Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility . Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils . Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line . Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres . These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture.

Br J Exp Pathol, 1987 Apr, 68(2), 157 - 65
Preparation of viable single cell suspensions of tracheal epithelial cells; Johnson NF et al.; This paper reports a procedure used for isolating the entire epithelial lining of the rat trachea . Isolated trachea was initially filled with 0.2% hyaluronidase and incubated at 37 degrees C for 30 min . Tracheas were flushed with medium and then reinflated with 0.5 microgram/ml cytochalasin B and re-incubated for 60 min . The tracheal lumens were again flushed and reinstilled with 24 iu/ml pronase and incubated for a further 30 min . The tracheas were flushed again and the cells removed enumerated and viability assessed by trypan blue dye exclusion . Cell yields (X 10(6)) from 30 consecutive Fischer 344 rats were 5.06 +/- 0.16 (s.e.m.) and the mean percentage of viable cells was 83.13 +/- 1.10 (s.e.m.) . This cell yield was close to the estimated tracheal cell population (5.3 X 10(6)) . The suspensions were predominantly single cells which apparently retained a normal ultrastructural appearance.

Photodermatol, 1987 Apr, 4(2), 66 - 72
Effect of UVA and PUVA on alloactivating and antigen-presenting capacity of human epidermal Langerhans cells; Mork NJ et al.; Human epidermal cell suspensions (EC), obtained with a suction blister technique and enzyme digestion, were irradiated with various doses of UVA with (PUVA) or without previous incubation with 8-methoxypsoralen (8-MOP) . EC were then cocultured with allogeneic T cells or pulsed with the soluble antigen purified protein derivative of tuberculin (PPD) for 90 min before being cocultured with autologous T cells . While low doses of UVA induced a small but significant increase in the PPD-specific T-cell response, both PUVA and higher doses of UVA induced dose-dependent reductions . The allogeneic T-cell responses were reduced with PUVA, as well as with UVA, in a dose-dependent fashion . PUVA was far more effective than UVA in reducing both allogeneic and antigen-specific T-cell responses . There were no differences between numbers of DR-positive cells in EC before, immediately after or 24 h after PUVA or UVA radiation, and quantitative determination of EC HLA-DR molecules using an indirect radioimmunoassay (RIA) technique did not reveal any difference between PUVA-treated and non-irradiated cells.

Gan To Kagaku Ryoho, 1987 Apr, 14(4), 1100 - 5
{Application of cytopathology in a sensitivity test for anti-tumor agents . I . An experimental study}; Wada Y et al.; Although the human tumor clonogenic assay (HTCA) is extremely reliable in determining clinical correlations, it is a complicated process requiring considerable time in order to obtain results . Thus, an experimental study on cytopathologic observation (cytologic assay) and comparative evaluation between it and HTCA were performed in order to establish a more rapid and accurate drug sensitivity test . Materials included Colon 26, a cell line established in our department, malignant effusion and surgical specimens . In carrying out HTCA according to the Hamburger-Salmon method, the cell suspension samples following exposure to anti-tumor agents (MMC, L-PAM, ADM, CDDP) were cultivated in test tubes for 3-8 hours and stained by the Papanicolaou and Giemsa methods . According to Tokita's criteria, when cellular changes showed as nuclear pyknosis and nuclear destruction were found to have increased significantly in comparison with a control group, the cells were judged to be sensitive . Very similar and parallel results were obtained between HTCA and cytologic assay in this study, with a significant correlation . Cytologic assay was proved to be an easy, rapid and accurate method for testing drug sensitivity and its clinical application can be expected in the future.

Lab Invest, 1987 Apr, 56(4), 381 - 93
Single cell studies on the immunological marker profile of plasmacytoid T-zone cells; Beiske K et al.; Plasmacytoid T cells (PTC) are known to home to thymic (T) zones in human lymph nodes and are characterized by their abundant, concentrically layered, rough endoplasmic reticulum . These cells have been found in reactive and neoplastic conditions . Three cases of PTC lymphomas have so far been reported . All of them were complicated by a myelomonocytic leukemia leading to the assumption of a functional relationship between PTC and the myeloid system . The immunologic phenotype of PTC, as revealed on frozen tumor tissue sections, comprised the expression of CD5 (T1), CD4 (T4), and HLA-DR, but not CD8 (T8) and CD2 (T11) and suggested an affiliation to the T cell system . Extending our previous report on one of these cases we here present the first study on the immunological marker profile of suspended PTC . The employment of unfractionated or PTC-enriched tumor cell suspensions rendered possible the application of a panel of monoclonal antibodies (moAbs) on both fixed and unfixed cells and enabled us to allocate various markers either to the intracytoplasmic or surface domain of this cell type . Our results suggest that PTC from our case rest in the G0/G1 phase of the cell cycle . They express the transferrin receptor, but not the Il-2 receptor (CD25) or the nuclear antigen Ki-67 . No T cell antigen was demonstrated on the surface of unfixed suspended PTC . Under these conditions only HLA-DR and a predominantly monocytic antigen (CD36/moAb 5F1) were identified . Fixed cells, however, showed a weak cytoplasmic reactivity for CD5 and two myelomonocytic antigens (CD15/moAb 1G10 and CD14/moAb My4) . Our findings do not sustain positive evidence for a T cell nature of PTC . Whether their phenotypical pattern indicates terminal differentiation with concomitant loss of T cell antigens or points to a cytogenetic relationship of PTC to the myeloid system, remains speculative . Until the cytogenesis of PTC is clarified we propose the noncommitted term "plasmacytoid T-zone cells" for this elusive cell type.

Blood, 1987 Apr, 69(4), 1249 - 54
In vitro tests that predict tumor-associated idiotype levels in the serum of patients with B cell lymphomas and leukemias; Miller RA et al.; The presence of circulating tumor idiotype interferes with the in vivo effectiveness of anti-idiotype antibodies . We developed two assays that permit identification of patients with high levels of serum idiotype without the need for first producing an anti-idiotype antibody . A cell suspension made from the tumor was cultured for seven days with or without phytohemagglutin (PHA) and/or phorbol myristic acetate (PMA) . Ig secretion in vitro by patients' tumor cells varied . In 4 patients, no secretion in vitro occurred, 5 patients had low levels, and 5 patients had high levels of Ig secretion . In three patients, Ig secretion occurred only after stimulation with PHA, PMA, or both . Spontaneous or induced immunoglobulin secretion in vitro is related to the levels of tumor idiotype secretion that exist in vivo . Eight patients with serum idiotype levels greater than 100 micrograms/mL (mean 265 micrograms/mL), had a minimum of 1.0 microgram/10(6) cells of idiotype secretion in vitro . Nine patients with serum idiotype levels less than 30 micrograms/mL (mean 3.7 micrograms/mL), had less than or equal to 0.5 microgram/10(6) cells of idiotype secretion in vitro . In another assay, the levels of IgM kappa and IgM lambda in patients' sera were compared with those in normal serum . An imbalance in the relative amounts of IgM kappa and IgM lambda indicated high levels of circulating idiotype in the serum, but this assay was less sensitive than the in vitro secretion assay and limited to IgM-secreting tumors . These assays will be useful for future clinical studies using anti-idiotype antibodies.

J Endocrinol Invest, 1987 Apr, 10(2), 117 - 21
Human medullary thyroid carcinoma in tissue culture; secretion of calcitonin and carcinoembryonic antigen; Oosterom R et al.; Monolayer cultures of medullary thyroid carcinoma (MTC) cells were prepared from 5 patients with familial and 4 with sporadic MTC . Basal calcitonin (CT) release decreased rapidly during 17 days in culture . Addition of extra calcium (2 mmol/l) or dibutyryl cyclic AMP (dBcAMP, 3 mmol/l) increased CT release in all investigated cultures (p less than 0.01) . No significant differences were observed between cell suspensions prepared from familial and those from sporadic MTC . Carcinoembryonic antigen (CEA) was found in 3 out of 5 MTC cell suspensions from patients with familial tumors and in 2 out of 2 sporadic tumors . Only two cultures secreted measurable amounts of CEA, which decreased with culture time but at a much slower rate than that of CT release . CEA release was not affected by the addition of calcium and dBcAMP . This study shows an absent relation between basal and stimulated CT and CEA release by cultured MTC cells.

Cancer Res, 1987 Apr 1, 47(7), 1820 - 4
Role of uridine triphosphate in the phosphorylation of 1-beta-D-arabinofuranosylcytosine by Ehrlich ascites tumor cells; White JC et al.; Pyrimidine nucleotide pools were investigated as determinants of the rate of phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) by Ehrlich ascites cells and cell extracts . Cells were preincubated for 2 h with 10 microM pyrazofurin, 10 mM glucosamine, 50 microM 3-deazauridine, or 1 mM uridine in order to alter the concentrations of pyrimidine nucleotides . Samples of the cell suspensions were taken for assay of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), cytidine 5'-triphosphate, guanosine 5'-triphosphate, deoxycytidine 5'-triphosphate (dCTP), and deoxythymidine 5'-triphosphate; then 1 microM {3H}ara-C was added and its rate of intracellular uptake was measured for 30 min . 3-Deazauridine lowered dCTP and stimulated ara-C uptake; however, pyrazofurin and glucosamine were potent inhibitors of ara-C uptake although they also decreased dCTP levels . Uridine stimulated ara-C uptake despite an increase in dCTP . A crude cytoplasmic extract was prepared by a procedure which permitted results of ara-C kinase assays to be expressed as pmol per min per 10(6) cells as in the cellular uptake studies . When assayed in the presence of mixtures of ribo- and deoxyribonucleoside triphosphates at concentrations close to their cellular levels, ara-C kinase activity closely approximated the cellular uptake rate for the five incubation conditions . Deletion of cytidine 5'-, guanosine 5'-, or deoxythymidine 5'-triphosphate from the assay mixture had little effect, while deletion of dCTP increased kinase activity 9-fold . Elimination of ATP also did not alter kinase activity in the presence of the remaining five ribo- and deoxyribonucleoside triphosphates; however, deletion of UTP reduced activity to 22% of the rate with the control mixture . When ara-C kinase was assayed with only 3 mM ATP, dCTP was a very potent inhibitor (50% inhibition concentration = 0.4 microM) . Inhibition was complete at 10 microM dCTP, a concentration below the intracellular dCTP level in control cells (25 microM) . With 0.9 mM UTP, enzyme activity was 2-fold greater in the absence of dCTP and the dCTP was 15-fold less potent as an inhibitor (50% inhibition concentration = 6 microM) . We conclude that the actual phosphate donor for the phosphorylation of 1 microM ara-C in Ehrlich cells is UTP and not ATP . These observations suggest that successful combination protocols aimed at stimulating ara-C uptake by means of a decrease in dCTP levels must simultaneously preserve or increase UTP pools.

Z Naturforsch {C}, 1987 Apr, 42(4), 333 - 42
Characterization of 2 beta (R)-17-O-acetylajmalan: acetylesterase--a specific enzyme involved in the biosynthesis of the Rauwolfia alkaloid ajmaline; Polz L et al.; A novel enzyme was isolated, partially purified (217-fold) and characterized from cell suspension cultures of Rauwolfia serpentina Benth . The enzyme catalyzes one of the late biochemical reactions in the biosynthesis of ajmaline by hydrolysis of 17-O-acetylated alkaloids of the ajmalan group forming the appropriate deacetylated compounds . This esterase exhibits an unusually high substrate selectivity and exclusively accepts acetylated ajmaline derivatives with the naturally occurring 2 beta (R)-configuration . The properties of the enzyme were determined showing an optimum pH at 7.5, an isoelectric point of pH 4.9 and a relative molecular weight of 33 +/- 2 kDa . Inhibition studies of enzyme activity point to the necessity of SH-groups . The esterase seems not to be inhibited by ajmaline, the end product of the pathway . The highest enzyme activities were observed in leaves and cell suspension tissues of the tribe Rauwolfieae which are known to synthesize ajmaline and its congeners . The specific function of the esterase in the biosynthesis of the later alkaloids was established.

Br J Cancer, 1987 Apr, 55(4), 437 - 42
Angio-immunoblastic lymphadenopathy: a clinical, immunological and molecular study; Ganesan TS et al.; Twenty four patients with angio-immunoblastic lymphadenopathy (AILD) presenting between 1974 and 1985 have been reviewed . Clinical features at presentation included rash, fever, lymphadenopathy and hepatosplenomegaly in 75% of patients . Polyclonal hypergammaglobulinaemia was seen in 19/20 patients; 5 had normal immunoglobulin levels . Combination chemotherapy with MVPP was the optimal treatment with 6/7 patients achieving complete remission . Duration of remission ranged from 9 months to 4 years and was significantly longer in patients achieving complete as opposed to partial remission . In 6 patients phenotype studies were performed on single cell suspensions and immunoperoxidase studies on frozen sections of 7 lymph nodes . There was a reversal of the normal T suppressor/helper cell ratio with a predominance of T suppressor cells . Loss of normal B follicles was observed histologically in all except one lymph node . Germline configuration of the beta B-chain of the T cell receptor was observed in lymph nodes of 4 patients with AILD, and a rearranged T cell receptor was observed in 1 patient in whom a second lymph node biopsy had shown alteration of the histological picture to that of T-zone lymphoma . Frozen sera of 15 patients were screened for antibodies to HTLV I and III and were found to be negative.

J Bacteriol, 1987 Apr, 169(4), 1632 - 8
Ammonium and methylammonium transport in Rhodobacter sphaeroides; Cordts ML et al.; Rhodobacter sphaeroides maintained intracellular ammonium pools of 1.1 to 2.6 mM during growth in several fixed nitrogen sources as well as during diazotrophic growth . Addition of 0.15 mM NH4+ to washed, nitrogen-free cell suspensions was followed by linear uptake of NH4+ from the medium and transient formation of intracellular pools of 0.9 to 1.5 mM NH4+ . Transport of NH4+ was shown to be independent of assimilation by glutamine synthetase because intracellular pools of over 1 mM represented NH4+ concentration gradients of at least 100-fold across the cytoplasmic membrane . Ammonium pools of over 1 mM were also found in non-growing cell suspensions in nitrogen-free medium after glutamine synthetase was inhibited with methionine sulfoximine . In NH4+-free cell suspensions, methylammonium (14CH3NH3+) was taken up rapidly, and intracellular concentrations of 0.4 to 0.5 mM were maintained . The 14CH3NH3+ pool was not affected by methionine sulfoximine . Unlike NH4+ uptake, 14CH3NH3+ uptake in nitrogen-free cell suspensions was repressed by growth in NH4+ . These results suggest that R . sphaeroides may produce an NH4+-specific transport system in addition to the NH4+/14CH3NH3+ transporter . This second transporter is able to produce normal-size NH4+ pools but has very little affinity for 14CH3NH3+ and is not repressed by growth in high concentrations of NH4+.

