Cell suspensions

Eur J Cancer Clin Oncol, 1989 May, 25(5), 777 - 83
Phenotyping of 76 human bladder tumors with a panel of monoclonal antibodies: correlation between pathology, surface immunofluorescence and DNA content; Hijazi A et al.; Phenotyping of 76 bladder tumors (11 grade I, 33 grade II and 32 grade III) has been carried out by flow cytometry on cell suspensions with simultaneous determination of DNA content and surface immunofluorescence using G4 and 5 new monoclonal antibodies (10D1, 7C12, 6D1, 3C6 and 12F6) directed against bladder tumor cells . Ten normal bladder samples were used as control . Antibodies 6D1 and 12F6 were specific for tumor cells whereas the others also labelled umbrella cells . Cells from grade I tumors were labelled with 10D1, 6D1, 7C12 and 12F6 antibodies, and cells of grade II tumors with 7C12 and to a lesser degree with 12F6 but not with 10D1 and 6D1 . Grade III tumor cells were specifically labelled with antibodies 3C6 and G4 . Reactivity of antibodies with tissue sections was well correlated with cytometry results, except for the antibody 3C6 . Finally, most of the cells stained by 3C6 and G4 were shown to have a DNA index greater than 1.0 . In conclusion cells of low grade tumors can be identified with 10D1 and 6D1 antibodies, and antigens recognized by 3C6 and G4 antibodies are mostly expressed by aneuploid cells.

Neurochem Res, 1989 May, 14(5), 391 - 7
Na+, K+-ATPase activity in cultured C6 glioma cells; Folbergrova J et al.; Rat C6 glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na+, K+-ATPase activity was determined in homogenates of harvested cells . Approximately 50% of enzyme activity was attained at 1.5 mM K+ and the maximum (2.76 +/- 0.13 mumol Pi/h/mg protein) at 5 mM K+ . The specific activity of Na+, K+-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay . Ten minutes' exposure of glioma cells to 10(-4) or 10(-5) M noradrenaline (NA) remained without any effect on NA+, K+-ATPase activity . Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity . The nonresponsiveness of Na+, K+-ATPase of C6 glioma cells to NA is consistent with the assumption that alpha (+) form of the enzyme may be preferentially sensitive to noradrenaline . Na+, K+-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2 x 10(-7) M concentration . In spite of the fact that Na+, K+-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme . Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na+, K+-ATPase may contribute to the previously reported inhibition by vanadate of Na+, K+-ATPase of the whole brain tissue.

Immunology, 1989 May, 67(1), 96 - 102
Immunoregulatory properties of bone marrow-derived cells in the iris and ciliary body; Williamson JS et al.; Iris and ciliary body of mouse eyes have been examined for the presence of bone marrow-derived cells possessing the capability of functioning as antigen-presenting cells (APC) . We have determined that iris and ciliary body contain significant numbers of cells bearing T200, indicating their bone marrow origin . Most of these express the F4/80 marker typically found on mature macrophages . However, approximately one-third of the cells express Ia and a similar number express Mac-1 markers . Virtually none of the cells express Thy-1 or surface immunoglobulin . Whole preparations of excised iris/ciliary body, or single cell suspensions prepared from these tissues were then assayed for their capacity to induce proliferation among allogeneic lymphocytes . It was discovered that iris/ciliary body tissues or cells did not function as alloantigen-presenting cells, although tissue and cells derived from the corneal limbus were allostimulatory . In addition, iris/ciliary body tissues and cells displayed the ability to suppress mixed lymphocyte reactions to which they had been added as regulatory cells . We conclude that normal iris and ciliary body contain bone marrow-derived cells that fail to function as alloantigen-presenting cells . However, cells were present that have the capacity to inhibit alloimmune lymphocyte proliferation . The strategic location of inhibitory cells in the tissues that line the anterior chamber of the eye raises the possibility that these cells may play a role in the phenomenon of immunological privilege that is characteristic of this site.

Eur J Immunol, 1989 May, 19(5), 955 - 8
Emigration of B cells from chicken bursa of Fabricius; Lassila O; The extent of emigration of cells from the bursa of Fabricius to the periphery was estimated . Per anum application of fluorescein isothiocyanate to label bursal cells in situ was used . Migrant cells can be visualized on frozen sections or cell suspensions of peripheral organs by their fluorescence . The data show that at 2-3 weeks after hatching about 5% of bursal cells leave the bursa per day . Since the bursal cells divide rapidly, this indicates that the vast majority (95%) of bursal cells die in situ . Cells that leave the bursa are surface IgM positive and go first to peripheral blood and later into B cell areas of spleen, thymus and cecal tonsils . The results are also discussed on the basis of their implication for the generation of antibody diversity in the chicken bursa.

Am J Med Sci, 1989 May, 297(5), 314 - 20
Endothelial mediation is necessary for subsequent hepatocyte uptake of transferrin; Soda R et al.; The authors previously have reported on the presence of transferrin (TF) receptors on liver endothelial cells and have shown that hepatic uptake of transferrin-iron (TF-Fe) complexes in the liver is mediated by the endothelium . We now provide evidence that this endothelial cell mediation may be necessary for hepatocyte uptake of TF-Fe complexes . Transport of TF-Fe from endothelial cell to hepatocyte was studied in mixed cell suspensions in which radiolabeled TF-Fe complexes were incubated at 37 degrees C with the two cell populations purified and then mixed in equal ratios . The mixtures were then refractionated at various times after incubation and cell-associated radioactivities measured . Radiolabeled TF was rapidly taken up by the endothelial cell fraction, but radioactivity began to decline in this fraction as it increased in the hepatocyte fraction . In double labeling experiments with 125I-TF-59Fe, both radiolabels moved across the endothelium in parallel fashion, indicating that Fe remains associated with TF during transcytosis . However, in hepatocytes the two radiolabels became dissociated, with Fe remaining cell-associated and TF being recycled . Hepatocyte uptake of processed TF was partially inhibitable by galactan and asialofetuin, indicating that hepatocyte uptake may occur via asialoglycoprotein receptors of hepatocyte.

J Biol Chem, 1989 Apr 15, 264(11), 6438 - 46
Association of glyceraldehyde-3-phosphate dehydrogenase with the plasma membrane of the intact human red blood cell; Rogalski AA et al.; Glycolytic enzymes have been observed to associate in vitro with membranes and cytoplasmic filaments in a variety of systems, but their distribution in vivo is contested . We have therefore examined the distribution of glyceraldehyde-3-phosphate dehydrogenase (G3PD) in the intact human erythrocyte using indirect immunofluorescence and affinity-purified rabbit antibodies to G3PD . Antibody specificity was demonstrated by immunoblotting as well as immunofluorescence experiments with ghosts specifically depleted of and reconstituted with G3PD . Anti-G3PD immunolabeling experiments utilized both fixed whole cells and fixed cell suspensions infused with 2.3 M sucrose, frozen and thick-sectioned . In all experiments a two-step fixation protocol was employed which ensured that cytoplasmic hemoglobin was retained when cells were subjected to Triton X-100 permeabilization, the anti-genicity of G3PD was preserved, and antibody penetration was complete . We used mixtures of biotinylated affinity-purified antibodies to G3PD and dichlorotriazinylaminofluorescein-labeled, affinity-purified antibodies to hemoglobin, followed by rhodamine-streptavidin, in double-label experiments . In both whole and sectioned human erythrocytes, G3PD staining was predominantly membrane associated while hemoglobin staining was diffusely distributed throughout the cytoplasm . In isolated ghosts, some G3PD was tightly bound to the membrane and was resistant to elution with phosphate-buffered saline and NAD+/arsenate . However, in immunolabeled rat reticulocytes and erythrocytes G3PD was cytoplasmic . Nucleated human blood cells and platelets also exhibited cytoplasmic G3PD . In approximately 10% of the human erythrocyte population G3PD was also cytoplasmic . These cells were flatter in shape and exhibited strong cytoplasmic immunolabeling for hemoglobin which was sometimes concentrated along the cell membrane; possibly, these cells were late reticulocytes or early erythrocytes . We conclude that G3PD is preferentially associated with the plasma membrane of human erythrocytes in a specific fashion.

Experientia, 1989 Apr 15, 45(4), 363 - 5
Regular oscillations in suspensions of a putatively chaotic mutant of Dictyostelium discoideum; Goldbeter A et al.; We have tested the light-scattering properties of suspensions of the Dictyostelium discoideum mutant HH201 derived from the mutant Fr17 . Previous studies indicated that HH201 and Fr17 possess highly irregular rhythmic properties which might represent aperiodic oscillations, i.e . chaos . We report that the former mutant can display regular oscillatory behavior . Possible explanations for this result are discussed, including that of a transition from chaotic to periodic behavior resulting from some parameter change or from strong intercellular coupling in cell suspensions.

J Urol, 1989 Apr, 141(4), 965 - 8
Cytotoxicity of high energy shock waves: methodologic considerations; Laudone VP et al.; In vivo and in vitro experimentation with high energy shock waves (HESW) is necessary to further our understanding of the biologic effects and potential application of this novel energy form . Factors are identified which are critical to the design and subsequent interpretation of HESW experimentation . First, the nature of the containment vessel and the presence or absence of acoustic interfaces are shown to significantly alter the outcome of cell suspension experiments . Second, the effects of HESW are shown to differ markedly for cells in suspension versus cells in tissue making comparisons between the two uncertain . Finally, the need for appropriate negative controls is demonstrated with in vivo experiments to control for the generalized toxicity which occurs when small animals are exposed to such an intense force distributed over a relatively large area . These findings affect the interpretation of previously reported work which investigated the cytotoxic potential of HESW.

Zhonghua Nei Ke Za Zhi, 1989 Apr, 28(4), 222 - 5, 252
{Correlation study of in vitro tests with clinical response to ALG therapy in patients with severe aplastic anemia}; Ji SQ et al.; Fourteen patients with severe aplastic anemia were treated with antilymphocyte globulin (ALG) . Eight were studied with co-culture of patient's lymphocytes with normal bone marrow cells . Suppression of CFU-C was prevented by pretreatment of T lymphocytes with anti-T lymphocyte McAb in four patients and concordance with clinical outcome was observed only in two patients . Conclusive in vivo therapy result for this correspondence are lacking . Seven patients received fetal liver cell suspension infusion 24-36 hours after completing ALG therapy and remission were "more complete" in three cases with good response . Response of treatment in the fourteen patients was as follows: eight had complete or partial responses and the remaining did not respond or died (42.8%).

Rev Fr Transfus Hemobiol, 1989 Apr, 32(2), 135 - 40
{HLA class II typing of peripheral blood B lymphocytes separated using monoclonal antibodies}; Lepage V et al.; The different methods proposed for preparation of B lymphocyte suspensions are not always simple, rapid and reliable . Presence of monocytes in the B cell suspension makes difficult HLA-DQ typing . For these reasons we propose a method using monoclonal antibodies (McAb) and complement to lyse the non B cells . Mononuclear cells, isolated from peripheral blood by Ficoll-Hypaque, are suspended in a mixture of anti-CD2, CD3, CD11b (OKM1 recognizing monocytes and granulocytes) McAb . This method of preparation of B cell suspension is rapid and provide better typing reactions than did the suspensions prepared by depletion of sheep red blood cell rosetting cells.

No To Shinkei, 1989 Apr, 41(4), 383 - 90
{Cell kinetic analysis of human brain tumors by bivariate flow cytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine}; Okuda Y et al.; Cell kinetics of 91 human brain tumors obtained from 88 patients were analyzed with the following two methods, 1) bivariate (two-color) flow cytometric measurement of cellular DNA content and amount of bromodeoxyuridine (BrdU) incorporated into cellular DNA, in 66 specimens, 2) immunohistochemical detection of BrdU incorporated S-phase cells, in 34 specimens . Patients were given an intravenous 1 hour infusion of 200 mg/sq . m . of BrdU 1-2 hours before the surgical removal . The excised tumor specimen was divided into several portions . One was fixed with 70% ethanol and embedded in paraffin, and another was digested mechanically and/or chemically to obtain a single cell suspension, and fixed in 70% ethanol . Paraffin-embedded tissue sections were stained by the peroxidase-antiperoxidase immunohistochemical method using anti-BrdU monoclonal antibody (MoAb) . Single cell suspensions were reacted with fluorescein isothiocyanate (FITC) conjugated anti-BrdU MoAb, or anti-BrdU MoAb and FITC-conjugated second antibody successively by the staining with propidium iodide, for flow cytometry (FCM) . Rates of S-phase fraction in single cell suspensions calculated by bivariate FCM were correlated well with labeling indexes (LI, i.e . the percentage of BrdU incorporated cells) calculated in tissue sections, but not with the result of analysis of DNA histogram by Dean's method . This discrepancy is probably due to large coefficient value in several samples . Histological malignancy of the tumors was reflected both in the proliferating index (PI, i.e . % S+G2M phase) calculated by bivariate FCM and the LI by immunohistochemical method . PI tended to be high in primitive neuroectodermal tumors and metastatic carcinomas, moderately high in gliomas, and low in benign tumor groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Br J Pharmacol, 1989 Apr, 96(4), 773 - 8
Na+ -K+ pump activity in rat peritoneal mast cells: inhibition by extracellular calcium; Knudsen T et al.; 1 . Pure populations of rat peritoneal mast cells were used to study cellular potassium uptake . The radioactive potassium analogue, 86rubidium, was used as a tracer for potassium for measurements of the activity of the cellular potassium uptake process . 2 . The ouabain-sensitive and the ouabain-resistant potassium (86rubidium) uptake of mast cells incubated in the presence of calcium, 1 mmol l-1, were very low, 52 and 147 pmol per 10(6) cells min-1 . 3 . Calcium-deprivation of the cells uncovered a large capacity ouabain-sensitive potassium (86rubidium) uptake mechanism . The activity of the uptake mechanism was decreased by reintroduction of calcium into the cell suspension, and it was dependent on cellular energy metabolism, temperature and pH . 4 . The potassium (86rubidium) uptake of mast cells incubated in a calcium-free medium occurs through an active and ouabain-sensitive mechanism that has the nature of an enzyme, and it is mediated by the Na+ -K+ pump located in the plasma membrane . It is demonstrated that the activity of the Na+ -K+ pump mechanism is inhibited by low concentrations of extracellular calcium (0.1-1.2 mmol l-1) . The possibility is discussed that calcium-deprivation may increase the pump activity by increasing the permeability of the plasma membrane for Na+.

J Bone Miner Res, 1989 Apr, 4(2), 259 - 68
Bone resorption by isolated human osteoclasts in vitro: effects of calcitonin; Murrills RJ et al.; Human osteoclasts were isolated from 12- to 17-week-old fetal tissue and from transiliac crest bone biopsies for an in vitro study of their biology . A hypodermic needle was used to flush either the fetal long bones or the trabeculae of the iliac crest bone biopsy with tissue culture medium and the resulting cell suspension sedimented briefly either onto the surface of plastic tissue culture dishes, for time-lapse microcinematography, or onto slices of devitalized bovine cortical bone for quantitative assay of bone resorption . The osteoclasts were motile, tartrate-resistant acid phosphatase positive and capable of excavating pits in slices of devitalized bovine cortical bone . Human calcitonin, at doses of 1 ng/ml and 1 microgram/ml, caused a 70% inhibition of bone resorption by human fetal osteoclasts over a 24 h period but had no apparent effect on the morphology or motility of either fetal or adult osteoclasts.

Int J Artif Organs, 1989 Apr, 12(4), 270 - 5
Endothelial cell seeding on PTFE vascular prostheses using a standardized seeding technique; Gerlach J et al.; A standardized method was developed for seeding endothelial cells (EC) in tubular vascular grafts . A rotational cell seeding device for tubular prostheses is presented and parameters influencing the kinetics of cell adhesion (rotation speed, graft diameter, cell suspension level, inoculated cell number) are reported . Seeding EC in 14 mm ID PTFE vascular grafts with rotation rate of 10 rph gave an adhesion rate of 80% in a homogeneous monolayer.

Cell Calcium, 1989 Apr, 10(3), 171 - 80
Measurement of cytoplasmic calcium concentration in cell suspensions: correction for extracellular Fura-2 through use of Mn2+ and probenecid; McDonough PM et al.; 1321N1 astrocytoma cells loaded with Fura-2 were found to continuously transport Fura-2 to the extracellular medium . To correct for extracellular Fura-2 fluorescence a protocol was developed in which Mn2+ was added to duplicate cuvettes of cells to quench extracellular Fura-2 at the beginning and end of the experimental time course . Since the export of Fura-2 was linear with time, two separate quench determinations allowed the amount of fluorescence from extracellular Fura-2 fluorescence to be estimated at every point in the time course and subtracted from the data . The uncorrected and Mn2+-corrected basal cytoplasmic calcium concentrations averaged 153 nM and 72 nM, respectively . The peak intracellular calcium concentrations following muscarinic stimulation with 300 microM carbachol averaged 1159 nM (uncorrected) and 889 nM (Mn2+-corrected) . Probenecid (2.5 mM) was found to block the export of Fura-2 from these cells and did not change the basal calcium concentration or the muscarinic calcium response.

Int J Radiat Oncol Biol Phys, 1989 Apr, 16(4), 1111 - 4
Toxicity of RSU-1069 for KHT cells treated in vivo or in vitro: evidence for a diffusible toxic product; Hill RP et al.; RSU-1069 is a highly effective hypoxic cell cytotoxin in KHT sarcomas treated in vivo . However, relative to the hypoxic cells, the oxic cells in the tumor appear more sensitive to the drug than would have been predicted on the basis of results with CHO (AA8-4) cells treated in vitro with the drug under oxic and hypoxic conditions . To examine possible reasons for this difference, suspensions of KHT cells were prepared from tumors growing in vivo, and treated with RSU-1069 in vitro under oxic or hypoxic conditions . The sensitivity of the KHT cells was similar to that of AA8-4 cells, regardless of whether the cells were obtained from untreated tumors or from tumors given 15 Gy in vivo just prior to the preparation of the cell suspension . We observed, however, that the sensitivity of both AA8-4 cells and KHT cells to drug treatment under hypoxic conditions increased with the density of the cells in the treated suspension . This result suggests the possibility that a diffusible toxic product may be released from cells . Such a product could contribute to the toxicity of the drug for oxic cells in tumors in situ.

Invest Ophthalmol Vis Sci, 1989 Apr, 30(4), 714 - 6
In vitro inhibition of lens epithelial cell growth by continuous wave Nd:YAG laser; Miyake K et al.; Bovine lens epithelial cells were suspended in MEM medium and subjected to continuous wave, low power, pulsed neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation . The temperature of each suspension was maintained at 36 degrees C . Laser applications ranged from 1 to 10 watts and from 100 to 2000 seconds, but the total dose to each of the epithelial cell suspension was 2000 J . Six to thirty-nine percent of the cells were dead immediately after irradiation . Surviving cells, cultured for 15 days, showed decreased attachment and failed to grow . These preliminary results suggest that the Nd:YAG laser may be used during cataract surgery to prevent subsequent lens epithelial cell proliferation and the resulting vision reduction and glare.

J Clin Lab Immunol, 1989 Apr, 28(4), 161 - 8
A cytotoxic monoclonal islet cell surface antibody from the NOD mouse; Pontesilli O et al.; Monoclonal antibody (Mab) 1.93B7 was obtained by fusion of spleen cells from a diabetic NOD mouse with P3X63Ag8.653 myeloma cells and screening for complement mediated lysis of rat insulinoma (RIN) cells . Immunofluorescence studies revealed that this Mab binds to RIN cells but not to the rat pituitary tumour line GH3 . The binding of Mab 1.93B7 to RIN cells was abolished by trypsin but not by neuraminidase treatment of the cells, suggesting that the antigen recognized is a protein . Mab 1.93B7 bound to approximately 30% of mouse (BALB/c) and rat islet cells which had been subjected to trypsin digestion and incubated as a single cell suspension for 12h to allow reexpression of trypsin sensitive antigens . Since Mab 1.93B7 is potentially pathogenic, as suggested by its reactivity to primary islet cells and its complement fixing capacity, we injected it into BALB/c and NOD mice . Cytotoxic activity against RIN cells was detected in the serum of the animals injected with Mab 1.93B7, but the Mab did not exert a diabetogenic action and failed to reverse diabetes when administered at onset in NOD mice . No modification of the course of spleen cell mediated transfer of diabetes in NOD mice was observed when the Mab was administered from the time of spleen cell inoculation to the appearance of glycosuria . The implications of the lack of an effect in vivo of Mab 1.93B7 under the conditions employed are discussed.

Crit Care Clin, 1989 Apr, 5(2), 331 - 52
Induction of tissue injury and altered cardiovascular performance by platelet-activating factor: relevance to multiple systems organ failure; Lefer AM; PAF is a phospholipid formed from the action of phospholipase A2 upon cellular membranes in response to immunologic or hypoxic stimuli . PAF does not exist in its active form as a storage product within cells, but is synthesized rapidly after phospholipase A2 activation . A potent lipid released by multiple cell types in mammalian systems, the emerging perspective is that PAF is a major endogenous mediator influencing the pathogenesis and outcome of ischemia and conditions of circulatory shock . These effects appear to be especially relevant to the syndrome of MSOF during critical illness . All of the major criteria for validation of a shock factor have been fulfilled for PAF . First, PAF has been measured in biological fluid of animals during shock states, although this is not an easy task since PAF is formed in minute amounts and is rapidly metabolized . Nevertheless, combinations of high pressure liquid chromatography (HPLC) and bioassay methods employing washed rabbit platelets have been successfully utilized in this regard . Second, synthetic PAF has been injected into cell suspensions, isolated tissues, and live animals, where it produces most of the effects attributed to endogenous PAF released by immunologic or hypoxic stimuli . These studies have shown that PAF exerts a variety of pathophysiologic actions, including (1) cardiodepression (that is, a negative inotropic effect), (2) reductions in systemic blood pressure, (3) leakage of fluid from the microvasculature, (4) bronchoconstriction, and (5) platelet aggregation . All of these actions of PAF can initiate or exacerbate shock and ischemic injury in multiple organ systems . Third, specific PAF receptor antagonists have been found to markedly attenuate the severity of endotoxic, anaphylactic, hemorrhagic, and traumatic shock, as well as acute myocardial ischemia . In all these conditions, a variety of PAF receptor antagonists (including PAF analogues and structurally dissimilar substances) have improved survival and have retarded pathophysiologic processes believed to be important in causing tissue injury . These processes include lysosomal membrane damage and proteolysis . Moreover PAF receptor antagonists attenuate the release of secondary toxic factors in shock, such as myocardial depressant factor . Thus, administration of specific PAF receptor antagonists early in the course of circulatory shock and organ ischemia may prove to be useful therapeutic agents in a variety of life-threatening disorders . In addition to having direct actions, PAF appears to function as a pivotal agent in a chain of mediators producing tissue injury . Recent evidence suggests that tumor necrosis factors (i.e., cachectin) stim

J Histochem Cytochem, 1989 Apr, 37(4), 509 - 13
Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders; Bardales RH et al.; We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL) . Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane . The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM) . The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases) . The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay . TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.

Int J Radiat Biol, 1989 Apr, 55(4), 705 - 15
A single-shot rapid-mixing device for radiobiological studies with mammalian cells; Hodgkiss RJ et al.; A single-shot rapid-mixing device is described for the rapid addition of solutions of radiation-modifying agents, to cell suspensions, at well-defined times relative to a pulse of radiation . The liquid injection system could be used to initiate or quench a wide range of chemical or biochemical reactions . The rapid-mixing device is based on a syringe driven by a stepper motor and can inject up to 2 cm3 liquid in less than 100 ms . The radiation source, a 4 MV Van de Graaff accelerator, provides an electron beam which is deflected from the beam dump on to the sample in two stages, providing a 10 ms radiation pulse . A digital delay circuit defines the interval between mixing and irradiation . The apparatus has been designed to study the kinetics of processes that occur over a time range extending from about 0.1 s to some minutes . It bridges the gap between the ranges available with conventional fast-mixing and those using standard X- or gamma-irradiation methods . The time resolution of the technique has been examined by following the timecourse of radiosensitization by oxygen in mammalian cells . The timecourse of radioprotection of aerobic mammalian cells by dithiothreitol has been measured using the technique.

Am J Clin Pathol, 1989 Apr, 91(4), 417 - 21
A method for measuring lymphocyte proliferation in mixed lymphocyte cultures using a nuclear proliferation antigen, Ki-67, and flow cytometry; Palutke M et al.; The mixed lymphocyte culture procedure using tritiated thymidine (3H-TdR) incorporation is time consuming and labor intensive, therefore costly . With the use of a fluorescent antibody to a human nuclear proliferation antigen, Ki-67, and flow cytometry, mixed lymphocyte cultures on 20 families of renal and bone marrow transplant patients and normal controls were performed . In this method for measuring lymphocytic proliferation, previously developed by the authors, the entire culture and staining procedures are performed in microculture plates . Finally, the cell suspensions are aspirated with a microsampler to be analyzed by a flow cytometer . Excellent correlation of the percentage of Ki-67-positive cells and the counts per minute (CPM) of 3H-TdR incorporated into the DNA was obtained . This method eliminates the use of radioactive labels, is less time consuming, and yields results two to three days earlier than the radioactive method . In addition, the authors dual-labeled the lymphocyte nuclei with Ki-67 and propidium iodide (Ki-67/PI) . This permitted the comparison of the appearance of nuclear antigen with the various phases of the cell cycle.

Exp Clin Endocrinol, 1989 Apr, 93(1), 61 - 8
Changes of dipeptidyl peptidase (DP IV) activity in the T lymphocytes of rats following administration of ACTH, dexamethasone and opiates; Koranyi L et al.; The aim of the work was to study the effect of glucocorticoids, opiates and stressful stimuli on dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) activity of T lymphocytes prepared from the thymus of intact and adrenalectomized rats . Four week old male rats of Wistar strain were used . The in vivo administration of ACTH, dexamethasone and morphine treatment resulted in an increase of DP IV activity in the cell suspension . In adrenalectomized rats ACTH treatment failed to modify the enzyme activity, however, pain or emotional stress resulted in an elevated DP IV activity . Morphine and D-Met2-Pro5-enkephalinamide resulted in a dose dependent activation of DP IV in T cells, an effect which could be modified by naloxone pretreatment . Our findings show that DP IV mechanisms in T cells are highly sensitive to exogenous and endogenous steroids, opiates and biologically active substances released in response to stress in rats.

J Clin Pathol, 1989 Apr, 42(4), 427 - 31
Cell suspensions from collagenase digestion of bone marrow trephine biopsy specimens; Ades CJ et al.; A technique for the extraction of cells from bone marrow trephine core biopsy specimens using collagenase digestion was assessed in 39 cases (33 diagnostic and six normal) . Diagnostically useful numbers of cells were extracted from all marrows . Morphological assessment of cytocentrifuge preparations of these cells gave a correct diagnosis in 23 (60%) of cases compared with 27 (70%) for the corresponding aspirated marrow smears . Phenotypic analysis using flow cytometry showed persistence of a range of surface membrane antigens following collagenase digestion . Increased autofluorescence was a problem in some cases . Cytochemistry, bone marrow culture, and cytogenetic analysis could also be carried out on these cells . It is concluded that this technique has useful diagnostic applications in cases of dry taps.

Am J Physiol, 1989 Apr, 256(4 Pt 1), G808 - 16
Secretagogue-induced protein phosphorylation and chloride transport in Caco-2 cells; Burnham DB et al.; The effects of vasoactive intestinal polypeptide (VIP), 16,16-dimethyl prostaglandin E2 (DMPGE2) and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on protein phosphorylation were studied in relation to stimulation of chloride transport in cell suspensions of the human colon epithelial cell line Caco-2 . In 36Cl-loaded cells, VIP and DMPGE2 within 1 min decreased cellular chloride content 35-40%, with half-maximal effects being elicited at 1.0 and 85 nM concentration, respectively . A similar effect on chloride content occurred after 10 min of treatment with 0.5 mM DBcAMP . For all three secretagogues, decreases in cellular chloride content were associated with increases in membrane permeability to chloride . DMPGE2 and VIP within 1 min, and DBcAMP within 10 min, increased the phosphorylation of an unidentified soluble protein of Mr = 42,000 and pI = 6.1, and of a protein of Mr = 20,200 and pI = 4.9 identified as myosin regulatory light chain . Between 10 and 30 min of stimulation, however, phosphorylation of the Mr = 42,000 protein and chloride transport activity remained elevated in DMPGE2- and DBcAMP-treated cells, whereas light chain phosphorylation returned to control level . No effect of secretagogues on phosphorylation was detected in the total particulate fraction or an integral membrane protein fraction . It is concluded that increased membrane permeability to chloride induced by cAMP-mediated secretagogues in Caco-2 is temporally associated with the increased phosphorylation of a Mr = 42,000 soluble protein.

Nippon Ika Daigaku Zasshi, 1989 Apr, 56(2), 187 - 95
{Suppressor T cells against delayed type hypersensitivity to Mycobacterium intracellulare}; Kitamura K; Mycobacterium intracellulare Mino strain (Mino) grows progressively in the organs of susceptible mice, such as C57BL/6, C57BL/10 or BALB/c . It is very difficult to induce acquired immunity against M . intracellulare in those susceptible mice . C57BL/6 (B6) mice show no or very weak delayed type hypersensitivity (DTH) to partially purified Mino antigens when sensitized with 10(7) living Mino subcutaneously . B6 mice pretreated with cyclophosphamide (CY) showed enhanced DTH to Mino, suggesting that suppressor mechanism exists in this system . Whether or not such a suppressor mechanism exists in the induction phase of DTH (A) and/or in the expression phase of DTH (B) was examined by cell transfer experiments . Recipients for testing (A) were B6 mice receiving CY 2 days before an immunization, and those for (B) were B6 mice receiving both CY and subcutaneous injections of Mino 3 weeks before the cell transfer . B6 mice which were intravenously injected with Mino a week or 6 weeks before preparing spleen cell suspension, were used as the donors . Single cell suspensions of the spleens were plated on tissue culture dishes and non-adherent cells were harvested after incubating for 60 min at 37 degrees C . These cells were used for cell transfer . In the induction phase experiment, recipient mice received the cells before sensitization, while they received the cells after sensitization in the expression phase experiment . DTH were markedly suppressed in the cell transferred groups as compared with the non-transferred groups . This activity disappeared by treatment of the transferred cells with antibody (alpha Thy-1 Ab) and complement.(ABSTRACT TRUNCATED AT 250 WORDS)

Agents Actions, 1989 Apr, 27(1-2), 93 - 6
Passive sensitization of human intestinal mast cells; Nolte H et al.; Dispersed human intestinal mast cells were used for passive sensitization experiments . Eight biopsies (9.7 +/- 1.2 mg/biopsy) of human duodenum were collected from non-atopic children (5) and adults (5) . The tissue was dispersed mechanically and enzymatically to yield single cell suspensions . The method produced 2 x 10(3) mast cells per mg wet weight of tissue in a purity of 2.8% . Passive sensitization of the mast cells was performed with the patients' own plasma and plasma obtained from atopic donors . The non-atopic mast cells were able to bind the allergen-specific IgE . In addition, passive sensitization with atopic donor-plasma enhanced the cell sensitivity and cell reactivity to anti-IgE challenge, but had no effect on the cellular response to the ionophore A23187 . The study shows that the enzymatic dispersion of human intestinal mast cells produces functionally intact mast cells with preserved Fc-receptors which can be passively sensitized.

Agents Actions, 1989 Apr, 27(1-2), 101 - 3
Histamine release from canine lung and liver mast cells induced by radiographic contrast media; Ennis M et al.; Radiographic contrast media are commonly used diagnostic aids to improve imaging, e.g . in computerized tomography . However, the routine application of these agents may cause adverse allergic/pseudoallergic reactions . In order to understand more completely the underlying mechanisms involved in these reactions, experiments on histamine release both in vivo and in vitro are necessary . Using canine mast cell suspensions from lung and liver, we have investigated the histamine release caused by six commonly used preparations . The dog is an ideal model for both in vitro and in vivo studies not only by virtue of its size but also because of its similarity to man with respect to e.g . cardiovascular reactions after drug-induced histamine release . The two non-ionic preparations (Solutrast, Ultravist) released little histamine from both cell types (ca . 4-6%) . The ionic contrast media (Angiographin, Hexabrix, Telebrix, Rayvist) dose-dependently released histamine from the liver cells and pulmonary cells (maximum release between 18-35%) . The liver cells (the liver is the shock organ in the dog) reacted more strongly to these agents than the pulmonary cells, thus providing further evidence for mast cell heterogeneity and the importance of selecting the appropriate mast cell model for the investigation.

Jpn J Cancer Res, 1989 Apr, 80(4), 341 - 7
Enhanced liver metastatic potential of alpha-fetoprotein-producing human gastric carcinoma after carbon tetrachloride-induced liver damage in nude mice; Sawada H et al.; The liver metastatic potential of alpha-fetoprotein (AFP)-producing human gastric carcinoma (NSC-3) was examined in male, BALB/c, nude mice . Metastatic nodules in the liver were produced by intrasplenic (IS) injection of tumor cell suspension prepared by trypsinization from subcutaneous NSC-3 tumor . The serum AFP level increased exponentially after IS injection along with the growth of metastatic nodules in the liver, and a positive correlation was observed between the estimated weight of metastatic nodules and serum AFP level . To investigate the effect of liver damage by carbon tetrachloride (CCl4) on the metastatic potential of NSC-3 cells injected intrasplenically, the mice were divided into 4 groups: Group 1 received IS injection of 1 x 10(6) of NSC-3 cells without CCl4 treatment; Groups 2, 3 and 4 received IS injection 7 days, 2 days and 1 day after CCl4 treatment, respectively . All mice were killed 64 days after IS injection . The incidence of liver metastasis was 80% in Group 1, but 100% in Groups 2, 3 and 4 . The mean numbers of metastatic nodules per liver were 4.2 in Group 1, 16.8 in Group 2, 18.0 in Group 3 and 44.5 in Group 4 . Significant differences in the mean numbers of metastatic nodules were observed between Group 4 and the other groups . It was clearly demonstrated that the metastatic potential of AFP-producing human gastric carcinoma cells (NSC-3) is enhanced in the situation prevailing after liver parenchymal cells are damaged by CCl4.

Exp Neurol, 1989 Apr, 104(1), 1 - 9
Nimodipine enhances growth and vascularization of neural grafts; Finger S et al.; The potential of the calcium channel antagonist, nimodipine, to enhance vascularization and growth of neural grafts has been investigated . Four groups of rats received unilateral 6-OHDA lesions of the nigrostriatal pathway followed 16-23 days later by intrastriatal grafts of embryonic ventral mesencephalon . The grafts were derived from (i) Embryonic Day 14 embryos (group E14), (ii) Day 17 embryos (group E17), and (iii) Day 20 embryos (group E20), all implanted within 90 min of preparation, or (iv) Day 14 embryos with a 6-h delay prior to implantation (group E14/6H) . Half the rats in each group received intragastric intubation with nimodipine daily for 14 days after transplantation surgery, whereas the other half received control intubations . The rats were killed by perfusion with formalin containing Indian ink 6 weeks after grafting . Nimodipine treatment enhanced the vascularization of the grafts in all four groups and induced a significant increase in the volume of grafts under the three suboptimal transplantation conditions (i.e., groups E17, E20, E14/6H) . The results suggest that vascularization of graft tissue is an important determinant of survival and growth of neural transplants prepared by the dissociated cell suspension technique and show that vascular perfusion of grafts can be enhanced by nimodipine.

Eur J Biochem, 1989 Mar 15, 180(2), 421 - 7
In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri; Santos H et al.; Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR . The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells . Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data . High levels of intracellular ATP up to 4 mM were detected . The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR . In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped . The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis . High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected . However, the methanol consumption rate was not affected.

J Biol Chem, 1989 Mar 15, 264(8), 4324 - 8
Activation of protein kinase C modulates the adenylate cyclase effector system of B-lymphocytes; Wiener E et al.; Antibodies to surface immunoglobulins activate inositol phospholipid hydrolysis in B-lymphocytes, but very little is known concerning their effects on cAMP levels . In other cells, products from the hydrolysis of phosphatidylinositol 4,5-bisphosphate can increase and/or potentiate cAMP accumulation . In this study we have examined whether goat anti-mouse IgM (mu-chain-specific) stimulates and/or potentiates increases in the cAMP levels of splenocytes from athymic nude mice . Goat anti-mouse IgM, by itself, stimulated a 60% increase in cAMP within 2 min . Pretreating the cell suspensions at 37 degrees C with anti-IgM produced opposite effects on the forskolin- and prostaglandin E1 (PGE1)-induced increase in cAMP . Anti-IgM (25 micrograms/ml) potentiated the rise in cAMP induced by 100 microM forskolin 76%, but it decreased the response to 50 nM PGE1 by 30% . Direct activation of protein kinase C (Ca2+/phospholipid-dependent enzyme) by 12-O-tetradecanoylphorbol 13-acetate and/or sn-1,2-dioctanoylglycerol resulted in a similar pattern of responses . A 3-min preincubation with 97 nM 12-O-tetradecanoylphorbol 13-acetate potentiated the forskolin-induced response from 1.7 +/- 0.1 to 4.3 +/- 0.6 pmol of cAMP/10(6) cells but reduced the PGE1 response from 0.98 +/- 0.06 to 0.51 +/- 0.03 pmol of cAMP/10(6) cells . Similarly, preincubating the cells for 3 min with 5 microM sn-1,2-dioctanoylglycerol increased the forskolin response from 1.7 +/- 0.1 to 5.1 +/- 0.2 pmol of cAMP/10(6) cells but reduced the response to PGE1 from 1.15 +/- 0.03 to 0.75 +/- 0.04 pmol of cAMP/10(6) cells . Thus, activation of protein kinase C by hydrolysis products of inositol phospholipids, 12-O-tetradecanoylphorbol 13-acetate, or exogenous diacylglycerols modified adenylate cyclase itself and sites upstream of adenylate cyclase such as the receptor or G proteins coupling the receptor to the cyclase . Furthermore, modification of the PGE1 response by anti-IgM provides a mechanism by which antigen can differentially regulate T- and B-cells responding to macrophage-produced prostaglandins during an immune response.

J Theor Biol, 1989 Mar 7, 137(1), 55 - 69
Magnetic potential and field gradients of a model cell; Mendz GL et al.; The magnetic potential within and outside a nucleated cell placed in a uniform magnetic field is described for a model consisting of two concentric diamagnetic spheres . The analytical description of the magnetic potential in and around a system consisting of a finite number of concentric diamagnetic spheres in a uniform magnetic field was derived . The solution was employed to calculate the field gradients outside and inside a model chicken red blood cell . The form and magnitude of the gradients provide a theoretical basis on which to discuss experimental results relating to the attenuation of signals obtained using proton nuclear magnetic resonance spectroscopy with chicken erythrocyte suspensions . The form of the magnetic field, field difference and field gradients in the external medium of a cell suspension was simulated for a distribution of spheres in a uniform magnetic field, such as might occur in an idealised dilute cell suspension.

