Cell suspensions

J Immunol Methods, 1990 Dec 31, 135(1-2), 181 - 9
A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens; Arunachalam B et al.; This paper describes the adaptation of a cellular enzyme-linked immunosorbent assay (CELISA) for the detection of antibodies to cell-surface antigens . This CELISA has the advantages of convenience and rapidity and is therefore ideally suited for the screening of a large number of hybridoma culture supernatants . The basic procedure involves the direct drying of cell suspensions onto the wells of enzyme immunoassay (EIA) plates and a subsequent EIA with appropriate blocking reagents . In order to overcome high background binding of primary antibodies to Fc receptors and of secondary antibodies to surface Ig (sIg), this method involves a blocking step consisting of unlabelled secondary antibodies . Once CELISA plates are prepared, they can be stored for a period of at least 6 months and hence this assay does not rely on the availability of fresh, viable cells for each assay . This assay is simple, reproducible and sensitive . The results can be assessed in an objective manner and can also be adapted for the detection of cellular antigens . This paper describes a CELISA for the detection of antibodies to blood group antigens and human leukocyte (HLA) antigens.

J Comp Neurol, 1990 Dec 22, 302(4), 729 - 38
Long-term survival and sprouting in culture by motoneurons isolated from the spinal cord of adult frogs; Kuffler DP; Motoneurons of adult frog spinal cord have been retrogradely labeled with the carbocyanine derivative diI . Spinal cords were then dissociated and the labeled motoneurons partially purified by centrifugation over a bovine serum albumin (BSA) density gradient . The resulting cell suspension was plated on a substrate of innervated muscle extracellular matrix (ECM) to which the motoneurons attached and extended processes . Labeled adult motoneurons survived for more than 4 weeks in a defined medium in the absence of added serum or growth factors . These cultures of adult motoneurons provide a favorable preparation for studying molecular factors that influence process outgrowth and synapse formation.

Nippon Gan Chiryo Gakkai Shi, 1990 Dec 20, 25(12), 2781 - 7
{Consideration of simultaneous combination chemotherapy--employing a sensitivity test in Dunn osteosarcoma and NR fibrosarcoma by intra-test tube contact of tumor cell suspension, and subcutaneous inoculation}; Inoue O et al.; Fundamental concepts of combination multi-drug chemotherapy have not been well recognized from the aspects of chemo-sensitivity test upon malignant tumors . A chemo-sensitivity test by in-vitro bioassay for Dunn osteosarcoma and NR fibrosarcoma was developed by us to study the simultaneous interactions between two anticancerous agents . 0.1 ml of cell suspension of either mouse sarcoma was immersed in 0.4 ml of RPMI 1640 cell culture medium containing an anticancerous agent such as Mitomycin (MC), Cyclophosphamide (CPM), Vincristine (VC), Bleomycin (BM), 5-FU, Adriamycin (ADM), Cisplatin (CDDP) or Methotrexate (MTX) in a test-tube, and incubated at 37 degrees C for 3 or 6 hours . Then, the sedimented cell suspension of 0.1 ml was inoculated subcutaneously in the dorsum of C3H mouse which provided 4 sites for 4 different sensitivity tests . In 3 weeks, sensitivities of the anticancerous agents were evaluated as positive sensitivity if no growth of the tumor was observed, or negative sensitivity if the growth of more than 10 mm in diameter was observed . Then, the determination of antitumorous effect on 2-drug combination out of the 8 anticancerous agents, were performed on each mouse sarcoma by the same method . In Dunn osteosarcoma or NR fibrosarcoma, the combination of 2 sensitivity-positive agents revealed no apparent synergistic effects . In any combinations of one sensitivity-positive agent with the other sensitivity-negative agent, except the combinations with CPM which possessed mighty antitumorous effect, apparent reduction of antitumorous effects was observed . The combination of 2 sensitivity-negative agents never produced any antitumorous effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1990 Dec 15, 272(3), 637 - 45
Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells; Li GD et al.; The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA) . Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished . Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx) . Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells . OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ({Ca2+}i), both of which were unaffected by PKC down-regulation . Except for that caused by vasopressin, the secretagogue-induced {Ca2+}i elevations were similar in control and PKC-depleted cells . The {Ca2+}i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented . These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion . Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment . PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations . At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment . The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses . However, changes in cyclic AMP production could not explain the difference . These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.

J Immunol, 1990 Dec 15, 145(12), 3992 - 7
Preferential proliferation of T cell receptor V gamma 3-positive cells in IL-2-stimulated fetal thymocytes; Leclercq G et al.; Thymocyte cell suspensions, prepared from mice at different ages, were cultured in vitro with human rIL-2 . This stimulation resulted in a cell population that contained almost 50% TCR-gamma delta-positive cells if thymocytes were taken from fetal day 17 until just after birth . Analysis of the variable (V gamma) region used by the TCR-gamma delta cells revealed that 90% of them expressed TCR-V gamma 3, and less than 5% expressed TCR-V gamma 2 . Cells positive for TCR-alpha beta were barely detectable . If fetal day 18 organ cultured thymus lobes, instead of a cell suspension, were stimulated with IL-2, no rise in the number of TCR-V gamma 3+ or TCR-delta+ cells was observed, whereas a partial outgrowth of TCR-alpha beta+ cells occurred . From day 1 after birth, the number of TCR-gamma delta cells recovered from an IL-2-stimulated thymocyte cell suspension dropped to reach a plateau of 15% of the total cell number, whereas TCR-V gamma 3+ cells became undetectable in older animals . TCR-alpha beta+ cells, on the other hand, quickly rose in cell number after birth . Kinetic analysis showed that the preferential outgrowth of TCR-V gamma 3+ cells in IL-2-stimulated fetal day 18 thymocyte cell suspensions was present from the onset of the culture; a significant proliferation of CD4 or CD8 single positive TCR-alpha beta cells was never observed . This lack of proliferation of TCR-alpha beta cells was not due to inhibition by the activated TCR-V gamma 3+ cells . Throughout the IL-2 culture, one-fourth of the TCR-V gamma 3+ thymocytes was positive for CD8 . Analysis of the DNA content and the IL-2 receptor (IL-2R) p55 expression showed that during the first days of culture the TCR-V gamma 3+ cells had a much higher proliferation rate than the TCR-V gamma 3- cells, although TCR-V gamma 3+ IL2R p55+ cells could not be detected . From day 3 to 4 of culture, the proliferation rate of TCR-V gamma 3+ cells equaled that of the rest of the cells and less than 20% of the TCR-V gamma 3+ cells expressed the IL-2R p55 . The biologic significance of our findings is discussed.

Biochim Biophys Acta, 1990 Dec 10, 1055(3), 234 - 9
Catabolism of geraniol by cell suspension cultures of Citrus limon; Berger RG et al.; The addition of geraniol to cell suspension cultures of Citrus limon resulted in the rapid formation of nerol, citronellol, geranic acid and citronellic acid . Concurrently, a transient accumulation of bound forms of branched chain fatty acids, and, with a few hours delay, of regular chain C2 to C12 fatty acids was elicited . A concerted action of combined alpha/beta-oxidation enzymes on the terpenic acids, followed by an enlarged acetyl CoA pool is suggested . Terpene catabolism in plants and in vitro plant cells is discussed.