Photodermatol, 1987 Apr, 4(2), 73 - 8
Phototoxicity of non-steroidal anti-inflammatory drugs demonstrated in vitro by a photo-basophil-histamine-release test; Przybilla B et al.; The phototoxic activity of the non-steroidal anti-inflammatory drugs (NSAID) acetylsalicylic acid, benoxaprofen, carprofen, diclofenac, indoprofen, ketoprofen, tiaprofenic acid, and of thiophene, a heterocyclic ring structure of the tiaprofenic acid molecule, was evaluated in vitro by a newly developed photo-basophil-histamine-release test (PBHRT) performed with human leukocytes . Cell suspensions incubated with 10(-6) to 10(-3) M of the test compounds were exposed to 1 to 100 J/cm2 UVA . Maximum photo-basophil-histamine-release was 4% with acetylsalicylic acid, 10% with benoxaprofen, 20% with thiophene, 28% with diclofenac, 39% with tiaprofenic acid, 40% with carprofen, 55% with ketoprofen, and not demonstrable with indoprofen . These results indicate that many NSAID can exert phototoxic effects, thus confirming clinical observations as well as other in vitro experiments . The PBHRT seems to be a promising new method for the identification of phototoxic compounds.

Agents Actions, 1987 Apr, 20(3-4), 284 - 7
Comparison of the release of various mediators from atrial and ventricular tissues of sensitized guinea-pig hearts; Ghanem NS et al.; By comparison with ventricular tissues, collagenase-dispersed cell suspensions obtained from atrial tissues of sensitized guinea-pigs showed a higher histamine content, a higher proportion of mast cells, and a higher release with antigen or antisera to IgG, IgG1 and IgG2 of the following mediators: histamine, thromboxane B2 and leukotriene C4.

Agents Actions, 1987 Apr, 20(3-4), 226 - 8
Isolation and sensitivity of human mesenteric mast cells to immunological and nonimmunological histamine releasers; Brzezinska-Blaszczyk E et al.; Since recent studies have emphasized that mast cells from different tissues within a given species may exhibit marked differences in their functional properties, we have now examined the effect of some immunological and non-immunological histamine releasers on human mesenteric mast cells . The mesentery derived from the patients subjected to gall-bladder surgery was dispersed by collagenase (concentration of enzyme--1 mg/ml, time of incubation--90 min, 37 degrees C) . The mesenteric cell suspension contained about 2% mast cells as identified by staining with toluidine blue . We observed that the mesenteric mast cells released histamine when challenged with anti-human IgE, but marked individual variations were observed . These cells had a low sensitivity to challenge with Concanavalin A and compound 48/80 (histamine release about 6%), but responded to ionophore A23187 and polymyxin B (histamine release up to 24% and 22% respectively).

Agents Actions, 1987 Apr, 20(3-4), 219 - 22
Isolation of mast cells from rabbit lung and liver: comparison of histamine release induced by the hypnotics Althesin and propanidid; Ennis M et al.; The enzyme collagenase was used to disperse rabbit lung and liver into their component cells . The resulting cell suspensions contained ca . 6.9% (lung) or 6.5% (liver) mast cells and were used in studies of histamine release without further purification . Both cell suspensions exhibited a low spontaneous release of histamine (ca . 6.6% lung, ca . 7.2% liver) . Both cell types responded to challenge with anti-rabbit serum with a maximum release of the amine of ca . 22% (lung) and ca . 45% (liver) . Concanavalin A challenge generally resulted in bell-shaped dose response curves, however some lung preparations did not respond . The rabbit cells were refractory to stimulation by Compound 48/80 and dextran . However a dose-dependent release of histamine was elicited after challenge with the detergents cremophor El, TN (12-hydroxystearic acid polymerized with ethylene oxide, degree of polymerization 15) and the hypnotics Althesin and propanidid . The maximum release observed depended on which cell preparation had been used . These results further emphasize the functional heterogeneity of mast cells from both different species and from different organs within the same species.

Biochem J, 1987 Mar 15, 242(3), 707 - 12
Toxic effects of ozone on murine L929 fibroblasts . Enzyme inactivation and glutathione depletion; Van der Zee J et al.; Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes . Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase . The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected . The localization of the lysosomal enzymes did not change during ozone exposure . After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined . The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone . These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell plays an important role in its susceptibility to ozone . The intracellular levels of reduced and oxidized glutathione were affected as well . The ATP content, however, proved to be insensitive to ozone exposure.

J Immunol, 1987 Mar 15, 138(6), 1744 - 9
Activation through CD3 molecule leads a number of human T cell clones to induce IgE synthesis in vitro by B cells from allergic and nonallergic individuals; Romagnani S et al.; Seventy-eight clones established from tonsillar T lymphocytes of two nonallergic children were tested under different experimental conditions for their ability to induce in vitro IgE synthesis by B cells from allergic or nonallergic donors . After 24 hr preactivation with phytohemagglutinin (PHA), 11 out of 32 CD4+ clones from the first and 17 out of 36 CD4+ clones from the second tonsil donor showed the ability to induce IgE synthesis in vitro by B cells from both allergic and nonallergic individuals, whereas none of 10 CD8+ clones nor T blasts of PHA-induced cell lines obtained from unfractionated T cell suspensions of the same tonsils had such an effect . Seven of the 11 T cell clones from the first tonsil donor active on IgE production after pre-activation with PHA also induced IgE synthesis in vitro by nonallergic and allergic B cells upon stimulation with anti-CD3 monoclonal antibody . Under the same experimental conditions, virtually all of the T cell clones able to induce IgE synthesis in vitro by target B cells showed the ability to stimulate IgG and IgM production as well . T cell clones were also established from the peripheral blood of a nonallergic donor and were tested for their ability to induce IgE synthesis in autologous B cells . After preactivation with PHA, seven out of 35 CD4+ clones induced the production of detectable amounts of both IgE and IgG in autologous B cells . The addition to the cultures of PHA-stimulated unfractionated T cells inhibited in a dose-dependent manner the IgE but not the IgG synthesis induced by an autologous helper T cell clone in autologous B cells . Taken together, these data indicate that a remarkable proportion of human T cell clones upon triggering of the CD3 molecular complex were able to provide help for the synthesis of IgE in B cells from both allergic and nonallergic individuals . The successful induction of IgE synthesis by single T cell clones was apparently related to the lack of concomitant suppressor activity to which IgE-producing cells appeared to be exquisitely sensitive.

Int J Cancer, 1987 Mar 15, 39(3), 338 - 42
Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5-azacytidine; Ishikawa M et al.; The effect of quercetin, a flavonoid derivative, on the transplantability (tumorigenicity) and metastatic behavior of mouse tumor cells was studied . BMT-11 c1-9 fibrosarcoma cells were treated in vitro with quercetin, and after cloning by limiting dilution, cell suspensions of each clone were injected subcutaneously (s.c.) into syngeneic C57BL/6 mice at a dose of 2 X 10(5) cells per mouse . Out of 17 clones examined, 8 were nontumorigenic in normal mice ("regressor" clones), whereas these clones were able to grow in immunosuppressed (600-rad-irradiated) mice . Furthermore, 1 out of 9 tumorigenic clones metastasized spontaneously to the lungs despite the very low metastatic potential of the parent BMT-11 c1-9 cells . In contrast, all 15 clones selected from the untreated parental line grew progressively in normal mice with no evidence of metastases . The appearance of both regressor and metastatic clones was also observed after treatment with a DNA hypomethylating agent, 5-azacytidine . These altered phenotypes resulting from treatment with both chemicals, however, were not necessarily stable if maintained in culture for several months . The data suggest that quercetin may be a useful new material for obtaining regressor or metastatic clones from parental tumor lines.

Biochim Biophys Acta, 1987 Mar 11, 927(3), 382 - 91
Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: differential effects of monensin; Jesaitis RK et al.; Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37 degrees C . Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-{3H}Phe for up to 30 min at 37 degrees C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 microM . 50% inhibition was observed at 1-10 microM monensin with no significant change in fMet-Leu-Phe dose dependency . Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent . The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected . Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology . Incubation of cell suspensions with up to 30 microM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation . Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations . These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production . They also are consistent with a role for receptor modulation and processing in the chemotactic response.

Biull Eksp Biol Med, 1987 Mar, 103(3), 356 - 8
{Clonal nature of fibroblast colonies formed by stromal bone marrow cells in culture}; Latsinik NV et al.; The clonal nature of CFUf-derived fibroblast colonies was tested in mixed cultures of CBA and CBAT6T6 bone marrow cells . Inoculation of marrow cell suspensions into flasks coated with poly-I-lysin has proved that no stromal aggregates were present among cells subjected to explantation . Marrow cell cultures depleted of macrophages and myeloid cells were used for chromosome analysis . The coincidence of karyotypes within a stromal colony was found in mixed cultures, which proves that CFUf-derived fibroblast colonies are cell clones.

Dev Biol, 1987 Mar, 120(1), 65 - 76
Sulfated glucuronic acid-containing glycoconjugates are temporally and spatially regulated antigens in the developing mammalian nervous system; Schwarting GA et al.; Monoclonal antibody 4F4, which was raised against a cell suspension of embryonic rat forebrain, reacts with acidic glycolipids and several high-molecular-weight glycoproteins in rodent brain . The major reactive glycolipid is maximally expressed at Embryonic Day 15 (E15) and is no longer detectable at Postnatal Day 14 (P14) in the rat . 4F4 antibody reacts with a glucuronic acid- and sulfate-containing lipid isolated from human sciatic nerve as well as with lipids from mouse and rat embryonic brain tissue . Although the glycolipid disappears postnatally, the immunoreactive glycoproteins continue to be expressed in brain until adulthood . Both sciatic nerve and embryonic brain glycolipids are hydrolyzed by glucuronidase/sulfatase treatment but are insensitive to all other glycosidases tested . In addition, the observed 4F4 reactivity with extracted glycolipids, glycoproteins, and tissue sections of embryonic brain is identical to the reactivity demonstrated by HNK-1 antibodies . Immunocytochemical studies in developing brain showed stage-specific distribution of this carbohydrate antigen . At E10 in the mouse, immunoreactivity is associated with the mantle layer of the neural tube . At E15 in the cortex, the most intense staining is associated with the molecular layer and the subplate, and weaker staining is seen in the intermediate zone and cortical plate, suggesting that the antigen is highly concentrated on postmigratory cells in the embryonic nervous system.

Exp Clin Endocrinol, 1987 Mar, 89(1), 1 - 6
Effect of hCG on protein synthesis and progesterone production in the bovine luteal cells; Kumar A et al.; The study was carried out to investigate whether the luteal steroidogenesis in response to tropic stimulus is mediated through de novo synthesis of protein(s) . Luteal cell suspension was prepared by collagenase-DNAse treatment of the bovine corpora lutea, weighing 4-6 g . Cells equivalent to 250 micrograms of protein were incubated in a total volume of 550 microliter of medium 199 with or without various test substances . Production of progesterone by the luteal cells was stimulated by hCG in a dose dependent manner . Incorporation of 3H-leucine into the cellular proteins was concomitantly enhanced . Emetine, cycloheximide and puromycin inhibited both basal and hCG stimulated protein synthesis as well as progesterone production in the cells . Thus, luteal steroidogenesis induced by hCG seems to be dependent upon de novo synthesis of a protein(s).

Cytometry, 1987 Mar, 8(2), 146 - 52
Method for detection and isolation of cholesteryl ester-containing "foam" cells using flow cytometry; Kruth HS et al.; Accumulation of cholesteryl ester within vascular cells is a defining characteristic of atherosclerotic lesions . Therefore, it is of interest to be able to monitor this critical event in the development of atherosclerosis . With this objective in mind, we have developed a method for the detection of cholesteryl ester-containing cells (i.e., foam cells) in cell suspensions prepared from enzymatically dissociated aortas . Cholesteryl ester in aortic cells was selectively stained with the fluorescent dye filipin . Because filipin binds to unesterified cholesterol but not to esterified cholesterol, it was necessary first to remove unesterified cholesterol from cells by ethanol extraction so that its presence would not interfere with the specific detection of cholesteryl ester . Then unesterified cholesterol made available by enzymatic hydrolysis of cellular cholesteryl ester could be specifically stained with filipin . The filipin-stained cell suspensions were analyzed using flow cytometry . With a flow cytometer it was possible to detect and sort cholesteryl ester-containing cells onto glass slides for microscopic analysis . Cell suspensions prepared from either grossly normal or atherosclerotic swine aortas contained cells with cholesteryl ester inclusions . As expected, these cells were more numerous in the atherosclerotic aortas . Cells with higher levels of fluorescence contained more numerous cholesteryl ester inclusions . Flow cytometric detection of cholesteryl ester-containing cells should be generally useful in studies of cellular cholesterol metabolism as well as in specific studies of cellular cholesterol accumulation in atherosclerotic vessels.