Int J Radiat Oncol Biol Phys, 1989 Mar, 16(3), 755 - 61
Misonidazole binding in SCCVII tumors in relation to the tumor blood supply; Olive PL et al.; Misonidazole (MISO) binding was examined in murine squamous cell carcinomas as a function of distance from the tumor blood supply . C3H mice bearing subcutaneous SCCVII tumors were injected intraperitoneally with 3H-MISO followed at various times later by intravenous injection or infusion of the fluorescent perfusion probe, Hoechst 33342 . Tumors were then excised, and single cell suspensions prepared for fluorescence activated cell sorting . Cells sorted on the basis of Hoechst 33342 fluorescence were examined for 3H-MISO content by liquid scintillation counting or autoradiography . MISO content in the 10% of cells most distant from the blood supply was as much as 8 times greater than the amount in the 10% of cells closest to the blood supply . The largest differentials in MISO content between dimly and brightly staining regions were obtained if (a) tumors greater than about 300 mg were used, (b) at least 3 hr were allowed for MISO metabolism and binding prior to analysis, and (c) cell sorting was performed on the basis of concentration of Hoechst (correcting for cell size) rather than on the basis of total fluorescence intensity per cell . As tumors enlarged, MISO content increased even in the cells closest to the blood supply suggesting a decrease in net tumor oxygenation perhaps caused by intermittent hypoxia.

Am J Reprod Immunol, 1989 Mar, 19(3), 92 - 8
Membrane fluidity of trophoblast cells and susceptibility to natural cytotoxicity; Szekeres-Bartho J et al.; This study examines the relationship between membrane lipid microviscosity and susceptibility of villous trophoblast to lysis by natural cytotoxic cells . Trophoblast-enriched cell suspensions prepared from term human placentae were treated with cholesteryl hemisuccinate (CHS)--a modulator of membrane lipid microviscosity . CHS-treated cells were more susceptible targets for natural lymphocyte cytotoxicity than were untreated controls . In binding experiments, increased binding of lymphocytes to CHS-treated target cells was found . Preincubation with progesterone prevented membrane rigidification by CHS . Progesterone, cortisol, and estriol restored the impaired resistance of CHS-treated trophoblast cells to lysis . We determined microviscosity and progesterone concentration in villous surface membranes, prepared from placentae from idiopathic spontaneous abortions and normal first-trimester pregnancies . An inverse relationship was found between progesterone content and microviscosity of the membranes . Microviscosity of the membranes from abortion placentae was significantly higher (P less than .01) and progesterone concentration was significantly lower (P less than .001) than those in the membranes of normal first trimester placentae.

Neuroendocrinology, 1989 Mar, 49(3), 262 - 6
Testicular interstitial cells as targets for peripheral benzodiazepines; Ritta MN et al.; We evaluated the 'in vitro' effect of a selective peripheral benzodiazepine (BZD) receptor agonist, Ro 5-4864, on basal and hCG-stimulated androgen production by testicular interstitial cell suspensions . Ro 5-4864 (10(-9)-10(-5) M) induced a significant increment of basal testosterone release into the medium . In addition, under conditions of hCG stimulation, Ro 5-4864 (10(-7) M) induced a potentiated response to the gonadotropin in a dose-dependent manner . The selective peripheral BZD antagonist PK 11195 fully prevented the stimulatory effect of Ro 5-4864 . On the other hand, clonazepam, a central BZD agonist, failed to affect androgen production significantly, whereas diazepam (10(-5)-10(-4) M), which binds to both central and peripheral BZD receptors, was able to induce a significant increment of basal and hCG-stimulated testosterone production . These results suggest that under our experimental conditions Ro 5-4864 exerts an effect on testicular steroidogenesis, presumably through binding to the previously described peripheral-type BZD receptor.

J Neurosci Methods, 1989 Mar, 27(2), 121 - 32
In vitro and in vivo transplantation of fetal rat brain cells following incubation with various anatomic tracing substances; Stoppinni L et al.; Implantation of fetal brain regional anlage into host brains ('brain transplantation') holds promise as a plausible treatment for certain human neurodegenerative disorders . Improvements in experimental brain transplantation techniques include: (1) utilization of brain cells in tissue culture as opposed to freshly prepared cell suspensions as a transplantation source, (2) prelabeling of fetal brain cells with inert, non-toxic tracer substances to allow subsequent (a) unequivocal identification of those cells as being fetally derived, and (b) anatomical and immunohistochemical identification of transplanted neurons, and (3) development of in vitro models for transplantation to allow physiological studies of connections formed between fetal neurons and host brain tissue . We examined the ability of brain cell suspensions derived from rat fetuses 15-17 gestational days old to accumulate and retain anatomic tracing substances, including Phaseolus vulgaris leucoagglutinin (PHA-L), rhodamine-labeled latex microspheres (RLM) and fluorogold (FG) . All tracers were rapidly accumulated by fetal brain cells, but only PHA-L and RLM were retained following implantation into adult hosts or in tissue culture in vitro . PHA-L-labeled fetal brain cells transplanted in vivo showed morphological characteristics similar to fetal neurons kept in tissue culture in vitro . RLM- or PHA-L-labeled fetal brain cells can be co-cultured with rat brain slices maintained in long-term roller culture . This in vitro system will allow identification and physiological or immunohistochemical study of interactions between fetally derived and host brain neurons.

Vopr Med Khim, 1989 Mar-Apr, 35(2), 121 - 3
{Activity of lysosomal proteinases in the liver, spleen, thymus and peritoneal macrophages after immunization of mice with thymus-dependent and thymus-independent antigens}; Vasil'ev AV et al.; Mice of the CBAxC97B/6 strain were immunized with sheep erythrocytes at a dose of 0.5 ml containing 5% and 25% of the cell suspension and with Vi-antigen at doses of 2 micrograms/ml and 10 micrograms/ml, respectively . Sheep erythrocytes caused dose-dependent stimulation of cathepsin B, C and H in spleen, whereas cathepsin B was activated 3.1-3.6-fold after administration of Vi-antigen . Functional state of the lysosomal proteolytic system did not alter in thymus in response to sheep erythrocytes, while Vi-antigen activated distinctly thiol-dependent proteinases . In peritoneal macrophages administration of sheep erythrocytes led to 2-5-fold decrease in activity of all the lysosomal proteinases studied (cathepsins A, B, C, D, H and L), however Vi-antigen exhibited direct dose-dependent effect on activity of cathepsins A, B, D and L . The data obtained suggest that T-independent reactions of the immune system were realized via thiol-dependent lysosomal proteinases.

Transplantation, 1989 Mar, 47(3), 449 - 50
Evidence that temporary complete occlusion of splenic vessels prevents massive embolization and sudden death associated with intrasplenic hepatocellular transplantation; Nieto JA et al.; It has been reported elsewhere that liver cell suspensions injected at several locations retain some proper hepatic functions, significantly improve the survival rate of rats with different models of acute fulminant hepatic injury, correct some congenital enzyme deficiency diseases, and improve liver function in cirrhotic animals . Among several locations, the splenic parenchyma has been shown to be the most suitable place for hepatocellular transplantation . Unfortunately, infusion of cells into the splenic pulp is not without risk . In fact, portal hypertension and hepatic embolizations have been described after intrasplenic transplantation of hepatocytes or pancreatic islets or fragments . In addition, pulmonary hepatocyte embolizations have been observed in rats with spontaneous (unpublished observations) or surgically induced portosystemic shunts . In this work, we evaluate the efficacy of temporary occlusion of splenic vessels to prevent hepatic and pulmonary embolizations after liver cell transplantation into the spleen in portal hypertension cirrhotic rats with portosystemic shunts.

Clin Orthop, 1989 Mar, (240), 270 - 80
Osteogenic stem cells and the stromal system of bone and marrow; Beresford JN; According to current hypothesis, cells of the osteogenic lineage, which includes both osteoblasts and chondroblasts, are derived from a stromal stem cell in the postnatal organism . That there exist osteogenic precursors in association with the soft, fibrous tissue of the marrow stroma is well established . An osteogenic tissue comprised of cartilage and bone is formed when marrow or marrow cell suspensions are cultured in vivo within diffusion chambers . Bone with a functional marrow organ is formed when marrow or marrow cell suspensions are transplanted heterotopically, e.g., under the renal capsule . Cultures of marrow stromal fibroblasts are readily established in vitro from single-cell bone marrow suspensions . Such cultures do not demonstrate overt differentiation in an osteogenic direction in vitro . When transplanted in vivo, however, they differentiate to form cartilage and bone in diffusion chambers and bone with a functional marrow organ when transplanted heterotopically . Single-cell bone marrow suspensions can be cultured in vitro under conditions that facilitate the formation of stromal fibroblast colonies . Circumstantial evidence supports the conclusion that each colony is derived from a single initiating cell termed a colony-forming unit-fibroblastic (CFU-F) . A proportion of CFU-F demonstrates extensive proliferative potential both in vitro and in vivo . In vitro the extensive proliferative potential of a subset of CFU-F has been shown to be associated with a capacity for extensive self-renewal . On transplantation in vivo, the progeny of a proportion of CFU-F has been shown to be capable of proliferating and differentiating into all the stromal cell lines necessary for the formation of bone and reconstitution of the hematopoietic inductive microenvironment . These findings provide strong circumstantial evidence to support the hypothesis that there are stem cells present within the marrow stroma that are capable of giving rise to cells of a number of different lineages, including those of the osteogenic lineage (chondroblasts and osteoblasts).

J Neurosurg, 1989 Mar, 70(3), 441 - 5
Implantation of dispersed cells into primate brain; Plunkett RJ et al.; Although several experimental therapies such as dopaminergic cell implantation in parkinsonian models and intratumoral placement of lymphokine-activated killer cells require intracerebral deposition of dispersed cell suspensions, a successful technique of needle implantation of cells into primate brain has not been demonstrated . The authors have sought to establish a stereotaxic technique to predictably deposit dispersed cells in primate brain . Human lymphocytes were cultured in recombinant interleukin-2, labeled with sodium 51 chromate (51Cr), and stereotaxically injected into the frontal white matter of six anesthetized rhesus monkeys . A 10-microliters aliquot of cell suspension (2 X 10(7) cells/ml) was deposited 16 mm deep to the dura at 5 microliters/min via Hamilton No . 22s or 26s needles . Five control aliquots were counted for each injection . Reflux out of the needle track was absorbed on gauze, and the recovered cells were counted . The animals were sacrificed 1 hour after implantation and the brain was removed and sectioned such that the cortex and white matter along the needle track were separate . The tissue sections were then counted . Recovery was expressed as the percentage of total injected radioactivity (cpm) that was present in each brain section . Two additional injected hemispheres were processed for autoradiography and histological study . Cell recovery in the brain (mean +/- standard deviation) was 87.2% +/- 13.9% (3.3% +/- 4.9% in cortex and 83.9% +/- 15.9% in white matter) . The autoradiograms and histological examination showed a dense accumulation of radioactivity (cells) at the target site and minimal radioactivity (cells) in the needle track . Accurate intracerebral deposition of dispersed cells in primates was achieved with the technique described . This knowledge permits reliable stereotaxic implantation of cells into the brains of nonhuman primates and humans for investigation and therapy.

Zh Obshch Biol, 1989 Mar-Apr, 50(2), 149 - 57
{Experimental embryologic, biochemical and molecular biology approaches to the identification of embryonic inducers}; Mikhailov AT; Problems of the mechanisms of embryonic induction in vertebrate development have been considered on the basis of author's experimental data . Though several polypeptide factors with certain inducing activity have been identified recently, molecular genetic mechanisms of their effect on embryonic target cells remains largely unclear . One of possible causes of very slow progress in this area of developmental biology is an inadequate system of biotesting of inducers at tissue level (ectoderm of early amphibian gastrulae) using histological criteria . A necessity for carrying out similar studies on cellular level and estimating effect of inducers using immunochemical and molecular biological methods has been postulated . Methods allowing to carry out biotesting of inducers on cell suspension or aggregate of a one type of embryonic cells have been proposed . New approaches, combining the methods of experimental embryology and molecular biology, to studies of embryonic inducers, receptors, and their mRNA, have been analyzed.

Eur Respir J, 1989 Mar, 2(3), 202 - 9
Increased generation of the arachidonic metabolites LTB4 and 5-HETE by human alveolar macrophages in patients with asthma: effect in vitro of nedocromil sodium; Damon M et al.; Alveolar macrophages (AM) are the principal resident phagocytes in the human lung, and play a major role in local defence against environmental agents . It is now known that during asthma these cells take part in the amplification of the inflammatory mechanism . It has been demonstrated in vitro that they can be activated to generate leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE), mediators with potent pharmacological properties . These two arachidonic metabolites were identified and quantified by reversed phase high performance liquid chromatography (HPLC) performed in cell suspensions, and in cell free supernatants . AM from asthmatics, after stimulation by the calcium ionophore A23187 or opsonized zymosan, released significantly (p less than 0.05) more LTB4 than those from healthy subjects . The increase in LTB4 release could be evidence for in vivo activation . On the other hand, the levels of 5-HETE in the AM from asthmatics were significantly (p less than 0.03) higher than those in cells from healthy subjects . This intracellular increase could be correlated with a greater migratory ability of these inflammatory macrophages, as observed for eosinophils . The clinical efficacy of nedocromil sodium may be partly related to the decreases in LTB4 releasability and intracellular 5-HETE levels observed only in AM from asthmatic patients.

Am J Physiol, 1989 Mar, 256(3 Pt 1), C598 - 607
Increase vs . decrease of calcium uptake by isolated heart cells induced by H2O2 vs . HOCl; Kaminishi T et al.; Adult rat heart myocytes were labeled rapidly with exogenous {45Ca2+} . Addition of 2.5 mM H2O2 to the heart cell suspension raised the content of rapidly exchangeable intracellular Ca2+ twofold, whereas addition of 1-30 mM HOCl decreased the Ca2+ content . The H2O2-induced increase in Ca2+ content was dependent on the medium Na+, pH, and temperature but was not significantly affected by addition of verapamil, diltiazem, amiloride, or 3-aminobenzamide . The {3H}ouabain binding to myocytes was suppressed by H2O2, whereas the Ca2+ efflux from myocytes was not influenced . An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, reduced Ca2+ content, implying that the H2O2-induced change in Ca2+ content was not directly related to ATP depletion . On the other hand, the H2O2-induced Ca2+ accumulation in myocytes was prevented by deferoxamine or o-phenanthroline . These results suggest that H2O2 inhibited Na+-K+-ATPase, resulting in an increase in intracellular Na+ concentration and stimulation of sarcolemmal Na+-Ca2+ exchange activity, which caused a transient net Ca2+ influx into myocytes . By contrast, HOCl decreased the Ca2+ content of the rapidly exchangeable pool below control levels and this action of HOCl was antagonized by 1,4-dithiothreitol . HOCl accelerated Ca2+ efflux from myocytes . Ca2+ uptake and Ca2+-ATPase of the isolated sarcoplasmic reticular (SR) fraction were highly sensitive to the action of HOCl . Ca2+ uptake by intracellular sites, studied with myocytes permeabilized with digitonin, was inhibited by both H2O2 and HOCl . Thus these results suggest that HOCl inhibits the SR Ca2+ pump, resulting in the observed acceleration of Ca2+ efflux from and decline in Ca2+ content of myocytes.

Hum Cell, 1989 Mar, 2(1), 70 - 3
{A significance of dual parameter flow cytometric DNA analysis using the anti-keratin antibody}; Ishikawa H et al.; Flow cytometric DNA analysis using the anti-cytokeratin antibody was carried out in order to estimate more reliable measurement in single cell suspension obtained from solid tumors . It was difficult to detect a DNA aneuploidy with DI of 2.0 by one parameter analysis of DNA . Whereas it could be detected easily by using dual parameter analysis of cytokeratin and DNA . And also, the pattern of DNA multiploidy could be selected for cytokeratin positive cell population by gate analysis.

Biol Reprod, 1989 Mar, 40(3), 466 - 74
Effects of lymphokines and immune complexes on murine placental cell growth in vitro; Armstrong DT et al.; Isolated murine placental cells obtained at Day 16 of allogeneic gestation (C3H x DBA/2J) were cultured for 3 days alone or in coculture with irradiated mouse splenocytes at the end of which 3H-thymidine was added for an additional 18-h culture to assess cell proliferation . Placental cell proliferation was significantly enhanced at spleen cell:placental cell ratios of 10:1 and 25:1 above that observed in the absence of added spleen cells . The stimulatory effect of irradiated allogeneic (C3H plus Balb/cJ) spleen cell cultures was significantly greater (approximately 2-fold) than that of isogeneic spleen cells (C3H alone) . Conditioned medium from murine spleen cells cultured with concanavalin A (ConA) to induce lymphokine production had dose-dependent inhibitory effects on proliferation when added to placental cell cultures over a range of concentrations from 10 to 40% (vol:vol) . Addition of pseudo "immune complexes" in the form of heat-aggregated human gamma globulin (AHGG) to culture medium failed to alter placental cell proliferation over a range of concentrations from 2 to 200 micrograms/ml either in the absence or presence of ConA-conditioned medium . In contrast to late-gestational stage placental cells, cell suspensions obtained from Days 8-9 murine ectoplacental cone (EPC) outgrowths, or from earlier stage placentas (Days 12-14) responded to low concentrations of conditioned medium from ConA-stimulated splenocytes with increased proliferation . The effect was less impressive on placental cells at gestational ages later than 12 days than on earlier stage preparations . On all placental cell suspensions tested, as well as EPC cells, a clear-cut inhibition of growth was observed at high doses of conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Exp Metastasis, 1989 Mar-Apr, 7(2), 187 - 99
Tumor cell settling and early invasion of the peritoneum; van de Molengraft F et al.; A Sewall Wright strain-2 guinea pig model producing malignant ascites after injection of a diethylnitrosamine-induced hepatocellular carcinoma cell suspension (Line-10) was used to demonstrate the multilayered settling of tumor cells on the peritoneal surface, frequently followed by the formation of papillary projections and the early invasion in a proliferating submesothelial tissue . At the border of tumor cells and the desmoplastic tissue the malignant cells changed their shape and generally two categories were recognized . Often multilayering, atypical flat cells covered the stromal tissue, while mostly rounded ones invaded using their branched penetration processes, being devoid of cationized ferritin, which was only present on the luminal sides of all cellular elements . Flattened malignant cells, penetrating processes and invading cells lost their microvillous surface pattern . The infiltrating cells were often only detectable with the monoclonal antibody 10 TL 40 and the anti-cytokeratin OV TL 12-5, demonstrating the need for immunohistochemistry in diagnosing solitary invading malignant cells in light microscopy . It appeared that still numerous mesothelial cells were found scattered deeply within the desmoplastic tissue . These former lining cells were identified by their junctions and the presence of remnants of basal lamina as well as by their microvillous 5'-nucleotidase activity.

J Immunol Methods, 1989 Feb 24, 117(2), 275 - 84
Detection and isolation of antigen-specific B cells by the fluorescence activated cell sorter (FACS); Hoven MY et al.; A method is described for the isolation of antigen-specific B cells from immunized and subsequently boosted mice . Antigen-specific B cells were stained by incubation with fluorescein isothiocyanate (FITC)-labelled antigen and then detected and isolated in a fluorescence activated cell sorter (FACS) . Ovalbumin (OVA) and Helix pomatia haemocyanin (HPH) were used as antigens in this procedure, yielding relative amounts of antigen-FITC-binding lymphocytes of 0.9 +/- 0.4% and 3.5 +/- 3.1% . The FITC-positive cells were visible as distinct cell populations in the FACS-generated histograms . All antigen-FITC-binding cells were B cells, as shown by double staining with phycoerythrin-conjugated anti-mouse Ig In addition, as tested in a spot-ELISA, the sorted, antigen-FITC-binding cell population contained almost the entire population of antigen-specific antibody-producing B cells . However, sorting had a negative influence on the antibody production capability of the sorted cells . Through washing of isolated spleen cells in the procedure before labelling with antigen-FITC proved to be essential for the specific detection of antigen-specific B cells, since staining without prior washing resulted in antigen-FITC binding to all B cells . This 'nonspecific' staining phenomenon was caused by the presence of antibodies, specific for the immunizing/boosting antigen, which were also present in the spleen cell suspension . These antibodies formed immune complexes with antigen-FITC and bound to Fc receptors present on all B cells, interfering in this way with any specific binding of antigen-FITC to sIg on the B cells.

Neurosci Lett, 1989 Feb 13, 97(1-2), 51 - 6
The rabbit retina: a suitable mammalian tissue for obtaining astroglia-free Müller cell cultures; Scherer J et al.; Monolayer cultures were prepared from two distinct parts of early postnatal rabbit retinae . Cell suspensions obtained from the developing medullary ray (MR) region contained neurons, Muller (glial) cells, and astrocytes, cells obtained from the remainder (peripheral) part of the retina contained neurons and Muller cells, but no astrocytes . Muller cells lack glial fibrillary acidic protein (GFAP) immunolabeling in situ but some of them acquire faint GFAP labeling in both types of cultures . Strongly GFAP-labeled cells, most likely astrocytes, were seen in MR cultures only . We propose that the periphery of the rabbit retina is ideal for obtaining astroglia-free Muller cell cultures to study their functional properties in vitro.

Clin Sci (Lond), 1989 Feb, 76(2), 183 - 7
Human whole-blood granulocyte aggregation in vitro; Fisher TC et al.; 1 . Aggregation assays are a commonly used technique for the study of granulocyte activation . These studies are usually performed using a pure cell suspension in buffer . This necessitates a separation procedure which is time-consuming and may modify the function of the cells . Interaction between different cell types is precluded . 2 . To avoid these disadvantages a method was developed which quantifies granulocyte aggregation in whole blood . Samples drawn from an incubated vessel before and after the addition of a chemotactic stimulus were fixed with formaldehyde to prevent disaggregation . Erythrocytes were then removed by chemical lysis and using an electronic cell-sizing device the number of single cells and aggregates could then be easily measured . 3 . Results from a group of volunteers showed a rapid and reversible response to a chemotactic tripeptide, with a fall in single granulocyte count and the appearance of doublets and triplets . Lymphocytes were unaffected . Intra-assay reproducibility was better than +/- 5% . 4 . Using this assay, a significant elevation in aggregability was observed in blood from patients after acute myocardial infarction . 5 . This novel technique, by avoiding the separation step, is faster, simpler and more physiological than previous methods, and as such is useful for both assays of drug action in vitro and the study of cell activation in disease states.

Am J Physiol, 1989 Feb, 256(2 Pt 1), C252 - 9
Regulatory volume decrease by cultured renal cells; Knoblauch C et al.; Volume regulatory responses of OK cells (a continuous epithelioid cell line from opossum kidney) are examined by electronic cell sizing and measurements of intracellular pH in cell suspensions . In response to a 40% reduction in osmolality, the cells swell and then subsequently shrink toward their starting volume . This regulatory volume decrease (RVD) is reduced by replacement of Cl- in the medium with acetate . Replacement of Cl- with NO3- accelerates the RVD . The RVD response is inhibited by 1 mM quinine or 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in the medium . The inhibitory effect of 100 microM DIDS (but not 1 mM quinine) is altered by replacement of Cl- by NO3- in the medium . Hypotonic challenge does not induce a DIDS-sensitive net flux of acid-base equivalents . Addition of (9 microM) valinomycin also inhibits the RVD response . It is suggested that the RVD response of OK cells involves activation of separate K+ and Cl- channels.

Exp Hematol, 1989 Feb, 17(2), 171 - 6
The concentration and resolution of primitive hemopoietic cells from normal mouse bone marrow by negative selection using monoclonal antibodies and Dynabead monodisperse magnetic microspheres; Bertoncello I et al.; High proliferative potential colony-forming cells (HPP-CFC) detected in clonal agar culture in the presence of the combined stimulus of colony-stimulating factor 1 (CSF-1) + interleukin 3 (IL-3) + interleukin 1 alpha (IL-1 alpha) are closely related to developmentally early progenitor cells capable of reconstituting the hemopoietic system of lethally irradiated mice following transplantation . Flow cytometric analysis and sorting of normal, unperturbed bone marrow has shown that HPP-CFC are B220- and 7/4-, whereas the committed progenitors of the macrophage lineage responsive to CSF-1 alone (CSFCSF-1) are B220- and 7/4+ . Negative immunomagnetic selection using an anti-7/4, anti-B220 antibody cocktail and second-antibody-coupled Dynabead microspheres to replace flow cytometry results in the highly reproducible and specific enrichment of HPP-CFC, and simultaneous resolution of HPP-CFC from CFCCSF-1 . The tenfold enrichment of HPP-CFC compared with unfractionated bone marrow cell suspensions was comparable to that obtained by fluorescence-activated cell sorting . Enrichment was achieved with negligible loss of HPP-CFC at the immunomagnetic bead selection step, and 65% of HPP-CFC were recovered . The method is rapid, highly reproducible, and efficient, and has wide application to the separation of rare hemopoietic cells from normal bone marrow.

Cancer Res, 1989 Feb 1, 49(3), 528 - 32
Antitumor activity of murine neutrophils demonstrated by cytometric analysis; Ackermann MF et al.; The cytostatic and cytolytic activities of activated polymorphonuclear neutrophils (PMNs) against YAC-1 lymphoma target cells were examined using multiparameter flow cytometric analysis . PMNs were resolved from tumor cells by 90 degrees light scatter . The number of surviving tumor cells was determined by adding a known concentration of fluorescent latex particles to the fixed cell suspension immediately prior to analysis and counting the particles simultaneously with the cells . Cell cycle progression of the YAC-1 target was studied by dual parameter analysis of DNA content and bromodeoxyuridine incorporation into tumor cell DNA either prior to or following addition of PMNs . The results indicate that activated PMNs effectively kill tumor cells within the first 24 h of coculture . However, between 24 and 48 h, tumor cells which escape destruction resume growth and eventually reach a growth rate greater than control cells.

Development, 1989 Feb, 105(2), 379 - 85
Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture; Bennett DC et al.; We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively . Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts . The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets . The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy . Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA) . Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME) . The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c . Neither line is tumorigenic in nude mice . Heterokaryons between the two lines can be constructed and form wild-type, black pigment . Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations . These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.

Scand J Immunol, 1989 Feb, 29(2), 193 - 201
Migration pattern of lymphocyte subsets in the normal rat and the influence of splenic tissue; Westermann J et al.; Lymphocyte subsets leave the blood and appear in the thoracic duct of normal rats at different rates . The aim of the present study was to investigate their migration pattern through blood, spleen, bone marrow, mesenteric lymph nodes, and Peyer's patches in normal Lewis rats and to study the role of the spleen using splenectomized and spleen-transplanted animals . Fluorescein isothiocyanate (FITC)-labelled thoracic duct lymphocytes (TDL) were injected intravenously into rats and after 15 min, 1, 6, and 24 h the percentages of B, T, T helper (TH) and T-cytotoxic/suppressor (TC/S) lymphocytes in the FITC+ cells were determined in cell suspensions by means of monoclonal antibodies . B and T lymphocytes are preferentially localized in different organs, e.g . B cells in Peyer's patches and T cells in mesenteric lymph nodes . The migration of TH lymphocytes differed from that of TC/S lymphocytes in all the organs investigated . In the late phase after injection the migration of B and TH lymphocytes was influenced by the spleen, since after splenectomy the number of injected B lymphocytes increased and that of TH lymphocytes decreased in all organs investigated except the bone marrow . Splenic autotransplantation could not normalize the disturbed migration.

J Immunol, 1989 Feb 1, 142(3), 1036 - 45
Effect of LAK cells against three-dimensional tumor tissue . In vitro study using multi-cellular human glioma spheroids as targets; Jaaskelainen J et al.; The anti-tumor mechanisms of local LAK cell therapy are difficult to study in vivo . We describe a method to study in vitro the action of LAK cells against three-dimensional tumor tissue . Spherical cell aggregates (spheroids) grown from human glioma cell lines H-2 and U-251 were labeled with 51Cr and then incubated for up to 24 h with LAK cells . After the incubation, most spheroids were still macroscopically identifiable, and the measured reduction of volume did not correlate to the extent of damage . LAK cells infiltrated into spheroid tissue slowly as a frontier which explains why the specific 51Cr release was clearly slower from spheroids than corresponding single cell suspensions . The infiltrated area was at 1 to 2 h very thin but by 8 to 12 h consisted already of several cell layers . Most H-2 spheroids became totally infiltrated by 16 to 24 h whereas in U-251 spheroids the infiltration usually remained peripheral . In accordance with the different extent of infiltration, H-2 spheroids were clearly more sensitive to LAK cells than U-251 spheroids: at E/T ratio 10:1 the mean specific 51Cr release by 24 h was 63 and 36%, respectively . A single exposure to LAK cells released 51Cr from H-2 spheroids approximately 12 h but over 24 h from U-251 spheroids . The spheroid model can be used to study the infiltrative capacity and cytotoxicity of LAK cells against three-dimensional tumor tissue, and the method may help to find an optimal mode of local LAK cell therapy, i.e., proper combination of lymphokines and LAK cells, and proper timing of their administration.

Tsitologiia, 1989 Feb, 31(2), 251 - 3
{A method of shaking-blotting--a simple and reliable means for obtaining direct chromosomal preparations from chorionic biopsies}; Baranov VS; The method proposed is based on a gradual fixation, and short-term hydration before softening and on a new technique of chromosome preparation . The latter is based on the sample softening in 60% acetic acid directly on the slide, its gentle shaking and spot-blotting, a careful spreading of the cell suspension on slide surface avoiding the usage of micropipets and other handlings causing metaphase plate breakage . The method provides a highly efficient karyotyping of human embryos within the 7-12th weeks of gestation.

Anal Quant Cytol Histol, 1989 Feb, 11(1), 67 - 71
Comparison of flow cytometric data obtained using fresh and paraffin-embedded lymphoid tissue; Grace J et al.; Flow cytometric (FCM) DNA analysis was carried out on 24 lymph nodes: 13 from benign reactive hyperplasias and 11 from non-Hodgkin's lymphomas . FCM was performed on two types of samples: (1) fresh cell suspensions and (2) suspensions prepared from formalin-fixed, paraffin-embedded sections . FCM of fresh samples detected aneuploidy in 23 of the 24 cases while FCM of paraffin-embedded samples detected aneuploidy in only 6 of the 24 cases . Those six cases were lymphomas considered histologically as having a poor prognosis . Only one case, a lymphoma, was euploid with both methods . The coefficients of variance determined in each case for both methods were found to be within "normal ranges," but were greater in the paraffin-embedded specimens . The results suggest that FCM DNA analysis of formalin-fixed, paraffin-embedded sections does not have as great a resolution capacity as does analysis of fresh cell suspensions, since the former failed to detect cell populations that had a small degree of aneuploidy (close to the 2n population).

Artif Organs, 1989 Feb, 13(1), 4 - 20
The physics of continuous flow centrifugal cell separation; Brown RI; The governing equations defining the separation of blood in continuous flow centrifuges are derived using an empirical model for the viscosity of blood . The effects of fluid shear on the separation process are addressed and further models are developed to permit estimations of shear rate during centrifugation . Simplified predictive equations for species specific separations are developed . Contributions due to shear enhanced diffusion are addressed and found to be negligible at the discontinuous interface between cells and plasma . Experimental results from an investigational centrifuge are presented and compared with theoretical predictions . The role of rouleaux formation in conventional centrifuges is discussed and known centrifugal separation characteristics are explained . The viscosity of blood is related to that for suspensions of rigid particles and an equation for the hydraulic permeability of red blood cell suspensions is derived.

Eur J Cell Biol, 1989 Feb, 48(1), 116 - 20
The hepatic asialoglycoprotein receptor selectively binds to some endogenous tissues; Treichel U et al.; The hepatic asialoglycoprotein receptor (ASGP-R) was isolated from various rat tissues or freshly prepared single cell suspensions and tested for the binding to endogenous tissues or specific cell types by indirect immunofluorescence . Inhibition with N-acetyl-D-galactosamine demonstrated specificity of binding . ASGP-R binds to mesodermal tissues and to selected cells of the majority of glandular tissues but not to lining epithelia . ASGP-R stains heart muscle but not skeletal muscle . In addition, ASGP-R stains spleen cells (52%), bone marrow cells (55%), thymocytes (62%), and a fraction of peripheral blood lymphocytes (29%), which was identified as B-lymphocytes . Five different rat tumors also showed binding of ASGP-R . The binding pattern and staining intensity of peanut agglutinin and soybean agglutinin were strikingly different although the binding specificity of these lectins is related to the ASPG-R . It is concluded that considerable numbers of endogenous binding sites for the hepatic ASGP-R exist in normal tissue, even on cells which pass the liver on circulation.

Plast Reconstr Surg, 1989 Feb, 83(2), 368 - 81
Historical review and present status of free fat graft autotransplantation in plastic and reconstructive surgery; Billings E Jr et al.; Free fat graft autotransplantation for soft-tissue replacement has been a neglected subject in recent years . In a review of the literature, investigations of the various uses of free fat autotransplantation in animals and humans provide an understanding of the problems associated with the use of fat as a free graft . Results of free fat autotransplantation were found to be quite unpredictable, with wide variations in the resulting bulk of the graft . Microscopic studies of this behavior led to controversy as to whether the graft ultimately was made of surviving graft adipocytes (cell survival theory) or host adipocytes (host replacement theory) . Studies revealed a "fibroblast-like" mesenchymal cell within adipose tissue that was believed to be an immature adipocyte precursor or preadipocyte . Further characterization of the preadipocyte and its complete differentiation was accomplished using tissue-culture techniques . These investigations provide evidence of the dynamic nature of adipose tissue that strongly supports the cell survival theory and gives explanation to the unpredictable behavior of free fat autografts . Many conditions treated by plastic surgeons require soft-tissue augmentation . Autogenous adipose tissue is the most appropriate and natural replacement material . With new culturing techniques, preadipocytes in a single cell suspension may provide an injectable soft-tissue replacement . This subject appears ripe for investigation.

Sheng Li Xue Bao, 1989 Feb, 41(1), 70 - 5
{The content of tyrosine in rat luteal cell and the changes caused by hCG, cAMP and progesterone}; Zhang Q et al.; The ovaries taken from the immature female rats primed with PMSG-hCG were digested with collagenase-DNAase solution to obtain the luteal cell suspensions . Luteal cells were then incubated with different doses of hCG, cAMP or progesterone for 1.5 hours . The contents of tyrosine in luteal cell suspensions were determined by thin layer chromatogram using dual-wavelength chromatogram scanner . It was found that the content of tyrosine in rat luteal cell suspensions was 1.41 + 0.24 nmol/L/10(6) cells after one hour incubation . hCG, cAMP and progesterone all could significantly increase the content of tyrosine in the suspensions, suggesting the release of "endogenous tyrosine" . The increase of tyrosine was not due to increased transformation of phenylalanine, the precursor of tyrosine, and increased protein synthesis, because both phenylalanine and cycloheximide failed to influence such increase.

Eur J Biochem, 1989 Feb 1, 179(2), 469 - 72
Proton translocation coupled to the oxidation of carbon monoxide to CO2 and H2 in Methanosarcina barkeri; Bott M et al.; Cell suspensions of acetate-grown Methanosarcina barkeri mediate the conversion of CO and H2O to CO2 and H2 . The reaction is coupled with the phosphorylation of ADP . Evidence is presented that CO oxidation by the cells is associated with the transient acidification of the suspension medium . Up to 2 mol vectorial protons were measured/mol CO oxidized when the transmembrane electrical gradient was kept low by the addition of valinomycin (20 nmol/mg protein) and KCl (200 mM) or of KSCN (50 mM) . No transient acidification was observed in the presence of the protonophore tetrachlorosalicylanilide which stimulated rather than inhibited CO oxidation . Proton extrusion remained unaltered when the proton-translocating ATPase was specifically inhibited by dicyclohexylcarbodiimide . The latter finding indicates that proton translocation is associated with CO conversion to CO2 and H2 rather than with ATP hydrolysis in the cells . The data substantiate that the coupling of CO oxidation with ADP phosphorylation in M . barkeri occurs via a chemiosmotic mechanism.

Immunology, 1989 Feb, 66(2), 190 - 5
Effects on rat T-helper cell proliferation by syngeneic epidermal cells exposed to IFN-gamma in vivo; Skoglund C et al.; In several conditions in the skin, characterized by T-cell infiltration, keratinocytes are induced to synthesize and express class II transplantation antigens . The biological significance of this induced expression is still not understood . In this study, class II antigens were induced on rat ear keratinocytes by local intradermal injections into the ear of rat recombinant interferon-gamma (IFN-gamma) . Epidermal cell suspensions prepared from these ears contained more than 50% class II-expressing cells, as judged by immunocytochemistry, compared with less than 5% in untreated epidermis . When comparing the capacity of these to different epidermal cell populations to stimulate a syngeneic PPD-specific T-helper cell line, it was found that IFN-gamma-exposed epidermal cells induced a lower T-cell response to PPD than did normal epidermal cells . This discrepancy could not be explained by either an infiltration of inflammatory cells into the epidermis of IFN-gamma-treated ears or by a difference in interleukin-1 production as determined in culture supernatants . The addition of indomethacin to cultures with IFN-gamma-exposed epidermal cells restored the T-cell response to PPD to that of normal epidermal cells, suggesting an inhibitory effect of prostaglandins . Our data indicate that epidermal cells exposed to IFN-gamma in vivo can suppress an antigen-specific T-cell proliferation.

Int J Radiat Oncol Biol Phys, 1989 Feb, 16(2), 415 - 35
Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells; Uckun FM et al.; The purpose of this study was to evaluate the individual radioprotective effects of 4 distinct purified recombinant human hematopoietic growth factors, namely recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF), recombinant human granulocyte colony stimulating factor (rG-CSF), recombinant human interleukin 1 (rIL-1), and recombinant human interleukin 2 (rIL-2) on human myeloid (CFU-GM) and erythroid (BFU-E) bone marrow progenitor cells . We demonstrate that (a) preconditioning with rGM-CSF, rG-CSF, or rIL-1 enables CFU-GM to repair sublethal radiation damage and renders CFU-GM less radiosensitive, (b) preconditioning with rGM-CSF or rIL-1 enables BFU-E to repair sublethal radiation damage, and (c) preconditioning with rIL-2 does not increase the radiation survival of CFU-GM or BFU-E . The effects of recombinant growth factors, in particular rGM-CSF, on the radiation damage repair, radiosensitivity, and proliferative activity of bone marrow progenitor cells resulted in a substantial increase in the mean numbers of progenitor cell-derived hematopoietic colonies in irradiated marrow samples . The effects of rGM-CSF on the radiation response of CFU-GM and BFU-E, and the effects of rG-CSF as well as rIL-1 on the radiation response of CFU-GM did not appear to require the presence of T-cells/T-cell precursors, NK-cells, B-cells/B-cell precursors, monocytes, macrophages, MY8 antigen positive non-CFU-GM myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, or glycophorin A positive erythroid cells since virtually identical results were obtained with unsorted marrow samples or highly purified fluorescence activated cell sorter (FACS) isolated progenitor cell suspensions . To our knowledge, this report represents the first study on recombinant human growth factor-induced modulation of the radiation responses of normal human bone marrow progenitor cells.

Inflammation, 1989 Feb, 13(1), 103 - 23
Guinea pig lung cells . Method of isolation and partial purification, identification, ultrastructure, and cell count; Pele JP et al.; Guinea pig lung cells (over 700 x 10(6) cells/lung) were obtained following gentle digestion of lung tissues with a solution of protease type VII (50 micrograms/ml) . The viability of these cells was over 86% as estimated by the trypan blue exclusion technique . The cell suspensions were elutriated into eight fractions, which were characterized by selected staining techniques (Alcian blue, esterase, and Papanicolaou) and by electron microscopy . Differential cell counts were done to establish the percentages of each type of cells in the overall population . Electron microscopy analyses of the cell populations have allowed the identification of most of the various isolated cell types and showed that the cellular organelles and ultrastructures were well preserved . These cell populations will be used for characterizing lung immunologic, metabolic, and endocrine functions, as well as for studying cell interactions.