J Comp Neurol, 1990 Dec 8, 302(2), 272 - 93
Regeneration of adult dorsal root axons into transplants of fetal spinal cord and brain: a comparison of growth and synapse formation in appropriate and inappropriate targets; Itoh Y et al.; Cut dorsal root axons regenerate into transplants of embryonic spinal cord and form synapses that resemble those found in the dorsal horn of normal spinal cord . One aim of the present study was to determine whether these axons also regenerate into and establish synapses within transplants of embryonic brain . A second aim was to compare the patterns of growth in embryonic brain and spinal cord transplants . Embryonic spinal cord or brain was transplanted into the lumbar enlargement of adult Sprague-Dawley rats, the L4 or L5 dorsal root was cut, and the cut root was juxtaposed to the transplant . The transplants included whole pieces or dissociated cell suspensions of embryonic day 14 (E14) spinal cord, or whole pieces of E14 neocortex, E18 occipital cortex, E15 cerebellum, or E18 hippocampus . One month later the regenerated dorsal root axons were labeled by immunocytochemical methods to demonstrate calcitonin gene-related peptide (CGRP) . CGRP-immunoreactive axons regenerated into all the transplants examined and formed synapses in the neocortex and cerebellum transplants in which they were sought . Synapses were far rarer in neocortex and cerebellum than we had observed previously in transplanted spinal cord, and the patterns of growth differed in transplants of spinal cord and brain . In solid transplants of spinal cord, regenerated axons remained relatively close to the interface with the dorsal root, branched, and formed bundles . Areas of dense ingrowth were separated by regions with few labeled axons . In transplants of brain regions, the regenerated axons were few, unbranched, and appeared as individual fibers rather than in bundles, but they were distributed widely in neocortex transplants . The results of quantitative studies confirmed these observations . The area fraction occupied by regenerated axons in solid spinal cord transplants was significantly larger than in occipital cortex or cerebellum transplants . Distribution histograms of the area occupied in transplants demonstrated that regenerated axons were distributed sparsely but homogeneously in transplants of brain, whereas spinal cord transplants were heterogeneous for regenerated axons and contained areas in which growth was dense or sparse . In contrast, several measurements of axon distribution, including area, longest axis, and length of lateral extension, indicated that CGRP-labeled axons spread more widely in occipital cortex transplants than in solid transplants of spinal cord or cerebellum . The results indicate that embryonic CNS tissues that are not normal targets support or enhance the growth of severed dorsal roots and suggest that the conditions that constitute a permissive environment for regenerating axons are relatively nonspecific.(ABSTRACT TRUNCATED AT 400 WORDS)

J Immunol Methods, 1990 Dec 5, 134(2), 153 - 61
A new method for removal of mononuclear phagocytes from heterogeneous cell populations in vitro, using the liposome-mediated macrophage 'suicide' technique; Claassen I et al.; In this study we present a new method for the elimination of mononuclear phagocytic cells from cell suspensions . By making use of liposome-encapsulated dichloromethylene diphosphonate we were able to effectively remove macrophages from spleen cell suspensions . This effect was not observed when using the free drug or control (PBS) liposomes . The use of this procedure has no effect on other cell types, as measured by growth, protein production, antigen presentation and antigen specific T cell proliferation, though PBS liposomes in very high doses were able to inhibit antigen presentation . The finding that lymphocytes are not affected by the liposome encapsulated drug suggests that the observed loss of lymphocytes in vivo, after intravenous dichloromethylene diphosphonate liposome treatment, may be due to damage inflicted by lysosomal enzymes released from dying macrophages . This method permits the removal of both macrophages and monocytes from heterogeneous cell populations (i.e., blood, lymphoid tissue suspension) in vitro with a very high rate of reliability . With the concentrations and incubation time used, no negative effects on other cell types were observed.

J Invest Dermatol, 1990 Dec, 95(6 Suppl), 121S - 124S
Interleukin-1 and interleukin-6 in psoriasis; Prens EP et al.; We report on the levels of expression of IL-1 and IL-6 in skin from psoriasis patients . Different approaches were pursued . Initially, the levels of IL-1 beta and IL-6 were measured in suction blister fluid from lesional and uninvolved skin from psoriasis patients, using a sensitive enzyme-linked immunoabsorbent assay (ELISA) and bio-assay . Skin sections were also examined for the presence of IL-1 and IL-6 using IL-1 beta- and IL-6-specific antibodies . Finally, the expression of IL-1 and IL-6 mRNA was determined in cultured keratinocytes (KC) and fibroblasts from psoriasis skin . Suction blister fluid from lesional and uninvolved psoriasis skin and from skin of healthy individuals did not contain detectable levels (greater than 100 pg/ml) of IL-1 beta . Blister fluid from psoriasis lesions contained low but significant levels of IL-6, whereas the serum levels of IL-6 in these patients was undetectable . Using cryostat skin sections and an IL-1 beta-specific monoclonal antibody (MoAb) in an indirect immunoperoxidase technique, a diffuse staining in the entire epidermis was observed in sections of uninvolved skin from psoriasis patients . In cryostat sections of psoriasis lesions, a faint diffuse staining of the epidermis and a pronounced "dot-like" intracellular staining pattern was observed . On the other hand, the same IL-1 beta-specific MoAb showed, in a indirect immunofluorescence technique using unfixed epidermal cells, bright membrane staining in epidermal cell suspensions from psoriasis lesions . Slightly elevated levels of IL-1 beta and IL-1 alpha mRNA were observed in cultured KC from psoriasis lesions as compared to those in normal KC and in the HEp-2 cell line . Very low levels of IL-6 mRNA were expressed in KC from psoriasis lesions and healthy individuals . Fibroblasts from psoriasis lesions expressed extremely low levels of IL-1 alpha and IL-1 beta, but high levels of IL-6 mRNA . The results point to a paradoxical situation in psoriatic skin: blister fluid from psoriasis lesions contains no IL-1 beta, whereas IL-1 beta is overexpressed on the plasma membrane and in the intracellular compartment of epidermal cells . This finding may help in explaining the observed absence of IL-1 in aqueous extracts of psoriatic scales . Because cultured KC from psoriasis lesions express minimal levels of IL-6 mRNA . dermal fibroblasts, probably together with the inflammatory infiltrate, may represent a major source of IL-6 in psoriasis lesions in vivo.

Surgery, 1990 Dec, 108(6), 1033 - 8; discussion 1038-9
Characterization of a simplified method of cryopreserving human parathyroid tissue; Saxe AW et al.; Cryopreservation of human parathyroid tissue plays an important role in managing difficult parathyroid disease . It also can permit investigators to conduct experiments without dependence on the operating room schedule . Availability of cryopreservation has been limited by the perceived need for expensive, complex equipment . We adapted a simple method of freezing cell suspensions to freezing human parathyroid tissue . Vials containing human parathyroid in culture media, dimethylsulfoxide, and patient serum were placed in a plastic rack in a metal pan containing prechilled (4 degrees C) ethanol and placed in a -70 degrees C freezer . We compared viability (trypan blue dye exclusion by collagenase dispersed cells) of tissue frozen in this manner to that of tissue frozen in a programmable liquid nitrogen freezer at 1 degrees C per minute, a cooling rate recommended for human parathyroid tissue . The viability of 30 patients' samples cooled in liquid nitrogen (average length of storage 5 months) was 74% +/- 15% and that of 64 patients' samples cooled in ethanol (average length of storage 26 months) was 71% +/- 15% . Viability of 19 samples of fresh tissue was 79% +/- 10% . Neither method had a statistically significant correlation between length of storage and viability . Successful cryopreservation with simplified technology may expand the availability of parathyroid tissue to meet both clinical and investigative requirements.

J Clin Microbiol, 1990 Dec, 28(12), 2739 - 43
Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction; van den Brule AJ et al.; A two-step polymerase chain reaction (PCR) procedure was used as a new screening strategy for the detection of human papillomavirus (HPV) genotypes in cervical scrapes omitting prior DNA extraction . Sample preparation consisted of a freeze-thaw step followed by boiling the cells before the PCR mixture was added . This pretreatment was as efficient and reproducible for HPV DNA amplification as DNA purification . By using crude cell suspensions, a prescreening of the samples with the general primer-mediated PCR method (GP-PCR) was performed to detect a broad spectrum of sequenced and still unsequenced HPV types at the subpicogram level . HPV-containing scrapes by GP-PCR were subjected to HPV 6, 11, 16, 18, 31, and 33 type-specific PCR (TS-PCR) to identify the sequenced HPV types . This direct GP/TS-PCR method was tested on a large group of cervical scrapes (n = 459) from women visiting a gynecologic outpatient clinic . The results were compared with HPV data obtained by a method using modified filter in situ hybridization and TS-PCR in which the PCR was mainly used to confirm HPV positivity . A substantially higher HPV prevalence rate was found by direct GP/TS-PCR strategy . The results indicate that GP/TS-PCR is a rapid, sensitive, and reliable detection method for HPV in cervical scrapes . The easy performance on crude cell suspensions makes this strategy applicable for large HPV-screening programs.