Brain Res Bull, 1987 Mar, 18(3), 337 - 43
Transplants of cholinergic septal explants reinnervate adult rodent hippocampus; Ezerman EB et al.; Embryonic septal-basal forebrain tissue was grown in explant culture for 1, 4 or 5 days prior to transplantation to the hippocampus of adult rats denervated of its septal input by a fornix/fimbria transection . The explant transplants were compared with transplants of septal cell suspensions . Analysis of fiber ingrowth by acetylcholinesterase (AChE) histochemistry at various timepoints post-transplantation shows little difference in the developmental time course or extent of cholinergic axon ingrowth into the host hippocampus between the suspension transplants and the explant transplants . Septal explants in culture for 5 days were as effective as those in culture for one day prior to transplantation in providing cholinergic reinnervation to the host hippocampus . Studies using antibodies to choline acetyltransferase (ChAT) and the fluorescent retrograde tracer, fast blue, confirmed that the AChE-positive cells in the transplants were cholinergic and that the innervation to the host hippocampus came from the transplanted cholinergic neurons . Thus the results demonstrate that embryonic cholinergic septal neurons can be maintained in short-term culture prior to transplantation without adversely affecting their ability to innervate an appropriate CNS target in vivo.

J Reprod Fertil, 1987 Mar, 79(2), 539 - 48
Progesterone production, LH receptors, and oxytocin secretion by ovine luteal cell types on days 6, 10 and 15 of the oestrous cycle and day 25 of pregnancy; Harrison LM et al.; Corpora lutea were collected from sheep on Days 6, 10, and 15 of the oestrous cycle and Day 25 of pregnancy and dissociated into single cell suspensions . Purified preparations of large and small luteal cells were prepared by elutriation on all days except Day 6 . Basal progesterone production by large cells was 6-8-fold higher than by small cells (36-65 vs 6-9 fg/cell/min) . Oxytocin secretion was maximal on Day 6 (1.0 fg/cell/min) and declined thereafter . The number of receptors for LH increased between Day 6 and Day 10 and the two cell types had an equal number of receptors on Days 10 and 15 (19,000-23,000) . Large cells on Day 25 of pregnancy had fewer receptors (12,000) than did small cells (26,000) . Progesterone secretion by small luteal cells from all days examined was stimulated by LH (0.01-1000 ng/ml) in a dose-dependent manner; maximum sensitivity to LH occurred on Day 10 . Despite the presence of receptors for LH on large cells, LH failed to stimulate progesterone production . Basal production of progesterone by large and small cells, and the response of small cells to LH, was not influenced by day examined . Re-combinations of large and small cells from Day 10 synergized to increase progesterone secretion . Prostaglandin E-2 (0.1-1000 ng/ml) did not stimulate progesterone secretion by large or small cells.

J Nutr, 1987 Mar, 117(3), 567 - 71
Impaired natural killer cell activity in iron-deficient rat pups; Sherman AR et al.; Natural killer (NK) cell activity was studied in iron-deficient rat pups . Pregnant dams were fed diets containing 6, 10 or 250 ppm Fe ad libitum from d 1 of gestation through d 21 of lactation . Two days post parturition litters were adjusted to seven pups each, and on d 17 the pups were injected intraperitoneally with 5 X 10(5) plaque-forming units of vaccinia virus . Following a 4-d incubation period, spleens were removed and the cell suspensions combined with YAC-1 target cells to measure cytolysis in a 4- and 16-h chromium release assay . Hematocrit levels of severe (6 ppm) and moderately (10 ppm) iron-deficient rat pups were significantly lower than that of controls . Similarly, body weight and spleen weights were significantly lower in iron-deficient pups than in control pups . Iron deficiency significantly impaired spleen NK cell activity when measured by two different effector:target ratios and assay time periods.

Br J Cancer, 1987 Mar, 55(3), 283 - 6
A comparison of three methods for the determination of the growth fraction in non-Hodgkin's lymphoma; Schrape S et al.; The proliferation rate of non-Hodgkin's lymphomas (NHL) was estimated by using 3 different methods . In cell suspension we determined the proportion of cells in cycle with the monoclonal antibody (Mab) Ki-67 and also in S-phase after the incorporation of bromo-deoxyuridine (BrdU) utilizing Mab anti-BrdU . In low grade lymphomas 3.5 +/- 1.6% of the cells were in cycle and 1.2 +/- 0.9% in S-phase, the corresponding values for high grade lymphomas were 22.5 +/- 18.7% and 8.9 +/- 7.8% respectively . Frozen sections of NHL were reacted with an antibody to the transferrin receptor (TR) and Ki67 as markers for proliferative activity . A high number of TR positive cells was found in low grade lymphomas of all histological types, whereas Ki67 positivity correlated closely with grading . With a few exceptions, low grade lymphomas contained less than 25% Ki67 positive cells within the tumour cell population . This observation is relevant to treatment strategies for low grade NHL.

Strahlenther Onkol, 1987 Mar, 163(3), 195 - 200
{In-vivo marking of human tumors using BUDR for flow-cytometry determination of kinetic cell parameters}; Karcher H et al.; The flow-cytometry results of solid, human tumours are reported which were labelled with Bromodeoxyuridine in vivo . The preparation of the single cell suspension is prescribed . The S-phase percentage could be determined exactly in all cases but one . It is a rapid procedure which gives results in one day and the in vivo labelling had no adverse effects.

Scand J Immunol, 1987 Mar, 25(3), 305 - 13
Release and functional characterization of the leukotriene D4-metabolizing enzyme (dipeptidase) from human polymorphonuclear leucocytes; Raulf M et al.; Polymorphonuclear leucocytes released LTD4-dipeptidase activity in a time-, calcium-, and cell number-dependent fashion . The LTD4-dipeptidase released from polymorphonuclear leucocytes (PMN) by incubation with calcium (0.91 mM) was detectable up to a cell concentration of 1 X 10(6)/ml and increased with higher concentrations . Maximal LTD4-dipeptidase activity within the extracellular environment was detected after 15 min of incubation (2 X 10(7)/ml) in the presence of 2-4.5 mM calcium or after 30 min, when stimulation was carried out with 0.91 mM calcium . The activity of the released LTD4-dipeptidase was modulated by various metal ions and other compounds . The addition of Mn2+, Co2+, and Zn2+ (final concentration 1 mM) enhanced the LTD4-dipeptidase activity, while Cu2+ led to a complete inhibition . In the absence of exogenous calcium EDTA inhibited LTD4-dipeptidase . Calcium up to a concentration of 5 and 10 mM decreased the dipeptidase activity . The LTD4-dipeptidase is not affected by bestatin, leupeptin, or N-ethyl-maleinimide (NEM) . The Km of LTD4-dipeptidase for LTD4 was 0.95 +/- 0.2 microM and Vmax was 737.5 +/- 112.5 pmol/min X mg protein (n = 3 +/- SEM) . The highest LTD4-dipeptidase activity was obtained at physiological pH values . LTD4-dipeptidase activity can also be released from other cell types, but the enzyme activity from human PMN exceeded that of other cells (e.g . human lymphocytes/monocytes and basophils (LMB) and human lung cell suspension).

J Gen Physiol, 1987 Mar, 89(3), 443 - 57
Cell suspensions from porcine olfactory mucosa . Changes in membrane potential and membrane fluidity in response to various odorants; Kashiwayanagi M et al.; A suspension of olfactory epithelial cells was prepared from porcine olfactory mucosa and the physiological functions of the suspension were examined . The membrane potential of the cell suspension, which was monitored by measuring the fluorescence changes of rhodamine 6G, was depolarized by an increase in the K+ concentration in the external medium . Various odorants depolarized the cell suspension in a dose-dependent fashion . The magnitude of depolarization by odorants was either unchanged or slightly increased by a reduction of the concentration of Na+, Ca2+, and Cl- in the external medium, which suggests that changes in the permeabilities of specific ions are not involved in depolarization by odorants . The application of various odorants to the cell suspension induced changes in the membrane fluidity at different sites of the membrane that were monitored with various fluorescent dyes {8-anilino-1-naphthalene sulfonate, n-(9-anthroyloxy) stearic acids, 12-(9-anthroyloxy) oleic acid, and (1,6-diphenyl-1,3,5-hexatriene)}, which suggests that the odorants having different odors are adsorbed on different sites in the membrane . On the basis of these results, a possible mechanism of odor discrimination is discussed.

Am J Physiol, 1987 Mar, 252(3 Pt 2), F412 - 22
Immunological segmentation of the rabbit distal, connecting, and collecting tubules; Poujeol P et al.; To obtain monoclonal antibodies (MAB) specific for the different cell types of distal and collecting tubules, BALB/c mice were immunized with cell suspensions highly enriched in cells from the distal segments of the rabbit nephron . Nine MAB were selected and cloned . Four groups could be identified on the basis of double-labeling immunofluorescence (IF) on frozen kidney sections and on microdissected tubules . In addition, binding specificity at the cellular level was studied by immunoelectronmicroscopy (IEM) for selected MAB . A single MAB (group 1) was specific for distal bright cells and a subpopulation of cortical ascending limb cells . Six MAB (group 2) reacted with connecting and collecting tubules . Five of these (group 2A) had similar binding patterns and reacted identically with the two tubular segments . The MAB studied by IEM was specific for connecting and principal cells . One antibody (group 2B) reacted with only a fraction of the cells associated with the connecting tubule (CNT), but with all cells of the cortical collecting tubule (CCT) . By IEM, this antibody was found to be specific for intercalated cells in CNT and bound both principal and intercalated cells of the CCT . Two MAB (group 3) reacted with antigen(s) expressed by the various terminal segments of renal tubule . MAB of groups 1 and 2A, which define distal bright cells and connecting-principal cells from the CNT-CCT, respectively, were used for cell fractionation experiments . Heterogeneous rabbit cortical cells were first incubated with the selected MAB . MAB-bearing renal cells were separated on plastic dishes previously coated with an affinity-purified goat anti-mouse immunoglobulin . Using these procedures it was possible to obtain highly purified subpopulations of distal, bright, or connecting-principal cells.

Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Mar, 51(3), 401 - 19
Flow cytometric analysis of the effects of 0.4 MeV fission neutrons on mouse spermatogenesis; Spano M et al.; (C57Bl/Cne X C3H/Cne)F1 male mice were irradiated with single acute doses of 0.4 MeV neutrons ranging from 0.05 to 2 Gy, and testis cell suspensions were prepared for cytometric analysis of the DNA content 2-70 days after irradiation . Various cell subpopulations could be identified in the control histogram including mature and immature spermatids, diploid spermatogonia and spermatocytes, tetraploid cells and cells in the S-phase . Variations in the relative proportions of different cell types were detected at each dose and time, reflecting lethal damage induced on specific spermatogenetic stages . The reduction of the number of elongated spermatids 28 days after irradiation was shown to be a particularly sensitive parameter for the cytometrical assessment of the radiosensitivity of differentiating gonia . A D0 value of 0.13 Gy was calculated and compared with data obtained after X-irradiation, using the same experimental protocol . In the latter case a biphasic curve was obtained over the dose range from 0.25 to 10 Gy, possibly reflecting the existence of some cell population heterogeneity . RBE values were estimated at different neutron doses relative to the radiosensitive component of the X-ray curve, and ranged from 3.3 to 4, in agreement with data in the literature . Genotoxic effects were monitored 7 days after irradiation by a dose-dependent increase of the coefficient of variation (CV) values of the round spermatid peak, reflecting the induction of numerical and structural chromosome aberrations, and 14 or 21 days after irradiation by the detection of diploid elongated spermatids, probably arising from a radiation-induced complete failure of the first or second meiotic division.

Exp Hematol, 1987 Mar, 15(3), 296 - 303
A new density gradient for the separation of large quantities of rosette-positive and rosette-negative cells; Dooley DC et al.; Allogeneic transplantation of peripheral blood stem cells will not be feasible until new techniques are developed for large-scale depletion of T-lymphocytes . Small quantities of cells can be depleted of T-lymphocytes by sheep erythrocyte rosetting and Ficoll-diatrizoate discontinuous gradient fractionation . However, when processing 10(9)-10(10) cells, the fractionation step is inefficient because of the limited capacity of Ficoll-diatrizoate . We therefore developed a new discontinuous gradient for the separation of large numbers of sheep red blood cell (RBC) rosette-positive and negative cells . The gradient was designed so that sheep cells and rosettes would not interfere with the banding of rosette-negative cells . In that way, nonspecific entrapment was reduced, and high cell capacity and yield were achieved . The system fractionated rosetted cell suspensions into four populations: free sheep RBC, rosetted T cells, nonrosetted T cells, and low-density non-T cells . The 15-ml gradient routinely separated 3-24 X 10(8) cells . Progenitor yield ranged from 83% to 99%, with 95% depletion of T-lymphocytes . The method is rapid, reproducible, and inexpensive . This preparative technique could prove useful clinically when large-scale separation of E-rosette-positive and negative cells is required.

Cell Tissue Kinet, 1987 Mar, 20(2), 227 - 31
Chemoattraction enrichment of thymus-homing bone marrow cells; Haar JL et al.; It has been reported that a population of cells from mouse bone marrow migrates to supernatant made from the incubation of minced fragments of new-born mouse thymuses and that the migrated population is enriched for immature lymphoid cells . In the present study, we show that this method enriches for thymic-homing cells . Migration-enriched cells were labelled with fluorescein isothiocyanate (FITC) and were injected into the tail veins of lethally irradiated mice . Cell suspensions of the thymuses from these experimental mice had 8.1 +/- 1.8% fluorescing cells compared to control mice given equal numbers of non-migration-enriched FITC labelled cells which had 2.4 +/- 1.7% positive cells.

Transfusion, 1987 Mar-Apr, 27(2), 125 - 33
Low ionic antiglobulin tests; Ahn JH et al.; Seven serologic procedures were studied to determine their respective value in compatibility and screening tests . All seven were significantly improved by the use of 4 volumes of serum, rather than 1, with 1 volume of red cell suspension, and a low-ionic antiglobin test (LIAGT) was distinctly superior to the other six procedures evaluated . In this test, during the incubation of serum and cells at 37 degrees for 20 minutes, ionic concentration was reduced 62 percent . However, after removal of all supernatant, the red cells were washed three times with an isotonic solution that provided 80 percent reduction in ionic concentration, and the washed cells were tested for their agglutinability with low-ionic (80% ionic reduction) anti-IgG antiglobin reagent . This modified LIAGT was usually more, and apparently never less, sensitive than a test described earlier and is expected to be associated with much less nonspecificity . The extreme sensitivity of LIAGT for many long-term frozen stored alloantiserums is a retained property of the modified test and has been associated with IgG aggregation during storage.