Cell Biophys, 1989 Feb, 14(1), 1 - 16
Energetics of cell-cell and cell-biopolymer interactions; van Oss CJ; The energy vs distance balance of cell suspensions (in the presence and in the absence of extracellular biopolymer solutions) is studied, not only in the light of the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory (which considered just the electrostatic (EL) and Lifshitz-van der Waals (LW) interactions), but also by taking electron-acceptor/electron-donor, or Lewis acid-base (AB) and osmotic (OS) interactions into account . Since cell surfaces, as well as many biopolymers tend to have strong monopolar electron-donor properties, they are able to engage in a strong mutual AB repulsion when immersed in a polar liquid such as water . The effects of that repulsion have been observed earlier in the guise of hydration pressure . The AB repulsion is, at close range, typically one or two orders of magnitude stronger than the EL repulsion, but its rate of decay is much steeper . In most cases, AB interactions are quantitatively the dominant factor in cell stability (when repulsive) and in "hydrophobic interactions" (when attractive) . OS interactions exerted by extracellularly dissolved biopolymers are weak, but their rate of decay is very gradual, so OS repulsions engendered by biopolymer solutions may be of importance in certain long-range interactions . OS interactions exerted by biopolymers attached to cells or particles (e.g., by glycocalix glycoproteins), are very short-ranged and usually are negligibly small in comparison with the other interaction forces, in aqueous media.

Gynecol Oncol, 1989 Feb, 32(2), 203 - 14
Characterization of a hormone-producing ovarian carcinoma cell line; Poels LG et al.; An ovarian carcinoma cell line (OTN 11) was produced from the ascitic fluid of a patient with a moderately to well differentiated papilliferous cystadenocarcinoma of the ovary . The cell line was characterized using electron microscopy karyotyping, immunohistochemical techniques with monoclonal antibodies against keratins as epithelial markers, and the monoclonal antibodies OV-TL 3 and OC 125 as ovarian carcinoma markers . These techniques revealed the epithelial and adenocarcinomatous nature of the cell line and the presence of ovarian carcinoma-related surface markers . The adenocarcinomatous nature of the cell line also became apparent after heterotransplantation of cell suspensions into nude mice and nude rats, in which adenomatous tumor structures were formed . These xenografts had the same ultrastructural and immunohistochemical properties as the cell line . Despite the adenocarcinomatous character of the tumor the cultured cells release estradiol into the culture medium . We may conclude that OTN 11 is an ovarian carcinoma cell line which has retained highly differentiated functions, such as the production of an ovarian hormone.

Experientia, 1989 Jan 15, 45(1), 96 - 8
Presence of benzodiazepine binding sites (receptors) and amplification thereof by imprinting in Tetrahymena; Csaba G et al.; Live Tetrahymena cells bound 3H-diazepam specifically, as demonstrated by autoradiographic evidence of displacement of about 25% of labeled diazepam in the presence of a 1000-fold amount of cold diazepam . The 3H-diazepam bound to membrane preparations isolated from untreated (control) cells was not displaced by cold diazepam, whereas cells involved in primary interaction (imprinting) with diazepam showed amplification and specificity of diazepam binding in both in vivo (cell suspension) and in vitro (pellicle) systems, as well as displacement of bound label in the presence of 1000-fold cold diazepam . It appears that diazepam induced imprinting and, consequently, also the formation of specific receptors in Tetrahymena.

J Biol Chem, 1989 Jan 15, 264(2), 973 - 80
Suppression of insulin release by galanin and somatostatin is mediated by a G-protein . An effect involving repolarization and reduction in cytoplasmic free Ca2+ concentration; Nilsson T et al.; The effects of galanin and somatostatin on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration {( Ca2+}i) were investigated using beta-cells isolated from obese hyperglycemic mice . Whereas insulin release was measured in a column perifusion system, membrane potential and {Ca2+}i were measured with the fluorescent indicators bisoxonol (bis-(1,3-diethylthiobarbiturate)trimethineoxonol) and quin 2, in cell suspensions in a cuvette . Galanin (16 nM) and somatostatin (400 nM) suppressed glucose-stimulated insulin release in parallel to promoting repolarization and a reduction in {Ca2+}i . The reduction in {Ca2+}i comprised an initial nadir followed by a slow rise and the establishment of a new steady state level . The slow rise in {Ca2+}i was abolished by 50 microM D-600, a blocker of voltage-activated Ca2+ channels . Both peptides suppressed insulin release even when {Ca2+}i was raised by 25 mM K+ . Under these conditions the inhibition of insulin release was partly reversed by an increase in the glucose concentration . Addition of 5 mM Ca2+ to a cell suspension, incubated in the presence of 20 mM glucose and either galanin, somatostatin, or the alpha 2-adrenergic agonist clonidine (10 nM), induced oscillations in {Ca2+}i, this effect disappearing subsequent to the addition of D-600 . The effects of galanin, somatostatin, and clonidine on {Ca2+}i were abolished in beta-cells treated with pertussis toxin . In accordance with measurements of {Ca2+}i, treatment with pertussis toxin reversed the inhibitory effect of galanin on insulin release . The inhibitory action of galanin and somatostatin on insulin release is probably accounted for by not only a repolarization-induced reduction in {Ca2+}i and a decreased sensitivity of the secretory machinery to Ca2+, but also by a direct interaction with the exocytotic process . It is proposed that these effects are mediated by a pertussis toxin-sensitive GTP-binding protein.

Brain Res, 1989 Jan 9, 476(2), 345 - 50
Minimal connectivity between six month neostriatal transplants and the host substantia nigra; McAllister JP 2nd et al.; The present study sought to determine if axonal connectivity is established between 6-month-old neostriatal transplants and the host substantia nigra . Cell suspensions of fetal neostriatum were transplanted into the adult rat neostriatum lesioned previously by kainic acid . Horseradish peroxidase injections into the ipsilateral ventral midbrain labelled the lesion site and the intact neostriatum extensively, but no appreciable anterograde or retrograde label was found within the graft . These results demonstrate a paucity of connectivity between neostriatal grafts and the host brain at a time when other investigators have described transplant-mediated recovery of function.

Avian Dis, 1989 Jan-Mar, 33(1), 163 - 7
Separation of avian heterophils from blood using Ficoll-Hypaque discontinuous gradients; Andreasen CB et al.; Rapid separation of avian heterophils from anticoagulated whole blood was achieved using Ficoll-Hypaque discontinuous gradients . An average of 14.4% of blood heterophils was harvested with a mean purity exceeding 99% . Heterophil viability, as determined by trypan blue dye exclusion, averaged 99.8% . The integrity of isolated heterophils was evaluated by cytochemical staining and ultrastructural examination . Cytochemical staining reactions of heterophils in whole blood and of isolated cell suspensions were similar . No ultrastructural abnormalities were observed . Using this procedure, viable intact heterophils were rapidly isolated from blood with an acceptable cell yield and purity for cell function studies.

Radiol Med (Torino), 1989 Jan-Feb, 77(1-2), 94 - 8
{Induction of liver metastases in the rat . An experimental model for research on magnetic resonance}; Pavone P et al.; The authors report their experience in the implantation of hepatic metastases in rat, for research in magnetic resonance (MR) imaging . Tumor cells were directly injected with a fine needle into an hepatic lobe, after enzymatic disaggregation of the cell suspension . Cryopreservation of the cell line is possible after the above-mentioned preparation . Tumor implants were observed in all the 20 animals studied, with size varying from 0.5 to 2.7 cm, according to the time elapsed between inoculation and sacrifice of the rat . On MR imaging the tumors presented the same signal intensity as the corresponding human pathology, on both T1- and T2-weighted sequences . The animal model can be used for the evaluation of the efficacy of MR imaging of the liver, and mainly for the assessment of new organo-specific contrast agents.

Magn Reson Imaging, 1989 Jan-Feb, 7(1), 1 - 8
Hepatic metastases: rat models for imaging research; Chen MC et al.; Improved rat liver tumor models with solitary or multiple metastatic tumors were developed for radiological imaging research . Unlike previous studies which employed trocar inoculation of tumor fragments, an enzymatically disaggregated cell suspension of mammary cancer was injected by fine needle either directly into the liver to produce solitary cancer nodules, or indirectly via the spleen or mesenteric vein to produce multiple liver metastases . Tumor size was proportional to the time elapsed after implantation . The operative mortality of direct liver, splenic parenchymal, and mesenteric inoculations were 8%, 4%, and 27%, respectively . MR tissue characteristics, image contrast, and pharmaceutical enhancement of these tumors closely resembles human hepatic metastases . The availability of reproducible, inexpensive animal models of metastatic cancer allows efficient evaluation of new liver imaging techniques.

J Orthop Res, 1989, 7(1), 22 - 7
The effects of demineralized bone matrix and direct current on an "in vivo" culture of bone marrow cells; Friedenberg ZB et al.; Bone marrow cells (BMCs) from rabbit femora and tibiae were grown in diffusion chambers implanted in rabbit muscle . At 42 days 80% of the BMC chambers exhibited cartilage formation within them . Demineralized bone matrix added to the marrow cell suspension in the chamber accelerated the appearance and increased the number of chambers with cartilage . Mineralization of the cartilage also occurred earlier in the chambers with bone matrix . In a second experiment, a 5-microA direct current cathode in the bone marrow chamber increased the number of chambers containing cartilage from 50 to 80% at day 25 . Mineralization also occurred earlier in the chambers with direct current.

Biomater Artif Cells Artif Organs, 1989, 17(3), 245 - 62
Dynamic evaluation of clotting phenomena in vitro and perfluorochemical oxygen transport across a membrane-bound thrombus model; Nguyen PD et al.; Citrated platelet-rich plasma was used to occlude 3-microns and 10-microns poresize Nuclepore membranes after recalcification as a thrombus model . Morphologic studies, using both light microscopy and scanning electron microscopy, indicated that over 90 percent of the number of pores available for filtration in hydrophilic and hydrophobic membranes were occluded either partially or completely . Results of transient and steady state pressure drop measurements supported the morphologic studies . It was found that the percentage of oxygen transported across the occluded membranes was greater for filtration of red blood cell suspensions diluted with a perfluorochemical emulsion than that of those diluted with Ringers . The findings in this study suggested that perfluorochemical emulsions might transport oxygen across a thrombus to maintain tissue viability during acute ischemic events.

Acta Med Hung, 1989, 46(2-3), 207 - 11
Altered filtrability of white blood cells after myocardial infarction; Bogar L et al.; Abnormal white blood cell rheological behaviour has been implicated as a cause of blood flow disturbances under conditions of ischaemia and reduced perfusion pressure . Accordingly, we have tested the mechanical properties of white cells following myocardial infarction by measuring the rate at which suspension of these cells cause plugging of Nuclepore filters . The number of clogging particles in a standard white cell suspension increased by the third day after infarction but subsequently decreased to the control levels . Since white cells can cause blockage of narrow blood vessels, it is assumed that such changes in cellular properties may influence the eventual extent of infarction.

Arch Dermatol Res, 1989, 281(5), 316 - 20
Induction of procoagulant activity in human epidermal cells; Schone A et al.; We have studied the induction of procoagulant activity (PCA) by lipopolysaccharide (LPS) in cultured human epidermal cells . Single cell suspensions of epidermal cells were prepared from surgical specimens and stimulated for 24 h with LPS (100 micrograms/ml) . PCA was determined by one-stage clotting assay . Stimulation of the epidermal cells with LPS resulted in a significant reduction of the clotting time (approx . 30%) as compared with the nonstimulated controls . Further analysis of the induced PCA showed that it did not require factors of the intrinsic pathway of the clotting cascade (factors XI and XII) . Similarly, PCA was not affected by factor IX-deficient plasma but required factors II, VII, and X for its full expression . PCA was inactivated by treatment with phospholipase C but not by heating to 56 degrees C . These data indicate that the epidermal cell PCA resembles tissue factor-like activity, activating the extrinsic clotting pathway . Elimination of Langerhans' cells from the epidermal cell suspension by antibody and complement-mediated lysis did not result in a reduction of PCA in the remaining epidermal cells, indicating that keratinocytes were most likely the producer cells . Induction of PCA on the cell membrane surface of epidermal cells may be an early event resulting in the initiation of a local inflammatory reaction.

Leuk Res, 1989, 13(9), 825 - 31
Studies on the suppression of HL-60 cell growth in vitro by low molecular weight suppressor of human fetal liver origin; Wu CT et al.; In human fetal liver there are at least two kinds of cell growth suppressor, arginase and a low molecular weight suppressor, both are cytotoxic towards HL-60 cells under the condition of in vitro culture, and mainly present in the supernatant of fetal liver cell suspension . As compared with arginase, the low molecular weight suppressor shows a preferential suppression on HL-60 cell growth rather than that of granuloid-macrophage progenitors of normal human bone marrow.

Leuk Res, 1989, 13(9), 799 - 809
Cytofluorometric analysis of thymic interdigitating cells from C57BL/6 mice prior and after leukemogenic X-irradiation; Sprecher E et al.; The thymus is populated by various Ia+ cell populations, including epithelial cells, macrophages and dendritic cells . Thymic cell suspensions were stained with an anti-Ia antibody and shown by cytofluorometry to contain a small number of strongly Ia+ cells characterized by a large diameter . The cell population was separated with the aid of the fluorescence-activated cell sorter (FACS) and characterized . They were shown to express high levels of membranal Ia antigens; they demonstrated ATPase activity and displayed the ultrastructural features characteristic of the previously described thymic interdigitating cells . C57BL/6 mice were submitted to various regimens of X-irradiation . Whereas exposure to a single dose of X-irradiation was followed by an increase in the percentage of strongly Ia+ cells, exposure to a leukemogenic regimen of fractionated X-irradiation led to a decrease in the percentage and absolute numbers of these cells in the thymus . Of the C57BL/6 mice that were irradiated with fractionated X-irradiation, 77% developed leukemia . Intravenous injection of syngeneic bone marrow one day following the last irradiation or protection of the femur during irradiation prevented both the appearance of leukemia and the disappearance of interdigitating cells . Therefore an inverse correlation between the presence of thymic dendritic cells and the incidence of leukemia in C57BL/6 mice could be demonstrated . These findings are discussed in relation to the putative role of dendritic cells in the thymus.

Exp Brain Res, 1989, 76(3), 639 - 45
Developmental expression of polypeptide PEP-19 in cerebellar cell suspensions transplanted into the cerebellum of pcd mutant mice; Chang AC et al.; Cerebellar cell suspensions were prepared from normal mouse embryos and implanted into the cerebellum of Purkinje cell degeneration (pcd) mutant mice, which are characterized by a virtually complete degeneration of Purkinje cells between postnatal day (P) 17 and P45 . The expression of immunoreactivity for PEP-19, a developmentally-regulated brain-specific polypeptide, was analyzed in normal mouse cerebellum, as well as in pcd mutants with or without grafts . In the normal cerebellum, PEP-19 immunoreactivity was present in Purkinje cells . In unoperated mutants, 45 days of age or older, Purkinje cells were absent . In grafted pcd mice, numerous PEP-19 immunoreactive, neuroblast-like cells were seen in the graft at 5 days after transplantation . By 9 days, large PEP-19 immunoreactive neurons were found in the host molecular layer; by 17 days after transplantation, such neurons displayed an extensive dendritic tree and resembled differentiated Purkinje cells . The vast majority of PEP-19 immunoreactive cells was located in the molecular layer of the host at 9 days after transplantation and beyond; nonetheless, the same cells extended axonal processes toward the graft, indicating an affinity for co-grafted (possibly deep nuclei) neurons . These results point to the ability of donor Purkinje cells for survival, migration into the host brain and morphological and chemical differentiation following transplantation to the degenerated cerebellar cortex of the recipient mutants.

Allerg Immunol (Leipz), 1989, 35(2), 123 - 32
Development of specific human mab's by a small scale electrofusion technique: the influence of some physical and chemical factors on hybridoma yield of human peripheral blood lymphocytes XCB-F7 fusions; Glaser RW et al.; A fusion chamber and an appropriate procedure are described which allow to fuse a sample of 15 to 25 microliters of cell suspension every two minutes . The cells can be observed throughout the process . They are not exposed to mechanical stress after the fusion pulse . Electrofusion between the heteromyeloma line CB-F7 and human mononuclear cells from peripheral blood of immunized donors is shown to provide stable hybridomas producing IgG against tetanustoxin . Pronase treatment, calmodulin, PEG, lanthanum and a number of variations in the fusion conditions were investigated as to whether they influence physical fusion of the cells, hybridoma yield, and immunoglobulin production.

Ultrasound Med Biol, 1989, 15(5), 451 - 60
Histopathology of shock wave treated tumor cell suspensions and multicell tumor spheroids; Brauner T et al.; L1210 mouse leukemia cell suspensions exposed to 500 shock waves (SW) in an experimental lithotripter (XL1, Dornier) revealed severe cellular damage . Apart from cell fragments and cellular debris, cells exhibited alterations of shape, vacuolization of the cytoplasm, perinuclear cisternae, swelling of mitochondria or rupture of the mitochondrial fine structure, and permeabilization of the cell membrane . Treatment of multicell tumor spheroids of both HeLa and EMT6/Ro cells in suspension with 500 SW resulted either in loss of peripheral cells and serious cellular damage in the outer regions or in a fragmentation of the spheroids . Many of the geometrically intact cells exhibited the same histopathological alterations as the suspended L1210 cells . Immobilization of the spheroids in agar or gelatine, however, prevented spheroids from being agitated and accelerated during SW-exposure . After treatment with 500 SW, spheroids immobilized in gelatine were not different from control cultures, as investigated with light- and electronmicroscopy . From our results we conclude that spheroids in suspension are subject to cavitation and liquid jet formation, causing not only acceleration and shearing forces but also collisions which account for the observed cell damage.

Parasitol Res, 1989, 75(8), 589 - 94
Adherence and surface properties of Tritrichomonas mobilensis, an intestinal parasite of the squirrel monkey; Demes P et al.; Adherence properties of the potentially enteropathogenic Tritrichomonas mobilensis were studied in vitro . Axenically cultivated trichomonads readily attached to isolated intestinal epithelial cells and mucus of the squirrel monkey . The kinetics and nature of T . mobilensis cytadherence were microscopically evaluated in cell-suspension assay using Chinese hamster ovary (CHO) cells and in microplate hemagglutination assay with human erythrocytes . Adherence of the parasites to target cells was concentration- and time-dependent; it was inhibited by sialic acid (N-acetylneuraminic or N-glycolylneuraminic acid) and sialyllactose . Neither trypsinization of the flagellates nor their exposure to low temperature (4 degrees C) affected their cytadherence capacities . The data indicate the presence of adhesin(s) with lectin properties on T . mobilensis . Agglutination of live protozoa by animal and plant lectins with various carbohydrate-binding specificities as well as the occurrence of an electron-dense cell coat on plasma membrane suggest marked glycosylation of the parasite surface.

Life Sci, 1989, 45(7), 623 - 30
Inorganic phosphate accelerates hemoglobin A1c synthesis; Kunika K et al.; The effects of inorganic phosphate (Pi), 2,3-diphosphoglycerate (2,3-DPG) and glucose-6-phosphate (G-6-P) on labile and stable hemoglobin A1c (HbA1c) synthesis were studied . After a 75 gram oral glucose administration, the rate of labile or stable HbA1c synthesis decreased in parallel to the decrease in plasma Pi concentrations . In in vitro incubations of red blood cell suspensions or hemoglobin preparations with glucose, Pi proportionally increased the rate of labile or stable HbA1c synthesis . The increase in 2,3-DPG caused by Pi explained only one fiftieth of the increased rate of labile HbA1c synthesis, and G-6-P did not affect HbA1c synthesis . The kinetic analysis of the effect of Pi showed the unchanged rate constant {K1}, the decreased rate constant {K-1}, and the increased rate constant {K2} . Based on these data it is concluded that Pi in its physiological range directly increases hemoglobin glycation by decreasing labile HbA1c dissociation and accelerating the Amadori rearrangement for stable HbA1c synthesis, and that Pi should be taken into account when using HbA1c to evaluate diabetic control.

Invasion Metastasis, 1989, 9(5), 298 - 309
Mechanisms of platelet activation by cultured human cancer cells and cells freshly isolated from tumor tissues; Grignani G et al.; We studied the effects on platelet function of cells isolated from freshly dissociated human tumor tissues (11 breast carcinomas, 9 colon carcinomas and 1 lymph node metastasis from melanoma) obtained at surgery as compared with cultured human tumor cells: namely, human melanoma 1402 cell line derived from a primary tumor and two lines derived from lymph node metastases (ME 7110/2 and Me 665/1) as well as a human hepatoma cell line (Hep G2) . The three melanoma cell lines activated platelets by producing ADP, as evidenced by the inhibitory effect of apyrase and by the direct measurement of the agonist in the supernatants of tumor cell suspensions; this production was much greater by the cells derived from metastases than by the cells derived from the primary tumor . On the other hand, aggregation induced by Hep G2 hepatoma cells was unaffected by apyrase and was inhibited by hirudin or concanavalin A, suggesting that the cells aggregate platelets by producing thrombin, probably through tissue factor activity of the cells themselves . Cells isolated from 16 of the 21 human tumor tissues possessed a potent platelet-aggregating effect, which was not inhibited by apyrase, hirudin or concanavalin A, but was virtually abolished by the cysteine protease inhibitors iodoacetic acid or p-hydroxymercuri-phenylsulfonate . Collectively, our data demonstrate that cells isolated from freshly dissociated tumor tissues activate platelets through tumor-associated cysteine proteinases rather than by the ADP- or thrombin-dependent mechanisms characteristic of cultured human tumor cell lines.

Z Med Lab Diagn, 1989, 30(3), 165 - 8
{Measuring chemiluminescence during blood preservation . 1 . Spontaneous chemiluminescence}; Lewin G et al.; A device for the measurement of spontaneous chemiluminescence (SCL) of biological fluids and cell suspensions according to Vladimirov (1966) description with using of contemporary equipment was constructed . Its dark impulse rate at room temperature of the photomultiplier was 1.5 s-1 . The volume of measuring cell was 2 ml . An application of the device to investigate the blood during its conservation (21 days at 4 degrees C) showed an absence of the SCL of whole blood . The mean values of plasma SCL were in agreement with literature data . There were no regular changes of plasma SCL during the storage of blood indicating unsuitability of SCL for the detection of blood injury during its conservation.

Neuroscience, 1989, 30(2), 313 - 30
Connectivity of striatal grafts implanted into the ibotenic acid-lesioned striatum--III . Efferent projecting graft neurons and their relation to host afferents within the grafts; Wictorin K et al.; Efferent projections of intrastriatally implanted striatal neurons have been studied using a combination of anterograde and retrograde axonal tracers . Adult rats subjected to a unilateral ibotenic acid lesion of the head of the caudate putamen received cell suspension grafts obtained from 14 15-day-old striatal primordia . Three and a half to 20 months after transplantation the rats received either intratransplant injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin or injections of fluorescent retrograde tracers . Fluoro-Gold and rhodamine-labelled latex beads, into the host globus pallidus and substantia nigra . Injections of Phaseolus vulgaris leucoagglutinin located entirely within the grafts labelled axons that ramified extensively within the tissue itself, as well as axons that extended caudally, across the graft host border, along the myelinated fascicles of the internal capsule to arborize in the medial parts of the host globus pallidus . A few axons also reached the entopeduncular nucleus . Injections of Fluoro-Gold into the host globus pallidus labelled large numbers of graft neurons, which had a prominent patchy distribution and were most abundant in the caudal portions of the grafts . Clear retrograde labelling was also seen after injection of Fluoro-Gold or rhodamine beads into the host substantia nigra, although the number of labelled graft neurons was 30-50 times lower than that seen after pallidal injections . Combined injections of Fluoro-Gold into the pallidus and rhodamine beads into the nigra showed that the vast majority of cells labelled from the nigra were also labelled by Fluoro-Gold from the pallidus . In some of the grafted and Fluoro-Gold-injected animals, the fetal donor tissue had been labelled with {3H}thymidine prior to transplantation . Many examples of neurons labelled with both {3H}thymidine and Fluoro-Gold were found after tracer injections into the host globus pallidus, and double-labelled neurons were identified also after Fluoro-Gold injections into the host substantia nigra . In several animals retrograde tracing was combined with labelling of host dopaminergic afferents (by tyrosine hydroxylase immunohistochemistry) and cortical afferents (by injections of Phaseolus vulgaris leucoagglutinin into the host frontal cortex) . Comparison of adjacent sections revealed a striking overlap between the patches of Fluoro-Gold-labelled graft neurons (labelled from the host pallidum) and the dense patches of tyrosine hydroxylase-positive terminals . In addition, many of the Fluoro-Gold-labelled cell patches received a high density of cortical afferents labelled by Phaseolus vulgaris leucoagglutinin.(ABSTRACT TRUNCATED AT 400 WORDS)

Acta Neurochir (Wien), 1989, 98(1-2), 77 - 89
Experimental transplantation gliomas in the adult cat brain . 1 . Experimental model and neuropathology; Wechsler W et al.; Tumours were produced in the adult cat brain by injection of the rapidly growing anaplastic rat glioma clone F98 in order to study their neuropathology, pathophysiology, regional biochemistry and magnetic reasonance imaging . We report here the neuropathological behaviour of cell suspensions in the basal ganglia and the left cerebral hemisphere one, two, three, four and six weeks after stereotactic implantation with respect to tumour growth, immunological tumour regression and alterations of the blood-brain barrier with associated vasogenic brain oedema . Injected cell suspensions produce consistently growing tumours during the first, second and third weeks . Tumour sizes varied according to the survival time and were only slightly dependent on the inoculated cell number, i.e., 3 and 6 x 10(6) tumour cells, respectively . Immunohistochemistry with respect to proteins of the cytoskeleton and other cell markers showed positive tumour cell immunoreactions for vimentin and S 100, but not for GFAP, Leu-7, Leu-M1 and MBP . While leucocyte infiltration is apparent after only one week, major tumour regression phenomena develop after three weeks in conjunction with severe lymphocytic reactions of the host, resulting in complete tumour rejection with scar gliosis after four and six weeks, respectively . This transplantation glioma model is accompanied by vasogenic brain oedema both within the tumour area and in the homolateral hemisphere . Immunohistochemistry of serum proteins, i.e . total serum protein, albumin and IgG reveals impairment of the blood-brain barrier after one week, reaching its maximum after two and three weeks . The oedematous changes decrease dramatically after four and six weeks, when most of the serum proteins are reabsorbed by cellular activities in the tumour scar . The vasogenic brain oedema in this xenogeneic glioma transplantation model may be enhanced by the immunological reactions in the brain.

Vasa, 1989, 18(2), 122 - 7
{Comparative studies of rheologic properties of erythrocyte concentrates in relation to a 6-week storage period in various preservative solutions}; Barras JP et al.; Buffycoat free red cell concentrates were stored at 4 degrees C for 42 days in CPD-A1 and two new red cell preservation solutions, PAGGS-Sorbitol and SAG-Mannitol . The rheological behaviour of the red cells was measured by viscometry of red cell suspensions (hematocrit 45%) at steady flow at various intervals during the storage period . The viscoelastic properties of the red cells were studied at oscillatory flow using an oscillation rheometer . The deformability of the red cells during storage was determined by measurement of the viscosity of hard packed cells at steady flow . The results show better red cell deformability during the entire storage period using the new solutions PAGGS-Sorbitol or SAG-Mannitol compared to the standard blood bank preservation solution CPD-A1.

Neuroscience, 1989, 29(3), 539 - 50
Restoration of the corticostriatal projection in rat neostriatal grafts: electron microscopic analysis; Xu ZC et al.; The corticostriatal projection in rat neostriatal grafts was studied by using the axonal transport of Phaseolus vulgaris-leucoagglutinin . The neostriatal primodia from 15-18-day embryos were used to make a cell suspension which was implanted unilaterally into the rat neostriatum 3-5 days after kainic acid lesion . Two to four months later, regions of the frontal cortex ipsilateral to the grafts were injected iontophoretically with Phaseolus vulgaris-leucoagglutinin . There were many Phaseolus vulgaris-leucoagglutinin labeled cortical fibers in the host neostriatum . Although the density of labeled fibers in the grafts was much lower than that in the surrounding host tissue, some fibers could be seen to enter the grafts and form terminal arborizations . The morphology of labeled fibers in the graft differed from that of corticostriatal fibers from the same injection but distributing in the host neostriatum . The labeled fibers in the host neostriatum arborized in an extended pattern, branching infrequently and making most of their synapses en passant at varicosities along their courses . The labeled fibers in the grafts made more dense arborizations with many short branches that formed clusters of terminals confined to small foci along their courses . The cellular composition and the structure of the neuropil in the neostriatal grafts were similar to that of the neostriatum . As those in the host, labeled corticostriatal terminals in the grafts contained densely packed round vesicles and made asymmetric synapses on dendritic spines, dendritic shafts and somata . A quantitative analysis, however, revealed that the distribution of postsynaptic elements of labeled boutons in the grafts was different from that in the hosts . More than 90% of the labeled cortical terminals in the host neostriatum contacted dendritic spines whereas only 47% of the labeled terminals in the grafts contacted spines, and 50% of them terminated on the dendritic shafts . The present study provides direct anatomic evidence to demonstrate the restoration of the corticostriatal projection in grafts . The difference in the distribution of postsynaptic elements in the grafts and the hosts may represent a response to the decreased innervation density of cortical inputs to the graft tissue, and may contribute to the recovery of corticostriatal responses by increasing the effectiveness of transmission by the fibers that do grow into the graft and form contacts there.

Eksp Onkol, 1989, 11(2), 39 - 41
{Effect of surgical treatment on the natural killer activity in lung cancer patients}; Ancheva M et al.; The NK activity was studied in 15 male patients with stage III of the lung cancer . All patients underwent operation . The NK activity was tested against H3 uridine-labeled K-562 tumour cells in the 4-hour cytotoxic test before and 8-14 days after the surgery . As control 15 healthy men were tested . The NK activity of mononuclear cells of the peripheral blood and nonadherent lymphocytes in the lung cancer patients before surgery was significantly decreased (P less than 0.001) . Surgery did not affect essentially the NK activity . The removal of monocytes from the mononuclear cell suspension of the peripheral blood caused enhancement of the NK activity only in the postoperative period (P less than 0.05) . Survival of the patients did not correlate with the cytotoxic activity of the NK cells before (r = 0.071) and after surgery (r = 0.275) . The postoperative cytotoxic activity of NK cells correlated better with survival of the patients.

Ann N Y Acad Sci, 1989, 554, 36 - 48
Molecular mechanism for the inhibitory action of interferon on hematopoiesis; Orlic D et al.; Spleen cell suspensions obtained from adult mice were separated by Ficoll/Hypaque and Percoll density gradient centrifugation . The enriched erythroblast populations were maintained in liquid culture medium for 8 hours with 10,000 units of murine interferon (IFN) alpha and beta . Exposure of these cell cultures to murine IFN alpha and beta resulted in a 48% to 70% increase in 2-5adenylate synthetase (2-5AS) activity . In parallel studies, adult mice were injected daily for 1 or 2 weeks with recombinant human IFN alpha A/D (rHuIFN alpha A/D) at a dose of 10(6) or 10(7) units/kg body weight . This treatment did not significantly affect body weight but did produce a mean 70% increase in spleen wet weight and a mean 46% increase in number of nucleated cells per spleen . This increase in number of splenic hematopoietic cells did not result in a corresponding increase in number of circulating cells . In fact, during this 1 to 2 week period the hematocrit dropped from 45% to 38% in mice injected with high dose rHuIFN alpha A/D . From these findings we propose that IFN induces an early 2-5AS activity in erythroblasts and megakaryocytes . This 2-5AS activity, which is known to inhibit protein synthesis in other cell systems, is thought to be responsible for the block or prolongation in blood cell maturation observed in the present studies.

Methods Enzymol, 1989, 168, 690 - 701
Assaying the reporter gene chloramphenicol acetyltransferase; Crabb DW et al.; These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity . We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA . The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase . The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved . This interference appears to be a common phenomenon . We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15 . The interference was particularly prominent in several neuroendocrine and hepatoma cells . We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference . Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested . It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr . The control assays were therefore run almost to completion, and were well beyond the linear range of the assay . Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product . After these studies had been performed, we found that others have also recommended heat treatment of the cell extract prior to CAT assay . We concur with this recommendation . We suggest that EDTA plus heat treatment of the cell extract should be incorporated into all CAT assay protocols, unless it has been previously determined that extracts of the cells used do not interfere . Furthermore, the heat treatment step should be used whenever the activity of promoter-CAT constructs is compared among different cell lines, as is often done to define tissue-specific expression.

Gematol Transfuziol, 1989 Jan, 34(1), 29 - 33
{Experimental study of hemodynamics and oxygen regimen in the body during replacement of massive blood loss with a suspension of erythrocytes in a solution of modified deionized gelatin}; Selivanov EA et al.; In experiments on dogs with acute massive hemorrhage the effectiveness has been studied of red blood cell suspension in glucose-saline solution N 8 prepared in the Central Research Institute of Hematology and Blood Transfusion, and in a preservative based on gelatin developed in the Leningrad Research Institute of Hematology and Blood Transfusion . The advantage of the colloid preservative has been proved by the parameters of macro- and microdynamics of the blood, oxygen-basic state, oxygen budget and rheologic properties of the blood.

Int J Rad Appl Instrum B, 1989, 16(2), 147 - 50
Monoclonal antibody internalization by tumor cells: an experimental model for potential radioimmunotherapy applications; Mariani G et al.; The monoclonal IgM 3G5, which reacts with the surface membranes of rat insulinoma cells RINm5F, was purified by HPLC and labeled with 125I using Protag-125; bovine IgG (bIgG) was similarly radiolabeled, and used as a control . 125I-3G5 was incubated with RINm5F cells either at 4 degrees C or at 37 degrees C . 125I-3G5 bound onto RINm5F cells growing in Petri dishes remained approx . constant over 44 h when incubated at 4 degrees C, whereas at 37 degrees C radioactivity was released back in the medium starting at 3 h (plateau at approx . 20 h) . At the end of incubation at 37 degrees C, activity in the medium included a high percentage of free 125I (15.69 vs 2.62% for bIgG, and 1% for 3G5 at 4 degrees C) . In a cell suspension experiment, cell-bound 125I-3G5 also remained constant over a 24 h incubation at 4 degrees C, whereas at 37 degrees C it decreased to 37.5% of its initial value (64.1% at 4 h) . Concomitant microautoradiography showed diffuse radioactive deposits within the RINm5F cells following incubation with 125I-3G5 (but not 125I-bIgG) at 37 degrees C . These results indicate that 3G5-IgM reacts with a surface antigen on the RINm5F cells, but is rapidly internalized by the cells: within the cells, this antibody undergoes some metabolic processing which results in the release of free 125I outside the cells.

Brain Res Bull, 1989 Jan, 22(1), 123 - 9
Intraspinal transplants of serotonergic neurons in the adult rat; Privat A et al.; Adult male Sprague-Dawley rats were made paraplegic by a complete transection of the spinal cord at lower thoracic level . One week later they were transplanted, below the level of the section, with a cell suspension prepared from the raphe region of 14-day embryos . After survival periods of 10 days to 1 year, the animals were sacrificed and the spinal cord processed for the immunocytochemical detection of 5-HT . Axons from grafted cells grew extensively into the grey matter of the host, and established axosomatic and axodendritic synapses in the anterior horn and intermediolateral column, similar to those of the intact animal . In addition, a group of transplanted animals was tested for sexual reflexes which are under the control of serotonin . It was found that ejaculation reflexes, which are absent in paraplegic rats, are restored in transplanted animals.

Bioelectromagnetics, 1989, 10(1), 85 - 98
Addition of magnetic field capability to existing extremely-low-frequency electric field exposure systems; Miller DL et al.; Magnetic field systems were added to existing electric field exposure apparatuses for exposing cell suspensions in vitro and small animals in vivo . Two horizontally oriented, rectangular coils, stacked one directly above the other, have opposite electric currents . This configuration minimizes leakage fields and allows sham- and field-exposure systems to be placed in the same room or incubator . For the in vitro system, copper plates formed the loop-pair, with up to 900 A supplied by a 180:1 transformer . Electric fields were supplied via electrodes at the ends of cell-culture tubes, eight of which can be accommodated by each exposure system . Two complete systems are situated in an incubator to allow simultaneous sham and field exposure up to 1 mT . For the in vivo system, four pairs of 0.8 x 2.7-m coils made of copper bus bar are employed . This arrangement is energized from the power grid via a 30:1 transformer; horizontal magnetic flux densities up to 1 mT can be generated . Pairs of electrode plates spaced 30.5 cm apart provide electric field exposure of up to 130 kV/m . Four systems with a capacity of 48 rats each are located in one room . For both the in vitro and in vivo systems, magnetic exposure fields are uniform to within +/- 2.5%, and sham levels are at least 2,500-fold lower than exposure levels . Potential confounding factors, such as heating and vibration, were examined and found to be minimal.

Neuroscience, 1989, 29(1), 209 - 23
The effects of nerve growth factor on the development of septal cholinergic neurons in reaggregate cell cultures; Hsiang J et al.; Recent studies suggest that nerve growth factor is present within the central nervous system where it may exert selective trophic effects on cholinergic neurons . We have measured the effects of nerve growth factor on septal cholinergic neurons in three-dimensional reaggregating cell cultures, a system which closely simulates the cellular environment in situ . Septal cells obtained from 15-day-old mouse embryos were dissociated into a single cell suspension and then allowed to reaggregate in culture in a rotary incubator shaker . After 17 days in culture, half of the reaggregates from a flask were sonicated for measurement of choline acetyltransferase activity, and the remaining reaggregates were processed for acetylcholinesterase histochemistry . Addition of nerve growth factor to medium containing septal reaggregates resulted in greater than a three-fold increase in choline acetyltransferase activity and in the number of acetylcholinesterase-positive cells, as well as an enhancement in the staining of acetylcholinesterase-positive fibers . All of these effects of nerve growth factor could be neutralized by antibodies to nerve growth factor . In order to evaluate the possible role of endogenous hippocampal-derived nerve growth factor, antiserum to nerve growth factor was added to the culture media containing septal-hippocampal coaggregates . After 21 days in culture, the presence of nerve growth factor antibodies did not qualitatively affect the pattern or density of cholinergic fibers observed . Synapse formation between cholinergic axons and hippocampal target cells was still in evidence as revealed by electron microscopy . However, there was a modest decrease in choline acetyltransferase activity (20%) and cholinergic cell number (30%) when compared with coaggregates grown in culture medium either without nerve growth factor antiserum or with non-immune serum . The magnitude of these effects was markedly less than the effects observed when exogenous nerve growth factor was added to septal cells grown alone in reaggregate culture . These results suggest that nerve growth factor may play a role during central cholinergic development, but that additional trophic mechanisms are likely to be required.