Fukushima J Med Sci, 1990 Dec, 36(2), 59 - 70
Isolation of rat megakaryocytes by immunomagnetic beads; Shikama Y; An immunomagnetic procedure was developed to purify rat megakaryocytes to homogeneity from flushed marrow cells . The cells from femurs, tibias, and humeri were initially centrifuged at low speed (800 rpm) to remove platelets and layered over a 1.050 g/cm3 Percoll density gradient . After washing at relatively high speed (1,500 rpm), rabbit anti-rat platelet serum (APS) was added to the cell suspension and incubated for 30 min at 4 degrees C . The cells were washed and subsequently treated with immunomagnetic beads coated with sheep anti-rabbit IgG antibody at room temperature for 10 min . Megakaryocytes were selectively isolated using a magnetic concentrator with a purity of 96.6 +/- 3.9%, recovery of 67.7 +/- 30.8%, and viability of 96.0 +/- 3.2%, although megakaryocytes accounted for 0.11 +/- 0.05% of starting marrow cells . Four to 7 x 10(6) megakaryocytes were obtained from 30 rats with a single population . This quantity provided us a possibility to characterize cytokine receptors . To determine if the nearly purified megakaryocytes were able to response to erythropoietin (Epo), one of purified promoting factors in megakaryocytopoiesis, the cells were incubated with 125I-labeled Epo at 15 degrees C for 90 min in the absence and presence of 100-fold excess unlabeled Epo . Autoradiographic analysis demonstrated the specific silver grains on the megakaryocytes, suggesting the presence of Epo receptors . Scatchard analysis revealed a single class of binding sites . These findings suggest that this method may be useful for megakaryocytic receptor studies of cytokines as well as the physiology or biochemistry of megakaryocytes.

Development, 1990 Dec, 110(4), 1091 - 9
Organotypic arrangement of mouse embryonic lung cells on a basement membrane extract: involvement of laminin; Schuger L et al.; The behavior of embryonic murine lung cells on a basement membrane extract (Matrigel) was investigated . Single cell suspensions generated by trypsinization of lungs removed from day 12 embryos were plated on Matrigel and cultured for up to one week . The basement membrane extract was used as a gel, and as a wet or dried film . In all of these instances, organotypic arrangement of the embryonic lung cells was observed . This process consisted of cell aggregation, sorting, polarization and formation of a tridimensional organization resembling embryonic lung . The maximal degree of organotypic development was obtained by using a thick gel; minimal reorganization was observed using a dried film . A rabbit polyclonal serum to laminin inhibited organotypic pattern formation while normal rabbit serum did not . Culture of lung cells on laminin gels promoted epithelial cyst formation but poor mesenchymal organization . By studying the behavior of epithelial and/or mesenchymal enriched cell populations on Matrigel, it was concluded that organotypic pattern formation on Matrigel required the presence of both cell populations . Cultivation of dissociated lung cells on a gel consisting of a mixture of collagens type I and III (Vitrogen-100) produced only cell aggregation . Cultivation of lung cells on a thin film of Vitrogen-100 or on uncoated tissue culture plastic produced monolayers of mesenchymal cells alone . Cultivation of lung cells in suspension also failed to induce organotypic arrangement even at maximal cell densities . The present study strongly supports a role for the basement membrane in the organotypic rearrangement of embryonic lung cells and subsequent in vitro cyst formation and budding of the reestablished epithelium . This, in turn, reinforces the concept of the basement membrane as a major regulator of organogenesis.

Bone Marrow Transplant, 1990 Dec, 6(6), 395 - 8
T cell depletion of human bone marrow using an oxidase-peroxidase enzyme immunotoxin; Ito H et al.; We have shown in a previous paper that an antibody-enzyme immunotoxin (eIT) constructed by chemically coupling the 097 monoclonal antibody to glucose oxidase and to lactoperoxidase (097 eIT) effectively eliminated T cells of human peripheral blood origin and killed cultured human thymoma cells . Here we tested its effectiveness against T cells present in human bone marrow cell suspensions contaminated by large numbers of erythrocytes . The T cell-depleting capacity of the 097 eIT was assessed by means of four different assay methods, three of which gave concordant results and indicated an effective depletion comparable in efficiency to published work . For example, by limiting dilution analysis assay of IL-2-producing T cells we found approximately 3 logs of T cell depletion . The growth of bone marrow stem cells (CFU-GM, CFU-E and BFU-E) was not affected by treatment with the 097 conjugates.

Recenti Prog Med, 1990 Dec, 81(12), 792 - 6
Serum suppressive factors may account for the reduced polymorphonuclear cell function in haemophilia; Serlenga E et al.; Haemophiliacs exhibit a broad range of immune defects . In this regard we have investigated the functional capacity of purified polymorphonuclear (PMN) cell suspensions in a group of Human Immunodeficiency Virus (HIV)+ or HIV- patients . Our results provide evidence for a significant reduction of PMN-mediated chemotactic responsiveness, phagocytosis and killing in haemophiliacs regardless of HIV infection . The depressed response does not reflect a PMN intrinsic dysfunction, since respiratory burst activity and lysosomal enzyme release from haemophilic PMN are unaffected in comparison to healthy donors . Quite interestingly the pretreatment of PMN from normal donors with either HIV+ or HIV- haemophilic sera gives rise to a reduction of PMN activity . Moreover, the suppressive effect is abrogated by serum heat inactivation . Taken together, these findings indicate a role for serum suppressive factors in the imbalance of PMN functional capacity in haemophilia regardless of HIV infection.

J Biol Response Mod, 1990 Dec, 9(6), 546 - 55
Immunotherapy with lymphokine-activated natural killer cells and recombinant interleukin-2: a feasibility trial in metastatic renal cell carcinoma; Hercend T et al.; Clinical immunotherapy trials have been performed recently where ex vivo interleukin-2 (IL-2)-activated peripheral blood mononuclear cells (i.e., the "LAK" cells) have been transfused in addition to IL-2 infusions . In such protocols, patients have received highly heterogeneous cell suspensions and the nature of the effector cells that may have contributed to tumor regression has remained unclear . In certain animal models, it has appeared that natural killer lymphocytes were the effector cell type responsible for tumor regression . To test whether NK cells could eventually be relevant for the treatment of human tumors, we have performed a feasibility trial where purified lymphokine-activated natural killer (LANAK) cells have been prepared and transfused to a limited series of renal cell carcinoma patients receiving IL-2 (continuous infusions at 3 x 10(6) U/m2/day) . Natural killer lymphocytes (1-2 x 10(6} were purified from peripheral blood mononuclear cells and expanded during 4-5 weeks in the presence of IL-2 on microtiter plates containing feeder layers cells . In vitro, the resulting LANAK cell suspensions were 100 times (range of 2 to 10(3} more efficient against Daudi target cells than their autologous LAK counterparts . Twelve patients were included; 9 received the two planned courses of treatment with LANAK cells and IL-2 . Overall toxicity was relatively moderate . Besides occasional chills, there were no apparent secondary effects due to cell infusions . The mean number of LANAK cells transfused per patients was 45.1 x 10(9), ranging from 7 to 125 x 10(9) . The biodistribution of LANAK cells was similar to that reported previously for LAK cells with no preferential localization to tumor sites . We conclude from this study that using well-defined populations of effector lymphocytes is a feasible cellular therapy approach that may lead to improved understanding and efficacy of the novel immunotherapy methods.

Exp Neurol, 1990 Dec, 110(3), 258 - 67
Formation of synaptic graft-host connections by noradrenergic locus coeruleus neurons transplanted into the adult rat hippocampus; Murata Y et al.; Transplants of cell suspension obtained from the locus coeruleus region of 13- to 14-day-old rat fetuses were implanted into the hippocampal formation of intact adult rats or rats from which the noradrenergic afferents to the hippocampus had been removed by bilateral 6-hydroxydopamine (6-OHDA) injections into the dorsal tegmental noradrenergic bundle . The growth noradrenergic axons into the host hippocampus from the implant was studied at 4-8 months after surgery by immunohistochemistry using antisera raised against tyrosine hydroxylase or noradrenaline . In the animals with an intact noradrenergic system the host noradrenergic afferents were removed by bilateral dorsal bundle lesions 2 weeks before sacrifice . Fine axon-like fibers (diameter about 0.3 micron) and thick dendrite-like fibers (diameter about 1.3 micron), labeled immunohistochemically, were abundant and spread far from the graft . By electron microscopy, immunolabeled axon-like fibers formed mostly symmetrical synaptic contacts with nonlabeled spines and shafts of dendrites in the host . Labeled dendrite-like fibers of presumed graft origin penetrated deep into the host neuropil and received abundant afferents from nonlabeled axon terminals . The extent of graft-derived noradrenergic axons and the synapses established with the host hippocampal neurons were similar in the chronically denervated animals and in the animals where the intrinsic noradrenergic afferents had been left intact until 2 weeks before sacrifice . The results show that implanted embryonic noradrenergic neurons are able to innervate the hippocampus in both the presence and the absence of an intact intrinsic noradrenergic innervation and that the ingrowing axons form abundant synaptic connections with the host hippocampal neurons under both conditions . Dendritic processes from the grafted noradrenergic neurons that extend deep into the host tissue may receive a reciprocal synaptic host afferent input.