Cell Tissue Kinet, 1987 Mar, 20(2), 171 - 80
Implications of disaggregation procedures on biological representation of human solid tumours; Costa A et al.; This study was designed to define some biological aspects of cell suspensions, obtained by mechanical or enzymatic disaggregations, and to verify whether single cell suspensions are representative of original solid tumours . The study was performed on a series of 25 human solid tumours including breast carcinoma, ovarian carcinoma and malignant melanoma . A higher cell viability and a loss of aneuploid subpopulations, or a lower fraction of aneuploid cells, were observed in enzymatically-released samples than in samples obtained by the mechanical procedure . Moreover, the proliferative activity, which was generally similar for the cell suspensions obtained by the two disaggregation procedures, was always markedly lower in the cell suspensions than in solid samples from the same tumour . In conclusion, the results from this study indicate that many changes, such as selective release of cell populations from the tumour matrix, damage and destruction of aneuploid and proliferating cells can be induced to various extents by different disaggregation procedures.

Arthritis Rheum, 1987 Mar, 30(3), 266 - 74
Modulation of prostaglandin E2 synthesis in rabbit synoviocytes; Rothenberg RJ; Prostaglandin E2 (PGE2) production by rabbit synoviocytes was markedly stimulated by hydroxyapatite only after a 60-minute delay . Release of 3H-arachidonic acid (C20:4) and 3H-PGE2 from cells with phospholipids that were prelabeled with 3H-C20:4 did not occur in parallel . At 240 minutes, phospholipase activity in sonicated cell suspensions had increased only 38%, while cyclooxygenase activity had doubled (109%) . This doubling, as well as the production of synoviocyte PGE2, was prevented by inhibiting the synthesis of protein . Cyclooxygenase is an inducible enzyme, and as such, it is a rate-controlling step in PGE2 production.

Blood, 1987 Mar, 69(3), 727 - 34
Erythrocyte deformation in shear flow: influences of internal viscosity, membrane stiffness, and hematocrit; Kon K et al.; The effect of shear force (depending on shear rate and viscosity of extracellular medium) and hematocrit of RBC suspension on RBC deformation was studied quantitatively using a cone-plate rheoscope with various kinds of cells, ie, partially hemolyzed (PH) cells, density-fractionated intact cells, and diamide-treated cells . The deformation index (DI) of ellipsoidally deformed cells was shown to be a function of beta gamma eta ex(eta ex/eta in)alpha, where gamma eta ex is applied shear stress, eta ex and eta in are external and internal viscosities, respectively, and alpha and beta are adjustable parameters related to the membrane viscoelastic properties . The increase of suspension viscosity at higher hematocrits (Hts) generally enhanced the ellipsoidal deformation of cells, in the same manner as increasing the suspending medium viscosity of a diluted cell suspension . The suppressing effect on cell deformation appeared above a certain Ht . When intact cells were mixed with glutaraldehyde-treated, hardened cells, the ellipsoidal deformation of intact cells was disturbed . The suppression of deformation probably occurred through disturbance of laminar flow-lines around intact cells.

Biochem Biophys Res Commun, 1987 Feb 27, 143(1), 212 - 7
Anomeric specificity of glucose effect on cAMP, fructose 1,6-bisphosphatase, and trehalase in yeast; Toyoda Y et al.; The addition of beta-D-glucose (final concentration, 50 mM) to a cell suspension of Saccharomyces cerevisiae in stationary phase caused a rapid 4-fold increase in the concentration of cAMP, while a 2-fold increase of cAMP was observed by the addition of alpha-D-glucose . beta -D-Glucose was also more effective than alpha-D-glucose in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase . These results, taken together with the previous report that alpha-D-glucose is transported more rapidly than beta-D-glucose in Saccharomyces cerevisiae, do not support the view currently proposed by some investigators that cotransport of D-glucose with protons causes the depolarization of the cell membrane, resulting in the activation of adenylate cyclase . The present data, however, provides supporting evidence for the view that cAMP-dependent protein kinase is implicated in the inactivation of fructose 1,6-bisphosphatase and the activation of trehalase.

J Biol Chem, 1987 Feb 25, 262(6), 2737 - 45
Reassessment of insulin effects on the Vmax and Km values of hexose transport in isolated rat epididymal adipocytes; Toyoda N et al.; Effects of insulin on the kinetic parameters of hexose transport in rat epididymal adipocytes were re-examined . The transport activity was assessed by measuring the rate of uptake of 3-O-{3H}methyl-D-glucose (MeGlc) under equilibrium exchange and zero-trans conditions . The incubation was carried out at 37 degrees C in an infant incubator . During the incubation, the cell suspension (25%, v/v, in a total volume of 48 microliter) was mechanically swirled at a rate of 600 rpm (r = 2 mm) . The swirling facilitated the rapid uptake of MeGlc without stimulating the basal transport activity by "mechanical agitation" . The basal and insulin-treated cells were incubated under identical conditions, except for the length of the incubation period . The incubation was terminated by the addition of 350 microliters of 1 mM phloretin, which inhibited transport in approximately 0.06 s . The time course of MeGlc uptake was consistent with the view that the process was a multiple-phase reaction . The initial phase of the reaction was completed when the intracellular distribution space of MeGlc was approximately 1% of the total cell volume . Insulin (10 nM) increased the Vmax value of MeGlc uptake 16-fold in equilibrium exchange experiments and 18-fold in zero-trans experiments . At the same time, the hormone decreased the Km value of MeGlc uptake from 11.7 to 5.4 mM in equilibrium exchange experiments and from 9.7 to 4.8 mM in zero-trans experiments . It is concluded that the major effect of insulin on MeGlc uptake is to increase the Vmax value, but the hormone has the additional effect of lowering the apparent Km value.

J Immunol Methods, 1987 Feb 11, 96(2), 233 - 7
Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens; Levitt D et al.; Fixation and immunofluorescent staining methods were developed for analyzing intracellular antigens with the cell flow cytometer . Fixing cell suspensions with 100% methanol provided best preservation of morphology, lowest fluorescent background staining and most intense specific immunofluorescence . Immunoglobulins present in B cell lines that were representative of different developmental stages could be distinguished quantitatively . Fluorescence histograms were compared with fluorescence microscope presentation of stained cells . Intracellular antigens that reacted with monoclonal antibodies could also be evaluated by flow cytometry . This method was utilized to assess plasmacyte development in mouse spleen cell cultures after stimulation with lipopolysaccharide.

J Surg Res, 1987 Feb, 42(2), 141 - 6
Adrenergic control of serotonin release from carcinoid tumor cells in vitro and in vivo; Gronstad KO et al.; Serotonin (5-HT)-producing human carcinoid tumors of midgut origin were transplanted to the anterior eye chamber of cyclosporine-treated rats . The release of 5-HT from in oculo transplants was studied after stimulation with adrenoceptor agonists applied locally to the eye . Chamber fluid was collected by micropuncture of the eye . 5-HT levels were determined by liquid chromatography with electrochemical detection . The release of 5-HT in cell suspensions of the same tumors was similarly studied after incubation with adrenoceptor agonists . In both experimental models activation of adrenoceptors caused release of 5-HT from carcinoid tumor cells . Individual variations in the type of response to adrenoceptor stimulation could be demonstrated for the different tumors . In two tumors there was good agreement between in vitro and in vivo findings with release of 5-HT at selective activation of beta-adrenoceptors with isoprenaline . In a third tumor there was a release of 5-HT at activation of alpha-adrenoceptors with norepinephrine in vitro . However, 5-HT release from this tumor in vivo was demonstrated at activation of beta-adrenoceptors . This finding may reflect different adrenoceptor populations on the tumor cell surface, each population activated to various degrees in the in vivo and in vitro situation.

Gan To Kagaku Ryoho, 1987 Feb, 14(2), 547 - 9
{Primary cultures of various differentiated human cells and their transfer (2) . The isolation of target cells from their specimen}; Yamane I; The specimen employed for the culture should be possibly rich of target cells in order to obtain the successful primary culture . When the target cells are located along the inner side of a duct forming tissue, they can be relatively easily isolated as major cell population in their whole dissegregated cells by means digesting the inner wall of ductive tissues . When the specimen supplied for the culture is so small and limited, the explant outgrowth culture method can be better applied for the culture . For the primary cultures of malignant cells, soft agar cell suspension method can be better available for the successful culture.

Exp Cell Res, 1987 Feb, 168(2), 431 - 8
A new method for isolating primary mesenchyme cells of the sea urchin embryo . Panning on wheat germ agglutinin-coated dishes; Ettensohn CA et al.; This paper describes a rapid and efficient way to isolate primary mesenchyme cells (PMCs) of the sea urchin embryo . The procedure involves three simple steps: Dissociation of mesenchyme blastulae in calcium-free artificial seawater . Incubation of the resulting cell suspension on dishes that have been coated with wheat germ agglutinin (WGA), to which the PMCs adhere more firmly than do other cell types . Gentle rinsing of the dishes to remove loosely attached cells, followed by more vigorous rinsing to remove PMCs . This panning procedure has been applied to embryos of three species of sea urchins, Lytechinus variegatus, L . pictus and Arbacia punctulata, and yields populations of PMCs that are 95-99% pure as determined by the proportion of cells that stain with fluorescein isothiocynate (FITC)-WGA and with a monoclonal antibody that binds specifically to PMCs . The yield of PMCs is 4-5 X 10(6) cells/100-mm dish, or 1-2 X 10(7) PMCs/ml of packed embryos . The principal advantages of this procedure are that it can be carried out rapidly and simply, and it yields pure populations of PMCs.

Pathol Res Pract, 1987 Feb, 182(1), 58 - 62
Flow cytometric analysis of the DNA profile of renal cell carcinoma; Ekfors TO et al.; DNA flow cytometry was applied to one hundred consecutive renal cell carcinomas from years 1974-1979 . Single cell suspensions were prepared from paraffin-embedded tissue . From 96 evaluable tumours 34 were considered diploid and 62 aneuploid . Ploidy did not correlate statistically significantly with local growth, venous invasion, nodal or distant metastatic spread, or nuclear grade of renal cell carcinomas . Also the survival of the patients with diploid and aneuploid tumours was not significantly different, but, excluding patients with distant metastases at the time of extrafascial nephrectomy, 83% of the patients with diploid tumour survived for five years, while only 57% of the patients with aneuploid tumour were alive after the same time interval . The difference between survival curves is statistically almost significant (p less than 0.05) . The cytologic nuclear grade predicted the prognosis more significantly (p less than 0.01), and the combination of these methods is recommended for the prognostication of renal cell carcinoma.

Pathol Res Pract, 1987 Feb, 182(1), 48 - 57
Pathology of tumours produced in syngeneic Fischer rats by fibroblast-like cells before and after transfection with oncogenes; Gao J et al.; Fischer rat cells before and after transfection with immortalizing and transforming genes produced tumours after s.c., i.p., or i.v . injection of cell suspensions and after s.c . implantation of cellular aggregates in the tail of syngeneic rats . Tumours were described histologically as fibrosarcoma-like . Virtually all tumours were considered macroscopically to be invasive because they adhered to the neighbouring tissues; in many tumours invasion was confirmed microscopically . All types of cells produced lung colonies (artificial metastases) after i.v . injection . Spontaneous metastases (from a primary tumour) were found with some tumours produced by cells before as well as after transfection . Differences in metastasis between various cell types could not be ascribed to variations in the periods of observation, in the minimum tumour-bearing periods, in the latency periods, or in the volume of primary tumours . We concluded that local invasion and spontaneous metastasis are usefull for the characterization of malignancy in experimental fibrosarcoma-like tumours . Since Fischer rat cells produced invasive and sometimes metastatic tumours before transfection, the present data do not show a role of immortalizing and transforming genes in the acquisition of invasiveness and metastatic capability.

J Appl Physiol, 1987 Feb, 62(2), 791 - 7
An in vitro capillary system for studies on microcirculatory O2 transport; Boland EJ et al.; An in vitro artificial capillary system has been developed for use in examining the O2 transport properties of free hemoglobin and erythrocytes . The artificial capillary was constructed by casting a thin film of transparent silicone rubber around a strand of tungsten wire that was 24 micron in diameter . After the rubber had polymerized, the wire was removed . Typical dimensions of the silicone rubber film were 170 micron thick, 1 cm wide, 5 mm long in the direction of flow, and a 27-micron lumen diameter . The artificial capillary bed was mounted on a microscope and perfused by either hemoglobin solutions or cell suspensions . Fractional saturation was measured as a function of axial position by a dual-wave-length microspectrophotometer, and the flow rate was regulated precisely by a syringe pump . O2 release experiments were carried out by suffusing the gas space surrounding the artificial capillary film with 100% N2 and perfusing with an oxygenated sample . O2 uptake experiments were carried out by suffusing the gas space with O2-N2 mixtures and perfusing with deoxygenated samples . The axial velocities were varied from 3 to 15 mm/s . The residence time (the time a particular red cell or hemoglobin molecule has spent in the capillary) for 50% oxygenation of a 4 mM (heme) deoxyhemoglobin solution was approximately 0.05 s at 37 degrees C when the gas space surrounding the capillary contained air . The corresponding time for 50% oxygenation of an equivalent red cell suspension was approximately 0.25 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Clin Pathol, 1987 Feb, 87(2), 276 - 81
Recognition of hairy cell leukemia in a spleen of normal weight . The contribution of immunohistologic studies; Burke JS et al.; A 68-year-old man with known hairy cell leukemia (HCL) underwent splenectomy; the spleen weighed only 140 g . Microscopic examination of the spleen revealed no clear diagnostic evidence of HCL, but there was an unusual and suspicious subendothelial lymphoid infiltration of the trabecular veins . Cell suspension studies also were not diagnostic because a monoclonal B-cell population could not be defined . Alternatively, when we focused on the subendothelial infiltrate in the frozen sections prepared for immunohistochemical studies, the findings indicated that the subendothelial cells were monoclonal B-cells (IgG, kappa) . Staining with monoclonal antibodies disclosed the phenotype Tac+, Leu-14+, Leu-M5+, B2-, BA-1-, and BA-2-, a phenotype characteristic of HCL . In this case, an unusual but nondiagnostic morphologic finding provided guidance for the correlative immunophenotypic analysis of the same site in frozen sections and thereby allowed a definitive diagnosis.