Exp Brain Res, 1989, 74(3), 512 - 26
Neural grafting to ischemic lesions of the adult rat hippocampus; Tonder N et al.; The purpose of this study was to examine the structural and connective integration of developing hippocampal neurons grafted to ischemic lesions of the adult rat hippocampus . The 4-vessel occlusion model was used to cause transient cerebral ischemia which damages CA1 pyramidal cells in the dorsal hippocampus, but spares nonpyramidal neurons and afferents in the area . One week later, cell suspensions were made from the CA1 region of fetal (E18-20) rats and injected stereotaxically into the lesion . The recipient brains were examined 6 weeks to 6 months later for survival, morphology, and intrinsic and extrinsic connections of the grafts . The methods used included cell stains, histochemical staining for acetylcholinesterase (AChE), immunocytochemical staining for neuropeptides (cholecystokinin (CCK), somatostatin (SS), enkephalin (Enk) and an astrocytic marker, glial fibrillary acidic protein (GFAP), as well as tracing by retrograde axonal transport of fluorochromes and light and electron microscopy of anterograde axonal degeneration . The grafts survived well (80%) and were often quite large . They were well integrated in the lesioned host brain area, contained both pyramidal cells and neuropeptidergic neurons and displayed a near normal GFAP immunoreactivity for astrocytes . The latter contrasted the dense gliosis of the host ischemic lesion . Judged by the AChE staining the grafts were innervated by cholinergic host septohippocampal fibers . Ingrowth of host hippocampal commissural fibers was demonstrated by Fink-Heimer staining for degenerating nerve terminals following acute lesions of the hippocampal commissures . At the ultrastructural level degenerating, electron dense terminals of host commissural origin were found even deep inside the graft neuropil in synaptic contact with mainly dendritic spines . A transplant efferent connection to the host brain was demonstrated by retrograde fluorochrome tracing and consisted of a homotypic projection to more posterior levels of the ipsilateral host CA1 and subiculum . Minor abnormal, efferent projections to the host dentate molecular layer were shown in Timm staining . We conclude that fetal CA1 neurons grafted to one week old ischemic lesions of the dorsal CA1 in adult rats become structurally well incorporated and can establish nerve connections with the host brain.

Cancer Immunol Immunother, 1989, 30(5), 277 - 82
Variations in lymphokine generation by individual lymph nodes draining human malignant tumors; Wen DR et al.; Individual lymph nodes draining tumors vary in their degree of immunological activity . Cell suspensions from tumor-free nodes located relatively near to tumors are spontaneously less reactive and respond poorly to exogenous stimulation by mitogens and lymphokines . Diminished spontaneous uptake of tritiated thymidine by lymph node cells not exposed to exogenous stimulation suggests that tumor-proximate immune suppression exists in vivo and is not purely a laboratory artefact . The present study was undertaken to explore that possibility further . Fluid in which cell suspensions from tumor-free nodes were prepared, and supernatants from short-term cultures of nodes located at different distances from tumors were compared for their capacity to inhibit the in vitro migration of the human lymphoblastoid cell line QIMR-WIL . Inhibitory activity of fluids from individual nodes was related to their position relative to the tumor and their immune competence, assessed by the responses to mitogens of cell suspensions prepared from them . Cell suspension fluids from 92/111 nodes (83%) significantly inhibited the migration of QIMR-WIL, at a level similar (44 +/- 14%) to that induced by the supernatants of mixed lymphocyte cultures (43 +/- 17%) . Fluids from the nodes of melanoma patients were more inhibitory than those from breast cancer patients (49 +/- 12% and 37 +/- 13%, respectively, P = 0.003) . The inhibitory activity of the different nodes of individual node groups varied significantly in 25 of 33 patients (76%), the node nearest the tumor generating least inhibitory activity (indexing the greatest immune suppression) in 20 of these 25 patients (80%) . The strength of migration-inhibitory activity was concordant with the responsiveness to mitogen stimulation in up to 14 of 18 patients (78%) . Studies of molecular size and heat stability indicated that the inhibitory factors had characteristics consistent with common migration-inhibitory lymphokines such as leukocyte-migration-inhibitory factor, macrophage-inhibitory factor and interleukin-2 . Our findings further support the hypothesis that lymph nodes nearest to tumors are relatively immune-suppressed in vivo.

Prog Neuropsychopharmacol Biol Psychiatry, 1989, 13(3-4), 453 - 67
Functional compensation afforded by grafts of foetal neurones; Iversen SD et al.; 1 . Adult neurones grafted to the adult nervous system show limited survival and growth potential . By contrast, certain foetal neurones of a defined age, when grafted to the damaged adult nervous system survive, and form a dense innervation of the host brain with morphologically defined synapses . Monamine containing neurones in the rat have these properties . In a number of model systems their ability to survive grafting to the adult brain has been demonstrated, the optimal conditions for their survival and the importance of endogenous growth factors continue to be investigated . 2 . Lesions of the ascending dopamine pathways to the dorsal and ventral striatum result in profound motor disorders . Dopamine-rich foetal neurones from the mesencephalon grafted as blocks of tissue or as cell suspensions provide a rich innervation to the dopamine-depleted target areas and reverse many of these motor impairments . Similar models have been developed for the cholinergic innervation from the basal forebrain to the hippocampus and cortex . Cholinergic depletion of these forebrain structures impair performance on spatial memory tasks, which are reversed by grafts of foetal basal forebrain neurones to cortex or hippocampus . 3 . The grafting model is a powerful tool with which to define the functional role of neurotransmitters in brain function . The neurological properties of foetal monoamine and cholinergic neurones which underlie their ability to afford functional compensation to the damaged nervous system and their vulnerability in certain degenerative diseases of the CNS in man remain obscure.

Cell Calcium, 1989 Jan, 10(1), 17 - 27
Glucose-induced changes in cytoplasmic free Ca2+ concentration and the significance for the regulation of insulin release . Measurements with fura-2 in suspensions and single aggregates of mouse pancreatic beta-cells; Arkhammar P et al.; The effects of glucose on cytoplasmic free Ca2+ concentration, {Ca2+}i, and insulin release were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice . Measurements of {Ca2+}i were performed in cell suspensions in a cuvette and in single cell-aggregates in a microscopic system, using fura 2 and quin 2 . Insulin release was studied from indicator loaded cells in a column perifusion system . In the presence of 1.28 mM extracellular Ca2+, an increase in the glucose concentration from 0 to 20 mM had two major effects on {Ca2+}i . Initially there was a decrease, which was immediately followed by a pronounced increase . At reduced extracellular Ca2+, or when Ca2+ influx was blocked, glucose induced only a decrease in {Ca2+}i . With increasing intracellular concentrations of indicator, the effects of glucose on {Ca2+}i were markedly reduced . Changes in {Ca2+}i, similar effects being obtained in the cuvette and microfluorometric measurements, were paralleled by changes in insulin release . Insulin release from indicator loaded cells did not markedly differ from that of non-loaded controls, either with respect to rapidity or size in the response to the sugar . The addition of 20 mM glucose increased the efflux of fura 2, an effect that was not related to insulin release . Permeabilization of indicator loaded cells demonstrated a substantial amount of fura 2 bound intracellularly . Although the effects of glucose on {Ca2+}i seemed to be similar in fura 2 and quin 2 loaded cells, the demonstrated leakage and possible intracellular binding should be considered before using fura 2 for measurements in pancreatic beta-cells.

Folia Med Cracov, 1989, 30(3-4), 123 - 34
{Possibilities of objective evaluation of local reactive cell infiltrates in transplantable tumors}; Chlap Z et al.; The studies on infiltrations consisting of reactive cells, i.e . lymphocytes, macrophages and neutrophils within and in the vicinity of neoplastic tissue were performed in order to elucidate the nature of the growth and to asses the role of these cells in human tumors . The objective methods for both qualitative and quantitative evaluation of the infiltrating cells are necessary to obtain comparable results . We report our studies on the numbers and types of reactive cells present in 3 transplantable murine tumors with different immunogenicity (SaL-1, LLC, MCA-Sa) . The cytological characterization of cells was performed as following: after enzymic digestion of the tissue the cell suspension was then passed through Millipore filters and dyed . The preliminary evaluation of the percentage of lymphocytes T found in the tumor infiltrations was performed using cytotoxicity test with monoclonal antithymocytic serum (Monoclonal Anti-Mouse Thy 1, 2) . We have shown that there are differences in the number and type of reactive cells in infiltrations of the three different tumor tested . With the increasing mass of tumors the percent of reactive cells decreases proportionally.

Neuroscience, 1989, 33(3), 605 - 16
First month of development of fetal neurons transplanted as a cell suspension into the adult CNS; Nothias F et al.; It has been demonstrated elsewhere that fetal thalamic tissue, when transplanted as a cell suspension into the excitotoxically neuron-depleted adult somatosensory thalamus, can grow, differentiate, and receive projections from host afferents . In the present study, we used the same paradigm to analyse the transplanted neurons during their morphogenesis, i.e . during the first month after transplantation . Using various anatomical criteria, at the light and electron microscope levels, we compared the development of transplanted neurons with the normal ontogeny of homologous neuronal populations . Confined solely to the mechanically lesioned area during implantation at seven days post-grafting, the transplant increased in size to occupy most of the previously neuron-depleted area by the third week after grafting . The final size of the transplant thus depended upon the size of the lesion . At seven days post-grafting, the neurons were small in size and the cellular density was high . At this immature stage few synaptic contacts were visible and the ultrastructure was characterized by large extracellular spaces . At 10 days post-grafting, the size of the neurons had increased and the cellular density had decreased . Both an extensive dendritic proliferation and a simultaneous active synaptogenesis could also be observed . All these events continued to evolve and during the third week the neuropil progressively acquired more mature ultrastructural characteristics . Synaptic contacts exhibiting characteristics comparable to those observed in the intact thalamus also became more numerous . At 20 days post-grafting, axonal myelination had started, the development of the graft apparently stopped and the various criteria had stabilized . Until that developmental stage, growth of grafted neurons compared to that of normal thalamic ones . At later stages, however, grafted neurons failed to grow larger and did not reach the size of the homologous population in the adult animal . It seems, therefore, that transplants of thalamic fetal neurons can be used as a tool with which to study thalamic neuronal development, within definable limits.

Neuroscience, 1989, 33(3), 435 - 62
Cholinergic system and memory in the rat: effects of chronic ethanol, embryonic basal forebrain brain transplants and excitotoxic lesions of cholinergic basal forebrain projection system; Arendt T et al.; Oral administration of ethanol (20% v/v) to male Sprague-Dawley rats for different periods of time up to 28 weeks resulted in profound reductions of acetylcholine content, in vitro synthesis and release of acetylcholine, choline uptake, activities of choline acetyltransferase, acetylcholinesterase and pyruvate decarboxylase, content of noradrenaline, serotonin and, to a lesser extent, dopamine throughout the brain . Changes were fully and partially reversible by a 4 weeks' ethanol-free period following a treatment of 8 and 18 weeks, respectively . They remained persistent, however, after 28 weeks of treatment . Performance in an eight arm-radial maze revealed a severe impairment in both spatial and non-spatial reference and working memory . A similar pattern of memory impairment was obtained after ibotenate lesion of the cholinergic basal forebrain projection system . In order to test whether this memory impairment depends on cholinergic deafferentation of the cortex, cholinergic-rich fetal basal forebrain cell suspensions were transplanted into cortex, hippocampus or both these sites in ethanol treated rats . Cholinergic-rich transplants, but not cholinergic-poor transplants, were effective in ameliorating impaired memory function and measures of cholinergic activity in the basal forebrain projection system . The behavioural efficacy of the basal forebrain grafts was well correlated with measures of both transplant volume and the degree to which they restored acetylcholine content at the transplant site; these transplants had no effect, however, on brain monoamine levels . The effects of the cholinergic-rich transplants into cortical and hippocampal sites were additive in their amelioration of performance in the radial maze . Similarly, ibotenate lesions of the sites of origin of the cholinergic projections to neocortex (in the region of the nucleus basalis magnocellularis) and hippocampus (the medial septal areas and nucleus of the diagonal band), respectively, were additive in their deleterious effects on maze performance . There were no qualitative differences in the susceptibility of the four different types of memory performance measured (spatial and non-spatial reference and working memory) to the effects of ethanol, ibotenate lesions of the cholinergic projection system, or cholinergic-rich brain tissue transplants . Thus, overall, the results indicate that the forebrain cholinergic system acts as a whole, without major functional differences between the projections originating in the medial septal area/diagonal band complex and the basal nucleus, and that it discharges a very general function in cognitive processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Biomed Biochim Acta, 1989, 48(10), 751 - 6
In vitro effects of ethanol and acetaldehyde on lymphoid cells as determined by 31P NMR spectroscopy; Walia AS et al.; We have used NMR spectroscopy to test the direct in vitro effects of ethanol and its metabolite acetaldehyde on a human leukemic T cell line (Molt-4) and normal murine spleen cells . The metabolic status of phosphate metabolites (phosphomonoesters including sugar phosphate, inorganic phosphate and ATP's) in cell suspension was monitored by 31P NMR spectroscopy . Spectral changes were observed in the intensity of inorganic phosphate and ATP . Addition of high concentrations of ethanol (400-1600 mM) to Molt-4 cells resulted in very little spectral change . However, the addition of 40 microM acetaldehyde resulted in substantially increased inorganic phosphate (Pi) signals, and decreased phosphomonoesters and ATP signals . Similar changes, but to a lesser degree, were observed with normal mouse spleen cells.

Acta Biol Hung, 1989, 40(1-2), 127 - 36
Effect of different cryoprotectants and transfer temperatures on the survival rate of hemp (Cannabis sativa L.) cell suspension in deep freezing; Jekkel Z et al.; Adequate cell dehydration is the precipitating element in the successful cryopreservation of plant cells and organs . This could be achieved by using different cooling rates, transfer temperatures and cryoprotectants . Experiments were performed to determine these critical points in the freeze preservation procedure of Cannabis sativa (L.) suspension cultures . The explants were frozen at a cooling rate of 2 degrees C/min, while the transfer temperatures were -10 degrees C, -20 degrees C, -30 degrees C, -40 degrees C and -50 degrees C . The applied cryoprotectants were the DMSO, glycerol, proline and PEG in different concentration . The highest viability (58%) was obtained by using 10% DMSO and at -10 degrees C transfer temperature . The optimum transfer temperature varied remarkably by different cryoprotectant concentrations indicating the importance of their interactions.

Acta Otorhinolaryngol Belg, 1989, 43(4), 297 - 320
Intercellular contacts between germinal center cells . Mechanisms of adhesion between lymphoid cells and follicular dendritic cells; Louis E et al.; Intercellular connections exist between germinal center cells especially between lymphoid cells and follicular dendritic cells (FDC) . Even after isolation, FDC remain associated to lymphocytes and are able, in a cell suspension, to establish new connections with others . Using human tonsillar cells or mouse lymph node cells we analysed these connections which were shown to be species-specific . Low temperature as well as absence of Ca++ and Mg++ in the culture medium reduced the adherence of fluorochrome-labeled lymphoid cells to FDC . Colchicine treatment did not impair the adherence, whereas cytochalasin B dit it; this was the first observation underlining the importance of microfibrils in FDC . Antibodies directed towards integrin molecules (LFA-1 alpha or beta chain, CD11a and CD18 respectively) reduced the adherence, others (anti-CR3 or anti-gp 150/95, CD11b and c respectively) did not influence it . Antibodies directed against MHC class II exerted no inhibitory action on the lymphoid cell adhesion to FDC . As, at ultrastructural level, gold-labeled immune complexes can be found between FDC and lymphoid cells, we examined the effect on cell adhesion of the addition of immune complexes to the cell suspensions . It only impaired the lymphoid cell adhesion when complement components were present . IgM complexes were then more inhibitory than IgG complexes . When antibodies against Fc IgG receptors (CD16) were added, the adhesion was strongly reduced whereas antibodies to Fc IgE (CD23) receptors had no influence . The antibody DRC1, specifically recognizing an antigen on human FDC reduced the attachment of cells to FDC . This antigen thus seems to play a role in the intercellular contacts; this is the first function ascribed to this FDC specific antigen.

Acta Leprol, 1989, 7(1), 13 - 7
In vitro studies on dermal leprosy granulomas: assessment of division and protein synthesis of cells; Narayanan RB et al.; Single cell suspension from dermal leprosy granulomas (10 tuberculoid and 10 lepromatous) was prepared and an assessment of the division and protein synthesis by the cells was made . The cells of tuberculoid granulomas showed a high incorporation of 3H-thymidine and 14C-leucine . On the contrary, the cells of the lepromatous granulomas exhibited poor division but their protein synthesis remained unimpaired . These observations suggest that the epithelioid cell granuloma of tuberculoid leprosy appears to be more active and secretory than the macrophage granuloma of lepromatous leprosy.

Thymus, 1989, 13(3-4), 205 - 12
Turnover of ED2-expressing macrophages in the thymus cortex of rats; Kampinga J et al.; To investigate the turnover of thymic ED2+ cortical macrophages, vascular thymus transplantation in RT7-congenic rats were performed . Thymus graft cell suspensions were analyzed using ED2 in combination with congenic markers . Immunohistology of thymus graft sections was performed to demonstrate the immigration and persistence of these macrophages at several time points after transplantation . In contrast to other mobile thymus cells like thymocytes and interdigitating cells, most ED2+ cortical macrophages showed a slow turnover rate . At 76 days after transplantation more than 30% of ED2+ macrophages were still of donor origin . The migration properties of these macrophages are discussed in relation to their presumed role in thymocyte maturation and proliferation.

Biorheology, 1989, 26(4), 771 - 84
Filterability of sickle cells as a function of pO2: role of physico-chemical factors; Kraiem A et al.; A rigidity index (RI) related to red blood cell deformability was measured by using the hemorheometre . The RI for 13 patients homozygous for sickle cell disease was 109 +/- 44 at 37 degrees C and at atmospheric pO2 . The filtration time curve as a function of pO2 is biphasic for sickle cell suspensions . The pO2 at which filtration time is maximum, pO2max., correlated with the rigidity index measured at atmospheric pO2 . This pO2max . value was very sensitive to small changes in physico-chemical parameters such as osmolality, pH, temperature, hematocrit, and cell density . Conditions which reduced the Hb S polymerization induced a leftward shift of pO2max. . The experimental curves are in agreement with theoretical models based on the presence of two abnormal cell types: filtrable "slow cells" and infiltrable "sickled cells".

Biorheology, 1989, 26(2), 247 - 59
Effects of aggregation on the flow properties of red blood cell suspensions in narrow vertical tubes; Murata T et al.; The flow properties of aggregating red cell suspensions flowing at low rates through vertical tubes with diameters from 30 microns to 150 microns are analyzed using a theoretical model . Unidirectional flow is assumed, and the distributions of velocity and red cell concentration are assumed to be axisymmetric . A three-layer approximation is used for the distribution of red cells, with a cylindrical central core of aggregated red cells moving with uniform velocity, a cell-free marginal layer near the tube wall, and an annular region located between the core and the marginal layer containing suspended non-aggregating red cells . This suspension is assumed to behave approximately as a Newtonian fluid whose viscosity increases exponentially with red cell concentration . Physical arguments concerning the mechanics of red cell attachment to, and detachment from the aggregated core lead to a kinetic equation for core formation . From this kinetic equation and the equation for conservation of red cell volume flux, a relationship between core radius and pressure gradient is obtained . Then the relative viscosity is calculated as a function of pseudo-shear rate . At low flow rates, it is shown that the relative viscosity decreases with decreasing flow and that the dependence of relative viscosity on shear rates is more pronounced in larger tubes . It is also found that the relative viscosity decreases with increasing aggregation tendency of suspension . These theoretical predictions are in good qualitative and quantitative agreement with experimental results.

Biorheology, 1989, 26(2), 143 - 51
A rheological study of packed red blood cell suspensions with an oscillating ball microrheometer; Tran-Son-Tay R et al.; Rheological properties of concentrated red blood cell suspensions are studied with a magneto acoustic microrheometer in which a ball is suspended in a vertically oriented cylindrical tube . The rheometer uses a conventional falling ball technique to measure steady state viscosity and a vertically oscillating, magnetically driven ball for viscoelastic measurements . The motion of the ball is tracked by ultrasound echo location in which sound waves are transmitted and received by an ultrasound transducer mounted at the base of the tube . The compact size of the rheometer allows rheological studies to be made with microliter quantities of opaque suspensions and permits sudden and accurate changes in temperature . Also, values for the adiabatic compressibility are evaluated from measurements of the speed of sound.

Henry Ford Hosp Med J, 1989, 37(3-4), 154 - 6
Adrenal cortex transplantation after bilateral total adrenalectomy in the rat; Scheumann GF et al.; An experimental animal model with adrenal cortex transplantation was developed to study adrenal cortex replacement therapy in patients with multiple endocrine neoplasia type 2 who have had bilateral adrenalectomy for pheochromocytomas . Adrenal cortex of syngenetic rats was isolated from the medulla by collagenase digestion and a defined sedimentation . The cell suspension of the cortical cells was implanted under the kidney capsule of untreated syngenetic rats . After two weeks the recipients were bilaterally adrenalectomized . Serum corticosterone levels were measured as an estimate of function of the grafts . All recipients were healthy throughout the observation period, whereas all adrenalectomized controls died within 18 days . Vital cortex cells could be demonstrated in the explanted grafts by immunohistochemistry . Corticosterone levels of transplanted animals were nearly normal (9.5 ng/100 mL +/- 0.4) compared to the controls (0.20 ng/mL +/- 0.06) . This animal model of adrenal cortex transplantation allows the separation of medullary from cortical cells . After transplantation, these cortical cells survived for eight weeks and were able to replace the adrenal cortex function.

Histochemistry, 1989, 92(4), 337 - 42
Induction of glutamine synthetase and transient co-expression with carbamoylphosphate synthetase in hepatocytes transplanted into fat pads of syngeneic hosts; Gebhardt R et al.; Isolated rat hepatocytes were transplanted into the interscapular and both anterior lateral fat pads of hepatectomized syngeneic rats . At various time points following transplantation, the fat pads were removed, fixed and embedded in paraffin . Serial sections were stained for glutamine synthetase (GS) and carbamoylphosphate synthetase (CPS) using specific antisera and the PAP technique . The initially low fraction of GS+-heptatocytes remained low up to the fourth day, then increased strikingly up to almost 100% and declined gradually after the 14th day . In contrast, the number of CPS+-cells declined continuously to about 30% after 28 days . If the animals were exposed to CCl4 prior to the isolation of the hepatocytes in order to reduce the number of GS+-cells in the initial cell suspension similar results were obtained and no difference in the probability of the colony formation was noted between this and the normal hepatocyte suspensions indicating that the appearance of the GS+-phenotype was not due to a selective survival of these cells . Analysis of the staining intensity of the transplanted hepatocytes revealed the appearance of two populations of GS+-hepatocytes, one with a strong and one with a weak staining, during the course of formation of larger nodules, while only a single weakly stained population could be discerned with respect to the staining for CPS . These results demonstrate that all hepatocytes or at least their descendents can be induced to express GS by the environmental conditions of the fat pads, and that GS and CPS can be co-expressed with an apparently reciprocal relationship.

Exp Brain Res, 1989, 77(3), 552 - 68
Degeneration and graft-induced restoration of dopamine innervation in the weaver mouse neostriatum: a quantitative radioautographic study of {3H}dopamine uptake; Doucet G et al.; A recently introduced quantitative radioautographic technique was used to characterize the striatal dopaminergic deficit in weaver mutant mice and to evaluate the extent of DA reinnervation resulting from cell suspension grafts of fetal ventral mesencephalic tissue . Brain slices from normal mice and unilaterally grafted weaver mice were incubated in {3H}DA, in the presence of desipramine and pargyline 3-5 months after graft surgery . Semi-thin sections from the fixed and resinembedded slices were subsequently exposed on tritium sensitive film and afterwards dipped in nuclear emulsion for light microscope radioautography . Alternative slices were embedded in Epon for post-embedding tyrosine hydroxylase (TH) immunocytochemistry . The grain density of the film radioautographs matched well the distribution of TH positive fibers . Both methods revealed an almost complete absence of DA axons in the dorsomedial quadrant of the weaver neostriatum and an increasing density of DA innervation towards the ventrolateral areas . In the light microscope radioautographs, only the ventral striatum (i.e . nucleus accumbens and olfactory tubercle) and a narrow ventral and periventricular zone of the caudate-putamen were covered by silver grain clusters typical of DA varicosity labeling . Such labeled varicosities were nevertheless found in reduced numbers the lateral portion of both nucleus accumbens and the olfactory tubercle . The remaining neostriatum was overlaid by diffuse silver grains . suggesting a deficient DA uptake and storage mechanism in the residual DA fibers in this region . Immunocytochemistry using antibodies specific for DA or TH provided further evidence that the residual DA innervation in the weaver neostriatum was biochemically defective . Weaver mice with grafts of ventral mesencephalic tissue in the right neostriatum showed an amphetamine-induced rotational bias to the contralateral side, which was not seen in the sham-operated animals . In contrast to the intrinsic weaver neostriatal DA innervation, DA fibers of graft origin exhibited the normal, clustered type of varicosity labeling . The computerized image analysis of silver grain density in film radioautographs was calibrated by counting these labeled varicosities in selected areas of light microscope radioautographs from the same sections . Results showed a mean DA reinnervation of neostriatal tissue surrounding the graft of about 20%, in some cases up to 80%, of the density seen in wild type mice, with a gradual decrease with distance up to 1-1.4 mm from the graft . The ventral parts of the neostriatum, which contained higher numbers of residual intrinsic DA fibers, were much more sparsely reinnervated than the dorsal and dorsomedial areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Virchows Arch B Cell Pathol Incl Mol Pathol, 1989, 57(3), 181 - 94
The morphology of the denuded epidermal basal cell layer of the hairless mouse after different preparation methods . A scanning and transmission electron microscopical study; Glaso M et al.; The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study . The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion . Trypsinization before removal of the suprabasal cells caused the basal cells to shrink . Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved . SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized . Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations . However, cell bridges, and occasional desmosomes were present . The cell interior demonstrated important cellular injury . If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated "regenerative" morphology, without junctions, similar to that observed in wound healing . Epidermal non-keratinocytes were seen only after trypsinization . Control experiments revealed that they adapted poorly to organ culture conditions . By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes . This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis . We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced "clear cells", whereby the neighboring cells appeared darker . This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells . The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation . The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with "foot pads" . Our study also shows that SEM of the epidermal basal layer might be feasible . Various artifacts, however, must be considered, depending on the denudation method used . We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis . Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry . Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t

Drug Metab Dispos, 1989 Jan-Feb, 17(1), 20 - 5
Activities of cytosolic and microsomal drug oxidases of rat hepatocytes in primary culture; Sherratt AJ et al.; Sensitive and specific chromatographic assays for the measurement of flavin-containing monooxygenase (N-oxygenase and S-oxygenase activities) and aldehyde oxidase activities in rat hepatocyte primary cultures were developed . Conditions for the measurement of enzymatic activities in rat liver cell cultures were first optimized using freshly isolated cell suspensions . Activities of the cytochrome P-450/P-448 isozymes in rat hepatocytes maintained in primary culture {assessed by the O-deethylation of 7-ethoxycoumarin (P-450/P-448) and the N-demethylation of N,N-dimethylaniline (P-450)} rapidly declined to 25% of the initial levels by 48 hr in culture . The flavin-containing monooxygenase system was considerably more stable in cell culture . Flavin N-oxygenase activity (assessed by the N-oxidation of N,N-dimethylaniline) declined slightly (10-15%) and remained almost constant over the 48-hr culture period, whereas S-oxygenase activity (assessed by the S-oxygenation of tetrahydrothiophen) gradually declined and stabilized at approximately 65% of its initial activity at 48 hr in culture . Aldehyde oxidase activity (assessed by the 1-hydroxylation of phthalazine) declined to approximately 20% of the initial value by 48 hr in culture . The differential stability of the microsomal and cytosolic drug oxidases in rat hepatocytes in primary culture demonstrates some of the limitations of this model for metabolic studies.

Vis Neurosci, 1989, 2(2), 189 - 98
Neurotrophic and behavioral effects of occipital cortex transplants in newborn rats; Haun F et al.; Cell suspensions of embryonic occipital cortex were transplanted into newborn rats with large unilateral visual cortex lesions . When the animals were adults, they were tested on a difficult visual discrimination, and subsequently their brains were analyzed for possible neurotrophic effects of the transplants on nonvisual cortical areas which normally form connections with the occipital cortex . Behaviorally, animals with lesions and transplants learn to discriminate between columns and rows of squares at a rate which is identical to normal rats while animals with lesions and no transplants are impaired . Volume and cell-density measures show that the transplants also rescue neurons in cortical area 8 that would normally degenerate following the cortical lesion . No such neurotrophic effect of the transplants is found in cortical area 24 or area 17 contralateral to the lesion . In rats with lesions and no transplants, there is a significant correlation between the amount of area 8 remaining after the lesion and trials to criterion on the columns-rows discrimination, a relationship that does not exist in transplant animals because of their normal learning curve and the consistent sparing of area 8 . Injections of HRP into the visual cortex contralateral to the lesion result in variable numbers of labeled cells within the transplant . However, there is no consistent relationship between the number of transplant cells which project to the opposite hemisphere and learning of the discrimination . It is suggested that the learning deficit following the lesion is largely attentional and that the sparing of cortical area 8 (which in rats may include the analog of the frontal eye fields present in the primate cortex) contributes to the sparing of function.

Acta Leprol, 1989, 7(1), 7 - 11
Isolation and characterization of cells in granulomas of nerves of leprosy patients; Kehrer D et al.; Single cell suspension from the granulomas in nerves of leprosy patients were prepared for an in vitro study of the properties of infiltrating cells . Nerve biopsies from 17 patients with tuberculoid (n = 9) and lepromatous (n = 8) leprosy cases were analysed . The granulomas were found to contain lymphocytes and macrophages . Lymphocytes were the predominant infiltrating cells in the tuberculoid nerves . In contrast, lepromatous nerves contained very few of these cells . The majority of lymphocytes in tuberculoid granulomas were activated T cells as they formed rosettes with sheep erythrocytes, exhibited esterase dots in the cytoplasm and expressed HLA-DR antigens . A small proportion of the lymphocytes also formed rosettes with EAC . Most macrophages from both the granulomas were mature macrophages as they were esterase positive, did not exhibit peroxidase activity and expressed HLA-DR antigens . The macrophages did not possess C3 surface receptors.

Acta Leprol, 1989, 7(1), 19 - 24
Comparison of the characteristics of infiltrates in skin and nerve granulomas of leprosy; Kumar V et al.; The characteristics of infiltrates in the dermal and neural granulomas from the same leprosy patients were compared by preparing a single cell suspension . Skin and nerve biopsies from 10 patients, 5 with tuberculoid and 5 with lepromatous leprosy were analysed . The granulomas contained lymphocytes and macrophages . Lymphocytes were the predominant infiltrating cells in the tuberculoid dermal and neural granulomas . A high proportion of lymphocytes in both the skin and nerve granulomas in these cases were activated T cells as they formed rosettes with sheep erythrocytes and expressed HLA-DR antigens . In contrast, lepromatous dermal and neural granulomas contained very few of these lymphocytes . Dermal and neural granulomas from both the types of leprosy contained mature macrophages as they were esterase positive, did not exhibit peroxidase activity and expressed HLA-DR antigens . These macrophages did not possess C3 surface receptors either . These findings suggest that the infiltrates in the skin and nerve granulomas of a given type of leprosy have similar characteristics.

Dev Comp Immunol, 1989 Summer, 13(3), 217 - 24
Leukocytes of rainbow trout (Oncorhynchus mykiss) pronephros: cell types producing superoxide anion; Plytycz B et al.; Fish pronephric and blood leukocytes yield a chemiluminescent response (CL) when stimulated appropriately . This response reflects the production of highly reactive oxygen derivatives which contribute to oxygen-dependent killing of targets such as pathogens and parasites . From suspensions of pronephric cells of the Rainbow trout, Oncorhynchus mykiss, we have obtained populations enriched for CL-positive cells . Four bands of cells were obtained using continuous gradients generated with 60% Percoll . The leukocytes of band II showed a very strong PMA-induced CL response, the magnitude of which was several times higher than that observed with equivalent numbers of cells form unseparated pronephric cell suspensions . Cells present in other bands were not significantly chemiluminescent . Flow cytometric analysis showed that band II contained large granular cells and small granular cells . Cytochemical analysis showed that this subpopulation was greatly enriched with neutrophils . Many band II cells adhere to glass whereas few band III cells do so . The glass-adherent cells loose their CL potential after a few days in vitro, whereas the nonadherent cells retain their CL responsiveness for at least a week in vitro.

Endocr Res, 1989, 15(1-2), 129 - 49
Isolation and biological activity of corticostatic peptides (anti-ACTH); Zhu Q et al.; Corticostatins (CS's) are a family of low molecular weight peptides, rich in arginine and cysteine with the ability to inhibit ACTH stimulated adrenocortical steroidogenesis . They show a high degree of specificity in that they do not inhibit the action of angiotensin II in the adrenal cortex . Four corticostatins have been isolated from rabbit lung extracts and peritoneal neutrophil extracts and one from human neutrophils . Among them corticostatin I, CSI, is the most potent with an I.D.50 of 25 nM . Corticostatin activity is different from other published inhibitory factors such as TGF-B and ANF, which inhibit basal and angiotensin II stimulated steroidogenesis . Other factors such as macrophage secreted products, and septic shock plasma factors which have not been fully characterized also have suppressive activity on ACTH induced adrenocortical steroidogenesis in vitro . It is not yet clear whether there is any relationship between corticostatins and these macrophage products and shock plasma factor . Corticostatins do not inhibit dbcAMP induced steroidogenesis, however they do inhibit the accumulation of cAMP in response to ACTH in rat adrenal cell suspensions . Binding studies show that CSI is a competitive inhibitor of ACTH, probably acting by blocking, the address recognition site of the receptor . There is wide variation in the potency of the corticostatins ranging from an ID50 of 100 ng/ml for CSI to a completely inactive analog, HP1 . In this paper we will describe the purification of the corticostatins and some recent results obtained on their mechanism of action.

Free Radic Biol Med, 1989, 7(1), 37 - 43
Evaluation of dibromonitrosobenzene sulfonate as a spin trap in biological systems; Samuni A et al.; In the present study dibromonitrosobenzene sulfonate (DBNBS) was examined for its suitability for spin trapping for ESR detection of superoxide radicals in biological systems . This nitroso spin trap recently has been reported to yield very persistent spin adducts with O2 . as well as with various carbon-centered radicals . In the present work the possible toxicity of DBNBS, the partitioning of its spin adducts into cells, and the stability of the adducts and the parent compound inside cells were studied . No significant toxicity was found . In cellular systems, however, DBNBS did not produce detectable adducts with O2.; it also did not detectably trap superoxide generated in the xanthine/xanthine oxidase system . Both DBNBS and a DBNBS adduct performed extracellularly and then added to cell suspensions were rapidly metabolized by cells . Intracellular spin adducts were not detected under any condition . Evidently, in spite of its promising features, DBNBS will not be useful for spin trapping radicals in cellular systems or for detecting superoxide radicals in any biological system.

Free Radic Biol Med, 1989, 6(5), 457 - 66
Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process; Toth KM et al.; Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro . DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro . By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro . Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury . DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes . Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells . In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO . Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions . In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers . Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.

Biomed Biochim Acta, 1989, 48(2-3), S5 - 10
Pathways and control of adenine nucleotide catabolism in anoxic rat hepatocytes; Van Den Berghe G et al.; Studies are reviewed that show that in isolated rat hepatocytes subjected to anoxia, the catabolism of AMP, leading to uric acid instead of to allantoin in normoxia, proceeds almost exclusively by deamination of AMP followed by dephosphorylation of IMP . Adenosine, which is nearly undetectable in normoxic cell suspensions, accumulates to a slight extent in anoxia . The regulatory properties of liver AMP deaminase and cytosolic IMP-GMP 5'-nucleotidase were found to provide protective mechanisms for the hepatic adenine nucleotide pool in hypoxia.

J Clin Invest, 1989 Jan, 83(1), 84 - 9
Vasopressin V1 receptors on the principal cells of the rabbit cortical collecting tubule . Stimulation of cytosolic free calcium and inositol phosphate production via coupling to a pertussis toxin substrate; Burnatowska-Hledin MA et al.; The effects of arginine vasopressin (AVP) on the cytosolic free calcium concentration ({Ca2+}f) were examined in freshly immunodissected rabbit cortical collecting tubule cells using fluorescent Ca2+ indicators fura-2 and indo-1 . The addition of AVP to a cell suspension resulted in a rapid and transient increase in the {Ca2+}f . The 1-deamino-8-D-AVP (dDVP), a V2 receptor agonist of AVP that stimulated adenosine 3',5' cAMP production in these cells, had no effect on {Ca2+}f and did not affect AVP-induced increase in {Ca2+}f . The AVP-induced increase in {Ca2+}f but not cAMP production was blocked by the V1 receptor antagonist, {1-(beta-mercapto-beta-beta-cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine} Arg8-vasopressin . The AVP-stimulated increase in {Ca2+}f appeared to be largely due to Ca2+ release from intracellular stores as reduction of extracellular Ca2+ with EGTA had little if any effect on the AVP-induced increase in {Ca2+}f . This AVP-induced increase in {Ca2+}f was associated with an increase in inositol-1,4,5-trisphosphate production and appeared to involve a guanine nucleotide-binding protein (G), since the pretreatment of cells with pertussis toxin for 4-6 h inhibited this effect . Finally, measurements of {Ca2+}f in single cells suggest that only the principal cells of the collecting tubules respond to AVP with an increase in {Ca2+}f . In summary, these results demonstrate that the principal cells of the cortical collecting tubule possess two distinct receptor systems for vasopressin, the well-known V2 receptor coupled to adenylate cyclase, and a V1 receptor system that leads to the mobilization of cytosolic calcium, coupled through a pertussis toxin substrate (G protein) to a production of inositol phosphates.

Clin Exp Metastasis, 1989 Jan-Feb, 7(1), 69 - 84
Tumorigenicity, invasiveness and metastatic capability of FR3T3 rat cells before and after transfection with bovine papilloma virus type 1 DNA; Coopman P et al.; Fischer rat FR3T3 cells were tested for tumorigenicity, invasive and metastatic capabilities before and after transfection, either with the entire bovine papilloma virus type 1 (BPV-1) genome or with a plasmid (pV69) containing a 69 per cent Bam H1-Hind III fragment of the BPV-1 genome as well as bacterial sequences . Cell lines were grouped as parental, pV69-transfectants, BPV-1 transfectants, in vitro derivatives, and in vivo derivatives . The tumorigenic, invasive and metastatic capabilities of these cell lines were examined in vivo through s.c., and i.p . injections of cell suspensions and through s.c . implantations of cellular aggregates into syngeneic rats . Invasiveness was tested in vitro through confrontations with embryonic chick heart fragments in organ culture . All cell lines including parental lines, were found to be invasive in vitro and tumorigenic in vivo; all tumors were invasive . It is, therefore, not possible to draw conclusions about the role of BPV-1 gene sequences in the acquisition of the invasive phenotype . Transfection with BPV-1 genes conveyed the metastatic phenotype upon parental FR3T3 cells, which were themselves found to be non-metastatic . With regards to this, no differences were found between BPV-1 transfectants compared with pV69 transfectants . Untransfected cells became metastatic also through passage in vivo as an s.c . tumor . The expression of the metastatic phenotype was not noticeably correlated with alterations of growth characteristics of the cell lines . We concluded that the implication of BPV-1 gene sequences in conveying the metastatic phenotype upon FR3T3, if any, was indirect, presumably through alterations of the host cell genome . Our experiments illustrate the need for long-term observations with parental cell lines before drawing conclusions about the role of oncogenes in the acquisition of the malignant phenotype.