Pflugers Arch, 1990 Dec, 417(4), 433 - 9
Calcium currents in the A7r5 smooth muscle-derived cell line; Marks TN et al.; We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patch-clamped cells, as well as cytosolic calcium ({Ca2+}i) in fura-2-loaded cell suspensions and monolayers . Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels . When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant . With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV . Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of approximately 1 microM . Isoproterenol (1 microM) or forskolin (10 microM) increased currents in approximately half of the cells tested . Forskolin (10 microM) increased single-channel activity in five of eight cell-attached patches . After cells had been quiescent for several weeks, cell suspensions showed changes in resting {Ca2+}i in response to DHPs and increased potassium . Most confluent monolayers of cells showed spontaneous transient elevations in {Ca2+}i . Bath application of Bay K 8644 increased the frequency and magnitude of these {Ca2+}i transients, whereas nifedipine abolished the transients . These data suggest that the {Ca2+}i transients were due to synchronous action potentials in electrically coupled cell monolayers.

Biotechniques, 1990 Dec, 9(6), 684, 686, 688 - 9
A multiwell cell settling and adherence chamber for morphology and differential counting; Leonard EJ et al.; A simple multiwell chamber is described that can be used to prepare randomly distributed cells on a microscope slide, suitable for morphological identification and differential counting . To the eight wells of the chamber are added 50-microliter volumes of cell suspension at concentrations of 10(3)-10(6) cells/ml . As the cells settle, fluid is slowly wicked away by a damp filter paper sandwiched between the microscope slide and the acrylic top plate of the multiwell chamber . Within 20-40 minutes, the cell monolayers on the slide are completely dry . The combined settling and bulk fluid removal results in a distribution of adherent cells that are sufficiently spread to exhibit excellent morphology after staining . If the chamber is centrifuged for 30 seconds at 50x g immediately after addition of cells, recovery of cells in the monolayer is virtually 100%, and as few as 50 input cells per 50 microliters can be detected . Agreement between predicted and observed differential counts of cell mixtures indicates that cells in the monolayer were distributed randomly.

Brain Res, 1990 Nov 26, 534(1-2), 83 - 93
Long-term survival of grafted cells, dopamine synthesis/release, synaptic connections, and functional recovery after transplantation of fetal nigral cells in rats with unilateral 6-OHDA lesions in the nigrostriatal dopamine pathway; Nishino H et al.; In animal models of hemi-Parkinson's disease, survival of grafted nigral cells, their synaptic connections, dopamine (DA) synthesis/release, and recovery from motor disturbances were investigated, and these were compared among 3 groups of animals raised for 3 months, 1 year and 2 years after the transplantation . Fetal nigral DAergic cell suspensions were transplanted in the ipsilateral caudate nucleus of rats with unilateral 6-OHDA lesions in the nigrostriatal DA pathway . Motor disturbances, assessed by methamphetamine-induced rotation, recovered partly in the 2nd week, significantly in the 4th week after the grafting, and remained stable thereafter . Many tyrosine hydroxylase (TH)-positive cells were detected along the grafting tracks . The number of TH-positive cells was similar in the 3 groups of animals . These TH-positive cells made synaptic connections in the host caudate . By in vivo microdialysis measurement, extracellular DA, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) around the grafted sites recovered to 30-100% of those of controls . No significant differences were observed in the concentration of DA, DOPAC and HVA among 3 groups of animals . They also responded to methamphetamine loading though the magnitudes were smaller . Using a TH cDNA probe, TH-positive cells were found to express TH mRNA in in situ hybridization-autoradiographic analysis . Data indicate that grafted fetal DAergic cells survive, synthesize and release DA, make synaptic connections in the host brain and ameliorate motor disturbances for over 2 years . There were no differences in these parameters among the 3 groups of animals, and no untoward side effects were observed even at 2 years after the grafting . Thus it was confirmed that the grafting of neuronal cells into the brain is a promising approach to restore disturbed function.

Brain Res, 1990 Nov 26, 534(1-2), 73 - 82
tGS ganglioside induces peculiar morphological features in grafted dopaminergic cells and promotes motor recovery in rats with unilateral lesions in the nigrostriatal dopamine pathway; Nishino H et al.; A cell suspension of substantia nigra from fetal rats was introduced into the ipsilateral caudate nucleus of rats with unilateral lesions in the nigrostriatal dopamine pathway, and effects of bovine total ganglioside (tGS) and monosialoganglioside (GM1) treatment on the morphological features of the transplanted cells and recovery from motor imbalance (rotation induced by methamphetamine) were investigated . Gangliosides (30 mg/kg) were administered intraperitoneally once a day for 2 weeks after transplantation to test animals while control animals received saline alone . tGS animals showed definite motor recovery in the 2nd week (P less than 0.05) while control and GM1 animals exhibited slight recovery only . At 6 weeks after transplantation, motor imbalance disappeared in all 3 groups . Tyrosine hydroxylase (TH) immunocytochemical staining revealed that in the 2nd week TH-positive cells in tGS animals had more primary dendrites and more large neurites (meganeurites) than did controls . TH-positive cells of all 3 groups often had spiny processes at that time . In the 20th week, TH-positive cells became more multigonal and had wider dendritic fields in all groups, and had less meganeurites and spines . Motor recovery of each animal was dependent on the number of TH-positive cells and no significant difference was observed in the number of TH-positive cells among the three groups . tGS treatment for 2 weeks without grafting induced immunohistologically no axonal sprouting in the substantia nigra, medial forebrain bundle, accumbens and caudate nucleus when the chemical lesions were complete . Data suggest that tGS induces hypertrophy but not hyperplasia of the transplanted nigral cells, and increases the morphological plasticity . This might be the basis for promotion of recovery in motor function after transplantation.

J Comp Neurol, 1990 Nov 22, 301(4), 520 - 34
Homotypic fetal transplants into an experimental model of spinal cord neurodegeneration; Nothias F et al.; Many neurotransplantation studies have dealt with the ability of solid fetal spinal grafts to develop in the previously traumatized spinal cord of a host . In neurodegenerative spinal diseases, however, motoneuronal death occurs in the absence of a trauma, i.e., in the absence of axotomy of afferent fibers . Lesioning the spinal cord with an excitotoxic agent may provide a useful neurodegenerative model . The present study has been undertaken to determine whether homotypic fetal neurons transplanted as a cell suspension are able to rebuild a neural circuitry . Emphasis is given here to the analysis of the development of transplanted motoneurons and host-graft connectivity . The lesion was made by kainic acid on the right side of the lumbar enlargement 1 week before transplantation . The fetal spinal cords were taken from rat embryos (gestational day E12-13) and transplanted as cell suspensions . Light- and electron-microscopic analysis demonstrated that the excitotoxic lesion extended over the entire spinal segment and was confined primarily to the ventral and intermediate horns, implying the death of all motoneurons with consequent paralysis and muscular atrophy of corresponding hindlimb . The lesion was characterized by a lack of neurons, glial proliferation, and sparing of fibers of passage and afferents . Two to fourteen months after surgery, the transplants were generally large, occupying most of the neuron-depleted area . The boundaries between the transplant and host tissue were clearly delineated by the higher cellular density of the graft and the particular cytoarchitecture, i.e., the cell suspension grafts did not display a laminar organization . Among the different neuronal populations within the transplant, one resembled motoneurons: large, typically Nissl-stained and immunoreactive for calcitonin gene-related peptide (CGRP) . No grafted neuron, however, extended an axon into the host ventral roots . Monoaminergic afferents from the host were studied using immunostaining for serotonin, noradrenaline, and tyrosine hydroxylase . These afferent fibers, thin and varicose, grew for a long distance and formed a network within transplants . Similarly, primary sensory CGRP-immunoreactive fibers (entering the graft from the dorsal host-graft interface) penetrated deeply into transplants . The response of cortico- and rubro-spinal afferents to the implantation of fetal tissue was different . After injection of WGA-HRP, a few anterogradely labeled cortical and rubral fibers entered only the most peripheral portion of transplants . In conclusion, our results indicate that fetal spinal neurons can be successfully transplanted into the adult neuron-depleted spinal cord.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem J, 1990 Nov 15, 272(1), 147 - 50
Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions; Lescure AM et al.; Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures {Proudhon, Briat & Lescure (1989) Plant Physiol . 90, 586-590} . This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate . It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin . It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis . It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration . However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.