Exp Mol Pathol, 1987 Feb, 46(1), 40 - 51
Flow cytometric quantification of cholesteryl ester-containing "foam" cells . I . Analysis of aortas from normolipidemic swine; Cupp JE et al.; We have quantified using flow cytometry foam cells of aortas from normolipidemic swine varying in age from 6 months to 12 years . These swine were maintained throughout their lives on a low-fat, cholesterol-free diet . Intimal-medial tissues removed from the swine aortas were enzymatically dissociated to prepare Formalin-fixed cell suspensions . Foam cells were labeled by specific staining of their intracellular cholesteryl ester using the fluorescent dye filipin . This was carried out by first removing cellular unesterified cholesterol with ethanol, then enzymatically hydrolyzing cellular cholesteryl ester, and finally staining with filipin the unesterified cholesterol derived from hydrolysis of cholesteryl ester . Results of flow cytometric analysis indicated that thoracic foam cell densities of female swine became more variable and tended to increase with age . It appeared that there were two subgroups of female swine with either high or low levels of thoracic foam cells . A similar finding was not observed for abdominal foam cells in female swine nor for thoracic or abdominal foam cells in the smaller number of older male swine . Abdominal foam cell densities in younger males, however, also appeared to be comprised of low and high foam cell groups . Foam cell densities did not correlate with serum cholesterol or triglyceride levels . However, a genetic basis for some of the variability in foam cell densities among these animals was suggested by the observation that females with high thoracic foam cell densities had greater commonality among ancestors than did females with low thoracic foam cell densities.

Am J Physiol, 1987 Feb, 252(2 Pt 2), R299 - 305
Age-related changes in circannual rhythms of lymphocyte blastogenic responses in mice; Brock MA; Blastogenic responses to T- and B-lymphocyte mitogens were tested in suspensions of splenocytes from 15- and 24- to 28-mo-old C57BL/6 mice and compared with analogous responses in young animals . The mice were housed under constant environmental conditions with alternating light-dark cycles (LD 12:12) . Single cell suspensions were cultured in vitro with mitogens, and the induced incorporation of tritiated thymidine by dividing cells was determined . Increases in periodicity of responses to concanavalin A and phytohemagglutinin by T cells and to lipopolysaccharide by B cells and lower mean levels of activation characterized rhythms in cells from 15-mo-old and senescent mice compared with young animals . Amplitudes of the rhythms were unchanged at 15 mo, but by 24 mo of age rhythmic responses of T but not B cells were damped . The separable effects of age on expression of circannual rhythms by T and B lymphocytes suggest another mechanism for imbalance in the immune system . Phases of depressed responses that are extended for several months in populations of older mice could provide increased opportunities for environmental assaults.

J Invest Dermatol, 1987 Feb, 88(2), 141 - 4
A model of human melanoma in cyclosporine-immunosuppressed rats; Goodman MM et al.; A human malignant melanoma maintained in athymic nude mice has been successfully implanted and grown in cyclosporine (Cys)-immunosuppressed Lewis rats . Suspended melanoma cells (10(6)) or solid tumor sections measuring 2-4 mm in diameter were implanted s.c . in rats receiving parenteral Cys doses of 15-50 mg/kg each day for 1 week, and 3 times per week thereafter . Eighty-five percent of solid tumor sections implanted in animals receiving 25 mg/kg resulted in tumor growth, whereas no tumors grew from cell suspension injection sites . The average maximum tumor growth rate was 2 cm3/day, with a doubling time of 8 days . Tumors retained pretransplant gross and microscopic morphology, karyotype, and labeling index . Possible advantages of this model over the athymic nude mouse include greater longevity, larger animal and tumor size, and less stringent aseptic environmental requirements . This model may prove useful for further study of the pathophysiology of melanoma and for testing of new antimelanoma therapies.

AJR Am J Roentgenol, 1987 Feb, 148(2), 415 - 7
Effects of MR imaging on murine natural killer cell cytotoxicity; Prasad N et al.; To determine the effect of MR imaging on the immune system, 21 male C57BL/6 X DBA/2 F1 mice were exposed to MR imaging at a field strength of 0.15 T for 2 hr . Another nine mice (controls) were sham exposed for the same amount of time . Mice were sacrificed and their spleens removed 24, 72, and 144 hr after the exposure (MR or sham) . Spleen cell suspensions were passed over nylon wool columns and then used as effector cells in a short-term natural killer cell cytotoxicity assay with 51Cr-labeled YAC-1 cells as target cells . The results showed no evidence of decreased cytotoxicity due to exposure to MR . On the contrary, at all three times after exposure and for all target-to-effector cell ratios, mean cytotoxicity was greater for MR-exposed groups than for sham-exposed groups . The results show that MR exposure has no adverse effect on the immune system, as evidenced by natural killer cell activity.

J Natl Cancer Inst, 1987 Feb, 78(2), 371 - 6
A human colon adenocarcinoma xenograft--radiation response, cellular composition, and tumor disaggregation; West CM et al.; The human colon adenocarcinoma cell line WiDr was xenografted and the tumor characterized . When athymic mice (NCR-nu) were inoculated with 10(6) cells, tumors appeared after 7-14 days with a 93-100% take rate and grew with an initial volume-doubling time of around 6 days . For optimizing the tumor disaggregation method, a comparison was made of two dissociation procedures and of different dissociation times . An enzyme cocktail (collagenase, DNase, pronase) resulted in total viable cell yields of 1-3 X 10(7) cells/g tumor tissue . Cell yield decreased with increasing tumor weight . Disaggregation with trypsin gave lower cell yields; and so, although the plating efficiencies (PEs) were higher, the enzyme cocktail was chosen for tumor disaggregation . On the basis of morphologic identification, cell suspensions prepared from WiDr tumors, by use of the enzyme cocktail for 2 hours, contained 49% malignant cells as well as a significant fraction of nonneoplastic cells . The major nonneoplastic host cell component was macrophage (33%); lymphocytes (13%) and granulocytes (5%) also were present . Host cells could be separated from neoplastic cells by centrifugal elutriation . By mixing various proportions of host and tumor cells, it was subsequently shown that the presence of host cells did not influence the malignant cell PE unless the cell suspensions contained greater than 90% host cells . Single-cell suspensions prepared from WiDr tumors, with use of the enzyme cocktail for 2 hours, were irradiated and then plated for survival (D0 = 1.5 Gy; n = 5) (D0, the 37% dose slope) . A comparison was made of the sensitivity to radiation, after the different dissociation methods . The radiation sensitivities after 1.5-hour trypsinization and 2- and 6-hour enzyme cocktail administrations were similar, but after 0.5 hour of trypsin, the cells were more sensitive to radiation.

J Lipid Res, 1987 Feb, 28(2), 152 - 61
Pantethine inhibits cholesterol and fatty acid syntheses and stimulates carbon dioxide formation in isolated rat hepatocytes; Cighetti G et al.; The effects of pantethine on cholesterol and fatty acid metabolism were investigated in isolated rat hepatocytes . Preincubation of the cells with pantethine induced a concentration-dependent decrease of the radioactivity incorporated into carbon dioxide and lipids in incubations with {2-14C}acetate . When pantethine and the labeled substrate were simultaneously added to the cell suspension, there was an enhancement of carbon dioxide radioactivity at short incubation time (5 min) whereas, at longer incubation time, values were comparable to those of controls; lipid radioactivity, instead, was dramatically reduced by pantethine even at short incubation time and decreased further during the incubation, being 23% of that of controls at 60 min . Analysis of the incubation medium showed that pantethine induced a concentration- and time-dependent release of acetate into the medium . Results of the effect of the acetate concentration on the incorporation of {2-14C}acetate radioactivity into CO2 and lipids in control hepatocytes allowed the conclusion that the above-described modifications induced by pantethine are only partially attributable to the dilution of the labeled substrate, and that catabolism of acetate to carbon dioxide is stimulated by the disulphide pantethine, whereas cholesterol and fatty acid syntheses are inhibited.

Infect Immun, 1987 Feb, 55(2), 490 - 3
Rapid parasite multiplication rate, rather than immunosuppression, causes the death of mice infected with virulent Plasmodium yoelii; Fahey JR et al.; Protective immunity against a lethal malaria challenge infection was passively transferred to naive recipient mice with spleen cells from donor mice bearing a lethal infection with the virulent YM strain of Plasmodium yoelii . Successful transfer of protection was contingent upon the elimination of residual, viable parasites from donor spleen cell suspensions prior to the infusion of cells . Passive transfer experiments failed to detect suppressor cells in the spleens of lethally infected mice because unfractionated spleen cells or T-cell-enriched spleen cells from mice infected with P . yoelii YM did not enhance parasitemias upon infusion into mice infected with cross-reactive nonvirulent P . yoelii 17X . We concluded that a form of protective immunity was generated during the course of virulent infection but that its expression was inconsequential because parasite growth apparently exceeded the capacity of the immune system to clear the infection.

Immunology, 1987 Feb, 60(2), 151 - 7
Allogeneic lymphocyte cytotoxicity (ALC) in rats: establishment of an in vitro assay, and direct evidence that cells with natural killer (NK) activity are involved in ALC; Rolstad B et al.; The evidence that NK cells can recognize and kill allogeneic lymphocytes has hitherto been based mainly on experiments in intact animals . Here we report results from an in vitro assay, showing allogeneic lymphocyte cytotoxicity in cell suspensions enriched for NK activity against tumour cells by Percoll gradient centrifugation of nylon-wool non-adherent cells . The addition of phytohaemagglutinin (PHA) to the NK-target cell cultures greatly enhanced the cytotoxic response against K562 and allogeneic, but not syngeneic, lymphocytes . The effector cells of ALC are present in the spleen of both euthymic and athymic nude rats, and to a lesser extent in the blood . ALC is augmented by interferon pretreatment of the effector cells, and by depleting the effector cell suspensions of all T cells and helper T cells with the monoclonal antibody MRC Ox19 and W3/25, respectively . Conversely, the activity was nearly abolished by depleting the cell suspensions of MRC Ox8+ cells reacting with rat cytotoxic T cells and NK cells . Furthermore, removal of residual B cells (Ox12+ cells) from the effector cells or attempts to block any putative antibody-dependent cellular cytotoxic mechanism in vitro with the monoclonal antibody Ox12 did not inhibit the NK activity against allogeneic lymphocytes nor against tumour cells . ALC in vitro did not discriminate between T and B or large and small lymphocyte targets . These characteristics of the ALC effector cells substantiate that they are present within the thymus-independent population of cells with NK activity, and are dependent on neither B cells nor immunoglobulin for their recognition and destruction of the target.

Exp Mol Pathol, 1987 Feb, 46(1), 52 - 63
Flow cytometric quantification of cholesteryl ester-containing "foam" cells . II . Analysis of aortas from cholesterol-fed swine; Cupp JE et al.; We have examined foam cell accumulation in abdominal and thoracic aortic segments of swine with experimentally induced atherosclerosis . Young (less than 1-year-old) male swine were divided into four groups that were fed control, lard (20%), lard (20%) plus cholesterol (1%), and regression (3 months cholesterol-lard followed by 3 months control) diets for 6 months . Aortas were removed from animals and enzymatically dissociated . Foam cells were detected by specifically staining their cholesteryl ester inclusions with the fluorescent dye filipin . Flow cytometry was used to quantify the number of foam cells in each aortic cell suspension . Cholesterol-lard feeding increased serum cholesterol levels 6-fold and induced a substantial increase in both abdominal and thoracic foam cell densities . Lesion development in cholesterol-lard-fed animals, as assessed by macroscopic evaluation of aortas, correlated with foam cell accumulation as determined by flow cytometry . Accumulation of foam cells was extremely variable from animal to animal and did correlate significantly with serum cholesterol but not with serum triglyceride levels . Interestingly, although serum cholesterol levels increased 1.5-fold in swine fed the lard diet, foam cells did not increase significantly as compared to control animals . Animals placed on a control diet following 3 months of the cholesterol-lard diet showed a trend toward lower foam cell densities as compared to animals fed the cholesterol-lard diet for the entire 6 months . Flow cytometric analysis of filipin-stained aortic foam cells provides a new means to evaluate atherosclerotic lesion development.

Biochim Biophys Acta, 1987 Jan 26, 896(2), 203 - 13
Passive electrical properties of the membrane and cytoplasm of cultured rat basophil leukemia cells . I . Dielectric behavior of cell suspensions in 0.01-500 MHz and its simulation with a single-shell model; Irimajiri A et al.; Frequency dependence of relative permittivity (dielectric constant) and conductivity, or the 'dielectric dispersion', of cultured cells (RBL-1 line) in suspension was measured using a fast impedance analyzer system capable of scanning 92 frequency points over a 10 kHz-500 MHz range within 80 s . Examination of the resulting dispersion curves of an improved reliability revealed that the dispersions consisted of at least two separate components . The low-frequency component (dispersion 1) had a permittivity increment (delta epsilon) of 10(3)-10(4) and a characteristic frequency (fc) at several hundred kHz; for the high-frequency component (dispersion 2), delta epsilon was smaller by a factor of 10(2) and fc = 10-30 MHz . Increments delta epsilon for both components increased with the volume fraction of cell suspension, while fc did not change appreciably as long as the conductivity of suspending medium was fixed . By fitting a model for shelled spheres (the 'single-shell' model) to the data of dispersion 1, the dielectric capacity of the plasma membrane phase (Cm) was estimated to be approx . 1.4 microF/cm2 for the cells in an isotonic medium . However, simulation by this particular shell model failed to reproduce the entire dispersion profile leaving a sizable discrepancy between theory and experiment especially at frequencies above 1 MHz where dispersion 2 took place . This discrepancy could not be filled up even by taking into consideration either the effect of cell size distribution actually determined or that of possible heterogeneity in the intracellular conductivity . The present data strongly indicate the need for a more penetrating model that effectively accounts for the behavior of dispersion 2.