Exp Brain Res, 1989, 75(1), 213 - 20
A primate model of Huntington's disease: cross-species implantation of striatal precursor cells to the excitotoxically lesioned baboon caudate-putamen; Isacson O et al.; Ibotenic acid was injected unilaterally into the baboon caudate-putamen (CP) to achieve a neural degeneration model in the primate, with a neuropathology similar to Huntington's disease . Four to six weeks later injections of cell suspensions of striatal precursor cells, obtained by dissection of the fetal rat striatal region (13-15 days gestational age), were made into the excitotoxically lesioned CP of 3 baboons immunosuppressed by Cyclosporin A . Morphological analysis indicated that in one of the baboons, which had the largest lesion of the CP and the shortest survival time (6 weeks after implantation), there was a surviving striatal implant . The implanted neurons grew in high densities in cellular aggregates within the host gliotic CP . These neurons had a neuronal size phenotypical for rat striatum, i.e . on average about a 25% smaller neuronal cell diameter than a similar population in the baboon caudate-putamen . Glial-fibrillary-acid-protein immunoreactivity was present on large astrocytes within the striatal implant, with a distinct border towards the lesion-induced astrogliosis of the host . Neuronal markers for acetylcholinesterase and Leu-enkephalin were distributed in a typical patchy manner in the striatal implants along with fiber staining for tyrosine-hydroxylase-like immunoreactivity (TH) possibly derived from afferent host dopaminergic axons . Some of these fibers in the implants came from intrinsic TH-positive neuronal somata, probably of neocortical fetal origin and transiently expressing the enzyme . In conclusion, the results indicate that neuronal replacement can be achieved by cross-species implantation of fetal striatal precursor cells to the previously neuron depleted primate CP under immunosuppression but that the survival and growth of such implants may be variable and subject to unfavourable trophic conditions.

Acta Med Leg Soc (Liege), 1989, 39(1), 457 - 61
Immunofluorescence detection of digoxin with monoclonal digoxin specific antibody; Sotonyi P et al.; The present study primarily focuses on the analysis of digoxin binding of the heart muscle cells . The primary aim of the investigation was to demonstrate the cardiac glycoside morphologically . The direct immunofluorescence staining technique with digoxin specific monoclonal antibody or Fab fragments and FITC or Texas-Red conjugated antisera are useful for morphological demonstration of digoxin binding and localization in cardiac cells . With the immunofluorescence method, linkage can be observed on the sarcolemma membrane and on the wall of capillaries and arterioles in myocardial cells treated by cardiac glycoside . The specificity of reaction is provided by the negative reaction of cells, not treated by digoxin . Intensity of reaction depends on concentration . The photometric measuring of fluorescence enables the quantitative analysis of cardiac glycoside . It shows the sensitivity of the method in that cardiac glycoside linked to the cell membrane can be detected in the upper sphere of a therapeutic dose . Application of the immunofluorescence method is manifold and relatively simple, and this quick method can be used in diagnoses and in the study of cardiac glycoside receptors of cell membrane . On the basis of our own experiments it is possible to study the kinetics of digoxin linkage . The use of this method is demonstrated for investigation of single cell suspension and cryostat sections.

Acta Histochem, 1989, 86(2), 111 - 5
Detection of islet cell specificity of monoclonal islet cell surface antibodies by means of double-staining immunofluorescence; Witt S et al.; We have generated monoclonal antibodies (mcab) reactive with islet cell surface antigens . 10 different mcab were characterized regarding their islet cell binding specificity by means of a modified double immunofluorescence test . At this assay, the monoclonal islet cell surface antibodies were visualized on rat islet cells by indirect immunofluorescence with fluorescein isothiocyanate-labelled antibodies against mouse immunoglobulin . The alpha and beta cell specificity was determined by indirect immunofluorescence using anti-glucagon or anti-insulin serum and a tetramethyl rhodamine isothiocyanate-labelled second antibody . The target islet cell suspension used contained 61% beta and 23% alpha cells . The monoclonal antibody K28D6 preferentially reacted with alpha cells . The binding of K29aC6 and K56aF3 indicates a high specificity against beta cells . The remaining 7 antibodies were reactive with alpha as well as with beta cells.

Biorheology, 1989, 26(2), 389 - 400
The importance of formaldehyde purity in studies on the electrokinetic charge of human red cells; Nordt FJ et al.; Electrokinetic measurements and rheological studies conducted in parallel have previously shown red cell surface charge to play a role in governing aggregative behavior and bulk flow properties of red cell suspensions . For these and other types of model investigations, aldehyde stabilized cells have been widely used . In this communication, the influence of the purity of formaldehyde was investigated . It was found that (a) the direct dissolution of commercially available paraformaldehyde in water or suitably buffered saline results in impure solutions which, if utilized in the fixation of human erythrocytes, produces cells which have significantly different electrophoretic properties from native cells; (b) the basis for the differences is the presence of metallic impurities in some commercially available paraformaldehyde preparations; (c) the impurities and thus the anomalous electrokinetic properties of the fixed cells may be eliminated by generating formaldehyde gas from paraformaldehyde by heating the latter to 203-210 degrees C; (d) alternatively, the impurities may be eliminated by addition of disodium ethylenediamine tetraacetate dihydrate to fixative solutions prepared directly from paraformaldehyde.

Arch Histol Cytol, 1989, 52 Suppl, 233 - 40
Expression of gastrointestinal endocrine tumours in culture systems; Ahlman H et al.; Human endocrine tumours were studied in in vitro systems (cell suspensions and tissue cultures) and in in vivo systems (tumour transplants to the anterior eye-chamber of immunosuppressed rats) . In the experimental systems the tumour cells were demonstrated to synthesize and secrete the same hormonal products as in the patient . Intraocular transplants of a gastrinoma secreted gastrin-17 into the chamber fluid . This molecule, normally not secreted in the rat, was also detected in the peripheral plasma of tumour-bearing rats . Intraocular transplants of midgut carcinoid tumours released serotonin (5-HT) at adrenoceptor stimulation, of a similar type as demonstrated in acute tumour cell suspensions . However, in tissue cultures genuine beta-adrenoceptors seemed to be modified, since pretreatment with beta-adrenoceptor antagonists or calcium deprivation did not prevent stimulated 5-HT release . Tachykinins were not liberated by adrenoceptor stimulation . In certain cultures of midgut carcinoid tumour cells, two different phenotypes developed: small rounded endocrine tumour cells with positive immunoreactions against 5-HT and tachykinins (TK), and large elongated neuron-like cells, which gradually lost 5-HT immunoreactivity, while TK immunoreactivity remained unchanged . These cultured tumour cells may produce an endogenous factor inducing transformation into a neuron-like phenotype . One candidate factor is nerve growth factor (NGF), since NGF-like immunoreactivity was demonstrated in cells of the endocrine phenotype.

Neuroscience, 1989, 30(3), 779 - 94
Development of dopamine innervation and turning behavior in dopamine-depleted infant rats receiving unilateral nigral transplants; Snyder-Keller AM et al.; Three-day-old rats were bilaterally dopamine-depleted with 6-hydroxydopamine and 3 days later cell suspensions derived from the dopamine-rich ventral mesencephalic area were injected into the right rostral striatum . The transplants rapidly developed a substantial innervation of one striatum, so that by 15 days after transplantation (21 days of age) animals rotated away from the reinnervated side in response to amphetamine . The amount of turning correlated with the extent of innervation of the striatum as determined by tyrosine hydroxylase immunocytochemistry . By 25 days post-transplantation (31 days of age), animals turned in response to stress as well as amphetamine, although this later-developing phenomenon was not associated with any significant change in the extent of dopamine innervation . A second group of animals was bilaterally dopamine-depleted at 3 days of age, but transplantation with nigral cell suspensions was delayed until maturity . Partial reinnervation of the rostral striatum occurred with this delayed transplant paradigm, and turning to both amphetamine and stress commenced at 15 days post-transplantation . In contrast to animals receiving transplants shortly after lesioning, these animals began to turn spontaneously at about 20 days post-transplantation . The serotonin hyperinnervation that occurs following dopamine depletion in infancy was not altered by dopamine transplants made at either time . Results from both groups of transplant animals suggest that dopamine-rich transplants can provide substantial innervation that exerts some functional control over the striatum . This occurs despite the fact that neonatally dopamine-depleted rats, unlike adult-lesioned animals, survive quite well in the absence of striatal dopamine . However, the degree of incorporation into existing circuitry, and the types of regulation that result, may vary considerably depending on the age at which the tissue is transplanted.

Virchows Arch A Pathol Anat Histopathol, 1989, 415(3), 243 - 51
Compaction stasis due to gravitational red cell migration and floatational plasma skimming . Reversal of the fahraeus effect due to pathological RCA-formation in plastic tubes and mesenteric venules; Gobel W et al.; A horizontally aimed microscope was directed at isolated rat mesentery preparations as well as artificial microchannels cast in polyester blocks . They were perfused with aggregating red cell suspensions containing about 7 g/l of bovine fibrinogen at various perfusion pressures . The effects induced by gravitational influences were monitored by measuring the local red cell concentration (video-densitometry) and velocity profiles (IPM-dual slit velocimetry) . At low perfusion pressures, sedimentation during maintained flow occurs, leading to a relative red cell slowing compared with plasma . Consequently, a progressive deposition of red cells at the bottom of vessels occurs and finally, blocking of the vessel by aggregated red cells is seen . Thus, the well-known phenomenon of compaction stasis must be attributed not merely to a transmural plasma loss but also to gravitationally induced haemoconcentration.

Exp Brain Res, 1989, 76(1), 75 - 87
Time-course of recovery of dopamine neuron activity during reinnervation of the denervated striatum by fetal mesencephalic grafts as assessed by in vivo voltammetry; Forni C et al.; In vivo voltammetry was used to monitor dopamine (DA) neuron activity during the course of reinnervation of the initially denervated caudate-putamen by grafted mesencephalic neurons . Fetal DA neurons were implanted as a cell suspension into the depth of the caudate-putamen in adult 6-hydroxy-dopamine-lesioned recipient rats . Recordings were performed over a period of 2.5-4 months, starting within a week after transplantation, using chronically implanted surface-treated multifiber carbon electrodes . The voltammetric method used in this study has generated considerable discussion centred on the ability of the multifiber electrodes to measure DA alone in vivo, but the results of previous studies have led to the conclusion that changes in the voltammetric signal most probably reflect dopaminergic terminal activity . It seems therefore possible to follow the time-course of changes in the voltammetric signal amplitude during the process of dopaminergic reinnervation of the host striatum from the grafts . A 6-hydroxydopamine lesion of the mesostriatal dopamine pathway caused a substantial (greater than 80%) reduction of the voltammetric signal within 8-10 days, and the low residual signal remained essentially unchanged for time periods up to at least 5 months in the non-grafted control rats . In 7 of 11 rats with DA-rich grafts there was a recovery of the signal amplitude to levels within, or close to, the range recorded from the striatum of normal intact rats . The increase was observed 6-8 weeks after grafting in the rats which had received the largest transplants, and at about 13-14 weeks after grafting in the rats which had received the smallest ones . The recovery of the signal amplitude, from baseline to maximal response, was quite rapid and typically developed between two or three recording sessions, i.e . over a period of one to two weeks . In contrast to the intact striatum, the recovered signal in the graft-reinnervated striata showed a progressive decline within one hour of sampling time at high sampling frequencies (1 per min to 1 per 3 min) . Grafted striata also showed a larger response to systemically administered amphetamine than did intact striata . Since the changes in the voltammetric signal recorded with the multifiber electrode mainly reflect dopaminergic terminal activity, the results provide evidence that the intrastriatal DA-rich grafts are spontaneously active, and that the grafted DA neurons can restore DA neuro-transmission in the reinnervated part of the host caudate-putamen to levels which are within the normal range.(ABSTRACT TRUNCATED AT 400 WORDS)

Biosensors, 1989, 4(2), 87 - 108
Apparatus for the electrical characterisation of conductive fluids; Blake-Coleman BC et al.; Non-invasive and fully automated conductimetric measurements of electrolyte and bacterial samples were achieved in a closed volume test cell, comprising a magnetic field coil and detector . By monitoring field induced currents in sample electrolytes the magnitude of the sample current was shown to vary as the inverse of the sample impedance . The impedance characteristic was shown to be that of an LCR resonant circuit . This characteristic is primarily a function of the applied frequency and the solution/cell properties being dependent on the solution conductivity and dielectric permittivity at any given concentration . Small changes in sample dielectric permittivity in the presence of a large background conductivity are shown to be significant . The apparatus described can provide fixed or swept frequency conductivity measurements in the range 1 kHz to 2.25 MHz with a lower conductivity sensitivity of 0.9 x 10(-3) Scm-1 . Bulk impedimetric characteristics of cell suspensions are derived by a two stage measurement.

Exp Brain Res, 1989, 75(1), 195 - 207
Intracerebral xenografts of dopamine neurons: the role of immunosuppression and the blood-brain barrier; Brundin P et al.; Fetal mesencephalic mouse tissue, rich in dopamine neurons, was xenografted as a dissociated cell suspension into the striatum of rats with unilateral 6-hydroxydopamine induced lesions of the mesostriatal pathway . The rats were either assigned to a 10-day, 21-day or 42-day Cyclosporin A (CyA) immunosuppression scheme, or given no immunosuppression . The functional effects of the grafts were followed over 6 months by monitoring changes in the recipient rats' amphetamine-induced turning behaviour . Without immunosuppression no grafts were functional at the end of the experiment . In the 10-, 21- and 42-day CyA treatment groups there was a significant reduction of rotational asymmetry at some timepoint following grafting in 26 of the 33 rats . However, by 6 months only 8 grafts remained functional suggesting that in several rats an immunological rejection took place following the termination of immunosuppression . This was supported by catecholamine histofluorescence analysis which revealed evidence of surviving grafts only in the few rats which had shown sustained functional graft effects at 6 months after grafting . In animals in which the grafts had undergone rejection, there was scar-like tissue in the striatum which appeared more extensive in rats that had lost their grafts after several weeks compared to rats in which the grafts were rejected at an early time-point . In a subgroup of the grafted animals the humoral antibody response against major transplantation antigens present on the grafted cells was investigated . All the studied rats were found to be immunized against the grafted mouse tissue following the intrastriatal implantation . This occurred irrespective of prior immunosuppressive treatment . In a parallel group of rats, the leakage of the blood-brain barrier was studied following intrastriatal implantation of a syngeneic fetal neural cell suspension . Evans Blue was infused into rats 3-12 days following transplantation surgery . At the early time-points there was a marked barrier leakage at the implantation site . This subsided with time such that there was minor leakage after 7-8 days and no leakage after 12 days . In summary, the results indicate the CyA is effective in promoting survival of intracerebral xenografts of fetal neural tissue, but that cessation of immunosuppressive treatment in most cases results in rejection of the grafted tissue . Temporary CyA treatment, even exceeding the time it takes for the blood-brain barrier to reform after transplantation surgery, is thus not sufficient to reliably support long term survival of xenografted dopamine neurons.

J Histochem Cytochem, 1989 Jan, 37(1), 83 - 9
Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies; Bardales RH et al.; The aim of this study was to compare the results of flow cytometric (FCM) determination of heavy and light chain cytoplasmic immunoglobulin (cIg) with those obtained by the peroxidase-antiperoxidase (PAP) method . Fifty-one patients, including five non-T-acute lymphoblastic leukemias, 16 B-chronic lymphocytic leukemias (CLL), 13 non-Hodgkin's lymphomas, seven hairy cell leukemias, four multiple myeloma/plasma cell leukemias, and six T-cell leukemia/lymphomas, as well as 12 normal controls, were studied . Saponin-permeabilized cell suspensions were indirectly stained with monoclonal antibodies and analyzed by flow cytometry . Acetone-fixed cytocentrifuge smears were stained for cIg by the PAP method . The results obtained indicate that: (a) detection of cIg by FCM is a feasible and useful technique to confirm the B-cell lineage of leukemias and lymphomas, particularly those characterized by low-density surface immunoglobulin, such as CLL; and (b) cIg detection by FCM and PAP staining are complementary methods to recognize with certainty the monoclonality of B-cell malignancies.

Fetal Ther, 1989, 4 Suppl 1, 104 - 7
Collection and use of fetal central nervous system tissue; Seiger A; A recent promising development in the field of central nervous system (CNS) tissue transplantation has suggested the use of human fetal CNS tissue from first trimester abortions for xenografting/explantation . Such experiments would certainly expand our knowledge of the normal developmental mechanisms in the human CNS, and allow studies of various indices of maturation and CNS function . However, the suggestion is looked upon with hesitance for ethical, legal and perhaps even for scientific reasons . The initial experiments have been very valuable, though, for our understanding of the structural and functional development of the human CNS, and several legal and ethical concerns have been addressed in working out the procedures for retrieving such tissue . This article tries to put our present knowledge in the right perspective of scientific achievements and potential, legal restrictions and ethical concernsPIP: Some of the recent experimental findings in research and development of fetal central nervous tissue transplantation in Sweden are described, all prerequisites for hope for treatment of human brain, spinal injury and degenerative disease, such as Parkinson's . First human embryonic CNS tissue has been successfully transplanted into immunosuppressed adult rodents, permitting studies of neuronal differentiation, growth, migration and outgrowth of neurites . Second, human CNS grafts have been maintained longterm and followed through structural and functional integration into host neuropil . Studies of the specificity of growth and functional impact are absolutely for potential human tissue transplantation . Using aborted tissue presents legal and ethical problems in some countries, even where induced abortion is legal . The tissue is viable if dissected and used within 2-3 hours . Cell suspensions and tissue fragments from spinal cord and dorsal root ganglia survive in vitro for weeks . The rodent eye chamber has been used as a model in studies of structural and functional differentiation of donor tissue: human CNS tissue survives, vascularizes, proliferates and differentiates for at least 6 months, but does not connect with the host CNS . Eventually experiments in human subjects will be needed to answer the question how does embryonic nervous tissue interact with mature central nervous system in impaired adults .

Med Dosw Mikrobiol, 1989, 41(3-4), 170 - 5
{Properties of phase I purified cellular antigen of Coxiella burnetii}; Kruszewska D et al.; An attempt was made to purify phase I cell suspension of Coxiella burnetii used as an antigen in diagnostic serological tests . Homogenised suspension of chick embryos infected with phase I Henzerling and "Z" strains, after preliminary purification from host cell contaminants of chick embryos was subjected to consecutive centrifugation in sucrose/uropoline gradient and to continuous 20-45% uropoline gradient . The fractions obtained from uropoline gradient centrifugation were applied as phase I antigen C . burnetii in the following tests: complement fixation and microagglutination . Only fractions containing protein were serologically active . They proved to be of similar specificity and sensitivity as the antigens obtained by standard method . Moreover, it was found that after formalin treatment of C . burnetii cells no soluble antigens are liberated which could be detected by complement fixation test.

Lab Delo, 1989, (10), 39 - 41
{A rheological method of diagnosis of multiple sclerosis}; Ierusalimskii AP et al.; A new method for the diagnosis of disseminated sclerosis is suggested, based on the determination of the red cell suspension viscosity within the range of physiologic temperatures . Three viscosity peaks (maxima): at 35, 37-38 (the physiologic), and at 40 degrees C have been detected, probably corresponding to thermotropic phase transitions in red cell membranes . The curves reflecting the eta (t degrees C) dependence in disseminated sclerosis are characteristic of this disease and may be used as a test for the laboratory diagnosis of disseminated sclerosis.

Cytobios, 1989, 58(234-35), 155 - 64
Monocyte- and cytokine-mediated effects on T immunoregulatory activity in the elderly; Antonaci S et al.; Aged individuals exhibit an impairment of T helper and/or T suppressor activity on B cell function in an antibody-specific induction system . Further evidence is now provided that soluble suppressive factors acting on monocytes play a key role in such deficits . In fact, overnight preincubation of isolated monocytes and supplementation of autologous lymphocytes reverses the immunoregulatory imbalance . The suppressive factors are also responsible for a decreased interleukin 2 (IL-2) synthesis since a similar pretreatment of cell suspensions or exogenous human IL-1 and/or IL-2 supplementation of aged cell cultures leads to a recovery of T regulatory effects on B cell differentiation . Similar effects are observed in the presence of thymopentin, a well known IL-2 inducer . Interferon alpha and gamma addition to cultures gives rise to a restoration of T immunoregulatory effects . These findings suggest that several mechanisms are involved in the depressed T immunoregulatory activity in the elderly.

Arch Microbiol, 1989, 152(3), 237 - 43
Characterization of sulfate transport in Desulfovibrio desulfuricans; Cypionka H; Uptake of 35S-labelled sulfate was studied with a new isolate of Desulfovibrio desulfuricans, strain CSN . Micromolar additions of sulfate (1-10 microM or nmol/mg protein) to cell suspensions incubated in 150 mM KCl at -1 degrees C were almost completely taken up and accumulated about 5,000-fold . Accumulation was not influenced by incubation in NaCl instead of KCl, by acidic pH (5.5) or by incubation under air for 10 min . In alkaline milieu (pH 8.5), after prolonged contact with air (2 h), or after growth with excess sulfate or thiosulfate as electron acceptor, the amount taken up was diminished approximately by half . Pasteurization inhibited sulfate uptake completely . With increasing concentrations of added sulfate (0.1 to 2.5 mM) the intracellular concentration increased only slowly up to 25 mM, and the accumulation factor decreased down to 8 . Sulfate transport was reversible . Accumulated sulfate was rapidly lost from the cells after addition of excess non-labelled sulfate or after addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) . The ATPase inhibitor dicyclohexylcarbodiimide (DCCD) specifically inhibited sulfate reduction but had no immediate influence on sulfate accumulation . Addition of the phosphate analogue arsenate (5 mM) was without effect . These results were not in favour of an ATP-dependent transport system . The K+-H+-antiporter nigericin (in 150 mM KCl) and the Na+-H+-antiporter monensin (in 150 mM NaCl) caused partial inhibition of sulfate accumulation, whereas the K+-transporter valinomycin (in 150 mM KCl) and the Na+-H+ exchange inhibitor amiloride (2 mM) were without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Lab Anal, 1989, 3(1), 26 - 33
Influence of methisoprinol (isoprinosine) on HIV-infected human lymphocytes: in vitro immunological, virological, and ultrastructural studies; De Simone C et al.; Methisoprinol (isoprinosine) is a synthetic compound with reported antiviral and immunomodulating properties . Results of the present study showed that methisoprinol at concentrations greater than or equal to 200 micrograms/ml reduces the p24 and gp120 viral antigen expression on the surface of human immunodeficiency virus (HIV)-infected lymphocytes and the reverse transcriptase levels . In addition, cell viability, the number of the CD4+ cells, and the CD4+/CD8+ cell ratio are higher in methisoprinol-pretreated cell suspensions than in untreated HIV-infected cell cultures . A quantitative freeze-fracture study on the density of the intramembranous particles (IMP) present on both fracture faces of the plasma membrane of lymphocytes has shown that pretreatment with methisoprinol induces a different molecular organization resulting in a nearly three times increase of IMP density.

Headache, 1989 Jan, 29(1), 46 - 8
Basophil histamine release and leukotriene (LTB4-LTC4) production in cluster headache; Martelletti P et al.; Histamine release and leukotrienes (LTB4 and LTC4) production from circulating basophil have been studied in 13 patients with episodic cluster headache (CH) during the remission phase of symptoms, and in 9 normal subjects . Cell suspensions of basophils were stimulated with scalar dilutions of anti-IgE, f-met-peptide and Ca2+ ionophore A23187 . Histamine was measured by an automated fluorimetric method; LTB4 and LTC4 with RIA in individual cases, and with Reverse-Phase HPLC in the two pools obtained from the supernatants of CH patients and controls . Mean values of histamine release in patients with CH were significantly lower when compared to those obtained in control subjects after stimulation with anti-IgE at the three dilutions used . LTC4 mean levels measured in CH patients were significantly lower than those of supernatants from controls after stimulation with 0.05 gamma/ml of A23187 . A reduction of LTC4 and LTB4 levels in CH patients was also observed in R-P HPLC, which showed different elution patterns in the two groups . The histamine release in individual cases was related to leukotriene production: LTC4 levels were significantly (p less than .05) higher in "high histamine releasers" than in "low histamine releasers" . Our results indicate that CH patients have complex abnormalities of histamine release and of leukotriene production during the painless phase of the disease.

Scand J Immunol, 1989 Jan, 29(1), 65 - 72
Increased natural killer cell activity and numbers of Leu-7 and Leu-11b (CD 16)-positive cells in bone marrow of children in remission from acute lymphoblastic leukaemia; Sorskaar D et al.; Natural killer (NK) cell activity and related markers were analysed in childhood acute lymphoblastic leukaemia (ALL) . Children with untreated ALL, children with active disease, and children in remission for less than 1 month and undergoing induction therapy had significantly lower NK cell activity in peripheral blood than the control group (P less than 0.05, P = 0.0005, and P less than 0.0025) . Patients in remission for 1-3 months and undergoing consolidation chemotherapy had normal NK activity (P greater than 0.05) . Children in complete remission for more than 3 months and undergoing maintenance therapy also had a normal NK activity in their peripheral blood (P greater than 0.05) . However, their bone marrow cells showed an increased NK cell activity (P less than 0.0005) . Cells positive for the Leu-7 marker were reduced in the peripheral blood from untreated children (P less than 0.025) and children in remission for less than 1 month (P = 0.025) . The percentage of cells from peripheral blood expressing the marker Leu-11b (CD 16) did not differ significantly from that of the controls (P greater than 0.05) . However, children in complete remission for more than 3 months had a higher number of bone marrow cells expressing the Leu-7 (P = 0.005) and the Leu-11b (CD 16) markers (P = 0.05) than controls . Stimulation of mononuclear cell suspensions with recombinant alpha interferon and recombinant interleukin 2 were shown to cause a normalization of the NK cell activity in peripheral blood and bone marrow.

Lung, 1989, 167(1), 1 - 10
Isolation of bovine type II pneumocytes in high yield and purity; Augustin-Voss HG et al.; A method has been developed for the isolation of bovine type II pneumocytes by enzymatic tissue dissociation and subsequent density gradient centrifugation . After mechanical defibrination, the crude cell suspension contains 71.9 +/- 26.6 X 10(7) cells (viability greater than 95%) with a type II cell purity of 39.4 +/- 10.9% . Due to their low buoyant density, bovine type II pneumocytes can be purified in a single step to 95.7 +/- 1.7% by discontinuous density gradient centrifugation on a 1.040 g/ml Percoll gradient . Isolated cells are identified by light and fluorescence microscopy that show their characteristic intracytoplasmatic surfactant granules . The fine structure of the surfactant lamellar bodies is examined in ultra thin sectioned and freeze-fractured type II pneumocytes . Isolation of bovine type II pneumocytes for the in vitro study of the surfactant system offers an alternative to the use of laboratory animals and provides an ideal system for the isolation of any desired cell number from one animal by simply increasing the size of the lung segment to be trypsinized.

Biochem Pharmacol, 1989 Jan 1, 38(1), 85 - 90
Evidence for intracellular superoxide formation following the exposure of guinea pig enterocytes to bleomycin; Turner MJ 3rd et al.; Spin trapping of free radicals during the exposure of guinea pig enterocytes to bleomycin (BLM) was investigated using an in vitro cell suspension . The spin traps employed in this study were 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 3,3-diethyl-5,5-dimethyl-1-pyrroline-1-oxide (DEDMPO) . The hydroxyl radical spin-trapped adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed with DMPO . In the presence of dimethyl sulfoxide (DMSO), the only 2,2,5-trimethyl-1-pyrrolidinyloxyl (DMPO-CH3) observed was that expected from hydroxyl radical formation by the decomposition of the superoxide spin-trapped adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) . Production of hydroxyl radical was not detected in the presence of DEDMPO, which is a nitrone that will spin trap hydroxyl radical, but not superoxide, at cellular concentrations . Thus, these data indicate that superoxide was produced during the exposure of guinea pig enterocytes to BLM and that DMPO-OH resulted from the cellular bioreduction of DMPO-OOH by glutathione peroxidase . Addition of superoxide dismutase to the in vitro reaction mixture indicated that superoxide production was intracellular.

Am J Clin Pathol, 1989 Jan, 91(1), 67 - 71
A combined cytochemical and immunocytochemical method for simultaneous visualization of cytoplasmic enzyme reactivity and cell surface antigens in cell suspensions; Gloghini A et al.; Immunocytochemical methods were used in combination with enzyme cytochemistry to visualize simultaneously cytoplasmic enzyme reactivity (for dipeptidyl{amino}peptidase {DAP IV}, acid phosphatase {AcP}, chloroacetyl esterase {CAE}) and cell surface antigens (Leu-3a, Leu-4, Leu-14, Leu-M1, OKT4, OKT8, OKB7) in cytospin preparations from cell suspensions of human reactive lymphoid tissues (four lymph nodes and three tonsils) . Different fixative solutions were tested . Enzyme and immunocytochemical reactions were carried out in different orders of sequence to establish which was the better direction for the combination of the two methods . The following immunocytochemical methods were tested: three stages, avidin-biotin complex, peroxidase-antiperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) (using both peroxidase and alkaline phosphatase as labeling enzyme) . Acetone or buffered formalin acetone gave the best results both for cytochemical and immunologic reactions . DAP IV and AcP reactivities could be visualized only when cytochemical reactions were performed before immunocytochemistry . CAE reactivity could be demonstrated either before or after immunocytochemistry . Cell surface antigens could be demonstrated with most immunocytochemical methods: however, the APAAP method was preferred for its sensitivity and effectiveness when combined with enzyme cytochemistry . By this approach, cells expressing only immunologic markers and cells expressing only cytochemical markers could easily be distinguished from those coexpressing both markers, because cytochemistry and immunocytochemistry could be combined without affecting the reactivity of each marker, and the reaction products did not hamper the interpretation of preparations.

Cancer Immunol Immunother, 1989, 28(1), 17 - 21
Auto-tumor recognition following in vitro induction of MHC antigen expression on solid human tumors: stimulation of lymphocytes and generation of cytotoxicity against the original MHC-antigen-negative tumor cells; Vanky F et al.; Expression of major histocompatibility complex (MHC) class I antigens was induced in eight out of nine freshly prepared tumor cell suspensions by exposure to interferon (IFN gamma) and tumor necrosis factor (TNF alpha) in vitro . The untreated, class-I-antigen-negative, and the treated, antigen-positive, cells of three tumors (one breast carcinoma, one plasmocytoma and one ovarian carcinoma) were compared for the capacity to stimulate autologous and allogeneic blood lymphocytes, to generate auto-tumor cytotoxicity and for sensitivity to the lytic effect induced in autologous mixed lymphocyte tumor cell culture (MLTC) . The MHC class I-negative cells did not stimulate, while the cells induced for expression of antigens did . On the other hand, when the autologous cytotoxic cells were generated in the MLTC by the class I antigen-positive tumor cells the class I-negative tumor cells were also damaged . Lysis of the class-I-positive tumor cells was abrogated by the W6/32 monoclonal antibody directed against the monomorphic part of the class I molecules.

Biochim Biophys Acta, 1988 Dec 16, 963(3), 401 - 13
Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells; Holtzman MJ et al.; To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid . Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry . In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol . By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha . Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase . Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity . In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2 . The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.

Biochem Pharmacol, 1988 Dec 15, 37(24), 4763 - 74
Immunochemical analysis of acetaminophen covalent binding to proteins . Partial characterization of the major acetaminophen-binding liver proteins; Bartolone JB et al.; A sensitive immunoassay for detecting acetaminophen (APAP) bound to proteins was developed using an affinity purified antibody directed against the N-acetylated end of the APAP molecule . Western blots of electrophoretically resolved liver proteins taken from mice given an hepatotoxic dose of APAP demonstrated that nearly 85% of the total detectable protein-bound APAP was covalently associated with proteins of 44 and 58 kD . Pretreatment of liver extracts with the sulfhydryl-specific reagent, N-ethylmaleimide (NEM), prior to derivatization with the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), greatly reduced immunochemically detectable APAP-protein adducts and indicated that the antibody detects protein-thiol conjugates of APAP . To investigate the basis of the binding selectivity in vivo, a variety of systems which yielded APAP-protein adducts were analyzed . Systems which activate APAP enzymatically, as in hepatocyte suspensions or in post-mitochondrial (S9) fractions fortified with an NADPH-regenerating system, resulted in a protein binding profile similar to that produced in vivo . Conversely, when extracts or cells were treated with chemically synthesized NAPQI, an alternative protein binding profile was obtained . Two-dimensional electrophoretic analysis of the reduced protein thiol (PSH) content of liver proteins using {3H}NEM labeling revealed that the 58 kD APAP-binding proteins were rich in PSH, whereas the major 44 kD binding protein had virtually no detectable PSH . Many PSH-rich proteins that were not arylated in vivo did bind NAPQI in vitro . However, the 44 kD proteins were not arylated when chemically synthesized NAPQI was added to homogenates or cell suspensions . The present data further suggest that, in addition to the amount and reactivity of free protein sulfhydryls, the cellular localization with respect to the cytochrome P-450 activation site may influence the susceptibility of proteins to NAPQI binding . These findings signal the need for caution in interpreting studies of APAP mechanisms that rely solely on NAPQI addition.

Eur J Biochem, 1988 Dec 15, 178(2), 519 - 25
Electron-transport-driven sodium extrusion during methanogenesis from formaldehyde and molecular hydrogen by Methanosarcina barkeri; Muller V et al.; Methanogenesis from formaldehyde or formaldehyde + H2, as carried out by Methanosarcina barkeri, was strictly dependent on sodium ions whereas methane formation from methanol + H2 or methanol + formaldehyde was Na+-independent . This indicates that the reduction of formaldehyde to the formal redox level of methanol exhibits a Na+ requirement . During methanogenesis from formaldehyde, a delta pNa in the range of -62 mV to -80 mV was generated by means of a primary, electron-transport-driven sodium pump . This could be concluded from the following results obtained on cell suspensions of M . barkeri . 1 . The addition of proton conductors or inhibitors of the Na+/H+ antiporter had no effect on sodium extrusion . 2 . During methanogenesis from formaldehyde + H2 a delta psi of -60 mV to -70 mV was generated even in the presence of proton conductors . 3 . ATPase inhibitors, applied in the presence of proton conductors, had no effect on primary sodium extrusion or generation of a delta psi . Evidence for a Na+-translocating ATPase could not be obtained.

FEBS Lett, 1988 Dec 5, 241(1-2), 149 - 53
Phorbol 12-myristate 13-acetate modulates the cAMP-induced light-scattering response of a Dictyostelium discoïdeum cell population; Thiery R et al.; The effect of phorbol 12-myristate 13-acetate upon the light-scattering response to cAMP of a D . discoideum cell suspension was investigated . It was found that the first spike of the cAMP-mediated light-scattering change (peaking at about 15-20 s after stimulation) was inhibited by the phorbol ester . This effect was concentration dependent with an half-maximum value for the inhibition of 4 nM . The inhibition was found to be maximal after a 10-20 min incubation time . The phorbol ester was shown to affect the dose-response relationship between the cAMP concentration and the relative amplitude of the light-scattering change, more by decreasing the number of cAMP receptors than by decreasing their apparent affinity for cAMP.

Scand J Immunol, 1988 Dec, 28(6), 667 - 73
T-cell response to purified protein derivative after removal of Langerhans' cells from epidermal cell suspensions containing keratinocytes expressing class II transplantation antigens; Tjernlund U et al.; In a previous study we observed that human epidermal cell (EC) suspensions containing HLA-DR-expressing keratinocytes showed an amplified T-cell response to purified protein derivative (PPD) . To evaluate further the possible immunological importance of class II transplantation antigens on keratinocytes we have compared the T-cell response to PPD in the presence of the following stimulator cells: EC suspensions from normal skin, or EC from tuberculin-reactive skin with or without removal of Langerhans' cells . The proliferation of purified T lymphocytes from peripheral blood in response to PPD in the presence of various concentrations of autologous EC was measured by {3H}thymidine incorporation on day 6 . In 3 experiments out of 4 the EC from tuberculin-reactive skin, containing 28-76% HLA-DR-expressing cells as judged by immunocytochemistry (which also revealed fairly numerous HLA-DQ/-DP-expressing keratinocytes and a slight increase in CD36- and CD4- but not CD1-expressing cells), induced a more pronounced T-cell response to PPD than did normal EC . This was not the case in the fourth experiment, in which a small number of HLA-DR-(15%) and few if any HLA-DQ-/-DP-expressing keratinocytes were found . Immunomagnetic removal of CD1-reactive Langerhans' cells from the tuberculin-reactive EC suspensions resulted in a reduction of the T-cell response to PPD, in most cases down to background level (T cells alone + PPD) . This study does not support the hypothesis that HLA-DR-expressing keratinocytes can in themselves act as antigen-presenting cells.

J Periodontol, 1988 Dec, 59(12), 811 - 5
Human gingival Langerhans cells as accessory cells in mitogen induced T cell responses; Newcomb GM et al.; Peripheral blood and gingival tissue was collected from patients undergoing surgery for the treatment of periodontal disease . Both unfractionated peripheral blood mononuclear cells and a T cell-enriched, monocyte-depleted lymphocyte population were isolated from the blood samples while a gingival epithelial cell suspension was prepared from the oral aspect of the gingival tissue . The blood cells (1 X 10(5)) were then co-cultured with the gingival epithelial cells (1 X 10(5) and 1 X 10(4)) in 200 microliters aliquots in microtitre trays for three days to see if the epithelial cells could act as accessory cells in the lymphocyte response to the mitogen, phytohaemagglutinin (PHA) . Lymphocyte proliferation was assessed by measuring the uptake of {6-3H} thymidine during the last 18 hours of culture . Removal of monocytes from peripheral blood mononuclear cells led to a significant, but not total, reduction in the proliferative response to PHA . When gingival epithelial cells were added to the T cells they restored their response to PHA in a dose dependent fashion . As Langerhans cells were the only potentially immunocompetent cells seen in the epithelial cell suspensions, it is concluded that these cells are acting as accessory cells in the T cell response to PHA.

J Invest Dermatol, 1988 Dec, 91(6), 547 - 52
Simultaneous colloidal gold immunoelectronmicroscopy labeling of CD1a, HLA-DR, and CD4 surface antigens of human epidermal Langerhans cells; De Panfilis G et al.; The simultaneous demonstration of three surface antigens of Langerhans cells (LC) within LC-enriched fresh epidermal cell suspensions from normal human skin was achieved, by means of a triple immunogold (IG) staining, using commercially available monoclonal antibodies (moAb) and immunoreagents, in a simple pre-embedding immunoelectronmicroscopy (IEM) procedure . As a result, suspended LC were triple-stained as follows: gold particles of 40 nm revealed the CD1 a antigen; gold particles of 20 nm revealed the HLA-DR antigen; and gold particles of 5 nm revealed the CD4 antigen . All the observed epidermal Birbeck granule-bearing LC were triple IG stained, thus simultaneously expressing the three surface differentiation antigens, which are therefore different from but coexisting with each other . The present investigation assesses the constant simultaneous expression by Birbeck granules bearing LC of not only CD1a and HLA-DR antigens, but also CD4 antigen . The occurrence is therefore excluded of both CD1a-positive HLA-DR-negative LC subpopulation and CD4-negative LC subpopulation, presumably due to the different sensitivity of the various procedures performed . The hypothetical occurrence of CD4-positive, CD1a-, and/or HLA-DR-negative LC subpopulations is ruled out . This study reaffirms indeed the high specificity and sensitivity of the IG-IEM method for a precise detection of the cell surface antigens of LC, and states the suitability of the IG labeling even for accurate multiple IEM stainings of LC.