Anal Biochem, 1990 Nov 15, 191(1), 144 - 55
Characterization of the efficiency of a cell separation process by the extent of elimination of a contaminating cell type; Bruyninckx WJ et al.; A stepwise approach to the selection of an appropriate technique for a cell separation problem is presented in which the preparative purification of cells is linked to their analytical separation . We have introduced the extent of elimination of a contaminating cell type from the cell type which one chooses to purify, as a separation parameter that characterizes the efficiency of a separation process independently of the relative cell composition of the starting material . In order to compare different separation techniques, a preparative fraction boundary needs to be chosen between the cell types . We defined this boundary in terms of the physical property on which the separation is based such that yield and purity of the isolated cell suspension are optimized simultaneously . With this analytical approach, it was found that a similar elutriation technique separated human and equine mononuclear cells equally well and that the separability of human monocytes and lymphocytes improved when the cells were separated by increasing the limiting sedimentation coefficient value of the elutriation chamber in small increments.

Anal Biochem, 1990 Nov 15, 191(1), 138 - 43
Use of thionitrobenzoic acid to characterize the stability of nitric oxide in aqueous solutions and in porcine aortic endothelial cell suspensions; Clancy RM et al.; Nitric oxide is an important vasodilator which can be biologically produced from leukocytes and endothelial cells . However, it is highly unstable, which is an obstacle to detection and quantitation . We have exploited the reactivity of nitric oxide with thiols to establish an assay based on oxidation of thionitrobenzoic acid (TNB) . The oxidation of thionitrobenzoic acid and the reaction with oxygen, which was measured by employing an oxygen electrode, were examined after the addition of nitric oxide solutions . The inhibition of aggregation of human platelets after challenge with 2.5 microM adenosine diphosphate was also investigated . These studies show the following properties of nitric oxide in aqueous solutions . (i) Nitric oxide is highly reactive to oxygen . (ii) Thiols react with a labile, highly reactive nitric oxide-oxygen product . (iii) Medium with very low oxygen content increases the life span of nitric oxide in aqueous solution . We also used the nitric oxide quantitation using TNB to study the metabolism of nitric oxide by porcine aortic endothelial cells and the results show that nitric oxide added to these cells in low oxygen content solution is stable . From these studies, we conclude that deoxygenated solutions stabilize nitric oxide . An important consequence of low oxygen content at localized tissue sites may be to augment biological effects mediated by nitric oxide.

J Biol Chem, 1990 Nov 15, 265(32), 19441 - 6
Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages; Hussaini IM et al.; Primary cultures of murine bone marrow macrophages (BMMs) were prepared from marrow cell suspensions . These cells expressed specific receptors that recognized the transformed conformation of human alpha 2-macroglobulin (alpha 2M) generated by reaction with CH3NH2 . alpha 2M receptor expression was regulated by colony-stimulating factor-1 (CSF-1) . The BMMs were deprived of CSF-1 for 6 h and then treated with different concentrations of the purified cytokine . After 18 h, binding of 125I-alpha 2M-CH3NH2 was examined at 4 degrees C . Analysis of the saturation isotherms and Scatchard transformations indicated that the KD was not affected by CSF-1 (1.9-2.4 nM), whereas the maximum specific radioligand binding capacity (Bmax) was increased from 5.6 x 10(4) receptors/cell in the absence of CSF-1 to 2.2 x 10(5) and 2.6 x 10(5) receptors/cell for BMMs treated with 1,000 and 10,000 units/ml CSF-1, respectively . The difference in total cellular protein after exposure to different levels of CSF-1 for 18 h was small (1.50-1.92 ng/cell) and not statistically significant . A 6-12-h lag phase was identified between the time of CSF-1 exposure and increased alpha 2M receptor expression . Cycloheximide completely blocked the increase in alpha 2M receptor expression when added simultaneously with the CSF-1; greater than 50% inhibition was observed when the cycloheximide was added up to 8 h later . The RNA synthesis inhibitors, actinomycin D and daunomycin, prevented increased alpha 2M receptor expression when added up to 4 h after the CSF-1, but had no effect at 8 h . At 37 degrees C, uptake and digestion of 125I-alpha 2M-CH3NH2 was increased in BMMs treated with 1,000 units/ml CSF-1 for 18 h compared with untreated cells . These studies demonstrate that CSF-1 increases the expression of alpha 2M receptors in BMMs through a pathway that requires new RNA and protein synthesis . We hypothesize that increased alpha 2M receptor expression may play an important role in cellular growth and differentiation.

J Biol Chem, 1990 Nov 5, 265(31), 19324 - 9
Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils; Green TR et al.; Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate . Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions . Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex . Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns . Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex . When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.

Orv Hetil, 1990 Nov 4, 131(44), 2431 - 3
{A case of hybrid acute leukemia in a a child}; Kiss C et al.; Hybrid acute leukaemia is characterized by the presence of lymphoid and myeloid markers in a single cell or in different blast cell subpopulations of the same patient . Authors report on a case of a 14-yr-old girl with hybrid acute leukaemia . Immunofluorescent analysis revealed CD 14, CD 10, CD 19 and HLA-DR antigens in the cell suspension isolated from peripheral blood of the patient . Because of the excess of FAB M1 type blast cells, the patient was treated according to IGCI-1984 protocol . Remission was not achieved despite combined cytotoxic treatment and patient died within 4 weeks following admission . The poor outcome of the disease agrees well with literature data . Hybrid acute leukaemia represents a challenge for the clinical science.

Anal Biochem, 1990 Nov 1, 190(2), 212 - 9
The use of monochlorobimane to determine hepatic GSH levels and synthesis; Fernandez-Checa JC et al.; We have used the specific reaction of monochlorobimane (mBCI) with GSH to analyze hepatic GSH, mBCI, itself nonfluorescent, forms a stable, fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases (GST) . When hepatocytes were labeled with mBC1 (100 microM) in Krebs-Henseleit buffer, the fluorescent signal recorded over time was directly proportional to the concentration of GSH . The HPLC analyses of hepatocytes that were preloaded with the dye indicated that GSH was the only thiol labeled . When the technique was applied to freshly isolated intact hepatocytes that contained different levels of GSH, a close correlation between the levels of GSH measured by the present method (mBC1) and the standard enzymatic recycling method was found . A similar agreement for the cytosolic and mitochondrial pools of GSH determined by the two methods was established . The fluorescent GSH-bimane adduct, once formed within the cell, was not released from the cell . In addition, we have applied this technique to determine directly the rate of synthesis of GSH in both cell-free conditions and in cell suspensions by monitoring the increase in fluorescent adduct when mBC1 is present in excess in the incubation.

Phys Med Biol, 1990 Nov, 35(11), 1575 - 83
Ion concentration and haematocrit as determinants of impedance in an erythrocyte suspension model of renal medullary tissue; Niewiadomski W et al.; In order to analyse the respective roles of ion concentration and fractional volume of the interstitial compartment as determinants of the impedance, Z, of renal medullary tissue, a model was needed in which both these factors could be varied independently . An array of blood cell suspensions ions in saline (different haematocrit values and different NaCl concentrations) was used for this purpose . It was found that: (i) up to a measuring frequency of about 10 kHz, the complex consisting of needle electrodes and 'tissue' can be regarded as serially connected resistances, R, and capacitances; (ii) the frequency range 3-10 kHz can be regarded as optimal since it simultaneously assures low electrode polarization and a negligible role of tissue capacitance; (iii) increasing the haematocrit had two consequences--a reduced contribution of polarization impedance to the total impedance measured and a decreased sensitivity of ion concentration measurement from R-1 (conductance); (iv) passive electrical properties of renal medullary tissue were close to those of a 75% haematocrit cell suspension; (v) since in high haematocrit suspensions the resistive component of impedance predominates, within the frequency range 3-10 kHz either conductance or admittance, Z-1, can be used as an index of ion concentration; and (vi) impedance changes in kidney tissue are primarily determined by fluctuations of ion concentration with a less important contribution from interstitial volume changes.