Eur J Pharmacol, 1987 Jan 13, 133(2), 215 - 23
Changes in membrane potential and phosphoinositides during alpha 1-adrenoceptor stimulation in smooth muscle cells of guinea-pig taenia caeci; Nelemans A et al.; The effect of a submaximal concentration of adrenaline (3-5 microM) was studied in taenia caeci smooth muscle cells . Membrane potential hyperpolarization was observed in intact muscle preparations and this response could be separated into two phases, depending on the state of a membrane-bound calcium compartment . The effect of alpha 1-adrenergic stimulation was also measured by {3H}inositol incorporation into phospholipid and inositol phosphate fractions of taenia cell suspensions both in the absence and presence of 2.5 mM extracellular calcium . In the absence of extracellular calcium the inositol phospholipids increased within 15 s after stimulation, followed by enhanced inositol phosphates . With calcium present there was a biphasic increase in the phosphatidylinositol 4,5-bisphosphate (PIP2) fraction with a simultaneous release of inositol phosphates . Lithium ions affected the incorporation of label into the lipids but not into the inositol phosphate fractions . These findings suggest that, in taenia caeci cells, alpha 1-adrenergic-induced membrane hyperpolarization resulting in muscle relaxation is associated with changes in the PIP2 content.

Brain Res, 1987 Jan 6, 400(2), 334 - 47
Restoration of rhythmic slow activity (theta) in the subcortically denervated hippocampus by fetal CNS transplants; Buzsaki G et al.; Solid pieces of the fetal septal region (SG) or hippocampus (HPC) were implanted in a cavity formed by aspiration of the fimbria fornix (FF) and the overlying cingulate cortex on one side in adult rats . In other lesioned animals cell suspensions obtained from the fetal septal area (SS) or the locus coeruleus region of the brainstem (LC) were injected stereotaxically into the deafferented host hippocampus . Six to 9 months after transplantation the animals had chronic recording electrodes implanted into both hippocampi . EEG and unit activity were monitored during running in a wheel, drinking and immobility . Unilateral fimbria-fornix lesions abolished rhythmic slow activity (RSA or theta, theta) in the ipsilateral hippocampus and no recovery was seen up to 9 months later in either the control FF-lesioned animals or in the rats with LC suspension grafts . Recovery of RSA, however, was observed in all animals with solid septal grafts and in some rats with solid HPC grafts . Similar to normal rats, RSA was present only during running and absent during drinking and sitting still . Coherence measurements of RSA between the transplanted and intact hemispheres resulted in high values (0.70-0.95) . Concurrent with RSA, interneurons and granule cells in the host hippocampus fired rhythmically at RSA frequency (6-9 Hz) . The depth profile and the antero-posterior distribution of the power of RSA correlated with the amount and distribution of the graft-induced acetylcholinesterase-positive reinnervation of the host hippocampus . In contrast to the animals with solid septal grafts, placed within the FF lesion cavity, the rats with intrahippocampal septal suspension grafts displayed only short duration bursts of RSA, and mainly during immobility . Based on these findings it is suggested that at least a proportion of the RSA 'pacemaker' cells of the host septum survives the transection of the fimbria-fornix fibers and that a graft of fetal septal or hippocampal tissue implanted into the lesion cavity may be capable of relaying this pacemaker activity to the host hippocampus . This effect may be due to the ability of the grafted tissue to promote the regeneration of new, direct or indirect, septo-hippocampal connections across the lesion cavity.

Eur J Pharmacol, 1987 Jan 6, 133(1), 83 - 8
Chloroquine is a muscarinic antagonist . Binding and dose-response studies with chick embryo cells; Schmidt H et al.; A muscarinic acetylcholine receptor is present on undifferentiated cells of the chick embryo . We show that, in the chick embryo, chloroquine binds to the muscarinic receptor site and behaves as a muscarinic antagonist . In competition studies performed with cell suspensions, chloroquine displaced the specific muscarinic ligand {3H}quinuclidinylbenzilate ({3H}QNB) from the receptor . The dissociation constant (KD) of chloroquine was calculated to be 6.0 X 10(-6) M . In saturation studies performed in chick embryo homogenate, chloroquine shifted the binding curve of {3H}QNB to the right (KD = 4.8 X 10(-6) M) . Dose-response curves were established by measuring the acetylcholine-triggered Ca2+ mobilization in cell suspensions by means of a fluorometric assay with chlorotetracycline . Chloroquine shifted the dose-response curve to the right . The inhibitory constant (KI) of chloroquine calculated from the dose-response curve was 1.5 X 1 10(-5) M . Our observations provide an explanation for the known interference of chloroquine with the muscarinic cholinergic systems in a variety of adult organs.

J Biol Chem, 1987 Jan 5, 262(1), 305 - 11
Erythrocyte anion transport of phosphate analogs; Labotka RJ et al.; The phosphate analogs are a series of chemically related small anions based upon tetrahedrally bonded phosphorus . Each compound is a mono- or disubstituted phosphorus oxyacid . These chemical substitutions lead to differences in the number and acidity of titratable protons, differences in molecular structures and charge distributions, and unique 31P, 19F, or 1H nuclear magnetic resonance spectra for each analog . These compounds include phosphate, phosphite, hypophosphite, fluorophosphate, thiophosphate, methylphosphonate, and dimethylphosphinate . NMR spectra were obtained from human erythrocytes suspended in buffers containing phosphate analogs . Intracellular and extracellular 31P and 19F chemical shifts of these anions were found to be nonequivalent, due to magnetic susceptibility differences between the two compartments, as well as to the transmembrane pH gradient . NMR spectroscopy was used to measure erythrocyte influx rates of the phosphate analogs, as well as the intracellular and extracellular pH changes that accompany influx, in red cell suspensions incubated for selected time intervals . Anion influx rates were found to vary over three orders of magnitude among the phosphate analogs . All analogs showed concentration-dependent influx rate saturation . The major determinant of influx rates was neither the molecular weight of the analog nor the net charge on the anion, but rather the structure of the anion . Phosphite (HPO2-3), the anion most closely resembling bicarbonate (a natural substrate for anion exchange) was found to have the highest influx rate.

Cell Tissue Res, 1987 Jan, 247(1), 227 - 31
Differences in myocyte subpopulations from segments of the thoracic aorta and their modifications with age and hypertension in the rat; Bodin P et al.; Smooth muscle cells obtained from three distinct segments of the thoracic aorta of both Wistar Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) taken at different stages of development were studied in terms of their volume, DNA content in single cell suspensions, and doubling time in primary cultures . The proliferative activity and mean cell volume of myocytes from WKY rats increase along the thoracic aorta in a gradient from the aortic arch to the diaphragm . The slope of this gradient is increased in SHR because of an extension of the area that contains myocytes with low proliferative activity in primary cell culture and large cell volumes in suspension . Tetraploid myocytes are found in both strains and their proportions are larger in SHR than in WKY, specifically after the onset of hypertension . However, they appear to be evenly distributed along the thoracic aorta with a size distribution that is included in that of the diploid cells from the same area . It is suggested that changes in the structural properties of the aortic-cell compartment, associated with maturation and hypertension, reflect quantitative changes in the relative proportions of several myocyte subpopulations within the aorta of the rat.

Am J Vet Res, 1987 Jan, 48(1), 68 - 71
Heparin-induced agglutination of erythrocytes in horses; Moore JN et al.; Heparin was administered subcutaneously 2 times a day for 4 days to 5 horses . An additional group of 5 horses was used as time-matched controls . Significant decreases in PCV, erythrocyte count, and hemoglobin concentration were observed during heparin therapy . The mean corpuscular volume (MCV) of the heparin-treated horses increased to a peak value of 66.1 fl on the last day of treatment . Erythrocyte creatine concentration and glucose 6-phosphate dehydrogenase activity increased moderately during the treatment . These data indicated that the rapid, profound increase in MCV during heparin therapy was not primarily a result of release of large immature erythrocytes from the bone marrow . A second experiment was subsequently performed, using 3 horses . These horses were given heparin 2 times a day, as was done in the first experiment . Saline wet mounts of erythrocyte suspensions were examined once a day for the presence of agglutination . Cell suspensions were examined with or without exposure to a dilute trypsin solution, and erythrocyte counts were done on each suspension, using an electronic cell counter . Agglutination of erythrocytes was evident on the first day of treatment and became more pronounced as treatment progressed . Exposure to trypsin solution reversed the agglutination . The apparent erythrocyte count decreased and MCV increased sharply in the samples processed normally, but there was little change in those suspensions exposed to trypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Stroke, 1987 Jan-Feb, 18(1), 59 - 62
Leukocyte rheology in recent stroke; Ernst E et al.; Eighteen patients with recent ischemic stroke were compared with an equal number of matched controls . Standardized suspensions of red cells as well as of red and white cells were filtered in a new filtration apparatus capable of discriminating between cell deformability and filter occlusion . Results show that red cell deformability, although slightly lower than in controls, is not significantly altered in stroke patients . Filter occlusion, however, was significantly higher in patients when red and white cell suspensions were filtered, but not when red cell suspensions were used, suggesting that white cell filterability is impaired after stroke, which could be due to decreased deformability and/or increased adhesiveness of leukocytes . Slowed white cell passage may also occur in the living microcirculation and may present an obstacle to nutritive flow in exchange vessels, possibly contributing to local ischemia and tissue necrosis after stroke.

Invasion Metastasis, 1987, 7(5), 275 - 83
Lung colonization and metastasis of murine mammary tumors: relationship to various characteristics of the primary tumors; Basolo F et al.; The ability to metastasize via the bloodstream of mammary tumors occurring in Balb/cfC3H and Balb/cfRIII mice (two substrains of identical Balb/c genotype carrying milk-transmitted C3H or RIII murine mammary tumor virus (MuMTV) infection, respectively) has been compared in MuMTV-free Balb/c virgin female recipients given intravenous tumor cell suspensions or subcutaneous solid tumor transplants from mammary tumor-bearing Balb/cfC3H and Balb/cfRIII breeding female donors . Tumor cell suspensions different for MuMTV inducing variant, growth rate, tumor size, and clinical duration, injected intravenously to Balb/c virgin female recipients, have been compared with respect to the foci of lung colonization induced in recipient hosts . The results obtained indicate that MuMTV variant, growth rate and clinical duration of the primary mammary tumor, but not the size of the primary tumor, significantly influence the lung colonization . Similar results were obtained with solid subcutaneous transplants of the same mammary tumors . The significance of these results for the understanding of the general mechanisms of tumor metastases is discussed.

Biorheology, 1987, 24(3), 297 - 309
Intrinsic viscoelasticity of blood cell suspensions: effects of erythrocyte deformability; More RB et al.; The intrinsic viscoelasticity of erythrocyte suspensions holds great potential for specifying the deformability of the individual, noninteracting cells in an oscillatory shear flow field . In order to extrapolate to zero cell concentration, the complex viscoelastic modulus was measured as a function of hematocrit using 2 Hertz oscillatory flow and a shear rate of 10/sec . This was done for both normal cells and cells with severely reduced deformability when hardened with glutaraldehyde . Suspension media were blood plasma, isotonic saline, and Dextran solutions . The real parts of the complex intrinsic visco-elasticities were obtained by an extrapolation using a regression fit to Huggins' equation . For normal cells in native plasma the values ranged from 1.7 to 2, increasing to the range 2.4 to 3.1 when the plasma was diluted with isotonic saline solution . For hardened cells the value obtained was near 3.5 . These results are compared with theories for suspensions of both rigid and deformable particles . Several theories for deformable particles predict an increase in intrinsic viscoelasticity with increases in the ratio of the viscosity of the interior of the particle to that of the suspending medium . This ratio controls the balance between rotational and deformational response of the cell in the flow field . The trends of these theories were observed in the measurements.

Arch Dermatol Res, 1987, 279 Suppl, S111 - 5
Histological study on the fate of haptenated epidermal cells injected intradermally in guinea pigs; Nakagawa S et al.; The percutaneous administration of in vitro haptenated epidermal cells (EC) has become established as a procedure to produce contact sensitivity (CS) in experimental animals for routine use . The cells have also been found to elicit a significant delayed-type skin reaction by intradermal test in the animals sensitized by painting the skin with the hapten . The fate of 2,4-dinitrophenylated (DNP) isogeneic epidermal cell suspensions (EC) injected intradermally was investigated histologically in intact or 2,4-dinitrochlorobenzene (DNCB)-sensitized strain 13 guinea pigs to study the role of the cells in CS . DNP-EC were found to proliferate actively in the dermis and formed EC nests with central keratinization and then elicited inflammatory reaction associated with necrosis of the epidermal structures 7 days after injection in the intact animals . DNP-EC injected intradermally into the animals which had received and reacted against DNCB underwent a suppression of EC proliferation . These findings are discussed in relation to the role of the haptenated EC in CS.

Biorheology, 1987, 24(2), 163 - 71
The apparent viscosity of aggregating and non-aggregating erythrocyte suspensions in the isolated perfused liver; Rogausch H; Vasodilated guinea pig livers were perfused with normal erythrocytes and echinocytes suspended in isoviscous high- and low-molecular-weight dextran solutions . The relative flow resistance of these suspensions and the oxygen uptake of the livers were then determined . The relative flow resistance of the echinocytes that were suspended in high-molecular-weight dextran, however, was significantly higher than that of any other red cell suspension . The oxygen uptake was independent of the perfusion media . It is proposed that high-molecular-weight dextran induces echinocytes to attach to one another, and that this clumping together, and shape-transformation of red cells, hinders their flow in the vasodilated liver.