Cancer Res, 1988 Dec 1, 48(23), 6708 - 14
In vitro and in vivo activation of B-lymphocytes: a flow cytometric study of chromatin structure employing 7-aminoactinomycin D; Stokke T et al.; The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry . Resting peripheral blood B- and T-lymphocytes (G0 cells) bound low amounts of 7-AMD (7-AMD- phenotype), while G1 REH cells and purified B-cells stimulated with anti-mu + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD+ phenotype) . 7-AMD binding increased up to threefold and the differences in binding between G0 and G1 cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1 . 7-AMD binding increased in parallel with autofluorescence and approximately linearly with time during the G0-G1 transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor . In cell suspensions from lymph node biopsies, the 7-AMD+ phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9) . The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD- cells . All tumors were diploid . The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (P less than 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity . Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (P = 0.07), while the light scattering (cell size) of G0/G1 cells was highly correlated to 7-AMD binding (P less than 0.001).

Vet Immunol Immunopathol, 1988 Dec, 20(1), 75 - 85
Intestinal absorption of colostral lymphoid cells in newborn piglets; Tuboly S et al.; Intestinal absorption of colostral lymphoid cells was studied in 23 piglets of four sows (sows A, B, C and D) . From the colostrum and blood of the sows the lymphoid cells were isolated with Ficoll-Paque and labelled with technetium (Na99mTcO4) . In the 7th hour after birth, 5-ml volumes of the cell suspensions were injected, following laparotomy, directly into the stomach (piglets of sow A) or into the jejunum (piglets of sow B), whereas piglets of sows C and D received the suspensions through a naso-oesophageal tube . Cryostat sections of duodenum, jejunum and lymph node samples of piglets killed by bleeding 8 h after the treatment were examined by autoradiography . It was found that lymphoid cells present in the colostrum of a piglet's own mother were absorbed from the digestive tract and, via the lymphatic vessels, were transported to the mesenteric lymph nodes . Electron microscopy revealed that absorption took place intercellularly . Colostral cells of sows other than a piglet's own mother were observed only in the epithelial layer of the mucous membrane . The lymphoid cells isolated from the sows' blood and heat-treated colostral lymphoid cells were not absorbed . The results indicate that in the pig, an animal having an epitheliochorial placenta, the colostral lymphoid cells are absorbed from the digestive tract and, hence, they can confer an active cellular immunity on the newborn piglets.

J Vet Pharmacol Ther, 1988 Dec, 11(4), 345 - 53
Comparative effects of etomidate and its fluoro analogue, R 8110 on testicular, adrenal and ovarian steroid biosynthesis; De Coster R et al.; The effects, of etomidate and of its fluoro analogue, R 8110, on adrenal, testicular and ovarian steroid biosynthesis were compared using cultures of guinea-pig adrenal, rat adrenal capsular, rat testicular and rat ovarian granulosa cells . At a concentration of 100 nM, etomidate inhibited the adrenal 11-hydroxylation of glucocorticoid and mineralocorticoid biosyntheses, producing a decrease in cortisol and corticosterone and an accumulation of 11-deoxycortisol and 11-deoxycorticosterone in guinea-pig adrenal and rat capsular adrenal cell suspensions, respectively . At higher concentrations (greater than 10(-6) M), etomidate also inhibited ovarian oestradiol production, testicular androgen formation and ovarian progesterone synthesis . The latter action suggests an effect on ovarian aromatase, on testicular 17 alpha/17,20-lyase activities and finally on cholesterol side-chain cleavage . The fluoro analogue of etomidate, R 8110, was ten times less potent as an inhibitor of 11-hydroxylation and affected progesterone formation only slightly in adrenal cell suspensions . Testosterone production was less affected by R 8110 than by etomidate . The increase of progestins suggests that the 17 alpha/17,20-lyase activities are the most sensitive testicular enzymatic reactions to R 8110 . For inhibition of ovarian oestradiol production, R 8110 was twenty times more potent than etomidate.

Exp Neurol, 1988 Dec, 102(3), 280 - 9
Fine structural correlates of vascular permeability of chromaffin cell transplants in CNS pain modulatory regions; Pappas GD et al.; Adrenal medullary tissue, bovine chromaffin cells, and PC12 cells were transplanted into the pain modulatory regions of the rat midbrain periaqueductal gray (PAG) or dorsal spinal cord . Fine structural studies of vascular permeability of these grafts revealed that in all three cases, the capillary endothelium of the graft vasculature was attenuated and fenestrated, unlike that of the surrounding host CNS tissue . The intravascular injection of the protein marker, horseradish peroxidase (HRP), enters the grafted tissue parenchyma and is found in the extracellular space of the surrounding host CNS . In contrast, control gelfoam transplants, which become vascularized, do not contain vessels with fenestrated endothelium and do not leak HRP . Since cell suspension implants do not contain endothelial cells, the vasculature of the grafts must be derived from the host . However, as their morphological characteristics are similar to those of the in situ adrenal medulla, it appears that the tissue environment of the graft influences the permeability properties of the vascular bed . The increased permeability to HRP is apparently permanent and most likely is due to the passage through endothelial cell fenestrae.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 9327 - 30
Suppression and induction of epileptic activity by neuronal grafts; Buzsaki G et al.; Fetal rat brain cell suspensions prepared from either the locus coeruleus region or hippocampus were implanted bilaterally into the subcortically denervated seizure-prone hippocampus of adult rats . Animals with locus coeruleus grafts were protected against picrotoxin-induced behavioral seizures and had significantly fewer interictal spikes . In contrast, in rats with fetal hippocampal grafts the incidence of interictal spikes was significantly higher than in lesion-only controls, and spontaneous behavioral seizures occurred in almost half of the animals . We suggest that neuronal grafting offers an alternative method for studying the mechanisms and control of epileptic brain activity.

Exp Hematol, 1988 Dec, 16(11), 916 - 21
Serotonin incorporation as a marker of murine megakaryocyte proliferation in liquid cultures; Vannucchi AM et al.; We have developed a new in vitro method for the quantitation of murine megakaryocyte proliferation that is based on the unique property of megakaryocytes to incorporate and store {14C}serotonin in cytoplasmic dense granules . The specificity of the assay was demonstrated by autoradiography of whole bone marrow cell suspensions, which showed evidence of grain accumulation only in megakaryocytes . Bone marrow cells were cultured in liquid cultures in the presence of a stimulator of megakaryocyte growth before the addition of 2.5 microM {14C}serotonin . The amount of serotonin incorporated in cells was evaluated after 3 h . Radioactivity peaked at days 6 and 7 and remained high until day 10; there was a linear relationship between the incorporation of serotonin and the number of cells plated . A dose-response curve between the incorporation of serotonin and the concentration of pokeweed mitogen spleen-conditioned medium (PWM-SCM) was observed, with inhibitory effects becoming predominant at the highest concentrations . The proliferation of megakaryocyte progenitors was also stimulated by partially purified interleukin 3, whereas both human recombinant erythropoietin and human recombinant granulocyte colony-stimulating factor (rG-CSF) failed to modify the incorporation of serotonin in comparison with unstimulated cultures . Finally, in parallel experiments we observed a significant correlation between the number of megakaryocytic colonies grown in agar and the radioactivity in liquid cultures . The method described herein is reproducible, sensitive, and easy to perform; it should be useful for the study and purification of factors affecting megakaryocyte proliferation.

Scand J Immunol, 1988 Dec, 28(6), 735 - 40
An altered response by psoriatic keratinocytes to gamma interferon; Baker BS et al.; To determine whether psoriatic keratinocytes differ from normal keratinocytes in their response to gamma interferon, epidermal cell suspensions from normal and from lesional and uninvolved psoriatic skin were cultured in the presence of gamma interferon and the induction of HLA-DR expression and inhibition of cell growth were measured . The addition of 10(2) units of gamma interferon/ml during a 7-day culture period significantly increased mean HLA-DR+ cell numbers in 21 epidermal suspensions of normal from 3.9 to 24.1% (P less than 0.0001), uninvolved psoriatic from 8.4 to 33.1% (P less than 0.0001), and to a lesser extent lesional psoriatic biopsies from 12.6 to 18.3% (P less than 0.01) . However, the increase in HLA-DR+ cell numbers in these latter cultures was significantly less than that observed in either normal or uninvolved psoriatic epidermal cell cultures (P less than 0.0001) . Furthermore, {3H}thymidine incorporation was substantially decreased by gamma interferon in 16 out of 22 (73%) cultures of normal epidermal cells; this decrease was statistically significant (P less than 0.01) . In contrast, only 4 out of 11 (36%) lesional and 9 out of 21 (43%) uninvolved psoriatic epidermal cultures showed comparable inhibition of proliferation . These findings suggest that psoriatic keratinocytes have an altered response to gamma interferon; this could explain the infrequency of keratinocyte HLA-DR expression in psoriatic plaques in vivo and may also contribute to the increased epidermal proliferation that characterizes this disease.

J Virol Methods, 1988 Dec, 22(2-3), 165 - 72
Use of heat inactivated viral haemorrhagic fever antigens in serological assays; Saluzzo JF et al.; Heating for 1 h at 60 degrees C completely destroyed the infectivity of sucrose-acetone-extracted antigen of Rift Valley (RVF) and Congo Crimean haemorrhagic fever (CCHF), as well as of RVF- and CCHF-infected mouse brain . These antigens could be successfully used, however, for complement fixation and IgM-capturing enzyme immunoassay . Vero E6 cell suspensions infected with hantaviruses such as Hantaan 76-118, Tchoupitoulas, SR 11, GB-B, CG 18-20, Hallnas, CG 13891, Seoul and Prospect Hill, as well as Vero cells infected with CCHF and RVF viruses, were completely inactivated after heating for 1 h at 60 degrees C . Indirect immunofluorescent antibody test results obtained on slides prepared with heat-inactivated cell suspensions correlated well with results obtained on slides prepared with unheated cell suspensions . Inactivation is a simple, rapid, economic and reproducible method for inactivation of hantaviruses and CCHF and RVF viruses, with preservation of the ability to react specifically with antibodies.

Neurosci Res, 1988 Dec, 6(2), 162 - 6
Reorganization of cerebellar cell suspension transplanted into the weaver mutant cerebellum and immunohistochemical detection of synaptic formation; Kohsaka S et al.; Dissociated cells prepared from the cerebellar primordia of normal 15-day mouse embryos were grafted into the cerebellum of 1-month-old weaver mutant mice which are characterized by degeneration of cerebellar granule cells during the early postnatal period . The growth of the grafted cells was investigated at 1 month after the operation . Implanted cells were highly developed to form a large mass of tissue in the host cerebellar folia . Histological examination revealed that a trilaminar cortical structure was partially developed in certain areas of the grafted tissue . The implanted granule-like cells were labeled with {3H}thymidine which was injected into the host, suggesting that the granule-like cells actively proliferate in the host cerebellum after the transplantation . In this area, strong immunoreactivity with synapsin I was detected indicating that the dissociated granule cells of the cerebellar primordia are able to develop a synaptic organization in the weaver mouse cerebellum.

J Invest Dermatol, 1988 Dec, 91(6), 603 - 5
Langerhans cells in S-phase in normal skin detected by simultaneous analysis of cell surface antigen and BrdU incorporation; de Fraissinette A et al.; We report on a double immunofluorescence staining for the detection of the Langerhans Cell (LC) population in S-phase . After trypsinization, the epidermal cell suspensions were enriched for LC and exposed to 10 microM BrdU for 2 h . Studies of BrdU labeled cells included the determination of their cell surface phenotype . Both membrane labeling and incorporated BrdU as revealed using anti-BrdU demonstrated the BrdU as revealed using anti-BrdU demonstrated the presence of LC in S-phase . We observed 6% of the epidermal LC in S-phase . This is a proof that LC are able to proliferate in the epidermal microenvironment . This technique, besides being rapid and free of radioactivity, allows the cytokinetic study of phenotypically defined LC in heterogeneous epidermal cell populations.

Am J Pathol, 1988 Dec, 133(3), 507 - 15
Analysis of a murine B cell lymphoma, CH44, with an associated non-neoplastic T cell population . I . Proliferation of normal T lymphocytes is induced by a secreted product of the malignant B cells; Willoughby PB et al.; A non-neoplastic T cell population associated with a murine monoclonal B cell malignancy, CH44, was analyzed . Immunofluorescence on cell suspensions and immunoperoxidase staining on tissue sections using monoclonal antibodies to the antigens Thy1.2, Ly-1, L3T4, and Lyt-2 confirmed the presence of both TH (Ly-1/L3T4+, Lyt-2-) and Tc/s (Ly-1/L3T4-, Lyt-2+) T cell subpopulations . The non-neoplastic T cells were present in both a 0.6 and 2.1 g CH44-bearing spleen . T cells, not normally in liver in significant numbers, were found in liver tissue when the CH44 tumor cells were present . These data implied an active proliferation of the T cell populations within tissues containing the malignant B cells . Supernatant from an in-vitro-adapted cell line of CH44 (CH44.LX) was tested for its ability to induce proliferation of normal murine splenocytes and thymocytes . As assayed by tritiated thymidine incorporation, both spleen and thymus cells proliferated in the presence of CH44.LX supernatant . Although supernatant from two of nine other B cell lines was able to stimulate the proliferation of spleen cells, only CH44.LX could induce proliferation of thymus cells . Supernatant from the seven other B cell lines and three hybridomas had no measurable effect on either splenocytes or thymocytes in this assay . It is hypothesized that the presence of a non-neoplastic proliferating T cell population associated with a neoplastic B cell lymphoma during in vivo passaging of the tumor is the result of effects derived from a secreted product of the malignant B cells . Whether the T cells have any effect on the growth of the malignant B cells is not known.

J Parasitol, 1988 Dec, 74(6), 985 - 92
Taenia taeniaeformis: cellular reconstruction of athymic mice and role of L3T4+ helper T lymphocytes in the early infection; Letonja T et al.; The role of T helper lymphocytes (L3T4+) in the early response to Taenia taeniaeformis metacestodes was investigated . Athymic BALB/c-nu/nu mice (susceptible) were inoculated intraperitoneally with the following cell populations from congenic BALB/c-nu+ + mice (resistant): (a) whole spleen single cells, (b) thymus single cell suspensions, or (c) spleen cells pretreated with anti-L3T4 monoclonal antibody before the injection . The mice were given 3 weekly injections of cells and then infected orally with 300 eggs 7 days after the last injection . Cryostat sections of the liver from the infected mice were examined at 6 days postinfection (PI) for parasite viability, the numbers of eosinophils, and L3T4+ T lymphocytes present within 100 micron of the parasite and for the presence of biotin in hepatocytes (involved in biosynthesis of fatty acids) around the parasite . The success of the cellular reconstitution of athymic mice with the lymphoid cells was measured by a T-cell mitogenic assay with concanavalin A (ConA) . The cellular reconstitution of athymic mice with a mixture of lymphoid cells from the spleen and thymus of BALB/c-nu/ + mice resulted in both parasite death and eosinophil infiltration . Reconstitution with mature splenic cells alone resulted in a greater parasite killing and eosinophil infiltration as compared to reconstitution with thymic cells . The better reconstitution with splenic cells was reflected in a greater mitogenic response to ConA.(ABSTRACT TRUNCATED AT 250 WORDS)

Hum Pathol, 1988 Dec, 19(12), 1434 - 43
Monoclonal antibody Leu-22 (L60) permits the demonstration of some neoplastic T cells in routinely fixed and paraffin-embedded tissue sections; Wieczorek R et al.; Monoclonal antibody Leu-22 (L60) detects a T cell-associated antigen which is stably expressed in routinely fixed and paraffin-embedded tissue sections . We investigated the utility of monoclonal antibody Leu-22 to immunophenotype routinely processed lymphoid neoplasms by determining its reactivity in 105 archival pathologic specimens of lymphoid neoplasia that had been previously immunophenotyped by standard cell suspension and frozen tissue section techniques . Monoclonal antibody Leu-22 reacted with 69% of T cell non-Hodgkin's lymphomas (NHLs), including cases belonging to each of the major clinicopathologic categories, and with 22% of B cell NHLs, but did not react with the Reed-Sternberg (RS) cells of Hodgkin's disease (HD) . We concluded that monoclonal antibody Leu-22 reacts preferentially but not exclusively with T cell NHLs . Therefore, we performed parallel analyses of the same 105 cases with monoclonal antibodies leukocyte common antigen (LCA), Leu-M1, LN1, and LN2, which detect various paraffin-resistant antigens, and of 80 of these cases with monoclonal antibody UCHL1, which detects a paraffin-resistant T cell-associated antigen . UCHL1 reacted with 61% of the T cell NHLs studied . Sixty-nine percent of T cell NHLs expressed the LCA+, Leu-22+ or Leu-M1+, LN1- phenotype and 47% of B cell NHLs expressed the LCA+, Leu-22-, Leu-M1-, LN1+ phenotype . These phenotypes had a false-positive rate of only 7% . The substitution of UCHL1 for Leu-22 or the combined use of UCHL1 and Leu-22 in this panel did not improve our ability to correctly predict the T cell phenotype of these lymphoid neoplasms . LN1 and LN2 reacted with 13% and 56% of T cell NHLs, respectively, and LN2 reacted with RS cells in 85% of cases of HD . In summary, our results demonstrate that the judicious use of monoclonal antibody Leu-22 in combination with other selected commercially available monoclonal antibodies permits the determination of the B cell or T cell origin of a high proportion of NHLs, and is helpful in the differential diagnosis between HD and NHL among cases that have been routinely fixed and paraffin-embedded.

Exp Neurol, 1988 Dec, 102(3), 290 - 7
Pharmacologic consequences of the vascular permeability of chromaffin cell transplants in CNS pain modulatory regions; Sagen J et al.; The transplantation of peripheral neural tissue into the CNS has been shown to alter blood-brain barrier (BBB) permeability to intravascularly injected proteins such as horseradish peroxidase . The pharmacological consequences of such BBB alterations following the transplantation of adrenal medullary tissue, isolated bovine chromaffin cell suspensions, or PC12 cell suspensions into the pain modulatory regions of the periaqueductal gray (PAG) or subarachnoid space of the lumbar spinal cord were studied using agents that normally do or do not readily pass the BBB . The injection of nicotine in animals with adrenal medullary or chromaffin cell transplants produces potent analgesia, most likely due to the stimulated release of opioid peptides and catecholamines from the transplanted cells . This analgesia could be blocked by nicotinic antagonist mecamylamine, which normally passes the BBB, but not by nicotinic antagonist hexamethonium, which normally does not readily pass the BBB . Furthermore, quaternary nicotinic agonists tetramethylammonium and 1,1-dimethyl-phenyl-piperazinium had no effect on pain sensitivity in animals with adrenal medullary implants . The Met-enkephalin peptide analog, D-Ala-Met-enkephalinamide, which normally does not alter pain sensitivity when injected systemically due to limited penetration to the CNS, produced analgesia in animals with adrenal medullary, bovine chromaffin cell, and PC12 cell implants in the PAG, but not in control gelfoam-implanted animals . This analgesia, as well as analgesia induced by nicotine, was completely blocked by naloxone pretreatment, but not by naloxone methobromide, a quaternary derivative of naloxone that does not normally pass the BBB.(ABSTRACT TRUNCATED AT 250 WORDS)

J Hepatol, 1988 Dec, 7(3), 293 - 304
'Albumin-receptor' uptake kinetics do not require an intact lobular architecture and are not specific for albumin; Nunes R et al.; When freshly isolated well-stirred single cell suspensions of rat hepatocytes were incubated with 5-600 microM {3H}oleate or {35S}sulfobromophthalein (BSP) in the presence of 150 microM bovine serum albumin (BSA), uptake of both ligands increased as a linear function of the total ligand concentration in the medium . By contrast, when the same ligand concentrations were incubated as 1:1 complexes with BSA, apparent saturation of ligand uptake was observed . Analogous results were obtained in incubations employing beta-lactoglobulin instead of BSA . In none of these studies did ligand uptake velocity correlate in simple fashion with the concentration of unbound ligand in the incubation medium . These studies establish that the basis for the kinetic observations termed the 'albumin receptor phenomenon' does not require an intact hepatic lobular architecture or space of Disse, and is not specific for albumin.

Br J Cancer, 1988 Dec, 58(6), 723 - 9
Phenotypic and genotypic heterogeneity of peripheral T-cell lymphoma; Smith JL et al.; A series of 21 phenotypically characterised T-cell lymphomas histologically defined as lymphocytic, lymphoblastic, immunoblastic, AILD type, pleomorphic, T-zone and Lennert's T-cell lymphoma, were investigated for T-cell receptor (TcR) and immunoglobulin (Ig) gene rearrangements . Phenotypic analyses of frozen sections and cell suspensions were heterogeneous and in many cases no single T-cell marker recognised all of the malignant cells . Data derived by staining with antibodies reactive with antigens in paraffin embedded tissue were consistent with T NHL in all cases except lymphoblastic lymphoma . TcR gene rearrangements were observed in lymphocytic, lymphoblastic and immunoblastic lymphoma, however, in the remaining 14 phenotypically and histologically defined peripheral T-cell lymphomas, 2 showed rearrangement of TcR gamma and beta genes consistent with T NHL and 2 showed Ig JH rearrangements only, suggestive of either reactive T-cell populations masking cryptic disease or presence of tumour populations with aberrant gene rearrangement and expression of T lineage antigens . No Ig or TcR gene rearrangements were found in the remaining 10 cases, in which morphologically identifiable tumour cells comprised 10-90% of the cell population . In 3/6 cases tested some CD3 positive cells failed to stain with WT31 or beta F1, monoclonal antibodies that recognise determinants on combined TcR gamma beta or TcR beta chains respectively . Whether these cases represent tumours arising from an undetermined cell of origin or polyclonal expansions of T-cells remains to be determined . Our results confirm the phenotypic heterogeneity of histologically defined peripheral T-cell lymphoma and indicate that in these particular histological subtypes gene rearrangement analysis can also yield heterogeneous results which may be unhelpful in determining cell lineage and clonality.

Biochemistry, 1988 Nov 29, 27(24), 8803 - 10
Physical basis of the effect of hemoglobin on the 31P NMR chemical shifts of various phosphoryl compounds; Kirk K et al.; The marked difference between the intra- and extracellular 31P NMR chemical shifts of various phosphoryl compounds when added to a red cell suspension may be largely understood in terms of the effects of hemoglobin on the 31P NMR chemical shifts . The presence of {oxy- or (carbonmonoxy)-} hemoglobin inside the red cell causes the bulk magnetic susceptibility of the cell cytoplasm to be significantly less than that of the external solution . This difference is sufficient to account for the difference in the intra- and extracellular chemical shifts of the two phosphate esters trimethyl phosphate and triethyl phosphate . However, in the case of the compounds dimethyl methylphosphonate, diethyl methylphosphonate, and trimethyl-phosphine oxide as well as the hypophosphite, phenylphosphinate, and diphenylphosphinate ions, hemoglobin exerts an additional, much larger, effect, causing the 31P NMR resonances to shift to lower frequency in a manner that cannot be accounted for in terms of magnetic susceptibility . Lysozyme is a protein structurally unrelated to hemoglobin and was shown to cause similar shifts to lower frequency of the resonances of these six compounds; this suggests that the mechanism may involve a property of proteins in general and not a specific property of hemoglobin . The effect of different solvents on the chemical shifts of the eight phosphoryl compounds provided an insight into the possible physical basis of the effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Res, 1988 Nov 15, 48(22), 6272 - 7
Clonal analysis of untreated non-Hodgkin's lymphoma utilizing immunoglobulin gene rearrangement and immunophenotype; Rudders RA et al.; We have prospectively examined 66 consecutive initial diagnostic lymph node biopsies from unselected patients suspected of having malignant lymphoma for clonal immunophenotypic and immunogenotypic markers . By morphological and cell surface phenotypic criteria 52 had non-Hodgkin's lymphoma derived from the B-cell lineage and in these we compared surface immunoglobulin criteria for clonality with immunoglobulin gene rearrangement as detected by JH, C kappa, and C lambda gene probes . We found that the addition of BglII and HindIII double digests to the standard BamHI and EcoRI restriction enzymes made it possible to detect rearrangement in the vast majority of lymphomas and that rearrangement of both JH alleles is the rule . A rearranged heavy and/or light chain gene was detected in 47 of 52 (91%) tumors and the JH probe alone detected rearrangements in 87% of tumors when multiple restriction enzymes were used . In contrast, surface immunoglobulin, the standard clonal marker for monoclonal B-cell malignancy, was either undetectable or did not exhibit light chain restriction in 29 of 52 tumors as detected by flow cytometric analysis . Further, in 24 of these 29 tumor DNAs we could detect an Ig gene rearrangement . In follicular (nodular) lymphoma which often gives ambiguous immunophenotypic results by cell suspension techniques, monoclonal gene rearrangements were detected in 16 of 18 tumor DNAs . Monoclonal surface immunoglobulin was detected in only 8 of 18 of this subset of cases . The 52 tumors were also analyzed for potential oligoclonality . We found that the use of BglII, a restriction enzyme that closely spanned the JH region, increased the sensitivity of detecting rearrangements and facilitated the identification of isotype switch variants . In only a single (1 of 52) tumor DNA were more than two rearranged bands seen with JH, C kappa, and C lambda probes, suggesting a multiclonal origin . Additional cases thought to potentially represent oligoclonality by immunophenotypic criteria proved to be isotype switch variants . We conclude that Ig gene rearrangement is an extremely sensitive method for defining monoclonality in lymphoma cell populations, particularly if multiple restriction enzymes are used, and that the vast majority of the clonal diversity seen in initial diagnostic biopsies is the result of isotypic switch within a given clone rather than true oligoclonality.

Brain Res, 1988 Nov 15, 473(2), 241 - 8
Fetal hippocampal cell suspensions ameliorate behavioral effects of intradentate colchicine in the rat; Tandon P et al.; Colchicine, a neurotoxin that preferentially destroys dentate gyrus granule cells and mossy fibers, was injected into the hippocampus of adult rats . Three weeks later, the rats were tested for colchicine-induced hypermotility after which they received fetal hippocampal explants . Locomotor activity was retested three weeks later, after which the rats were trained over a period of four weeks on a food-reinforced, spatial, working memory task in an 8-arm radial maze . Fetal hippocampal explants were found to attenuate significantly the colchicine-induced hypermotility and spatial learning deficits . Histological observations showed the presence of surviving hippocampal explants in both the lesioned and the control rat brains, suggesting that the presence of viable implants facilitates the recovery of behavioral function in rats with spatial memory deficits.

Biochem Biophys Res Commun, 1988 Nov 15, 156(3), 1152 - 9
Spatial distribution and temporal change of cytoplasmic free calcium in human platelets; Tsunoda Y et al.; Our digital imaging microscope equipped with a microspectrofluorometer revealed in single resting human platelets the existence of continuous Ca2+ gradient increasing towards the plasma membrane (frequency; 100%) and discontinuous ones (Ca2+ plateaus) in the endoplasmic regions (frequency: 70%) . An average cytoplasmic free Ca2+ concentration ({ Ca2+}i) in a whole cytoplasm was 72 +/- 7 nM, ranging from 30 nM in the lowest to 150 nM in the highest region just beneath the plasma membrane . When stimulated with thrombin, {Ca2+}i uniformly increased to the average {Ca2+}i of 300 nM and these gradients disappeared . This {Ca2+}i transient was followed by the sustained increase in {Ca2+}i in both single cells and cell suspension.

Gene, 1988 Nov 15, 71(1), 217 - 23
Polyadenylation of histone H3 and H4 mRNAs in dicotyledonous plants; Chaboute ME et al.; The histone H3 and H4 genes are shown to be expressed in both Arabidopsis plantlets and transitory multicellular suspension . The 5'- and 3'-ends of the H4 mRNAs have been localized on two H4 genes previously sequenced, H4A748 and H4A777 . S1-nuclease mapping and reverse-transcriptase-primer-elongation experiments revealed the existence of two start points for transcription, located 31 and 37 nucleotides downstream from the TATA-box . The 3'-end of the mRNA corresponding to H4A748 was localized at 177 nt after the stop codon . The other gene, H4A777, most probably is not expressed . In addition to a long 3'-untranslated region, the H4 mRNA was shown to be polyadenylated in both plantlets and cell-suspension . This observation was extended to the H3 mRNAs of Arabidopsis and of two other dicots, tobacco and sunflower . Previous results on maize H3 and H4 mRNAs suggest that polyadenylation is a common feature for histone mRNAs in higher plants.

J Comp Neurol, 1988 Nov 15, 277(3), 391 - 402
Behavioral and anatomical correlates of immunologically induced rejection of nigral xenografts; Carder RK et al.; Cell suspensions derived from the ventral mesencephalon of CD-1 mice were unilaterally transplanted into the striatum of neonatal Sprague-Dawley rats that had been bilaterally dopamine depleted . Thirty-eight percent of the grafts survived . Tyrosine-hydroxylase-immunoreactive neurons within the transplant innervated the host striatum with a dense fiber plexus . The grafts appeared to exert some degree of functional control over motor behavior in that these animals made contralateral rotations in response to amphetamine and tail pinch . In order to provide additional evidence that the motor behavior is associated with the transplant itself, the graft was removed . This was achieved by using a mouse skin graft to provoke an immunological response against the transplanted neural tissue . The immunological response resulted in the specific loss of the transplant with little or no damage to the surrounding neural tissue . The amount of rotation observed after tail pinch and amphetamine injection was severely affected by neural graft rejection . The loss of turning was associated most directly with the loss of tyrosine hydroxylase immunoreactivity within the transplant rather than with the massive reduction of tyrosine-hydroxylase-positive fibers in the ipsilateral host striatum . These data suggest that dopamine cells in mouse nigral grafts play an essential role in eliciting rotational behavior in neonatally dopamine depleted rats . They also show the value of skin grafting as a technique for specifically removing neural xenografts.

J Immunol Methods, 1988 Nov 10, 114(1-2), 187 - 90
Assessment of anti-HLA antibodies in sera being tested for platelet reactivity by a platelet lymphocyte immunofluorescence test (PLIFT); Muylle L et al.; An indirect immunofluorescence test using a platelet mononuclear cell suspension is described . The test is a simple and sensitive method to assess the contamination by anti-HLA antibodies of sera being tested for platelet reactivity and eliminates the need to support two independent test systems such as lymphocytotoxicity and immunofluorescence testing . The test could be of value in routine platelet serology testing and platelet crossmatching.

Nippon Seikeigeka Gakkai Zasshi, 1988 Nov, 62(11), 991 - 1001
{Anticancer drug screening test for human osteosarcoma transplanted into nude mice}; Mochizuki K; Osteosarcoma tissue was transplanted into the subcutis of nude mice, and an anticancer drug was injected into the abdomen of the mice . The effects of Cis-platinum, SF-1739HP, Melphalan, Peleomycin and Aclacinomycin were tested . Only Cis-platinum had previously been used in the treatment of osteosarcoma . As the conventional method of evaluation, tumor weight change was recorded along the time course . As a new evaluation method, toluidine-blue was added into the tumor cell suspension prepared from the tumor tissue in the back of nude mice . By the intensity of staining, the tumor cells in suspension were classified into 3 categories; strongly-positive, weakly-positive and negative . Results of evaluation by the staining method were similar to those by measurement of tumor weight . Cis-platinum proved to be most effective, followed in decreasing order by SF-1739HP, Melphalan, Pepleomycin and Aclacinomycin . In conclusion, the staining method is simple and useful for screening the anti-cancer effects of drugs.

Brain Res, 1988 Nov 1, 463(2), 341 - 5
Rapid growth of host afferents into fetal thalamic transplants; Nothias F et al.; Fetal cell suspension grafts grow and differentiate when implanted into adult rat CNS areas previously neuron-depleted using an excitotoxin . There is some controversy in the literature concerning the timetable of establishment and possible extent of host-graft connections in these experimental conditions . The present study was undertaken to analyze the development of adult host monoaminergic afferents into a transplant formed by fetal thalamic neurons in the previously excitotoxically lesioned thalamus . It is demonstrated that both norepinephrin- and serotonin-immunoreactive fibers are present in the transplant as soon as 8 days after grafting . At those times, immunoreactive fibers exhibit morphological characteristics typically associated with immature stages . After longer survival time, up to 4 weeks after grafting, immunoreactive fibers are numerous in the transplant and exhibit morphological features comparable to those observed in the adult thalamus . These results demonstrate the rapid ingrowth of some fiber systems of the adult host into the transplant and suggest that grafted fetal cells can be functionally integrated into the host circuitry as soon as a few weeks after grafting.

Mutat Res, 1988 Nov-Dec, 209(3-4), 145 - 7
DNA damage in stomach, kidney, liver and lung of rats treated with atrazine; Pino A et al.; The genotoxic activity of atrazine, a widely used triazine herbicide, was assayed by the DNA alkaline elution technique in rats given orally a single high dose or repeated daily doses . DNA breaks (and/or alkali-labile lesions) were detected in cell suspensions obtained from stomach, kidney and liver, but not in those from lung.

Mutat Res, 1988 Nov, 194(3), 183 - 91
DNA double-strand damage and repair following gamma-irradiation in isolated spermatogenic cells; Coogan TP et al.; Various cell types in spermatogenesis exhibit differential sensitivity to radiation-induced DNA damage . The investigation of DNA radiosensitivity in vitro is complicated by the heterogeneous population of male germ cells (MGC) present in isolated single-cell suspensions . In the present investigation, the neutral elution technique was used to assess gamma-irradiation-induced DNA double-strand damage (DSD) in spermatogonia and preleptotene spermatocytes (SG/PL), pachytene spermatocytes and spermatid spermatocytes, as well as in MGC . In addition, the capability of these cell types to repair DNA double-strand damage was investigated . Based on the well established timing of the rat spermatogenic cycle, the DNA of specific cell populations was labeled using tritiated thymidine . DNA from labeled cells was determined isotopically, whereas total DNA was quantitated using a fluorometric method . DSD was induced in a dose-dependent manner in the heterogeneous population as well as in the labeled cell populations . SG/PL were more sensitive to gamma-irradiation-induced DSD than either the heterogeneous MGC population, pachytene or spermatid spermatocytes . Each cell type exhibited a similar capability to repair DSD following exposure to 3000 rad; repair was rapid (maximal within 45 min) and incomplete (less than 40%) . Only pachytene spermatocytes exhibited significant repair following exposure to 6000 rad . Since a difference in sensitivity to radiation-induced DSD was demonstrated, the capability of each cell type to repair a similar initial frequency of strand damage was investigated . SG/PL, pachytene and spermatid spermatocytes differed in their capability to repair similar levels of strand damage . However, the difference in dose required to achieve equal damage may have contributed to other cellular effects, thus altering repair . In summary, a model is described that permits the evaluation of genotoxic responses in specific populations of spermatogenic cells within a heterogeneous cell suspension . The ability of specific cell types to repair gamma-irradiation-induced DNA double-strand damage is demonstrated.

Cell Immunol, 1988 Nov, 117(1), 152 - 64
Anti-Ia monoclonal antibody (10-2.16) inhibits lymphocyte-high endothelial venule (HEV) interaction; Manolios N et al.; Lymphocyte egress from the vascular compartment into the lymph node (LN) parenchyma occurs at the postcapillary venules, termed high endothelial venules (HEVs) . Lymphocyte adhesion and migration through the HEVs is a receptor-mediated, energy-dependent, process . The aim of this study was to investigate the role of MHC Class II antigen expression on lymphocyte-HEV interaction in normal (CBA) and autoimmune (MRL/l) mice . Using the HEV binding assay, lymphocyte adhesion to LN sections pretreated with monoclonal antibody (MAb; 10-2.16) was decreased compared to diluent (mean of the differences +/- standard deviation; xd +/- SD: 0.749 +/- 0.22, P less than 0.0075)- and myeloma immunoglobulin-pretreated controls (xd = 0.462 +/- 0.13, P less than 0.005) . Similar inhibition of binding was found in MRL/l LN sections pretreated with MAb 10-2.16 . Binding inhibition was concentration dependent, but total inhibition was never achieved . Several other anti-Ia MAb's were used, but failed to inhibit lymphocyte attachment . Lymphocyte binding to control sections treated with MAb's against MHC Class I antigen, plasminogen activator (PAM-3), anti-thrombin III (AT-IIIm), and MECA-325 antigen was not significantly different from diluent controls . LN cell suspensions pretreated with MAb 10-2.16 bound normally to LN sections . By contrast, MAb to lymphocyte homing receptor (MEL-14) inhibited lymphocyte adhesion . The role of Class II antigens in lymphocyte-HEV interactions is discussed.

Exp Cell Res, 1988 Nov, 179(1), 243 - 52
Control of the aggregation factor-aggregation receptor interaction in sponges by protein kinase C; Gramzow M et al.; By means of immunobiochemical and immunocytological techniques it was found that the aggregation factor (AF) from the sponge Geodia cydonium is stored in vesicles of spherulous cells . During the reaggregation process of dissociated cells, the AF which is present extracellularly was determined to be bound to the cell-surface-associated aggregation receptor (AR) only during the initial phase (0-5 h after addition of the AF to the single cell suspension) . At later stages (20 h), the AF colocalized with extracellular structures, e.g., collagen and glycoconjugates . Immobilized to nitrocellulose, the AR, a molecule with Mr of 43.5 kDa, displayed its binding affinity to the AF only if it was isolated from early aggregates (5 h) . The transition of the AF-susceptible to the AF-deficient state of the plasma membrane was mimicked in vitro by incubation of plasma membranes from early aggregates with purified protein kinase C . This conversion to the AF-deficient state could be prevented by the protein kinase C inhibitor staurosporine . Together with earlier findings, which revealed that the AR is phosphorylated by protein kinase C, we propose that in the sponge system this enzyme controls intercellular processes involved in morphogenesis.

Cancer Res, 1988 Nov 1, 48(21), 6137 - 44
Establishment of a human megakaryoblastic cell line (T-33) from chronic myelogenous leukemia in megakaryoblastic crisis; Tange T et al.; A megakaryoblastic cell line, termed T-33, was established from the peripheral blood of a patient with Philadelphia chromosome-positive chronic myelogenous leukemia in megakaryoblastic crisis . T-33 cells have been maintained in RPMI 1640 medium containing 10% fetal calf serum in a single cell suspension with a doubling time of 24-36 h for over 2 years . Giemsa-banded karyotypes were female hyperdiploid with a modal chromosomal number of 51, all cells including Philadelphia chromosome . The cells showed strong positivity for periodic acid-Schiff and alpha-naphthyl acetate esterase, and weak for alpha-naphthyl butyrate esterase, but were negative for myeloperoxidase . Flow cytometric analysis of cell surface markers showed the existence of HLA-DR, MY-7, MY-9, and a platelet antigen (Yukb), and no markers for T- or B-lymphocytes . Most of the cells fixed with acetone were positive for Factor VIII, platelet glycoprotein IIb-IIIa, IIIa (Yukb), and Ib, but negative for glycophorin A and hemoglobin . Ultrastructural platelet peroxidase was demonstrated in 2-3% of cells and the percentage of positive cells increased up to 20% after the treatment with 12-O-tetradecanoylphorbol-13-acetate . The cells contained small dense granules negative for platelet peroxidase, their number increasing threefold after 12-O-tetradecanoylphorbol-13-acetate treatment . Such treated cells frequently showed a complex of the demarcation membrane in the cytoplasm . T-33 responded thrombin to exhibit calcium influx . This cell line may be useful for the study of the early stage of megakaryocytic differentiation in human megakaryopoiesis.