Vet Microbiol, 1990 Nov, 25(2-3), 229 - 40
Grouping of Actinobacillus pleuropneumoniae strains of serotypes 1 through 12 on the basis of their virulence in mice; Komal JP et al.; Variations in virulence among strains of different serotypes of Actinobacillus pleuropneumoniae were detected on intraperitoneal or intranasal inoculation in mice . In general, strains of serotypes 1, 5, 9, 10 and 11 were found to be highly virulent and those of serotypes 2, 3, 4, 6, 7, 8 and 12 to be less virulent . However, a few strains of serotype 5 caused low mortality in mice while some strains of serotype 3 and 7 were found to be highly virulent . Highly virulent strains of A . pleuropneumoniae were invasive and appeared in the blood within 3 to 6 h of intranasal inoculation . The type specific antigen as detected by the coagglutination test was distributed in lungs, liver, heart and spleen after intraperitoneal inoculation whereas it was mostly concentrated in the lungs after intranasal inoculation . Lowest concentration of boiled whole-cell suspension of A . pleuropneumoniae showing limulus amebocyte lysate activity was variable and independent of the serotype . Mortality caused by boiled whole cell suspension was also variable and serotype independent.

In Vitro Cell Dev Biol, 1990 Nov, 26(11), 1073 - 8
Emergence of flat cells from glia in stationary cultures of embryonic chick neural retina; Moyer M et al.; When embryonic retina is dissociated into a single cell suspension and maintained in stationary culture, a population of flat cells is found on the culture dish . We have carried out a morphologic and immunologic study of the emergence of this population in vitro . Ten- and fourteen-day-old chick embryo retinas were dissociated with trypsin, seeded on glass cover slips for various times, and prepared for scanning electron microscopy (SEM) and immunofluorescence (IF) for Vimentin, an intermediate filament protein . SEM indicates that the characteristic flat cell morphology is initiated in some cells in as little as 30 min after the start of the culture . Not all of the cells that attach flatten . As incubation proceeds, small clusters of cells that had formed in suspension attach to the substrate, and flat cells emerge from them . The flattened cells are positive for Vimentin by IF within 10 min of attachment . The percent of fluorescent cells found on the substrate is constant during the time in culture . This suggests that flat cells do not attach first, followed by neural cells, but that the neural cells and flat cells attach to the dish at the same rate . When aggregates that had formed in suspension attach to the substrate, they are anchored by flat cells that migrate out of the aggregate . Since Vimentin appears in the cultured cells within 10 min, it is unlikely that it has been newly synthesized . Thus, the same cells that contained Vimentin in the retina now express it as flat cells . This supports the hypothesis that flat cells derive from the same cells in the retina that give rise to Muller cells . We have also observed the emergence of a population of cells with short (0.5 micron) microvilli that appear within 8 h of culture . They seem to be a distinct subpopulation of the cells on the upper portion of attached clusters.

Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8736 - 40
Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy; Szilvassy SJ et al.; Although hematopoiesis is known to originate in a population of very primitive cells with both lymphopoietic and myelopoietic potential, a procedure for enumerating such cells has to date not been available . We now describe a quantitative assay for long-term repopulating stem cells with the potential for reconstituting all hematopoietic lineages . This assay has two key features . The first is the use of competitive repopulation conditions that ensure not only the detection of a very primitive class of hematopoietic stem cells but also the survival of lethally irradiated mice transplanted with very low numbers of such cells . The second is the use of a limiting-dilution experimental design to allow stem cell quantitation . The assay involves transplanting limiting numbers of male "test" cells into lethally irradiated syngeneic female recipients together with 1-2 x 10(5) syngeneic female marrow cells whose long-term repopulating ability has been compromised by two previous cycles of marrow transplantation . The proportion of assay recipients whose regenerated hematopoietic tissues are determined to contain greater than or equal to 5% cells of test cell origin (male) greater than or equal to 5 weeks later is then used to calculate the frequency of competitive repopulating units (CRU) in the original male test cell suspension (based on Poisson statistics) . Investigation of this assay system has shown that all three potential sources of stem cells (test cells, compromised cells, and the host) can under appropriate circumstances contribute to long-term hematopoietic regeneration, thus establishing both the competitive pressure of hematopoietic stem cells in the cotransplanted compromised population and in the host, and the need to use genetic markers to track the specific contribution of the injected test cells . Analysis of the frequency of CRU in test marrow suspensions that varied widely in their CRU content gave similar values when endpoints of either 5 or 10 weeks posttransplantation were used and when either recipient marrow or thymus was used to identify progeny populations . In addition, repopulation of marrow and thymus was found to be associated in most mice injected with limiting numbers of test cells . These findings are consistent with the conclusion that the assay is highly selective for a very primitive, totipotent, reconstituting hematopoietic stem cell and should therefore be particularly useful in future gene therapy-oriented research as well as for more basic studies of hematopoietic stem cell regulation and differentiation.

Endocrinology, 1990 Nov, 127(5), 2104 - 10
Intercellular propagation of individually programmed growth bursts in FRTL-5 cells . Implications for interpreting growth factor actions; Derwahl M et al.; Five methods are commonly used to quantify FRTL-5 cells' and other thyrocytes' growth in vitro and the impact of growth inhibiting or stimulating maneuvers: Total cell count, mitotic index, DNA measurement, total {3H}thymidine incorporation, and the fraction of {3H}thymidine labeled cells . All of them assess cell growth as though all cells were homogeneous with an identical response to growth factors . We demonstrate here that this assumption is not valid . Rather, some intrinsically growth-prone cells appear to pass a growth signal to neighboring cells so that variably sized colonies of synchronized cells within each cluster growing from monodispersed cells are formed . This is true for FRTL-5 cells growing in vitro in monolayers and in three-dimensional, collagen embedded spheroids . The pattern is the same when cell suspensions or collagen-embedded spheroids are implanted onto nude mice . Patches with alternating high and low growth become particularly prominent in the large tumor-like organoids grown from monodispersed cells in nude mice . The pattern much reminds of similar observations in growing intact thyroids . Since there is no significant correlation between the fraction of {3H}thymidine labeled cells and the size of two- or three-dimensional clusters in any experiment, growth of signal-spreading cells is assumed to occur in leaps and bounds . Growth velocity in each subclone of a cell population depends on the mean interval between bursts of replications and on the number of cells synchronized by cell-to-cell diffusion of the growth signal emanating from one dividing cell . Thus, growth-promoting and growth-inhibiting factors may not only act on the mean interval between successive growth bursts, but they may also change cell-to-cell spreading of growth signals.

Obstet Gynecol, 1990 Nov, 76(5 Pt 1), 783 - 7
Production of prolactin by cultures of isolated cells from human first-trimester decidua; Hamaguchi M et al.; An enriched fraction of first-trimester decidual cells that synthesize and release prolactin (PRL) was obtained by discontinuous Percoll gradient (20-50%) centrifugation of collagenase type I- and deoxyribonuclease I-dispersed cells (3 mg/mL and 50 micrograms/mL, respectively) . Centrifugation of the cell suspension yielded three major bands aggregating at the density interfaces . The fraction of the 30-40% Percoll interface contained enlarged decidual cells and constantly secreted significant amounts of PRL into the medium for at least 10 days . The fraction of the 40-50% Percoll interface contained fibroblastic cells and secreted a small amount of PRL into the medium . Cells in the other fractions did not attach to the plastic dishes in 48 hours . Under the influence of progesterone (100 ng/mL), the cultured decidual cells retained their capability of PRL production for at least 10 days because no decline of the secretion rate was observed . The culture system established by the present study is satisfactory for investigating decidual cell functions, including the regulatory mechanisms of PRL production.