Leuk Res, 1987, 11(8), 725 - 30
Blood and spleen haematopoiesis in patients with myelofibrosis; Douay L et al.; Blood nucleated cells collected by leukapheresis and spleen cell suspension from patients with myelofibrosis with myeloid metaplasia (MMM) were studied for their haematopoietic capacity . Using committed progenitor cell assays (CFU-GM, BFU-e) and a one-stage long-term liquid stem cell system, we have shown: (1) a preferential expansion of the circulating committed progenitor cell pool above the more primitive stem cell compartment; (2) the absence of any development of a stromal adherent layer in long-term cultures of peripheral blood nucleated cells suggesting the self-sustaining capacity of the circulating primitive stem cells; (3) that the spleen is only a production site of committed progenitor cells but does not generate primitive stem cells; (4) the presence, in the spleen, of stromal progenitor cells . We conclude that the peripheral blood primitive stem cells in patients with MMM are not of splenic origin.

Exp Brain Res, 1987, 67(1), 195 - 215
Spatial learning and memory following fimbria-fornix transection and grafting of fetal septal neurons to the hippocampus; Nilsson OG et al.; The ability of intrahippocampal grafts of fetal septal-diagonal band tissue, rich in developing cholinergic neurons, to ameliorate cognitive impairments induced by bilateral fimbria-fornix transections in rats was examined in three experiments using the Morris water-maze to test different aspects of spatial memory . Experiment 1 . Rats with fimbriafornix lesions received either septal cell suspension grafts or solid septal grafts; normal rats and rats with lesions alone were used as controls . Sixteen weeks after surgery, the rats' spatial learning and memory were tested in the water-maze using a place test, designed to investigate place navigation performance, in which rats learned to escape from the water by swimming to a platform hidden beneath the water's surface . After 5 days of training, the rats were given a spatial probe test in which the platform was removed from the tank to test spatial reference memory . Experiment 2 . The same rats used in Exp . 1 were tested in a delayed-match-to-sample, working memory version of the water-maze task . The platform was located in one of two possible locations during each trial, which was composed of 2 swims . If the rat remembered the location of the platform on the 2nd swim of a trial, it should find the platform more quickly on that swim, and thereby demonstrate working memory . Experiment 3 . Prior to receiving fimbria-fornix lesions, normal rats were trained in a modification of the water-maze task using alternating cue navigation and place navigation trials (i.e., with visible or non-visible escape platforms) . The retention and reacquisition of the place task and the spatial probe test were examined in repeated tests up to 6 months after the lesion and intrahippocampal grafting of septal cell suspensions . The effects of central muscarinic cholinergic receptor blockade with atropine were also tested . Normal rats performed well in both the place and spatial probe tests . In contrast, rats with fimbria-fornix lesions only were unable to acquire or retain spatial information in any test . Instead, these rats adopted a random, non-spatial search strategy, whereby their latencies to find the platform decreased in the place navigation tasks . Sixty to 80% of the rats with septal suspension or solid grafts had recovered place navigation, i.e., the ability to locate the platform site in the tank, in Exp . 1 and 3, and they showed a significantly improved performance in the working memory test in Exp . 2 . Atropine abolished the recovered place navigation in the grafted rats, whereas normal rats were impaired to a lesser extent.(ABSTRACT TRUNCATED AT 400 WORDS)

Comp Biochem Physiol B, 1987, 87(2), 361 - 6
Structure and oxygen equilibrium of the three coelomic cell hemoglobins of the echiuran worm Thalassema mellita (Conn); Vinson CR et al.; 1 . The three coelomic cell hemoglobins from Thalassema mellita have been isolated to purity; the two major components have dimeric structure while the third minor component has monomeric structure . 2 . Acid-urea Triton gel electrophoresis of the isolated hemoglobins identified three polypeptides among the three hemoglobins, one of the dimeric hemoglobins is a heterodimer (pI = 4.9) with one polypeptide sharing identity with the monomeric hemoglobin (pI = 6.3), while the other dimer is a homodimer (pI = 4.5) consisting of the third polypeptide . 3 . SDS gel electrophoresis suggests that the two dimeric hemoglobins have interpolypeptide disulfide bonds . 4 . Coelomic cell suspensions and lysed coelomic cells have PO2 at half saturation (P50) of 2.5-3.0 mmHg and cooperativity values (n) of 1.5-1.93 . 5 . All three isolated hemoglobins have higher oxygen affinities and lower cooperativity values (P50 = 1-2 mmHg, n = 1-1.3) than lysed coelomic cells suggesting some heterotrophic and homotrophic interactions.

Gynecol Obstet Invest, 1987, 23(4), 261 - 6
Increase of progesterone production in human and rat luteal cells by beta-adrenergic stimulation; Horvath EJ et al.; The effects of the beta 2-adrenergic agonist hexoprenaline were studied on the progesterone production of rat and human corpora lutea and compared to hCG-induced hormone production . Human corpora lutea were obtained from healthy patients, rat corpora lutea were harvested on day 6 of pseudopregnancy . Corpora lutea were digested by trypsin and homogeneous luteal cell suspension (6 X 10(5) cells/ml) was incubated for 2 h . Hexoprenaline and hCG were added to the medium and progesterone production was measured by RIA . Hexoprenaline or hCG dose-dependently increased the progesterone production of rat luteal cells and of human cells in mid- and late luteal phase . Moreover, hexoprenaline further increased the hCG-induced hormone production . The stimulatory effect of hexoprenaline could be prevented by propranolol . It is supposed that beta 2-adrenergic stimulation induces an increase in progesterone production of luteal cells and potentiates the effects of gonadotropic hormones.

Oncology, 1987, 44(3), 150 - 5
Human tumor clonogenic assay for carcinoma of the lung . II . Factors that influence colony formation in soft agar; Kanzawa F et al.; The human tumor clonogenic assay (HTCA) has potential value for studies of both the chemosensitivity and biology of human tumors . However, many technical problems including low plating efficiencies and the preparation of sufficient numbers of viable cells remain . In this study, an improved method for disaggregation of solid tumors increased the yield of single cells . Consequently, more than 10 anticancer drugs could be tested in 94 of 168 specimens (56%) . Removal of peripheral blood lymphocytes from cell suspensions derived from effusions also improved colony formation . Adequate growth for sensitivity testing (greater than 30 colonies/plate) was obtained in 122 cases (73%), inadequate growth for drug evaluation (5-29 colonies/plate) in 29 cases (17%), and no colony formation (less than 5 colonies/plate) in 17 cases (10%) of the 168 viable samples . The cloning efficiencies of cells derived from primary tumors (median 0.015%) were higher than those of cells derived from metastatic tumors (0.012%), and they varied with the location of the metastatic site . Cloning efficiencies varied markedly from specimen to specimen, and were unaffected by tumor histology, grade of differentiation, patient age, stage of disease, or prior chemotherapy . The HTCA is promising as a potential tool for studying the biology of tumors.

J Cancer Res Clin Oncol, 1987, 113(4), 400 - 1
A rapid and simple procedure for dissociation of tumor tissue from the human colon; Kemmner W et al.; A rapid procedure for dissociation of colon tumor tissue which combines enzymatic and mechanical methods is described . Using this method a cell suspension almost free of cell aggregates is obtained which is further characterized by high cell yield and excellent cell viability.

Dev Comp Immunol, 1987 Winter, 11(1), 179 - 90
A comparative study of the GVH-R in the chick embryo: effects of various allogeneic cells and various administration routes; Desveaux-Chabrol J et al.; Allogeneic cells inoculated into immunologically immature chick embryos induce a Graft-Versus-Host Reaction (GVH-R), one of the manifestations of which is splenic enlargement . Splenomegaly varies with the composition of allogeneic cell suspensions, route of inoculation and age of recipient embryos at inoculation and autopsy . We have compared the splenomegaly induced by lymphoid cells from spleen or bursa of Fabricius with that induced by peripheral blood lymphocytes, after these cell preparations were either grafted on the chorioallantoic membrane (CAM) at 9 or 13 days or injected intravenously at 13 days . We confirm our previous findings that bursa cells grafted on the 9-day CAM induced splenomegaly as efficiently as other cell preparations . On the other hand, bursa cells did not mediate such an effect after they were injected at 13 days . On the whole, a hierarchy of decreasing efficiency is observed from PBL to spleen and finally to bursal cells . It appears that, as the recipient embryo ages, this hierarchy becomes more marked.

Cancer Immunol Immunother, 1987, 24(3), 272 - 4
Expression of HLA-DR antigens on tumour cells does not contribute to skin reactivity to autologous cholesteryl hemisuccinate (CHS)-treated tumour cells in patients with metastatic melanoma; Munzarova M et al.; Skin tests with autologous cholesteryl hemisuccinate (CHS)-treated and untreated cells were performed in ten metastatic melanoma patients . In the majority of cases evident reaction was noted with CHS-treated cells (9/10) while the reaction with untreated cells was mostly negative (7/10) . Tumour cell suspensions used for skin tests were characterized for reactivity with monoclonal antibody TAL 1B5 detecting the HLA-DR alpha chain . There were no differences between CHS-treated and untreated cells with respect to HLA-DR expression and no correlation was found between grade of skin reaction to CHS-treated cells and the proportion of HLA-DR positive cells in the injected cell sample.

Invasion Metastasis, 1987, 7(1), 30 - 40
Demonstration of site-specific tumour growth by a 50% end point assay; Willmott N et al.; Tumour growth arising after injection of cell suspensions derived from four commonly used experimental tumours has been examined both in lung tissue and at the SC site . From tumour incidence data TD-50 values were computed for growth at the two sites and compared statistically . It was observed that Mc7 and Sp24 cell suspensions grew better in lung tissue than at the SC site, whereas the converse was true for Walker 256 cell suspensions . It would appear that immunogenicity, cell size, capacity to elicit platelet aggregation and immediate arrest patterns in lung tissue are individually inadequate to explain why certain tumour cells exhibit a potential for growth in lung tissue greater or less than at the SC site.

Dev Biol Stand, 1987, 66, 307 - 13
Alternatives for harvesting cells grown on microcarriers: effects on subsequent attachment and growth; Lindskog U et al.; In order to facilitate the subpassaging of cells in microcarrier cultures for scaling up culture volumes, alternative procedures for harvesting cells from microcarriers were investigated . A variety of enzymes including trypsin and dextranase were tested separately and in combination to evaluate cell viability after detachment, recovery, and subsequent attachment and growth of cells following inoculation of the cells in the next microcarrier culture . Treatment of confluent microcarriers with dextranase detached cells efficiently and with high viability and in addition totally digested the dextran-based matrix of Cytodex, thus avoiding the possible need to separate the harvested cells from the microcarriers . Dextranase alone resulted in cells harvested as sheets, but if used in combination with trypsin, a single cell suspension of harvested cells was obtained.

Dev Biol Stand, 1987, 66, 273 - 7
Use of a dynamic filtration method for separation of animal cells; Rebsamen E et al.; For solving the problem of concentrating and washing of viable cell suspensions a dynamic filter device has been evaluated . Results show that concentrating of cells under sterile conditions can be performed . With a laboratory scale filter (Sulzer-Biodruckfilter BDF-01) a 9-fold concentration was achieved, the filtration speed was about 15 l/h . The viability did not change significantly . Details of experiments are given and advantages are discussed.

Exp Lung Res, 1987, 12(4), 311 - 29
Repopulation of denuded tracheas by Clara cells isolated from the lungs of rabbits; Hook GE et al.; An experimental approach was examined to study the growth and differentiation potential of different epithelial cell types isolated from the airways of adult rabbits . Clara cells isolated from the lungs of rabbits and a mixed cell population obtained from rabbit tracheas were injected (separately) into denuded rat tracheas which were then grafted into nude mice . Epithelial cells were obtained from rabbit tracheas by digestion of tracheal epithelium with proteases . The resulting cell suspension contained mucous cells, Claralike cells, ciliated cells, basal cells and a small number of inflammatory and unidentified cells . This mixed cell suspension was inoculated into rat tracheas denuded of their own epithelium which were then grafted subcutaneously onto the backs of nude mice . The tracheas were recovered two weeks later and were prepared for light and electron microscopy . These grafts were found to be lined by a tall columnar pseudostratified mucociliary epithelium, indicating that the rat tracheal grafts transplanted into nude mice supported the growth and differentiation of tracheal epithelial cells . Similarly Clara cells, isolated and purified to 85.9 +/- 2.8% (n = 9) purity from rabbit lungs by a combination of centrifugation and elutriation procedures, were inoculated into rat tracheas which were transplanted into nude mice . Two weeks later the grafts were recovered and prepared for histological and ultrastructural examination . These tracheas were lined with a low cuboidal epithelium reminiscent of bronchiolar epithelium . The epithelium was composed of ciliated cells and Claralike cells rich in smooth endoplasmic reticulum but containing only a few secretory granules . These results indicate that the tracheal graft model is suited to examine the differentiative potential of specific cell types isolated from the airways . Furthermore, they show that Clara cell isolates are able to establish an epithelium resembling bronchiolar epithelium in denuded tracheas while tracheal cells containing at least four different epithelial cell types are able to establish a mucociliary epithelium resembling the pseudostratified tracheal lining.

Arch Virol, 1987, 94(1-2), 169 - 73
Serial passage of murine K-papovavirus in primary cultures of mouse embryo cells . Brief report; Greenlee JE et al.; Murine K-papovavirus was serially passaged 10 times in primary cultures of mouse embryo cells, using cell suspensions and media from infected cultures to inoculate fresh flasks at each passage level . Titers of K virus infectivity in cell suspensions and media rose during serial passage, but viral hemagglutinating activity was not detected in cells or media before the 10th passage . Although the infectivity of K virus for mouse embryo cultures was enhanced by serial passage, lethality of the virus for suckling mice was lost . The present study represents the first successful serial transmission of K virus infection in vitro.