Cancer Res, 1988 Nov 1, 48(21), 5947 - 52
Metabolism of SR 4233 by Chinese hamster ovary cells: basis of selective hypoxic cytotoxicity; Baker MA et al.; The metabolism of SR 4233 (3-amino-1,2,4-bentotriazine-1,4-dioxide), recently reported as highly toxic to hypoxic cells in vitro, was studied by using suspensions of Chinese hamster ovary cells . The rates of formation of two known reduction products, the 1-oxide and the unoxygenated 3-aminobenzotriazine, were measured in aerobic and hypoxic cell suspensions for drug treatments producing both hypoxic and aerobic cytotoxicity . Formation of the 1-oxide and a small amount of the 3-aminobenzotriazine occurred preferentially in hypoxic suspensions . These metabolites were relatively nontoxic to either aerobic or hypoxic cells, implying another mechanism of toxicity . The activation of SR 4233 by single electron transfer, hypothetically forming a toxic drug radical, was explored . Aerobic stimulation of oxygen consumption in respiration-inhibited cells and malondialdehyde release from aerobic cells in the presence of SR 4233 indicated the formation of active oxygen species during drug activation . Increased malondialdehyde release in hypoxic cells and its attenuation by the hydrogen donor, dimethylthiourea, implied the presence of an oxidizing radical . Unlike the nitroimidazole, misonidazole, hypoxic metabolism of SR 4233 did not deplete intracellular glutathione or result in increased binding of drug metabolites to cellular macromolecules . These results are consistent with macromolecular damage caused by an oxygen sensitive, nonbinding, drug-free radical intermediate with oxidizing properties as the mechanism of selective hypoxic toxicity of SR 4233.

Brain Res Dev Brain Res, 1988 Nov 1, 44(1), 59 - 72
Immunocytochemical studies on the development of astrocytes, Müller (glial) cells, and oligodendrocytes in the rabbit retina; Schnitzer J; Glial markers, namely antibodies to glial fibrillary acidic protein (GFAP), vimentin, galactocerebroside (GC), 04 antigen, and 08 antigen, were used to study the development of neuroglial cells in the postnatal rabbit retina . In histological sections radially oriented Muller cells were detectable at birth . They were weakly vimentin-positive and their labeling intensity increased during further development . Few vimentin-labeled astrocytes, situated in the nerve fiber layer, were detectable at birth, but of these few many were also GFAP-positive . The number of GFAP-positive astrocytes, all of which co-labeled with vimentin antibodies, increased during the following days . From postnatal day (P) 9-10 onward, the vimentin labeling seen in GFAP-positive astrocytes began to decline . In the adult rabbit most GFAP-positive astrocytes were only weakly vimentin-stained; some astrocytes even lacked detectable amounts of vimentin . Thus, astrocytes were found to be strongly vimentin-positive at birth, and strongly GFAP-positive but weakly vimentin-labeled by adulthood . Such a transition was not observed for Muller cells, which lacked detectable amounts of GFAP at all postnatal stages studied . In single cell suspensions, cells positive for the cell surface markers 04 antigen and GC were first detected at P6-7, 08 antigen-labeled cells were found one day later . Thus, the third class of neuroglial cells in the rabbit retina, the oligodendrocytes, seems to develop about one week later than astrocytes and Muller cells.

Biull Eksp Biol Med, 1988 Nov, 106(11), 559 - 61
{The action of tuftsin on the reaction of macrophage-suppressor formation in vitro and in vivo}; Voevodin DA; A cell suspension consisting of nonadhering and adhering spleen cells in the ratio 30:1 was incubated in a 10(-4) M tuftsin's solution during 15-30 min . The addition of 10(7) cells incubated in tuftsin syngeneic recipients resulted in the suppression of the immune response of the latter to sheep red blood cells . It was noted that this effect may be induced by using adhering cells only of intact donors and only when incubated together with nonadhering cells . The addition of tuftsin one hour after transplantation of the nonadhering spleen cells resulted in the suppression of the immune response . It was proposed, that suppression effect released through generation of the macrophage-suppressors.

Carcinogenesis, 1988 Nov, 9(11), 1943 - 51
Modulation of natural killer activity by 12-O-tetradecanoylphorbol-13-acetate and benzoyl peroxide in phorbol ester-sensitive (SENCAR) and resistant (B6C3F1) mice; Updyke LW et al.; Following two weeks of topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) at 2, 4 and 8 micrograms/mouse on alternate days (7X total) or benzoyl peroxide (BZP) at 10, 20 and 40 mg/mouse, natural killer (NK) activity was determined in local (lymph nodes draining the lower dorsal region) and systemic (spleen) lymphoid tissue in phorbol ester-sensitive (SENCAR) and resistant (B6C3F1) mice . SENCAR mice, sensitive to tumor induction by TPA in two-stage chemical-induced carcinogenesis protocols, demonstrated suppression of NK activity in the spleen (no significant change in lymph nodes) and substantial dose-dependent increases in cell numbers in these organs after topical exposure to TPA . B6C3F1 (C57BL/6 X C3H F1) mice, reported to be resistant to TPA-induced promotion, demonstrated significant increases in NK activity in lymph nodes/spleen with an increase in cell numbers in the draining nodes only . Unlike the C57BL/6 parental strain, B6C3F1 mice are also reported to be resistant to promotion with BZP . Significantly, studies in this laboratory indicated that B6C3F1 mice dosed with BZP demonstrated increased NK activity in the spleen as was observed after dosing with TPA . These data suggest that alterations in NK activity as a result of exposure to tumor promoters may, in part, account for the resistance of particular strains of mice to tumor development . In both SENCAR and B6C3F1 mice, the blastogenic response of spleen cell suspensions isolated from TPA-dosed animals to phytohemagglutinin (PHA), a T cell lectin, was suppressed in a dose-dependent manner; BZP had no effect on spleen cell responses in either strain . Blastogenic responses of lymph node cells to PHA were enhanced in both strains of mice after topical application of TPA and BZP . Therefore, alterations in lymphoid cell responsiveness to PHA appeared unrelated to the reported sensitivities of SENCAR and B6C3F1 mice to tumor promotion.

Neuron, 1988 Nov, 1(9), 791 - 803
Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning; Barres BA et al.; Two different monoclonal antibodies to the Thy-1 antigen, T11D7 and 2G12, were used to purify and characterize retinal ganglion cells from postnatal rat retina . Although Thy-1 has been reported to be a specific marker for ganglion cells in retina, retinal cell suspensions contained several other types of Thy-1-positive cells as well . Nevertheless, a simple two-step "panning" procedure allowed isolation of ganglion cells to nearly 100% purity . We found that postnatal ganglion cells differed in antigenic, morphological, and intrinsic electrophysiological characteristics, and that these properties were correlated with one another . Minor variations of this panning protocol should allow rapid, high yield purification to homogeneity of many other neuronal and glial cell types.

Int J Cell Cloning, 1988 Nov, 6(6), 392 - 403
Comparison of an antimetabolic assay and an antiproliferative assay, both using 3H-thymidine incorporation, to test drug sensitivity of human tumors; Zaffaroni N et al.; Two assays based on the inhibition of 3H-thymidine incorporation into DNA were used to measure either the antimetabolic or the antiproliferative effects of anticancer drugs . A direct comparison of the two assays was made with cell suspensions obtained from 11 ovarian cancers and 22 malignant melanomas . Drugs with different effects on cell cycle phases were tested by both assays, for a total of 53 drug comparisons . When the sensitivity indices specific for each system was used, a significant association (p less than 0.01) was noted between the two assays . The agreement of both assays in defining in vitro sensitivity or resistance was 100% for ovarian cancer . For melanoma, 97% of samples resistant to the antimetabolic assay were also resistant to the antiproliferative assay; whereas, only 45% of samples sensitive to the antimetabolic assay were sensitive to the antiproliferative assay.

Biochem J, 1988 Nov 1, 255(3), 789 - 94
Inhibition of gastric acid secretion by epidermal growth factor . Effects on cyclic AMP and on prostaglandin production in rat isolated parietal cells; Hatt JF et al.; Histamine (0.5 mM) stimulated the cyclic AMP content of cell suspensions containing greater than 80% parietal cells . Epidermal growth factor (EGF) inhibited this stimulatory effect of histamine, but had no effect on basal cyclic AMP content . The half-maximally effective concentration of EGF for inhibition of histamine-stimulated cyclic AMP was 3.9 nM . The equivalent measurement for the inhibition of histamine-stimulated aminopyrine accumulation was 3.0 nM . Aminopyrine accumulation was measured because it provides an index of the secretory activity of the cell . The cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibitory effect of EGF on cyclic AMP content . This effect of IBMX was not caused by its ability to raise cellular cyclic AMP content in the presence of histamine . Prevention by IBMX of the inhibitory action of EGF on histamine-stimulated aminopyrine accumulation had been shown previously {Shaw, Hatt, Anderson & Hanson (1987) Biochem . J . 244, 699-704} . EGF stimulated prostaglandin E2 (PGE2) production in the cell fraction containing greater than 80% parietal cells, with the half-maximally effective concentration being 7.5 nM . EGF was ineffective in stimulating PGE2 production if the cell fraction was depleted of parietal cells (12%), or if 0.5 mM-histamine was added to the enriched parietal-cell fraction . In conclusion, EGF may inhibit histamine-stimulated acid secretion by decreasing the cyclic AMP content of parietal cells . This effect could be mediated by an increase in cyclic AMP phosphodiesterase activity, but it is unlikely to involve an effect of EGF on parietal-cell prostaglandin production.

Am Rev Respir Dis, 1988 Nov, 138(5), 1268 - 75
Alkaline phosphatase: a marker of alveolar type II cell differentiation; Edelson JD et al.; In an effort to identify type II cells by a method independent of staining phospholipid inclusions, we evaluated a histochemical technique for alkaline phosphatase activity in normal rat lung, in freshly isolated type II cells, and in primary culture of type II cells . In the adult rat alveolus, alkaline phosphatase staining selectively identified type II cells, although nonciliated bronchiolar (Clara) cells and loose perivascular connective tissue also stained for alkaline phosphatase activity . In cell suspensions of type II cells and other dissociated lung cells, alkaline phosphatase staining correlated closely with the modified Papanicolaou technique and was particularly useful in distinguishing type II cells from alveolar macrophages . To determine if alkaline phosphatase was related to the differentiated phenotype of type II cells, we studied conditions known to affect other type II cell functions . When type II cells were cultured on plastic substrata, the intensity of alkaline phosphatase staining decreased with increasing time in culture . To quantitate the apparent decrease in alkaline phosphatase activity, we used a biochemical assay to study the expression of alkaline phosphatase by type II cells . The specific activity of alkaline phosphatase in type II cells declined with increasing time in tissue culture on plastic substrata . Alkaline phosphatase activity was maintained, however, by culturing cells on Englebreth-Holm-Swarm (EHS) tumor matrix . Cells that had reduced levels of alkaline phosphatase activity following 48 h of culture on plastic substrata could be "rescued" by removing them from the plastic substratum and reculturing them for 48 h on EHS matrix . Alkaline phosphatase activity was also increased by culturing type II cells in the presence of cAMP or sodium butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol Methods, 1988 Oct 26, 113(2), 157 - 63
Transendothelial chemotaxis in vitro of human monocytes; Darby H et al.; Porcine aortic vascular endothelial cells were grown to confluence on microporous PTFE membranes and incorporated into a two-compartment chemotaxis assembly . Human peripheral blood mononuclear cells (20-25% monocytes) were placed in the upper compartment and zymosan-activated human serum (ZAHS) as chemoattractant in the lower compartment . Over a 3 h period monocytes migrated across the endothelialized membrane and adhered to a collecting filter sited in the lower compartment . Addition of ZAHS to the cell suspension in the upper compartment virtually abolished the migration response whilst only minimal leucocyte migration was supported by the bare unendothelialized PTFE membrane . The extent of transendothelial monocyte chemotaxis was dependent upon the concentration of chemoattractant in the lower compartment and upon the cell density of the suspension containing monocytes . A confluency test of fluid flow across the endothelialized filter showed that monocyte migration could take place without disturbing endothelial barrier function . The culture system is easy to assemble and the method provides experimental versatility.

J Immunol Methods, 1988 Oct 26, 113(2), 193 - 203
Enrichment of murine splenic natural killer (NK) cells by the sequential elimination of non-NK cells; Lemieux S et al.; A simple and reliable three-step procedure to enrich for murine endogenous splenic NK cells is described . The method is based on the sequential elimination of non-NK cell subsets by standard and inexpensive techniques executed in a specific order . First, macrophages and other adherent cells are eliminated by incubation on plastic surface . Secondly, the T cells are excluded from the multicellular aggregates formed by agglutination of the remaining cells with wheat germ lectin . Thirdly, after dissociation of the aggregates with N-acetyl-D-glucosamine and osmotic lysis of erythrocytes, NK cells are separated from other nucleated cells by nylon wool filtration . C57BL/6 spleen cells were used to establish the enrichment procedure . Usually their NK cell activity is intermediate but occasionally either low or high NK cell activity was observed in input cell suspensions . The NK cell activity recovery and the degree of enrichment varied inversely with the initial NK cell activity level of the input cell suspension . When initial NK cell activity was intermediate, it was enriched 10-30-fold . Experiments were done to establish if suppressor cells, and nylon wool-adherent, naturally activated NK cells, putatively present in input cells, could have been responsible for the abnormal initial NK cell activity detected in some C57BL/6 spleen cell suspensions and for the variations in the degree of enrichment achieved by the method here described . Either no or negligeable suppressor cell activity was noted in the cell fractions normally discarded at each step of the procedure . On the other hand, nylon wool-adherent NK cells were eliminated during the fractionation of spleen cells with higher than average initial NK cell activity and would account for the lower NK cell enrichment obtained in these conditions.

J Biol Chem, 1988 Oct 15, 263(29), 14703 - 11
Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell; Muallem S et al.; Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol . The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin . The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells . Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units . The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-{phenyl-methoxy}-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity . In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units . This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion . In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA) . This compound also blocked changes in pHi due to changes in extracellular Na+ . Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane . The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation . In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively . Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached . Thereafter, acidification continued without further change in pHi . To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl- . DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90% . Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.

J Immunol, 1988 Oct 15, 141(8), 2656 - 60
A monoclonal antibody reacting with distinct adhesion molecules defines a transition in the functional state of the receptor CD11b/CD18 (Mac-1); Altieri DC et al.; CD11b/CD18 (Mac-1) is a member of the leukocyte integrin family, a group of receptors that have been implicated in various effector functions and cellular collaboration in the immune response . It has been shown previously that CD11b/CD18 on cells of monocyte and myeloid lineage appears to undergo rapid activation and acquire new functional receptor specificities after exposure to selected agonists such as adenosine diphosphate (ADP) . We now show that ADP induces a reconformation of the CD11b/CD18 receptor with exposure of new epitopes characteristics of this activated state . By direct binding studies, flow cytometry, and immunoprecipitation experiments, it has been found that the mAb 7E3 reacts with CD11b/CD18 only after ADP-stimulation of the cell suspension . The activated state of CD11b/CD18 induced by ADP and recognized by 7E3 can also be recapitulated by agonists inducing transients in cytosolic Ca2+ such as the chemoattractant FMLP . Moreover, this process of receptor activation does not involve quantitative mobilization of the subcellular storage pool of CD11b/CD18 to the plasma membrane . Because 7E3 also recognizes a qualitative, ADP-mediated activated state of the platelet adhesion receptor GP IIb/IIIa, it is suggested that transients in cytosolic Ca2+ might represent early secondary events for a general pathway of rapid activation of integrin receptors and, as such, represent important signals for cellular interactions in the immune response.

Cancer, 1988 Oct 15, 62(8), 1539 - 55
T-cell-rich lymphoproliferative disorders . Morphologic and immunologic differential diagnoses; Winberg CD et al.; To differentiate peripheral T-cell lymphomas (PTCL), the authors evaluated the results of T11 monoclonal antibody studies on consecutive cell suspensions prepared from 509 lymph nodes from various lymphoproliferative disorders (LPD) . They used T11 (CD2) positivity to identify those LPD in which the content of T cells was high . There were 266 (52%) cell suspensions which contained more than 50% T11-positive cells . More than 75% of the following non-Hodgkin's lymphomas had over 50% T11-positive cells: diffuse mixed cell (DM), diffuse atypical poorly differentiated lymphocytic and lymphoblastic lymphomas; mycosis fungoides; and true histiocytic lymphoma . Eleven cell suspensions had more than 90% T11-positive cells; four were involved by B-cell lymphomas . The cell suspensions prepared from nine of 14 diffuse large cell lymphomas of the T-cell type had more than 50% T11-positive cells . Of these, three of five cases of the polymorphous subtype had fewer than 50% T11 cells, but six of seven lymph nodes of the clear-cell type had more than 50% T11-positive cells . Each of seven DM samples of the T-cell type contained over 50% T11 cells; none had a polymorphous appearance . In the 112 cases of reactive LPD studied, more than 75% of cases of necrotizing lymphadenitis, dermatopathic lymphadenitis, angioimmunoblastic lymphadenopathy, and those with lymph nodes with no specific reactive pattern had more than 50% T11-positive cells . The authors' findings indicate that T11 positivity is a reliable T-cell marker in reactive and neoplastic LPD except for those cases of PTCL with a polymorphous appearance; these tend to lose T11-expression . A multi-parameter diagnostic approach is required in the following LPD: (1) PTCL which are T11-negative; (2) PTCL of small lymphocytic type having an unremarkable T-cell phenotype; (3) SIg-negative B-cell lymphomas which are rich in nonneoplastic T cells; (4) non-Hodgkin's lymphomas with minimal disease which are rich in reactive T cells; and (5) polymorphous large cell proliferations.

Brain Res Bull, 1988 Oct, 21(4), 601 - 5
Cell-sized microspheres in the hippocampus show cleavage planes and passive displacement; Wells J et al.; Fluorescent microspheres (6 or 10 micron in average diameter) dispersed in fluid were injected into the hippocampus, neocortex or striatum . In the hippocampus the microspheres were located in one of three cleavage planes . Cleavage planes were found above the alveus, in the obliterated hippocampal fissure and on the hilar side of the dentate granule cells . When the injections were made into the infragranular cleavage plane, the adjacent granule cells degenerated, presumably because the cavity separated the axons from their cell bodies . Some microspheres were passively displaced beyond the boundary of the injection site . If the microspheres gained access to the subarachnoid space, some of the displaced microspheres were found at considerable distances from the injection site . There were no cleavage planes in neocortex or striatum but there was passive displacement of microspheres into the host parenchyma . In cell suspension transplants, the passive displacement of cells should be distinguished from migration and the possibility of a widespread distribution of transplanted cells needs to be considered.

J Steroid Biochem, 1988 Oct, 31(4B), 685 - 90
Aromatase-inhibitors of the androstenedione-type activate the 17 beta-hydroxysteroid-dehydrogenase in the rat testis tissue suspension model; Schroder H et al.; In an in vitro rat testis cell suspension model, the metabolism of tritiated testosterone, dihydrotestosterone and androstenedione was investigated . In the presence of aromatase inhibitors like 1-methyl-androsta-1,4-dien-3,17-dione (SH 489) and 4-hydroxy-androstenedione, the metabolism was shifted towards 17-keto forms . The same effect was observed in the presence of androstenedione, the parent compound of the two aromatase inhibitors . The consequent consideration of the whole labelled steroidal spectrum in each experiment leads to the conclusion that androstenedione and the derived aromatase inhibitors activate the 17 beta-hydroxysteroid-dehydrogenase in a product activating manner . Our results imply that aromatase inhibitors may regulate the intratissular levels not only of estrogens but also of other hormonally active steroids like dihydrotestosterone and 5-androstenedione.

Pharmacol Toxicol, 1988 Oct, 63(4), 266 - 73
In vitro oxidation of mercury by the blood; Hursh JB et al.; A method is described for studying the in vitro oxidation of mercury vapour by red blood cells at short times and with diminishing mercury vapour concentrations . It is found that for 40% red blood cell suspensions and 37 degrees at concentrations greater than about 6 ng mercury vapour/ml, the oxidation rate is zero order, and that at lower concentrations the rate changes to first order . The effect of temperature and of added hydrogen peroxide are studied . Results are considered in terms of the generally accepted belief that the catalase-compound I system is the main path of oxidation . If the results obtained in vitro in these experiments apply in vivo to man, it follows that inhaled mercury is carried in the blood to the brain and other organs primarily as dissolved vapour rather than as inorganic mercury ions.

Cancer Genet Cytogenet, 1988 Oct 1, 35(1), 5 - 20
Multiple karyotypic abnormalities, including structural rearrangements of 11p, in cell lines from malignant melanomas; Heim S et al.; Cell lines were obtained from three malignant melanoma patients by culturing cell suspensions from tumor biopsies . A total of six lines (I to VI) were established . One line each was established from the first two cases . Lines III and IV were established from two different methyl cellulose colonies derived from the primary tumor of case 3; line III was from a non-pigmented and line IV from a pigmented colony . Cloning of line IV resulted in two highly malignant (IV Cl 1 and IV Cl 3) and one less malignant (IV Cl 2) clone . Clone IV Cl 1 was inoculated intracardially in nude mice and gave rise to adrenal and brain metastases . Lines V and VI were derived from such metastases . Multiple structural and/or numerical chromosome abnormalities were detected in all lines . Line I had 57-61 chromosomes, with structural changes affecting 1p, 2p, 3q, 7p, 7q, 11p, 14q, 17q, and 22q, as well as one unidentified marker . Line II had 40-48 chromosomes, with structural changes of 1p, 1q, 4q, 5p, 6p, 8p, 11p, 11q, 14p, 20p, and two unidentified markers . Line III had 45 chromosomes, 6q+, del(11p), and a centric fusion between chromosomes 14 and 15 . Line IV had 45-46 chromosomes . The clonal changes included rearrangements of 1p, 9p, 11p, and the centric fusion of chromosomes 14 and 15 . Line V was pseudodiploid and contained aberrations of 1p, 9p, 11p, 14q, 20q, an isochromosome for 21q, and an unidentified marker . Finally, the pseudodiploid line VI had changes of 9p, 11p, centric fusion of chromosomes 14 and 15, and an unidentified marker . Although no single identical aberration was shared by all six lines, structural abnormalities of 11p were invariably present and, hence, might constitute a common cytogenetic feature in melanoma development . The most consistent difference between the amelanotic and melanotic lines derived from case 3 was the presence of a 6q+ marker in the former and a 9p+ marker in the latter.

Brain Res, 1988 Oct 1, 471(2), 225 - 34
Adhesion of neural cells to extracellular matrix constituents . Involvement of glycosaminoglycans and cell adhesion molecules; Werz W et al.; Single cell suspensions of early postnatal mouse cerebellum adhere to substrate-bound culture supernatants of the teratocarcinoma cell line PF-HR9 and can be inhibited to adhere by antibodies to the neural cell adhesion molecules L1 and N-CAM . Adhesion can also be inhibited by the glycosaminoglycans heparin and heparan sulfate, and less by chondroitin sulfate or hyaluronic acid . Heparinase treatment of cells, but not of HR9 substrate, reduces adhesion . Adhesion does not appear to be mediated by laminin, a constituent of HR9 extracellular matrix, since L1 and N-CAM antibodies do not interfere with cell adhesion on EHS sarcoma laminin as substrate and since antibodies to EHS sarcoma laminin partially inhibit adhesion to HR9 extracellular matrix which contains laminin . Of the other extracellular matrix constituents analysed in HR9 culture supernatants (collagen type IV, a heparan sulfate proteoglycan and fibronectin) none could be shown to promote adhesion, when coated as substrate, suggesting that yet unidentified compounds are responsible for L1- or N-CAM-mediated cell adhesion . These experiments show for the first time that extracellular matrix constituents can act as binding partners for the neural cell adhesion molecules L1 and N-CAM.

J Pharmacol Exp Ther, 1988 Oct, 247(1), 349 - 54
Gentamicin-induced increases in cytosolic calcium in pig kidney cells (LLC-PK1); Holohan PD et al.; LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were tested as a model system for assessing the role of calcium in gentamicin-induced nephrotoxicity . Cell viability was evaluated by a vital dye exclusion procedure, and intracellular free calcium {Ca2+}i was measured employing Fura-2 fluorescence . Exposing cell suspensions (10(6)/ml) to concentrations of the drug, which had no apparent effect on viability, produced a rapid and prolonged increase in intracellular {Ca2+} . The perturbation of calcium homeostasis could be blocked by the addition of mepiperphenidol, an inhibitor of the organic cation transport system . We propose that LLC-PK1 cells are an appropriate model to study drug-induced nephrotoxicity . Gentamicin disrupts calcium homeostasis and causes plasma membrane alterations . Since mepiperphenidol blocked the gentamicin-induced Ca2+ increases, the data suggest that aminoglycosides enter the cell via the organic cation transporter.

J Lab Clin Med, 1988 Oct, 112(4), 418 - 25
Characterization of type II alveolar epithelial cells by flow cytometry and fluorescent markers; Rochat TR et al.; Type II alveolar epithelial cells play a crucial role in maintaining the structure and functions of pulmonary alveoli . A number of techniques have been described to isolate type II cells for in vitro studies; however, type II cell suspensions isolated by each technique are still contaminated by macrophages or monocytes . The present studies describe the use of flow cytometry to accurately characterize the composition of these cell suspensions . With freshly isolated type II cell suspensions, type II cells could be distinguished from macrophages and monocytes by two methods: (1) the combination of natural fluorescence and orthogonal light scatter, or (2) the use of monoclonal antibodies OX-1 (directed against a common leukocyte antigen present on rat macrophages and monocytes) and PKK-1 (directed against cytokeratin intermediate filaments present in type II cells) . With cultured type II cells, the combination of natural fluorescence and orthogonal light scatter did not distinguish between type II cells and macrophages or monocytes; however, the monoclonal antibodies OX-I and PKK-1 continued to distinguish between these cell types . As an example of the use of these techniques, the methods were used to define the sequential expression of class I and II major histocompatibility antigens on both type II cells and on macrophages or monocytes in the same cell preparations . These methods are of potential value in isolating pure populations either of type II cells or of macrophages or monocytes by cell sorting and in accurately identifying the cells present in type II cell suspensions or cultures.

Exp Hematol, 1988 Oct, 16(9), 741 - 7
Analysis of human hemopoietic progenitor cells for the expression of glycoprotein IIIa; Kanz L et al.; Human hemopoietic progenitor cells were examined for the expression of glycoprotein IIIa (GPIIIa) . This protein, which forms the beta-subunit of the GPIIb/IIIa receptor for cytoadhesive proteins as well as the beta-subunit of the vitronectin receptor, represents the most sensitive cell surface marker so far identified for the megakaryocytic lineage . Bone marrow cells were fractionated by a discontinuous Percoll gradient to separate cells that form megakaryocytic colonies in culture (1.05 greater than rho less than 1.077 g/ml) . Density centrifugation was followed by indirect immunopanning to select for an enriched population of progenitor cells depleted of most of the mature cells of the myeloid, lymphoid, and erythroid lineages . This cell suspension was labeled with antibodies directed against determinants of GPIIIa and analyzed using a fluorescence-activated cell sorter (FACS) . Fractions of cells were sorted and analyzed for the ability to form hemopoietic colonies in culture . Our study demonstrated that megakaryocytic progenitor cells (CFU-M) as well as granulocyte-macrophage colony-forming units (CFU-C), erythroid colony-forming units (BFU-E), and mixed lineage colony-forming units (CFU-GEMM) express HLA-DR antigens but lack GPIIIa . Therefore GPIIIa represents a marker that is not present on hemopoietic progenitor cells, but is expressed on the progenies of CFU-M . In view of the importance of GPIIIa as a component of receptors for cytoadhesive proteins, this finding may help to elucidate the adhesive interactions between early hemopoietic cells and bone marrow interstitium.

Scand J Gastroenterol, 1988 Oct, 23(8), 931 - 4
Prostaglandins and CCl4-induced liver cell mortality . The effect of the prostaglandin analogue enprostil; Bang S et al.; Prostaglandins have been reported to reduce the carbon tetrachloride (CCl4)-induced liver cell injury in rats . The object of the present experiments was to examine the effect of the prostaglandin E2 analogue enprostil on the survival of isolated liver cells exposed to CCl4 . Liver parenchymal cells were isolated from rat livers by collagenase perfusion and released into a 'suspension' buffer . Aliquots of the cell suspension were incubated with 1 micrograms or 0.5 microgram CCl4, and to parallel test suspensions 20 ng enprostil was added 5-10 min before CCl4 . Incubation was performed on ice, at room temperature, and at 37 degrees C . The average percentage of dead cells after CCl4 treatment was significantly reduced by pretreatment with enprostil at room temperature (1 microgram CCl4: 69 +/- 21% and 44 +/- 13%, respectively) and after 10 min of incubation at 37 degrees C (1 microgram Cl4: 56 +/- 25% and 37 +/- 27%; 0.5 microgram CCl4: 51 +/- 33% and 29 +/- 18%, respectively) . When the liver cell mortality approximated 100% after long-term incubation at 37 degrees C, no protective effect of enprostil was observed.

Int J Biol Markers, 1988 Oct-Dec, 3(4), 221 - 32
Methods of simultaneous visualization of cytoplasmic enzyme reactivity and cell surface antigens (by cytochemistry combined with immunocytochemistry) in individual hematopoietic and lymphoid cells; Gloghini A et al.; Enzyme cytochemistry alone, and more recently, immunocytohistochemistry have been satisfactorily used by hematologists and hematopathologists for the study, diagnosis and classification of human hematological and lymphoproliferative disorders . To enhance the potential of these techniques, the possibility of combining immunocytohistochemical techniques with enzyme cytohistochemistry with simultaneous visualization of both reaction products has been examined by some investigators . This approach has been applied to normal, reactive and neoplastic material using mainly cell suspensions and frozen sections, with the aim of improving cell identification in specimens containing different cell types, of determining the cytochemical profiles of well-defined lymphocyte subpopulations and of establishing the cell surface phenotypes of cells that are positive for certain enzymes . In this paper, published reports on this subject are reviewed and compared with the experience of our study group.

Transplantation, 1988 Oct, 46(4), 558 - 63
Elimination of leukemia cells from human bone marrow using floating beads; Hirn-Scavennec J et al.; A new in vitro immunophysical method of removing leukemia or lymphoma cells from autologous bone marrow is described . This new technique makes use of low-density polypropylene beads (density: 0.91) coated with a monoclonal antibody anti-CALLA (antibody ALB2) . To ascertain its ability to selectively remove human B/pre-B hematopoietic cells, this technique was applied to normal human bone marrow cell suspensions contaminated with 1-5% of tumor cells . Samples were incubated with the floating beads at 4 degrees C on a rotating wheel for 60 min, followed by a 10-min decantation period, after which the beads bearing the tumor cells floated on the surface, whereas unbound normal marrow cells remained in suspension and were easily recovered free of beads . To demonstrate the feasibility of our method, 2 types of assays were carried out, one using target cell radiolabeled with 111indium, and the other a clonogenic assay . The first assays were to calibrate the different parameters (cellular density, quantity of beads, incubation time) with tumor cell lines: Namalwa (CALLA+) and Molt 4 (CALLA-) . These 2 cells lines being able to clone, it is hard to envisage clonogenic assays . In this case, it is very hazardous to evaluate correctly the remaining clonogenic units of Namalwa cells . It is why radiolabelling assays were used for these first experiments . The second assays were to study a model close to the clinical setting and to control the safety of the beads on normal bone marrow cells . In this case, the mixture experiments in which only Namalwa cells were able to clone were evaluated with clonogenic assays, which are more sensitive than radiolabeling assays . A 3- to 4-log reduction of tumor load was achieved with 1-step treatment, and an average of 5-log depletion was obtained by repeating the process twice, as ascertained by the clonogenic assay . Viability, average recovery of nucleated cells, and stem cells potential following the purge were excellent.

Blood, 1988 Oct, 72(4), 1261 - 8
A novel leukemia cell line, MR-87, with positive Philadelphia chromosome and negative breakpoint cluster region rearrangement coexpressing myeloid and early B-cell markers; Okamura J et al.; We developed a Philadelphia chromosome (Ph) positive cell line, designated MR-87, from a 4-year-old boy with Ph+-acute leukemia . MR-87 cells grew in single cell suspensions with a doubling time of 120 to 144 hours . Both MR-87 and original leukemia cells were positive for myeloperoxidase (MPO) and myeloid antigen CD13 . These cells exhibited the early B-cell phenotype, ie, terminal deoxynucleotidyl transferase+, Ia+, CD19+, and CD10+ . Rearrangement of the immunoglobulin heavy chain was confirmed in both . Approximately 80% of MR-87 cells coexpressed CD13 and lymphoid antigens CD10 or CD19, as confirmed by a two-color analysis . Simultaneous expression of MPO and CD19 on a single MR-87 cell was demonstrated at ultrastructural level . Thus, MR-87 is a Ph+ leukemia cell line exhibiting a hybrid phenotype . The breakpoint cluster region (bcr) was not rearranged in the MR-87 cells and subsequent analysis using antisera revealed that these cells expressed a novel protein, P190c-abl, which was immunoprecipitated with anti-abl and anti-phosphotyrosine antibodies . The MR-87 line will be most useful for investigating the biology and pathogenesis of Ph+ bcr- acute leukemia.

Immunol Cell Biol, 1988 Oct-Dec, 66 ( Pt 5-6), 361 - 7
Mg2+-dependent adenosine triphosphatase: an enzyme marker for ovine T lymphocytes; Alders RG et al.; Sheep T lymphocytes showed a cell surface magnesium-dependent adenosine triphosphatase (Mg2+-ATPase) reaction, which is reported to be characteristic of human B lymphocytes . In cryostat sections of lymph nodes, spleen and thymus, Mg2+-ATPase positive regions closely matched those labelled by sheep pan T monoclonal antibodies (Moab) . An Mg2+-ATPase reaction was also found in fibroblastic recticulum cells of T cell regions in lymph nodes . Double labelling of cells from peripheral blood and peripheral lymph for Mg2+-ATPase and the pan T marker showed that 78% of the lymphocytes were positive for both of these markers . In cell suspensions enriched for B lymphocytes the percentage of cells positively labelled was decreased to 37% . Samples of each cell population which were labelled with a pan T Moab and analysed by flow microfluorometry revealed T cell levels which were not significantly different from those obtained by histochemical or immunohistochemical techniques . Less than 1% of lymphocytes positive for heavy and light chains of immunoglobulin (Ig) G were labelled with Mg2+-ATPase . Veiled cells in lymph and monocytes showed a cytoplasmic Mg2+-ATPase reaction.

Am J Physiol, 1988 Oct, 255(4 Pt 2), F666 - 73
Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells; Seri I et al.; Dopamine, generated locally from L-dopa, inhibits Na+-K+-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium . To determine whether locally formed dopamine inhibits Na+-K+-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate (QO2) and 86Rb uptake in renal cortical tubule cell suspensions . L-Dopa (10(-4) M) did not affect ouabain-insensitive QO2 or mitochondrial respiration . However, L-dopa inhibited ouabain-sensitive QO2 in a concentration-dependent manner, with half-maximal inhibition (K0.5) of 5 x 10(-7) M and a maximal inhibition of 14.1 +/- 1.5% at 10(-4) M (P less than 0.05) . L-Dopa also blunted the nystatin-stimulated QO2 in a concentration-dependent manner, with a K0.5 of 5 x 10(-8) M and a maximal inhibition of 21.8 +/- 1.2% at 10(-5) M (P less than 0.05), indicating that L-dopa directly inhibits Na+-K+-ATPase activity and not sodium entry . Ouabain-sensitive 86Rb uptake was also inhibited by L-dopa (16.0 +/- 2.4%, P less than 0.05) . Carbidopa (10(-4) M), an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive QO2 and 86Rb uptake, indicating that dopamine rather than L-dopa was the active agent . The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

J Invest Dermatol, 1988 Oct, 91(4), 358 - 62
Enrichment of unlabeled human Langerhans cells from epidermal cell suspensions by discontinuous density gradient centrifugation; Teunissen MB et al.; In this report we introduce an alternative procedure for enrichment of human epidermal Langerhans cells (LC) from epidermal cell suspensions of normal skin . By means of discontinuous Ficoll-Metrizoate density gradient centrifugation, a fraction containing high numbers of viable, more than 80% pure LC was recovered, as judged by CD1a expression . The purity of the LC-enriched fraction appeared to be dependent on the percentage LC in the crude epidermal cell suspension . LC enriched by this method retained their accessory and antigen-presenting capacities, as determined in the Concanavalin-A induced T-cell response, in the allogeneic mixed leukocyte reaction and in the antigen-specific T-cell proliferation assay in vitro . The great advantage of this method is that it is simple and rapid and that the isolated LC are unlabeled.

Neurosci Lett, 1988 Sep 23, 92(1), 21 - 6
Intraretinal transplantation of fluorescently labeled retinal cell suspensions; del Cerro M et al.; Dissociated cell suspensions of neonatal neural retina, labeled with the fluorescent dyes Fast blue or Fluoro-gold, were transplanted into the retina of normal adult rats or of rats affected by late stage phototoxic retinopathy . Light microscopy showed good survival, differentiation, and integration of the transplants, as well as permanence of the label up to 100 days . The results indicate that the transplantation of dissociated, fluorescently labeled retinal cells has a number of advantages over the transplantation of solid fragments of retinal tissue, previously performed by ourselves and others . The following are some of the most immediate procedural advantages: the number of transplanted cells can be assessed, the transplanted cells are in a more intimate contact with the host tissue and therefore integrate better with the host, and the fluorescent tags permit precise determination of the survival and distribution of the transplanted cells.

J Immunol Methods, 1988 Sep 13, 112(2), 219 - 26
Immunoadsorption of T cells onto glass beads using tetramolecular complexes of monoclonal antibodies; Thomas TE et al.; Tetramolecular monoclonal antibody complexes were used to selectively cross-link a subset of human peripheral blood T cells (CD8 positive) to glass beads coated with hapten (fluorescein) modified bovine serum albumin . Tetramolecular antibody complexes were prepared with anti-CD8 (Leu 2a), anti-FITC mouse IgG1 monoclonal antibodies and monoclonal rat anti-mouse IgG1 . Optimum conditions for depletion of CD8 positive cells from peripheral blood mononuclear cell suspensions were determined with 1 ml columns . 90-99% of the CD8 positive cells could be removed with 0-17% non-specific adsorption of CD8 negative cells . The weakest link in this system was the bond between hapten-modified albumin and the glass beads . These results indicate that tetramolecular antibody complexes are useful for the specific immunoadsorption of cells to a defined affinity matrix.