Am J Physiol, 1990 Nov, 259(5 Pt 1), C715 - 22
PT pretreatment inhibits 48/80-induced activation of Ca(+)-permeable channels in rat peritoneal mast cells; Kuno M et al.; We recently reported that the secretagogue, compound 48/80, activated Ca2(+)-permeable channels of mast cells possibly via a second messenger {Kuno, Okada, and Shibata . Am . J . Physiol . 256 (Cell Physiol . 25): C560-C568, 1989} . The effects of pretreatment with pertussis toxin (PT) on compound 48/80 (48/80)-induced activation of the Ca2(+)-permeable channel have now been investigated by measuring Ca2+ signals of cell suspensions using the Ca2+ indicator fura-2 and by recording Ba2+ currents through the channel using the patch-clamp technique . In the presence of extracellular Ca2+, the fluorescence change was biphasic, with an immediate rise and a delayed peak at room temperature . The delayed peak, mainly due to Ca2+ entry through plasma membranes, was greatly reduced by pretreatment with PT . The quenching of the fluorescence by 48/80-induced Mn2+ influx was also decreased by PT, whereas the Ca2+ transients due to Ca2+ release from the intracellular stores apparently did not change . In patch-clamp recordings from cell-attached patches with pipettes containing isotonic Ba2+, the 48/80-induced Ba2+ currents were either suppressed or delayed in the PT-treated cells, under conditions where degranulation was absent . These results suggest that PT-sensitive GTP-binding protein is involved in activating the Ca2(+)-permeable channel in mast cells during stimulus-secretion coupling.

Vet Pathol, 1990 Nov, 27(6), 397 - 403
Spleen cell population changes and hemolytic anemia in F344 rats with large granular lymphocyte leukemia; Stromberg PC et al.; A spontaneous large granular lymphocyte leukemia from a F344 rat was transplanted to 36 syngeneic recipients to study the interactions among leukemia, T lymphocytes, and the development of immunemediated hemolytic anemia . Six rats were euthanatized at biweekly intervals, and spleen weight, total spleen cellularity, and differential spleen cell counts were correlated with hemograms and osmotic fragility . Sequential changes in splenic architecture were correlated with hematologic parameters . Monoclonal antibodies defining all T lymphocytes (W3/13), T helper-inducer cells (W3/25), and T suppressor cells (OX-8) were used to identify T cells in immunocytochemical techniques on spleen sections, as well as in fluorescence activated cell sorter analysis of spleen cell suspensions . The onset of hemolytic anemic at 7 weeks after transplantation coincided with the first detection of tumor cells in the spleen and peripheral blood . Tumor cells first accumulated in the marginal zones, and then they infiltrated the red pulp sinusoids . Although the leukemia caused dispersion of the splenic lymphoid tissue, there was no significant lymphopenia, and the relative number of helper (W3/25+) and suppressor (OX-8+) lymphocytes did not change . Because the induction of anemia was a relatively early event in splenic involvement, we concluded that anemia was unrelated to disruption of lymphoid architecture; furthermore, it does not appear to be caused by changes in the numbers of regulatory T lymphocytes.

Arch Biochem Biophys, 1990 Nov 1, 282(2), 437 - 42
Purification of chorismate synthase from a cell culture of the higher plant Corydalis sempervirens Pers; Schaller A et al.; Chorismate synthase (EC 4.6.1.4) was purified from a cell suspension culture of Corydalis sempervirens almost 1000-fold to near homogeneity . The subunit Mr estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 41,900 . The Mr of the native enzyme was estimated to be 80,100 by gel filtration, suggesting a dimeric structure . Antisera directed against the 41.9-kDa protein also reacted with the native enzyme . Further confirmation of the identity of the purified protein was obtained by sequence comparison of a tryptic peptide with known sequences of the Escherichia coli and Neurospora crassa chorismate synthases.

Zhongguo Yao Li Xue Bao, 1990 Nov, 11(6), 560 - 3
{Permeability of 5 methemoglobin formers through red cell membrane}; Ye L et al.; By measuring the methemoglobin formation, the permeabilities of some cyanide antidotes passing through mouse erythrocyte membrane were studied . K3Fe(CN)6(0.1 mol/L) did not permeate the red cell and no methemoglobin formed . To the red cell suspension, adding PAPP 0.07 mmol/L, an useful cyanide antidote, no methemoglobin was found . On the contrary, PHAPP, the metabolite of PAPP, transported into the cell readily and reacted with hemoglobin to form methemoglobin quickly . DMAP and NaNO2 passed through the red cell membrane easily . With comparable amount of methemoglobin formation, the concentration of NaNO2 was about 200 times as much as that of DMAP . A comparison of the anticyanide potency of DMAP and NaNO2, the permeability rate constant, the half time and activation energy were measured as: 0.217 and 0.0506/min; 3.2 and 13.7 min; 17.1 and 50.2 kJ/mol, respectively . Owing to its ready permeability and formation of methemoglobin, DMAP is a better antidote than NaNO2 against cyanide poisoning.

J Biochem Biophys Methods, 1990 Nov-Dec, 21(4), 285 - 8
Membrane-related thermo-osmotic effect as measured by medium conductivity in isotonic cell suspensions; Antonov P et al.; Changes in temperature result in changes in cell-membrane permeability for water and solutes . At isotonic conditions this thermo-osmotic effect is relatively small and remains indistinguishable by the ordinary impedance methods . The thermo-osmotic effect can be investigated in cell suspensions in the beta-dispersion region (high-frequency electric field with an intensity of 100-300 V/cm, peak-to-peak) . The current through the cell suspension of Nicotiana tabacum protoplasts (or human erythrocytes) is a nonlinear function of temperature jump and/or cooling rate . In experiments carried out in a microchamber with thermostatted electrodes at supra-zero temperatures quantitative data were obtained for the thermo-osmotic phenomenon in cell suspension as well as for individual cells.

Chem Pharm Bull (Tokyo), 1990 Nov, 38(11), 3175 - 6
Possible application of polyamine graft copolymer to targeting drug delivery; Muramatsu N et al.; Polystyrene microspheres were coated with polyamine graft copolymer and mixed with rat lymphocyte suspension . In the mixture of T and B cells, the microspheres formed large aggregates, while they were fairly well dispersed in the T cell suspension . This result was understood to be the result of preferential adsorption of B cells to the microspheres, indicating it would be possible to deliver drugs to B cells alone with these copolymer-coated microspheres.

Microvasc Res, 1990 Nov, 40(3), 317 - 26
Adhesive interaction of erythrocytes in vitro in multiple myeloma; Lee MM et al.; The early stage of rouleaux formation, which can be observed on a microscope slide, was assessed quantitatively for pairs of cells forming adhering doublets . Previous work on normal cells in normal plasma has been extended to blood samples from patients with multiple myeloma, a blood disorder with a large component of high molecular weight immune plasma protein . Cine film records were obtained for each of 19 pairs of cells which made adhesive contact in a cell suspension diluted by myeloma blood plasma . Analysis, restricted to the events of sliding contact between cells, revealed an approximate doubling of peak sliding velocity, a marked reduction in time to half complete sliding interaction (13 compared to 56 sec for normal cells), and a high variance when comparing sliding velocity as a function of position of overlap . We invite consideration that circumstances of low or zero fluid shear rate that can occur in the venous side of the circulation might have a transient but profound affect on local blood viscosity.

J Cell Sci, 1990 Nov, 97 ( Pt 3), 433 - 8
Ehrlich ascites tumour cells show tissue-specific adherence and modify their shape upon contact with embryonic fibroblasts and myotubes; Wojciak E et al.; Adhesiveness of Ehrlich ascites tumour (EAT) cells to glass, to mouse peritoneal membrane, living and aldehyde-fixed mouse embryo fibroblasts and chick embryo fibroblasts, myoblasts and myotubes was investigated . The ascitic EAT cells (and leukaemia L1210 cells) did not adhere to glass and peritoneum but readily adhered to embryo fibroblasts, myoblasts and myotubes . The attachment was followed by cell spreading and migration . Fixation of fibroblasts or myogenic cells with aldehydes did not prevent ascitic cells from attaching but reduced the rate of spreading . Only direct interaction of ascitic cells with embryo myoblasts or fibroblasts induced changes in tumour cell adhesiveness followed by cell spreading and locomotion . These results are discussed in relation to an observation that ascitic cells growing as a cell suspension intraperitoneally grow as a solid tumour when injected subcutaneously.