Ann Clin Lab Sci, 1987 Jan-Feb, 17(1), 1 - 7
Lymphomas: membrane markers and cell flow cytometric diagnosis; Glassman AB et al.; Lymphocytes are characterized by membrane markers which, in part, reflect biological and functional activity . This is particularly true for T lymphocyte subsets identified by monoclonal antibodies . The B lymphocytes can be identified but in a more general manner . It has been proposed that the use of these markers will aid in the differential diagnosis of a variety of lymphomas . The objective of this work was to evaluate the use of monoclonal antibodies (MAb-s) and surface immunoglobulin (sIg) analysis in a cell flow cytometer (CFC) as methods to identify and classify lymphomas . The cell flow cytometric findings were then evaluated in light of the histopathologic diagnosis (HPD) . Fifty-eight (58) patients with a variety of lymphomas and benign lymph node disorders were studied . Lymph node tissue samples were obtained after surgical removal and appropriately prepared for evaluation in a CFC . Results showed that the variety of hyperplasias and reactive follicular lymphadenopathies could not be characterized by the technique or application of CFC alone . Both B cell and T cell lymphomas could be recognized and differentiated by MAb-s and/or light chain monotypism using sIg's in a CFC, but morphologic and clinical information were required for diagnostic confirmation . Hodgkin's disease could not be identified by CFC because of the lack of a specific identifiable marker . Cell flow cytometry provides an easy and rapid adjunct to the diagnosis of a variety of lymphomas . At the present time, membrane markers in cell suspensions from tissues (lymph nodes) identified by MAb-s and sIg's in a CFC cannot be used to provide definitive diagnoses for reactive lymphadenopathies, Hodgkin's disease or some classes of lymphomas.

J Neurooncol, 1987, 4(4), 337 - 44
Human non-malignant and malignant brain tumor derived cell cultures: proliferation and sensitivity to natural human fibroblast (beta) interferon; Cook AW et al.; Twenty-one human brain tumor biopsies were processed by mechanical and enzymatic methods to produce mixed cell suspensions . Cultures were prepared in small plastic flasks, and primary outgrowth occurred in 16/21 cultures . The period required for primary outgrowth ranged from 3 days to 14 days . We established serial propagation with 15/16 of the primary cultures . Sensitivity to HuIFN-beta was determined between passages 3 to 12, using a microassay based on cell viability (uptake of a supravital stain, neutral red) . Extracted dye was quantified in acidic-methanol using the MR580 Microelisa Autoreader (Dynatech) . We observed a broad range of responsiveness to the drug among the 12 cell-strains tested . Thus, 4 cell strains were relatively sensitive; 4 were resistant to 10(4) IRU/ml of purified HuIFN-beta . Four cell strains exhibited a level of responsiveness that was intermediate to that of these two groups . During propagation of these biopsies, cytopathology suggestive of paramyxovirus-infection appeared in 4 of the cell-strains . This characteristic was not uniformly associated with high sensitivity to human beta interferon which is a very potent, naturally occurring antiviral substance . Our results support the concept that information concerning sensitivity to HuIFN-beta and other cytostatic agents may be rapidly obtained using microcultures of brain tumor cultures in conjunction with supravital stain uptake studies . Additionally, these results suggest that further clinical studies with beta interferon should be undertaken to define the parameters which determine successful in vivo application.

Histochemistry, 1987, 86(3), 241 - 8
Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs . Standardization of incubation conditions; Ginsbach C et al.; Filipin is used for ultrastructural cytochemical localization of cholesterol in biological membranes . It binds to unesterified 3 beta-hydroxy-sterols forming 25 nm complexes which are readily recognized in freeze-fracture replicas . Since most investigations with filipin have been performed in isolated cells (tissue culture, cell suspensions etc.) we have investigated the conditions for reproducible labeling of cholesterol in membranes of parenchymatous organs . Vibratome sections of rat kidney fixed by glutaraldehyde perfusion were incubated in filipin and freeze-fracture replicas were prepared using standard techniques . The concentration of filipin, the thickness of vibratome sections and the incubation time and temperature were varied over a wide range . Optimal results were obtained with 50 micron thick tissue slices incubated in 400 micrograms/ml of filipin for 46 h at room temperature . Under these conditions lysosomes were consistently labeled while mitochondria and the endoplasmatic reticulum were negative . Peroxisomes showed a little or no labeling at all while the nuclear envelope was heavily labeled in some cells being negative in others . The method described here should be useful in investigation of the role of cholesterol in function of biological membranes in parenchymatous organs and compact tissues.

Cancer Detect Prev, 1987, 10(5-6), 347 - 52
Plasma CEA in large bowel carcinoma: which patients should be followed by regular postoperative measurements? Preliminary follow-up results in 100 patients with different tumor DNA-ploidy patterns; Rognum TO et al.; In 100 patients with large bowel carcinomas, the tumors were divided into a distinctly aneuploid (AN) group (63) and a near diploid (ND) group (37) by flow cytometric (FCM) DNA quantitation of cell suspensions . Preoperative plasma CEA levels were determined in all patients . Thirty-eight patients with AN and 28 patients with ND tumors were operated on for cure and had normal plasma CEA levels postoperatively . These two groups had regular CEA plasma measurements as part of the clinical follow-up . In the AN group, 12 of 15 patients have had recurrence preceded by CEA elevation . In the ND group, however, only one of eight recurrences was preceded by a rise in CEA level; the one with elevation also had increased plasma CEA prior to operation . It thus seems that a low CEA output of ND tumors explains many of the "false-negative" CEA measurements in disseminated cases . It is concluded that, in addition to patients with an elevated preoperative plasma CEA level, all patients with aneuploid tumors should be subjected to repeated plasma CEA measurements as part of the follow-up program.

Microvasc Res, 1987 Jan, 33(1), 1 - 14
The development of a filtration system for evaluating flow characteristics of erythrocytes; Acquaye C et al.; A complete description of the pathophysiology of sickle cell disease requires a physiologically meaningful measurement of red cell deformability . We have designed and built a system which allows one to determine filtration characteristics of erythrocytes . A dilute red cell suspension is forced through a 3.0-micron polycarbonate Nuclepore membrane with a constant positive pressure of 20 mm Hg . Under these conditions blockage of the pores in the polycarbonate membrane is insignificant and flow is linear . We use the relative number of cells filtered through the membrane as a means of approximating the means deformability of cells in the suspension . Using this system we have compared erythrocytes from various mammals and shown that our technique is sensitive in detecting not only differences in cell deformabilities between mammalian species but also changes in cell deformability of human red cells due to exchange transfusion and application of drugs . There was a positive correlation between cell filtrability and percentage cell recovery (coefficient of correlation, 0.65) and a negative correlation between cell size and filtrability (coefficient of correlation, -0.61) . The filtrabilities of normal volunteers and sickle cell disease patients were found to be 71.8 +/- 6.6 and 53.6 +/- 5.0%, respectively . This system is sensitive and reliable, and should be useful in evaluating both the contribution of filtrability to the viability of red cells in vivo and potential therapeutic agents for sickle cell disease.

Int J Microcirc Clin Exp, 1987, 5(4), 335 - 45
Hematocrit fluctuations within capillary tubes and estimation of Fåhraeus effect; Secomb TW et al.; Experimental and theoretical approaches were used to study hematocrit fluctuations in blood flowing along a uniform microvessel . In the experimental studies, human blood cell suspensions were passed along glass tubes with inside diameters 9.8 micron to 16.8 micron . A characteristic pattern of hematocrit fluctuation was observed in the neighborhood of white blood cells, the cell being preceded by a 'plasma gap' with reduced hematocrit and followed by a 'train' of increased hematocrit . The passage times of trains and plasma gaps and the hematocrits within the plasma gaps were determined by microphotometry . From these data, train hematocrits were deduced, expressed as equivalent discharge hematocrits . They ranged from the feed hematocrit to a value of more than 0.8 and were found to vary inversely with white cell velocity at a given flow rate . A theoretical model was developed which relates train formation to the Fahraeus effect . The Fahraeus effect is the reduction of tube hematocrit (HT) below discharge hematocrit (HD) which occurs in capillary tubes because the mean velocity of the red blood cells (VRBC) is higher than the mean bulk flow velocity (VB) . The ratio of these velocities decreased with increasing hematocrit, and it is shown that train hematocrit is sensitive to this hematocrit-dependence . Increased hematocrit in trains behind slowly moving white cells is associated with reduced red cell velocity in the trains . From the dependence of train hematocrit on white cell velocity, the variation of Fahraeus effect with hematocrit was deduced . The results were shown to be consistent with a model for the Fahraeus effect in which VRBC/VB varies linearly with discharge hematocrit HD . In addition, the Fahraeus effect was found to be approximately independent of vessel diameter over the range examined.

Leuk Res, 1987, 11(5), 475 - 80
Immunohistochemical classification of acute leukemias using peripheral blood smears; Vago JF et al.; Immunophenotypic classification of the acute leukemias (AcL) is of well documented value in those of lymphoid or uncertain origin and of increasing importance in those of nonlymphoid origin . Most of these studies have been performed on viable cell suspensions . To study the efficacy of a simpler immunohistochemical approach to the classification of the acute leukemias requiring only peripheral blood smears, 15 AcL (including three CGL-BC) were studied using an immunoalkaline phosphatase method and a panel of anti-lymphoid and anti-myeloid monoclonal antibodies . Routine cytochemistries were also performed (Sudan black, PAS) . Using immunohistochemistry, five cases marked as common ALL (four were undifferentiated by cytochemistry, one ALL), eight cases as ANLL (all ANLL by cytochemistry) and two cases marked only with anti-HLA-DR (AUL by cytochemistry) . These results show that immunophenotypic analysis of AUL, ALL and ANLL can be successfully performed even when only air dried peripheral blood smears are available.

Biomed Chromatogr, 1987, 2(3), 91 - 4
Quantitative determination of adriamycin in rat hepatocytes using a volatile extraction buffer, HPLC and fluorescence detection; Mahdadi R et al.; A rapid and sensitive isocratic technique is described for the determination of concentrations of adriamycin and two of its metabolites, adriamycinol and adriamycinone, in freshly isolated rat hepatocytes . The drugs are easily and efficiently extracted from the cells with an organic mixture (chloroform-n-butanol) after proteolytic digestion with trypsin . Mean recoveries from spiked culture medium cell suspension are greater than 96% . The within run and day-to-day coefficients of variation are less than 7.5%.

Immunopharmacol Immunotoxicol, 1987, 9(1), 87 - 100
Enhancement of immune response of murine Peyer's patches by a diet supplemented with yogurt; De Simone C et al.; The first line of defense against pathogens that enter the host by the oral route involves the Peyer's Patches (PP) . For centuries many populations of the mediterranean basin have empirically administered soured milk (yogurt) to prevent and treat diarrhoea and entero-colitis . Recent reports have offered evidence in favour of a possible influence of yogurt on the host's immunocompetence . Scope of the present study was to evaluate the influence of a diet supplemented with yogurt on the PP from BALB/c mice . The results reported here suggest that yogurt feeding potentiates the host's cell-mediated immune response by increasing the percentage of B lymphocytes and the PHA and LPS-induced proliferative responses of PP cell suspensions.

Cancer Immunol Immunother, 1987, 25(3), 161 - 8
Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells; Ames IH et al.; We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells . Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h 51Cr-release cytotoxicity assays with syngeneic NK cells . Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient . Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7 . Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied . NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ) . Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells . We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice . While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.

Nat Immun Cell Growth Regul, 1987, 6(1), 37 - 44
Spontaneous growth of colonies with mature T lineage phenotype from T-cell-depleted bone marrow precursors in a patient with a lymphoproliferative disorder of the large granular lymphocytes; Pistoia V et al.; Bone marrow cells from a patient with pancytopenia and a lymphoproliferative disorder of large granular lymphocytes (LGL) were cultured and tested for their hemopoietic colony-forming potential . Neither erythroid nor granulocyte-macrophage colony formation could be obtained from unfractionated, or LGL-depleted bone marrow cell preparations . However, a spontaneous growth of lymphoid colonies was observed after culturing LGL-depleted (T3-) bone marrow cell suspensions for 25 days . Pooled colonies expanded with recombinant interleukin-2 yielded a population composed predominantly of mature T cells (T3+, Leu 6-) . These findings suggest that some (T3-) T cell precursors may mature in the bone marrow and that, in our patient, LGL may have exerted a suppressor effect on this maturational process.

Blood, 1987 Jan, 69(1), 173 - 9
Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics; Jackson CW et al.; We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions . Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients . The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added . Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma . Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes . All maturation stages of morphologically recognizable megakaryocytes were aggregated . The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated) . Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin . A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions . The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population . We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics . This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.

Arch Dermatol Res, 1987, 279(4), 236 - 40
Delayed-type skin reaction to 2,4-dinitrophenylated epidermal cells in guinea pigs with contact sensitivity to 2,4-dinitrochlorobenzene; Nakagawa S et al.; Contact sensitivity (CS) induced by hapten has been thought to be analogous to delayed-type hypersensitivity, such as the Mantoux reaction, because of outstanding similarities between the two phenomena . It can be suggested that animals with CS respond also to intradermal injection of the conjugate of hapten and protein as well as to epicutaneous application of hapten . However, evidence against this has been reported . In the present experiments, delayed-type skin reaction (DSR) was successfully obtained in JY1 strain guinea pigs sensitized by painting the skin with 2,4-dinitrochlorobenzene using in vitro dinitrophenylated epidermal cell suspension (DNP-EC) as antigen for a delayed intradermal test . The experiment using anti-Ia alloantiserum and complement showed that the elicitation of DSR is due to the presence of Ia-positive cells (presumably Langerhans cells) among DNP-ECs . The delayed intradermal test with the conjugates such as haptenated ECs in the animals with CS is considered to be an experimentally useful way of analysing the antigen in the sensitivity.

J Recept Res, 1987, 7(5), 667 - 78
Glucocorticoid receptors in murine erythroleukaemic cells; Hammond KD et al.; Glucocorticoid receptors in murine erythroleukaemic cells were studied in relation to hexamethylene bisacetamide (HMBA) induced differentiation . Specific binding of dexamethasone was measured . A single class of saturable, high affinity binding sites was demonstrated in intact cells; with cell homogenates or fractions binding was low and could not be reliably quantified . Receptor binding in whole cell suspensions was lower in cells which had been treated with HMBA (36.5 +/- 8.2 pmol/g protein) than in untreated controls (87.9 +/- 23.6 pmol/g protein); dissociation constants were similar in treated (2.7 nM) and untreated cells (2.5 nM) . Dexamethasone, hydrocortisone, corticosterone and progesterone competed with tritium-labelled dexamethasone for receptor binding sites; cortisone, deoxycorticosterone and oestradiol had little effect.

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