J Biol Chem, 1988 Sep 5, 263(25), 12373 - 7
Mass spectrometric determination of the inorganic carbon species assimilated by photoautotrophic cells of Euphorbia characias L; Rebeille F et al.; The chemical forms of inorganic carbon, CO2 or HCO3-, incorporated during photosynthesis in photoautotrophic Euphorbia characias cell suspension cultures were determined in experiments using 13CO2 and a mass spectrometry technique . From the equations of the CO2 hydration reaction, a kinetic model was first developed, and the effect of photosynthesis on the external CO2 concentration was simulated . It was predicted from this model that CO2 and HCO3- uptakes could be differentiated by recording only the CO2 variation rate in the external medium, successively in absence then in presence of an exogenous carbonic anhydrase activity . The results obtained with either CO2-grown or air-grown photoautotrophic cells were in good agreement with the model and demonstrated that CO2 was the sole species taken up during photosynthesis . In addition no accumulation of inorganic carbon within the cells was observed in the light . Similarly, in dark, CO2 was the only species released by respiration in the external medium.

Int J Radiat Oncol Biol Phys, 1988 Sep, 15(3), 699 - 702
Use of a colorimetric microtiter (MTT) assay in determining the radiosensitivity of cells from murine solid tumors; Wasserman TH et al.; We assessed the use of a colorimetric assay for determination of radiosensitivity for cells taken directly from murine solid tumors . The assay uses microtier plates and measures the ability of viable cells to reduce a tetrazolium salt (MTT) to an insoluble form, a formazan salt . We established the dependency of the assay on the cell number and time of assay for two murine tumors (EMT-6 and RIF-1) . We compared the MTT assay to the standard clonogenic assay and had good agreement of surviving fraction after radiation doses of 2 and 4 Gy . It is possible, therefore, to adapt the MTT assay for use with cell suspensions prepared directly from fresh murine tumors . This may provide a methodology for the determination of the clinical radiosensitivity of tumors including fresh clinical tumor specimens.

Arch Surg, 1988 Sep, 123(9), 1073 - 8
Clinical implications of procoagulant and leukoattractant formation during intraoperative blood salvage; Bull MH et al.; Experiments using 21 dogs and red cell salvage equipment (Haemonetics Cell Saver, Haemonetics Corp, Braintree, Mass) were employed to study the formation and potency of procoagulant and leukoattractant material during experimental autologous blood salvage . Washed red cell suspensions were found to include toxic degradation products that had been released from a deposit of platelets and white cells adherent to the centrifuge bowl wall . When reinfused, these toxic products resulted in a "salvaged blood syndrome" of intravascular clotting and pulmonary damage . The pulmonary arterioles showed leukocyte margination and tangled fibrin skeins with occlusive thrombi . Intra-alveolar and perivascular hemorrhages, along with extensive pulmonary edema, were also observed . The formation of procoagulant and leukoattractant material could be markedly decreased when the red cell salvage technique incorporated the following precautions: (1) minimal dilution with saline (normal plasma protein levels), (2) a low calcium level, and (3) minimal platelet activation (avoidance of the aspiration of clotted blood just before processing).

Proc Natl Acad Sci U S A, 1988 Sep, 85(17), 6342 - 6
Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression; Henthorn P et al.; The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells . When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates . Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining . The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements . The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive . Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

J Neuroimmunol, 1988 Sep, 19(3), 177 - 89
Characterisation of microglia isolated from adult human and rat brain; Hayes GM et al.; A method has been developed to isolate microglia from adult human and rat brain cell suspensions by rosette formation via Fc receptors . Immunocytochemical characterisation of the cells immediately following isolation and after 7-10 days in vitro with a panel of monoclonal antibodies has demonstrated that microglia from adult brain have the phenotypic characteristics and phagocytic capacity of mononuclear phagocytes, but lack the hydrolytic enzyme, non-specific esterase . The ability to isolate rapidly a purified population of microglia from adult brain provides a means for investigating mechanisms of activation and differentiation of tissue macrophages, which could elucidate their role in inflammation of the central nervous system.

Cell Immunol, 1988 Sep, 115(2), 471 - 80
Normal murine and porcine embryos recruit NK cells to the uterus; Croy BA et al.; Decidual NK cells, indistinguishable from those found in lymphoid tissues, are present in cell suspensions prepared from maternal decidua of random-bred mice between Days 6.5 and 10.5 of first gestation . The stringency of the correlation between NK cells and normal embryos during successful pregnancy is unknown . Our previous finding that active NK cells were unable to mediate lysis of fresh embryonic tissues at any stage during gestation suggests that if NK cells play a functional role in normal pregnancy it would be a noncytolytic role . Before studies on the function of uterine NK cells were undertaken, evidence that the association of NK cells with normal embryos is widespread was sought by assessing NK cell activity in cell suspensions from decidua of syngeneically mated mice, from decidua of multiparous, random-bred mice, and from the endometrium of pigs during first pregnancy . Neither parity nor maternal-fetal compatibility changed the pattern of high levels of decidual NK cell activity early in pregnancy followed by decline . Porcine NK cell activity was not detected in uterine cells isolated from cycling pigs but in pregnant animals it gradually increased during the preattachment period and reached levels greater than those in blood, postattachment (Day 28) . Some of this activity was hormone dependent but sustained increases in NK cell activity required the presence of an embryo . These studies demonstrate that the association of functional NK cells with normal embryos is widespread during early pregnancy.

Int J Hyperthermia, 1988 Sep-Oct, 4(5), 567 - 70
New culture tube with an inside wall devised for studies of short-term hyperthermia; Kageyama K et al.; We devised a new kind of culture tube with which a cell suspension could be warmed through both the inside and outside walls . Use of this tube makes it possible to measure the effects of short-term hyperthermia more accurately than with the conventional tubes, which is heated through the outside wall only . Temperature curves rose significantly faster in the double-walled tube than in the conventional one . The accelerated heating efficiency of the new tube was also seen by the enhanced inhibition of {3H}thymidine incorporation into the DNA of Ehrlich ascites tumour cells grown in these tubes at the same final temperature as in a conventional tube.

Invest Radiol, 1988 Sep, 23 Suppl 1, S153 - 6
Effect of water-soluble iodinated contrast media on pressure-flow relationship of red cell suspension; Kumazaki T et al.; The effect of radiological contrast media on blood flow through a vascular network was investigated, taking physical and physiological conditions such as osmolality into account . The perfusion of the bullfrog's hind limbs was performed, with a slight modification of the vertical tube method . The effect of contrast media on red cell deformability was studied by perfusion with erythrocyte suspensions in glutaraldehyde-fixed hind limbs . The echinocytic shape change induced by metrizamide and hypertonic iothalamate solutions caused a marked increase in resistance to flow . When the perfusion with erythrocyte suspension was performed using intact hind limbs, the pressure-flow relationship was influenced by contrast media effects on both red cell deformability and the vascular bed . Ioxaglate had less rheologic effect on the pressure-flow relationship than metrizamide or iothalamate . It could be concluded that contrast media should be isotonic, of low viscosity and chemotoxicity, and that ioxaglate was preferable to metrizamide and iothalamate at equal iodine content.

Hum Pathol, 1988 Sep, 19(9), 1001 - 7
Double labeling immunohistologic and flow cytometric analysis of human B cells with particular reference to Leu-8 expression; Poletti A et al.; Previous results have shown that human lymphocyte subpopulations are heterogeneous as to Leu-8 expression . In the present study, we performed a heretofore unreported immunohistologic analysis and flow cytometric double labeling investigation focused on Leu-8+ and Leu-8- human B cells, with special reference to their expression of other B cell lineage antigens (Leu-14, B1, OKB7 or B2, OKB2, BA-1, BA-2, IgD, and IgM) or of a functional marker of cell proliferation (Ki-67) . Immunohistologic analysis was performed on frozen sections of nine normal or reactive lymph node and tonsil biopsy specimens tested with either single or paired antibodies, the latter procedure (double labeling) being directed at revealing positively subtracted Leu-8+ or Leu-8- cells expressing a given marker depending on the antibody staining sequence used . Dual flow cytometry with paired antibodies was performed on cell suspensions from four normal or reactive lymph nodes and tonsils . As to the follicular district, immunohistologic analysis suggests that germinal center (proliferating) B lymphocytes, identifiable in cell suspension as Leu-8-B1+ and representing 80% of the B1+ cells, are Leu-8- and Leu-14+, BA-2+, OKB7+, and IgM+, being rarely IgD+ . Most lymphocytes located in the mantle zone (resting cells), identifiable in cell suspension as Leu-8+B1+ and representing the remaining 20% of the B1+ cells, are Leu-8+, Leu-14+ . OKB7+, OKB2+, BA-1+, IgD+, and IgM+ . Furthermore, from our results, there is indirect evidence to support the existence of a Leu-8+BA-2+ lymphocyte subset located in the mantle zone . Immunohistologic study of four lymph nodes by Leu-8 and Ki-67 antibody (that recognizes a nuclear, cell proliferation-associated antigen absent only in the Go phase of the cell cycle) showed that these markers are mutually exclusive and added further evidence that Leu-8 antigen is a marker of resting lymphocytes.

Cancer Res, 1988 Sep 1, 48(17), 4799 - 803
Selective modulation of antibody response and natural killer cell activity by purine nucleoside analogues; Priebe T et al.; Analogues that are poor substrates for adenosine deaminase or purine nucleoside phosphorylase may mimic immunodeficiencies associated with the enzyme deficiencies, and their activities may be directed toward selected lymphocyte subpopulations . Four analogues were studied for their effects on primary antibody response to either a T-dependent (sheep erythrocytes) or T-independent (trinitrophenyl-conjugated Escherichia coli lipopolysaccharide) antigen as well as effects on T-cytotoxic and natural killer cell activities in mice . The nucleosides were: an adenosine analogue, tubercidin; two deoxyadenosine analogues, 2-chloro, 2'-deoxyadenosine and 2-fluoroadenine arabinoside-5'-phosphate; and a deoxyguanosine analogue, 9-beta-D-arabinosylguanine . Drugs were given i.p . once daily for 3 consecutive days . Immune responses were determined in spleen cell suspensions 1 day after the last dose . Tubercidin inhibited both T-cytotoxic and natural killer cell activities at doses that did not reduce primary antibody response, whereas the reverse was true for 2-chloro, 2'-deoxyadenosine and 2-fluoroadenine arabinoside-5'-phosphate . At higher doses, T-cytotoxic lymphocytes appeared to be more sensitive than natural killer cells to the deoxyadenosine analogues . 9-beta-D-Arabinosylguanine did not selectively inhibit the immune responses at doses that clearly reduced the yield of spleen lymphocytes . Assuming the analogues mimic endogenous nucleosides, the results suggest that natural killer cells are more sensitive to adenosine than are those cells responsible for primary antibody response, whereas the reverse is true for deoxyadenosine.

Derm Beruf Umwelt, 1988 Sep-Oct, 36(5), 147 - 52
{Determination of the irritating potential of environmental substances in an epidermal cell suspension in vitro}; Wilke B et al.; A simple in vitro screening method was established, which allows estimation of the effect of environmental products on cell alteration or irritative potential . Primary cell suspensions, prepared from the epidermis of guinea pig ears through trypsination (0.2%) over night at 4 degrees C, were used . The cell suspension (3 mill./ml) and test substances were mixed in equal portions (500 microliters) and incubated at room temperature for approximately 90 min . The degree of cell alteration was determined by the trypan blue exclusion test . Chemically defined substances as well as disinfectants were employed . The cell alteration was clearly dependent upon substance concentration as well as incubation time . On the basis of the mean alteration time (AT50) measured in vitro, and the mean irritation time (IT50) determined in the patch test in vivo, the substances were ranked according to strength of cell alteration or irritative potency in vitro and in vivo . A good correlation (Spearman's rank correlation, rs = 0.76) was found between the in vitro and in vivo results . Thus, this method is suited for the screening of chemical products especially in industrial laboratories.

Behav Neural Biol, 1988 Sep, 50(2), 229 - 39
Effects of physostigmine and d-amphetamine on the behavior of rats with selective fimbria-fornix lesions and intrahippocampal fetal septal cell transplants; Cassel JC et al.; Female Long-Evans rats were given electrolytic lesions of either the medial fimbria bilaterally (Fi, n = 24), the dorsal fornix (Fo, n = 24), or both structures (FF, n = 24) at 31 days of age . Ten rats were given sham-operations . Ten days later, half the rats with lesions received bilateral intrahippocampal grafts of embryonic septal cell suspensions (FiT, FoT, FFT, respectively) . As already reported in a separate publication (J.C . Dalrymple-Alford, C.R . Kelche, J.C . Cassel, G . Toniolo, V . Pallage, & B.E . Will, 1987, Experimental Brain Research, 210, 115-128), 7 months after transplant surgery, grafted rats were found to be more impaired in an eight-arm radial maze than nongrafted rats . The present report concerns a pharmacological study carried out in the same rats 11 months after grafting . We examined the effects of ip injections of physostigmine (0.01, 0.05, 0.10 mg/kg) and then of d-amphetamine (1.6 mg/kg), as compared with baseline control injections of saline . Just prior to the drug treatments, performances of grafted and nongrafted rats did not differ significantly, but impairments in grafted rats reappeared during subsequent no-injection and saline control trials . Physostigmine failed to affect significantly the performances in rats of any group . d-Amphetamine improved performances in grafted rats with medial fimbria lesions, impaired performances in grafted rats with dorsal fornix lesions, and did not change performances in grafted rats with both lesions, as compared with their respective nongrafted counterparts . Histological analysis revealed variable reinnervation of the host structure and substantial graft-induced lesions of the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)

Biophys J, 1988 Sep, 54(3), 471 - 88
On the mechanism of injury to slowly frozen erythrocytes; Pegg DE et al.; When cells are frozen slowly in aqueous suspensions, the solutes in the suspending solution concentrate as the amount of ice increases; the cells undergo osmotic dehydration and are sequestered in ever-narrowing liquid-filled channels . Cryoprotective solutes, such as glycerol, reduce the amount of ice that forms at any specified subzero temperature, thereby controlling the buildup in concentration of those other solutes present, as well as increasing the volume of the channels that remain to accommodate the cells . It has generally been thought that freezing injury is mediated by the increase in electrolyte concentration in the milieu surrounding the cells, rather than reduction of temperature or any direct action of ice . In this study we have frozen human erythrocytes in isotonic solutions of sodium chloride and glycerol and have demonstrated a correlation between the extent of damage at specific subzero temperatures, and that caused by the action at 0 degrees C of solutions having the same composition as those produced by freezing . The cell lysis observed increased directly with glycerol concentration, both in the freezing experiments and when the cells were exposed to corresponding solutions at 0 degrees C, showing that the concentration of sodium chloride alone is not sufficient to account quantitatively for the damage observed . We then studied the effect of freezing in anisotonic solutions to break the fixed relationship between solute concentration and the volume of the unfrozen fraction, as described by Mazur, P., W . F . Rall, and N . Rigopoulos (1981 . Biophys . J . 653-675) . We confirmed their experimental findings, but we explain them differently . We ascribe the apparently dominant effect of the unfrozen fraction to the fact that the cells were frozen in, and returned to, anisotonic solutions in which their volume was either less than, or greater than, their physiological volume . When similar cell suspensions were subjected to a similar cycle of increase and then decrease in solution strength, but in the absence of ice (at 20 degrees C), a similar pattern of hemolysis was observed . We conclude that freezing injury to human erythrocytes is due solely to changes that occur in the composition of their surrounding milieu, and is most probably mediated by a temporary leak in the plasma membrane that occurs during the thawing (reexpansion) phase.

Biol Reprod, 1988 Sep, 39(2), 245 - 53
Effects of prostaglandin F2 alpha-induced luteolysis on the populations of cells in the ovine corpus luteum; Braden TD et al.; Receptors for prostaglandin (PG) F2 alpha in the ovine corpus luteum are localized on large steroidogenic luteal cells . Therefore, it was hypothesized that during luteolysis, the first demonstrable effects of PGF2 alpha would occur in the population of large luteal cells . To test this hypothesis, the numbers and sizes of large and small luteal cells, fibroblasts, capillary endothelial cells, and pericytes were determined in corpora lutea collected 12, 24, or 36 h (6 animals/group) following administration of PGF2 alpha on Day 10 postestrus and from untreated ewes on Days 10 and 12 postestrus . The numbers and sizes of luteal cells were determined after enzymatic dissociation of the luteal tissue into single cell suspensions and by morphometric analysis of luteal slices . Serum levels of progesterone decreased (p less than 0.05) within 12 h of treatment, indicating that luteolysis was induced . Recovery of the two types of steroidogenic luteal cells following enzymatic dissociation was different (p less than 0.05) . Recovery of both steroidogenic cell types decreased with time after PGF2 alpha treatment, suggesting that they had become more fragile . As determined by morphometry, the number of large luteal cells was not different at any time point examined; however, by 36 h after treatment, the average diameter of large luteal cells had decreased (p less than 0.05) . In contrast, by 24 h after treatment, there was a decrease in the number of small luteal cells (p less than 0.05) but no change in their diameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Br J Cancer, 1988 Sep, 58(3), 330 - 4
Interactions of doxorubicin and cis-platin in squamous carcinoma cells in culture; Kohno N et al.; Doxorubicin (DXR) has a positive inoculum effect and penetrates poorly into the core of multicellular tumour spheroids (MTS) . Cis-platin (DDP) displays neither of these characteristics . We evaluated whether combining these 2 agents would influence the cell kill effect at a tumour mass level . MTS were produced from a PC-10 squamous lung carcinoma cell line . MTS were exposed to either drug first for 1 h with different intervals between exposure . Cells were then trypsinized to a single cell suspension and subjected to clonogenic assay . Combination effects were analyzed by median effect plot analysis . The more MTS ml-1 medium, the lower the cell kill effect of DXR . Simultaneous exposure to the 2 drugs was synergistic . DXR exposure first followed by DDP was less efficacious than, or the same as, the simultaneous exposure . In contrast, DDP followed by DXR was more efficacious with the best cell kill at a 1 h interval between each drug . This phenomenon was observed even at non-toxic doses of DDP . The fluorescent microscopic study of DXR indicated that prior exposure of MTS to DDP resulted in increased DXR penetration into the MTS core leading to heightened synergism with this sequence . These data suggest that the proper combination of DXR plus DDP should be in sequence with DDP first . Clinical, toxicological and pharmacological trials of DDP administration first, followed by DXR, are warranted.

Respir Physiol, 1988 Sep, 73(3), 363 - 78
Functional properties of hemoglobin in human red cells: II . Determination of the Bohr effect; Kister J et al.; Parameters of the Bohr effect (delta log P50/delta pHi) for human normal red blood cell suspensions have been calculated between intracellular pH (pHi) 5.5 and 8.5, in order to test how these quantities compare with those measured in dilute human hemoglobin solution (Hb) . For a precise comparison between red cell and Hb solutions data, the cells were fractionated to reduce cellular heterogeneity and depleted of their 2,3-diphosphoglycerate (DPG) content . The P50 values were related to pHi . From the value of the Donnan equilibrium factor rH+ and the cellular water content at each extracellular pH (pHe), the variations of the intracellular chloride concentration were calculated . This quantity exhibited a three fold change between pHi 5.5 and 8.5, with extracellular chloride concentration {Cl-}e fixed at 140 mM . In the absence of DPG, chloride is the main allosteric effector of Hb and increases the alkaline Bohr effect . When all these interacting factors are accounted for, the oxygen affinity and Bohr parameters for red cell suspensions become identical to those observed for dilute Hb solutions . These results indicate that the hydration of the Hb molecules in the highly concentrated red cell milieu is not much different from that of nearly ideal solutions.

J Trace Elem Electrolytes Health Dis, 1988 Sep, 2(3), 145 - 8
A method for determination of zinc in mononuclear leucocytes; Bro S et al.; A method for the determination of the concentration of zinc in a well-characterized fraction of mononuclear leucocytes from human blood is described . Leucocytes were separated from whole blood by use of the one-step sodium metrizoate/Ficoll procedure . The cell suspensions obtained by the separation procedure had the following composition (mean, range) (N = 15): 82% (70-85%) lymphocytes, 15% (10-15%) monocytes and 4% (2-10%) neutrophils . The ratio of platelets to leucocytes was 1:1 (1:1-1.5) . Cell pellets were sonicated before zinc analysis by flame atomic absorption spectrophotometry . 10 healthy volunteers (5 males and 5 females) aged 21-45 years, median age 33 years, were studied . The concentration of zinc in mononuclear leucocytes was 1.14 +/- 0.14 mumol/10(10) cells or 156 +/- 8 mumol/g protein (mean +/- SD).

Transplantation, 1988 Sep, 46(3), 418 - 25
Postirradiation recovery of lymphoid cells in the rat; Farnsworth A et al.; Whole-body irradiation has been extensively used to ablate immune responsiveness in rodent recipients in adoptive allograft assays . This study was undertaken to determine the relative radioresistance and the tempo of regeneration, following whole-body irradiation, of cells involved in the allograft response . Six distinct cell populations have been identified in the lymphoid tissues of rats subjected to sublethal whole-body irradiation . The relative representation of these subpopulations was significantly different from that in nonirradiated controls . NK cells, macrophages, and plasma cells, which are present in very low numbers in cell suspensions prepared from normal lymphoid tissues, made up a significant proportion of the residual/regenerating population in the tissues of rats recovering from whole-body irradiation . More significantly perhaps, the mature T cell populations showed a significant increase in the T cytotoxic/suppressor to T helper cell ratio . These observations support the suggestion that a number of the cell types within the mixed cell population observed in the rejecting indicator grafts of irradiated recipients in adoptive allograft assays are host derived . The finding that the T cytotoxic/suppressor population is apparently more radioresistant than the T helper population supports a conclusion that graft rejection in irradiated recipients, restored with pure populations of T helper cells, may not be directly mediated by the injected cells but may be the result of collaboration between these and host-derived cytotoxic cell populations.

Ann Inst Pasteur Immunol, 1988 Sep-Oct, 139(5), 545 - 56
{Lymphocyte system and relative resistance of inbred mice to Trypanosoma brucei brucei}; Makumyaviri AM et al.; The distribution of B-(Ig+) and T-(Thy1.2+, Lyt1+, Lyt2+) lymphocyte subsets in murine lymphoid organs was analysed by immunofluorescence (FACS) on cell suspensions throughout a primary infection of C3H/He (susceptible) and CBA/Ca (subtolerant) inbred mice with metacyclic Trypanosoma brucei brucei EATRO 1125 . A direct correlation was observed between (1) the level of first-peak parasitaemia, (2) its subsequent control by the host and (3) survival time . In the course of the infection, the overall population of spleen and lymph node lymphocytes was subject to polyclonal activation whilst the proportion of differentiated B and T subsets decreased accordingly . No correlation was found between modulation of the lymphocyte system and susceptibility to trypanosomiasis.

Eur J Immunol, 1988 Sep, 18(9), 1323 - 8
Characterization of T cell receptors on resident murine dendritic epidermal T cells; Steiner G et al.; Cell lines derived from Thy-1+ dendritic epidermal cells (Thy-1+DEC) display a marked heterogeneity in T cell receptor (TcR) expression including CD3-associated alpha/beta, C gamma 1/delta or C gamma 2/delta and C gamma 4/delta TcR . In order to investigate whether this heterogeneity is primarily imposed by in vitro culture conditions or, alternatively, is already present within the epidermis, we studied TcR expression by Thy-1+DEC in situ and on freshly isolated epidermal cell suspensions greatly enriched for Thy-1+DEC . Immunolabeling experiments showed that resident Thy-1+DEC are CD45+, Thy-1+, CD3+, TcR V beta 8-, CD5-, CD4- CD8-, CD25- lymphocytes . Immunoprecipitation of lysates from 125I-surface-labeled Thy-1+DEC-enriched epidermal cells with anti-CD3 epsilon, anti-C gamma 1,2,3 and anti-C gamma 4 antibodies, respectively, and subsequent analysis of the precipitates by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only CD3-associated 35 kDa/45 kDa gamma/delta heterodimers . The demonstration of TcR heterodimers on resident Thy-1+DEC strongly implies that these cells are functional T cells . The selective expression of C gamma 1/delta (C57BL/6) and C gamma 1/delta or C gamma 2/delta (C3H/He) TcR makes these cells useful for the study of gamma/delta TcR function.

Endocrinology, 1988 Sep, 123(3), 1619 - 30
Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization; Velkeniers B et al.; A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland . After dissociation, cells were centrifuged on a Percoll gradient . Three layers were recovered . The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization {with DNA complementary to PRL or to GH messenger RNA (mRNA)} . Both methods yielded identical values . PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL) . A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones . A third layer (layer 3: higher density) was enriched in GH cells to 93% (representing, however, only 10% of the initial pituitary GH cells) . In addition, PRL and GH were measured by RIA in culture medium and in cell lysates . Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of {35S}methionine . These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells . Cells in layer 2 contained and produced more GH than PRL . PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population . In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide . Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.

Brain Res, 1988 Sep 1, 471(1), 39 - 47
Characterization of Ca2+-dependent and -independent aggregation mechanisms among mouse cerebellar cells; Fischer G et al.; To gain insight into the cellular and molecular mechanisms underlying cell interactions in the early postnatal mouse cerebellum, Ca2+-dependent and -independent aggregation mechanisms were characterized using single cell suspensions under conditions that allow discrimination between the two mechanisms . When cerebellar cells were derived from newborn to 10-day-old mouse cerebellum, both mechanisms were active and showed no major change in activity during this time period . Mg2+ could not replace Ca2+ in the Ca2+-dependent mechanism . In contrast to the Ca2+-independent mechanisms, the Ca2+-dependent mechanism was inactive at low temperatures, suggesting a necessity for molecular rearrangement within the surface membrane during aggregation . Neuraminidase, chondroitinase, heparinase or hyaluronidase treatment of cells did not influence the aggregation of cells under Ca2+-dependent and -independent conditions . Chondroitin sulfate inhibited and hyaluronic acid stimulated the Ca2+-dependent mechanism, whereas chondroitin sulfate only slightly and hyaluronic acid strongly inhibited the Ca2+-independent one . Dextran sulfate slightly inhibited both mechanisms, whereas heparin and fucoidan, a complex sulfated carbohydrate, did not influence cell aggregation, while they strongly inhibited attachment of cells to laminin . The polycation poly-L-lysine slightly stimulated the Ca2+-independent mechanism, but inhibited the Ca2+-dependent one . Interestingly, chondroitin sulfate and hyaluronic acid strongly stimulated cell aggregation under conditions where both mechanisms were almost destroyed or inactive . Dextran sulfate showed only a small effect under these conditions . These observations indicate that different molecular mechanisms are active in cell-cell versus cell-extracellular matrix interactions and suggest a hitherto unknown complexity in molecular mechanisms during early postnatal cerebellar development.

Biokhimiia, 1988 Sep, 53(9), 1462 - 6
{Regulation by the protein kinase C activator phorbol ester of calcium channels in polymorphonuclear leukocytes}; Grigorian MR et al.; The Quin fluorescence in gamma-hexachlorocyclohexane-stimulated polymorphonuclear leukocytes is rapidly increased, which points to the increase in Ca2+in concentration during leukotriene B4 synthesis in leukocytes . An addition of EGTA and calcium antagonists (nifedipine, verapamil, diltiazem) to cell suspensions does not affect the basal level of internal Ca2+ but results in the inhibition of the gamma-hexachlorocyclohexane-induced Ca2+ increase . Two mechanisms of calcium homeostasis regulation in neutrophils are proposed . One of them, cAMP regulation, is coupled with a potent inhibiting effect of prostacyclin, an adenylate cyclase activator, on Ca2+in increase in stimulated neutrophils . The other one is the activation of protein kinase C catalyzed by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate . The experimental results suggest that such an activation blocks Ca2+ influx into the cells via the closure of Ca2+ channels . The synergism of action of the above mechanisms in the regulation of calcium homeostasis in neutrophils is demonstrated.

J Rheumatol, 1988 Sep, 15(9), 1326 - 33
Characterization of human synovial mast cells; Kopicky-Burd JA et al.; Human synovium obtained at arthroplasty from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were characterized by assessing mast cell morphology, content and function . Histological studies confirmed significant numbers of mast cells in both RA and OA synovium . Electron microscopic data support the morphologic similarity between human synovial mast cells and human mast cells in lung and intestine . Likewise, synovial mast cells do not appear to be functionally different from pulmonary or intestinal mucosal mast cells . Mast cell suspensions with a cellular histamine content of 4.3 +/- 0.5 pg/cell (mean +/- SEM) released histamine following provocation with anti-IgE and calcium ionophore but not compound 48/80, f-met peptide or bradykinin . Prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) were also released in response to anti-IgE . Auranofin inhibited anti-IgE provoked histamine, PGD2 and LTC4 release while gold sodium thiomalate, cromolyn and indomethacin had no effect on histamine release . Theophylline inhibited anti-IgE induced histamine release only at concentrations greater than or equal to 10(-3) M . Our study argues against functional or morphologic mast cell heterogeneity of human intestinal, lung and synovial origin and suggests that mast cells may have a pathogenic role in both RA and OA.

Biochem Biophys Res Commun, 1988 Aug 30, 155(1), 524 - 9
Structure of a novel human granulocyte peptide with anti-ACTH activity; Singh A et al.; We report the purification, structure and biological properties of a peptide of novel sequence from human granulocytes that inhibits ACTH stimulated synthesis of corticosterone in rat adrenal cell suspensions . The peptide HP-4 is homologous to a previously described human granulocyte peptide HP-1 that has no anti-ACTH activity.

Brain Res, 1988 Aug 16, 458(1), 1 - 19
Serotonin neurons grafted to the adult rat hippocampus . I . Time course of growth as studied by immunohistochemistry and biochemistry; Daszuta A et al.; The maturation and growth of fetal serotonergic raphe neurons have been studied immunohistochemically and biochemically between 1 week and 5 months after grafting to the hippocampal formation in 5,7-dihydroxytryptamine-pretreated adult rats . The average number of surviving neurons in each group was 1800, which is equivalent to approximately 20% of the potential number of serotonin neurons contained in the grafted cell suspension . The fetal raphe cells, which were taken from 12-14-day-old embryos, had developed strong serotonin immunoreactivity at 1 week after transplantation, and the number of serotonin cells present at 1 week was similar to that found at later time points . Fiber outgrowth was demonstrable already at 1 week but the serotonin-positive fibers were restricted to the areas close to the graft . Single fibers, however, could be traced for distances of up to 500-800 microns into the host hippocampus and dentate gyrus . At later time points, the graft-derived serotonin-immunoreactive fiber network extended to cover the entire hippocampal formation . At the longest postoperative time point, 7 weeks and 5 months, some of the animals exhibited extensive hyperinnervation patterns throughout the dorsal parts of the hippocampus and the dentate gyrus . Consistent with these immunohistochemical observations, supranormal serotonin levels developed with time after transplantation in the grafted hippocampi from an average of 5% of normal at 1 week, to 28% of normal at 3 weeks, 146% of normal at 7 weeks, and 216% of normal at 5 months . Although the recovery of 5-hydroxyindoleacetic acid (5-HIAA) paralleled that of serotonin (5-HT), the increase in the metabolite concentrations was less than that of the amine, indicating a change in the turnover or metabolism of serotonin in the grafted neurons over time . Thus, the 5-HIAA:5-HT ratio was higher than normal at 3 weeks post-grafting (when the host hippocampus was only partially reinnervated); it was similar to normal at 7 weeks, and it tended to be lower than normal in the hyperinnervated specimens at 5 months' survival . A regression analysis revealed a significant inverse correlation between the hippocampal 5-HT concentration and the 5-HIAA:5-HT ratio in the graft-reinnervated hippocampal formation . In conclusion, the grafted serotonergic raphe neurons, in contrast to other types of aminergic neurons, exhibited a prominent tendency to form extensive hyperiinnervation patterns in the previously dennervated host target.(ABSTRACT TRUNCATED AT 400 WORDS)

J Comp Neurol, 1988 Aug 15, 274(3), 449 - 63
Fetal homotypic transplant in the excitotoxically neuron-depleted thalamus: light microscopy; Peschanski M et al.; One month after an in situ injection of kainic acid into the ventrobasal thalamic complex (VB), the lesioned area is totally depleted of neurons . The present study has been undertaken to determine the cytoarchitecture and connectivity of the nucleus constructed by fetal thalamic neurons implanted into the excitotoxically lesioned area . Adult rats received an injection of kainic acid inducing a total neuronal depletion of the right lateral thalamus (including both the nucleus reticularis thalami and the lateral portion of the ventrobasal complex) . One month later, homotypic neurons were taken from the dorsal thalamic primordium of rat embryos (gestational age 15-16 days), dissociated, and injected into the lesioned area as a cell suspension . After 2-4-month survival, the cytoarchitecture of the neonucleus formed by the grafted neurons within the previously neuron-depleted area was analyzed . Additionally, connectivity was analyzed in seven rats in which dorsal column nuclei and/or cortical projections to the area were labeled anterogradely with either 3H-leucine or wheat-germ agglutinin conjugated to HRP, and the animals were perfused and processed following various histological procedures (Nissl staining, autoradiographic processing, and histochemistry for visualization of peroxidase) . Fetal neurons grew, differentiated, and progressively occupied the previously neuron-depleted area of the adult host CNS . They organized themselves into a neonucleus with particular cytoarchitectural features including 1) the existence of two concentric zones--a central zone containing neurons and glial cells and a marginal zone only filled with a band of glial cells, 2) an increase in cellular density compared to the intact thalamus, 3) the grouping of neurons in spherical clusters, and 4) apparent polymorphism of neuronal somata . Lemniscal and corticothalamic afferents originating from the host were observed in the neonucleus when the fetal neurons had been implanted correctly into the lesioned area but not when they had been misplaced into either normal thalamic tissue or the internal capsule . The afferents labeled from either the dorsal column nuclei or the somatosensory cortex were, however, less dense in the neonucleus than in the normal thalamus . These results are discussed with regard to the normal cytoarchitecture and connectivity of the ventrobasal complex of the rat thalamus.

J Neurosurg, 1988 Aug, 69(2), 228 - 33
Stereotaxic implantation of dispersed cell suspensions into brain . A systematic appraisal of cell placement and survival; Plunkett RJ et al.; The application of several recent advances in cell biology, brain implantation, and cell-mediated tumor immunotherapy requires successful and reproducible placement of viable cell suspensions into brain . Stereotaxic implantation is being used to inject cytotoxic lymphocytes into gliomas and to replace dopaminergic cells in parkinsonian models . Systematic assessment of the factors that influence success in implantation of cell suspensions into solid tissues is needed . A model was developed for investigation of stereotaxic implantation using radiolabeled rat lymphokine-activated killer (LAK) cells . Anesthetized rats received microliter injections of cell suspension into the right caudate nucleus . The injection volume, cell concentration, infusion rate, and needle size were varied systematically . The animals were sacrificed 1 hour after injection; the brain was removed and sectioned, and the radioactivity was counted . Three aliquots of the suspension were injected into counting tubes for control analysis . Recovery of radioactivity was expressed as the percent of mean counts per minute (cpm) in the right frontal lobe/mean cpm in the three control tubes . To assess the viability of implanted cells, the right frontal region was mechanically dissociated in media and centrifuged, and the pellet and supernatant were counted . By using small needles and slow infusion of volumes of 10 microliters or less, 85% to 90% of the radioactivity was recovered in the caudate nucleus . At least half of the implanted cells were viable . Consistent, accurate implantation of dispersed cells into brain over a range of volumes, cell concentrations, infusion rates, and needle sizes was achieved.

Diabetes, 1988 Aug, 37(8), 1123 - 8
Use of liposomes to introduce substances into pancreatic islet cells; Welsh N et al.; The liposome technique is widely used to transport substances that cannot normally traverse the plasma membrane into the cell . The interactions of liposomes with the plasma membrane of pancreatic islet cells have not previously been studied . We evaluate the suitability of the liposome technique for introducing substances into the pancreatic beta-cell to which the cell membrane is impermeable . Liposomes were synthesized with an ether-injection method, and the cell-liposomal interactions were investigated by means of radioactive labeling and the fluorescent aqueous space marker 6-carboxyfluorescein . Experiments were performed on freshly isolated mouse pancreatic islets and on free islet cell preparations . With fluorescence microscopy, liposomes were observed to fuse spontaneously with islet cells, and the corresponding internalized volumes were quantified with spectrofluorometric measurements . The liposome association with islets and islet cell suspensions, as assessed by radioactive labeling, was found to increase with the liposome concentration . The effects of liposome membrane lipid composition on the fusion rate were found to be decreased in the presence of glucolipid . In addition, polyethylene glycol failed to affect the liposomal uptake . Freshly isolated islets incubated with liposomes containing glucose 6-phosphate were observed to release slightly more insulin than islets incubated with "empty" liposomes . In conclusion, liposomes fuse spontaneously with islet cells in vitro, and the uptake of liposomes is regulated by the lipid composition of the liposomal bilayer and the amount of liposomes present . The function of the beta-cell can be altered with the liposome technique, e.g., by addition of biologically active molecules such as glucose 6-phosphate.

Eur J Immunol, 1988 Aug, 18(8), 1209 - 15
Frequency analysis of functional Ig C epsilon gene expression in the presence and absence of interleukin 4 in lipopolysaccharide-reactive murine B cells from high and low IgE responder strains; Savelkoul HF et al.; Nonresponder SJL mice produce low levels of antigen-specific IgE after immunization, compared to responder strains . Young athymic BALB/c nude mice are unable to produce antigen-specific or total IgE in their serum . These mice also have very numbers of background IgE-secreting cells in their lymphoid organs . High-responder BALB/c mice do have substantial numbers of background IgE-secreting cells while low-responder AKR mice show intermediate numbers . Similar differences were found when analyzing lipopolysaccharide (LPS)-reactive B cells in cell suspensions of spleen and bone marrow in limiting dilution cultures . Limiting dilution analysis of T cell-depleted splenic B cell cultures revealed that the defective IgE production in SJL mice is not due to an intrinsic B cell defect . This defect can be substantially overcome by addition of exogenous interleukin 4 (IL4) to these cultures . Furthermore, it was shown in limiting dilution cultures that SJL thymocyte feeder cells were able to suppress IgE production by LPS-activated high-responder BALB/c B cells . The addition of IL4 or neutralizing antibodies against IL4 or interferon-gamma to these cultures helped to overcome this suppressive effect to a large extent . We conclude that different IgE responder types are caused, at least in part, by a defective IL4 production or by a defect in the TH2 system that is functionally detectable at the level of thymocytes.

Gamete Res, 1988 Aug, 20(4), 437 - 58
Trout Sertoli and Leydig cells: isolation, separation, and culture; Loir M; Trout testes at various stages of maturation were dissociated by perfusion at 12 degrees C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin . This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes) . Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml) . Sertoli and Leydig cells were prepared by a two-step separation method: 1) the testis cell suspension was separated by sedimentation at unit gravity into "isolated cell" and "cell cluster" populations; 2) these populations were fractionated by isopyknic centrifugation in Percoll gradients . In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes) . These various cell populations were cultured in modified Leibovitz L15 medium for 10-15 days . When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin . Leydig cells remained 3 beta-HSD positive and produced progesterone and 17 alpha, 20 beta-OH progesterone for at least 11 days . This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.

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