J Immunol, 1990 Nov 1, 145(9), 2854 - 61
In vivo ultraviolet-exposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressor-inducer T cells; Baadsgaard O et al.; In vivo UV exposure of human epidermis abrogates the function of CD1+DR+ Langerhans cells and induces the appearance of CD1-DR+ Ag-presenting macrophages . Epidermal cells from UV-exposed skin, in contrast to epidermal cells from normal skin, potently activate autologous CD4+ T cells, and, in particular, the CD45RA+ (2H4+) (suppressor-inducer) subset . We therefore determined whether UV-exposure in humans leads to a T cell response in which suppression dominates . Autologous blood T cells were incubated with epidermal cell suspensions from in vivo UV-irradiated skin . After activation, repurified T cells were transferred in graded numbers to autologous mononuclear cells (MNC) stimulated with PWM and the resultant IgG production analyzed by ELISA . Relative to T cells activated by unirradiated control epidermal cells, T cells activated by UV-exposed epidermal cells demonstrated enhanced capacity to suppress IgG production (n = 6; p less than or equal to 0.03) . Within the T cell population, CD8+ cells stimulated by UV-exposed epidermal cells could be directly activated to suppress PWM-stimulated MNC Ig production if IL-2 was provided in the reaction mixture . The suppressive activity was also transferable with purified CD4+ T cells stimulated by UV-exposed epidermal cells (n = 10; p less than or equal to 0.01), and was radiosensitive . Suppression was decreased when PWM-stimulated MNC were depleted of CD8+ T cells before mixing with CD4+ T cells activated by UV-exposed epidermal cells, suggesting indirect induction of CD8+ Ts cells contained within the responding MNC populations . Indeed, physical depletion of CD45RA+ cells resulted in total abrogation of the suppressor function contained in the CD4+ T cells . Activation of suppressor function was critically dependent on DR+ APC contained in UV-exposed epidermis . The data suggest that UV-exposure modulates cutaneous APC activity in humans, as in mice, such that the dominant immune response is tilted toward suppression . These mechanisms in normal individuals may function to dampen responses to UV-induced endogenous Ag that are pathogenic in autoimmune disorders . However, these mechanisms might also facilitate the growth of UV-induced skin cancers.

Appl Microbiol Biotechnol, 1990 Nov, 34(2), 198 - 202
Production of recombinant human erythropoietin in Bowes melanoma cells in suspension culture; Keay L et al.; Recombinant DNA technology was used to insert a fetal liver genomic library ApaI fragment encoding for human erythropoietin (Epo) into Bowes melanoma cells . The cells expressed the erythropoietin gene, and Epo was secreted into the culture medium together with the normally-secreted tissue plasminogen activator . Attempts to grow the cells in glass spinners in Dulbecco's medium supplemented with fetal bovine serum produced cell aggregates growing in suspension . When calcium-free suspension culture media (Joklik, DME-S, McCoy 5A-S) were used, single cell suspension cultures were obtained and high Epo production observed . When attempts were made to scale up the small glass spinners, poor growth or Epo production occurred unless the vessels were aerated . This was shown to be because of the drop in pH, possibly due to CO2 accumulation, rather than due to oxygen depletion . It was shown that a semi-continuous operation could be achieved in aerated 8-1 spinners fitted with either a conventional stirrer or a vibromixer agitator . The system was scaled up to a 100-1 stainless steel vessel fitted with a vibromixer agitator . This system was operated for over 4 months with weekly harvests producing over 100 million units of Epo in about 1000-1 of culture fluid . Interference by the serum proteins with downstream purification of the hormone from the culture fluid made the use of serum-free media highly desirable . Studies showed that the Epo was produced in serum-free systems containing peptones.

J Biotechnol, 1990 Nov, 16(3-4), 297 - 303
Two stage cultures for the production of berberine in cell suspension cultures of Thalictrum rugosum; Kim DI et al.; Two stage culture systems were examined in cell suspensions of Thalictrum rugosum which produce berberine in a growth associated manner . The utilization of indole 3-acetic acid (IAA) as a growth regulator, in place of 2,4-dichlorophenoxyacetic acid (2,4-D), at various growth phases was investigated . Although the enhancing effect was clear when it was used separately, in combination with 2,4-D effect of IAA was suppressed and medium change from 2,4-D to IAA was detrimental to product formation . The decrease of berberine production in this two stage culture may be due to the loss of conditioning factors associated with medium replacement.

Biochim Biophys Acta, 1990 Oct 5, 1028(2), 201 - 4
Determination of cell membrane passive electrical properties using frequency domain dielectric spectroscopy technique . A new approach; Bordi F et al.; To take into account the highly irregular surface morphology of cell membranes we have analyzed impedance measurements of biological cell suspensions in general terms using a fractal description of the surface roughness of the cell membrane . This analysis has been applied to human erythrocytes in different solutions of alkaline metal salts and to human lymphocytes, since these cells present a different surface irregularity . The passive electrical properties (dielectric constant and electrical conductivity) deduced from conductivity measurements at radiowave frequencies have been discussed on the basis of the fractal dimension of the membrane surface.

Exp Hematol, 1990 Oct, 18(9), 995 - 1001
Long-term culture of canine marrow: cytogenetic evaluation of purging of lymphoma and leukemia; Carter RF et al.; We established and maintained long-term cultures of marrow from normal dogs and dogs with lymphoma or leukemia by single inoculations of mononuclear cell suspensions . Media containing only horse sera (as opposed to horse and fetal calf sera) and catalase (for antioxidative effect) supported improved culture viability, as indicated by increased recovery of progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) and the release of abundant erythroid cells in the cultures for up to 3 weeks . CFU-GM were maintained for at least 3-4 weeks of culture . Culture appearance, cell counts, and assays of CFU-GM were used to compare the culture kinetics of tumor-involved marrow to normal marrow specimens . Cultures of marrow with extensive tumor involvement tended to be less viable, apparently due to a relative lack of competent progenitors . To investigate whether canine long-term marrow culture provided a purging effect similar to the loss of tumor cells noted in human long-term cultures of marrow from patients with chronic myelogenous leukemia (CML) or acute myelogenous leukemia (AML), we established long-term marrow cultures from 28 dogs with histologically confirmed untreated lymphoma or leukemia . Eleven of these dogs had cytogenetically marked tumor cells in the marrow at the initiation of culture . In six dogs with lymphoma and one dog with acute monocytic leukemia (AMoL) French-American-British classification (FAB) M4 leukemia, we could detect no cytogenetic evidence for persistence of the tumor clones in individually plucked or pooled CFU-GM grown from 3-week-old long-term cultures . In one case of AML (FAB M2), 80% of CFU-GM recovered from long-term cultures at 4 weeks still contained an extra metacentric marker chromosome associated with the continued presence of the leukemic clone in the cultures . Our documentation of a purging effect for some tumors supports the use of this canine model system in the investigation of autologous marrow transplantation with long-term cultured cells for humans with lymphoma and leukemia.

Prostaglandins, 1990 Oct, 40(4), 373 - 82
Impaired generation of prostaglandins from isolated gastric surface epithelial cells in portal hypertensive rats; Arakawa T et al.; We studied generation of prostaglandins E2 and 6-keto F1a by surface epithelial cell isolated from the gastric mucosa of portal hypertensive and sham-operated rats . Oxygenated cell suspensions containing 80 +/- 3% of surface epithelial cells were incubated for 30 min at 37 degrees C and the concentration of prostaglandin E2 and 6-keto-prostaglandin F1a in medium was measured by radioimmunoassay . Viability of the cells was assessed with Fast green exclusion at baseline and after 30-min and 60-min incubation . Within 30 minutes the surface epithelial cells obtained from portal hypertensive rats generated 22.0 +/- 1.6 (mean +/- SE) pg prostaglandin E2 and 40.7 +/- 4.7 pg 6-keto prostaglandin F1a, per 10(6) cells . These were significantly less than prostaglandin generation by cells obtained from sham-operated rats . The viability of the surface epithelial cells from portal hypertensive rats was also significantly reduced compared with sham-operated rats after 60 minute incubation . Reduced ability of the surface epithelial cells to generate prostaglandins may be one mechanism for increased susceptibility of portal hypertensive gastric mucosa to injury by noxious agents.

J Natl Med Assoc, 1990 Oct, 82(10), 697 - 9
Sodium-22 influx into erythrocytes from diabetic hypertensive patients on maintenance hemodialysis; Gambhir KK et al.; We have studied the percentage of 22Na+ uptake in cell suspensions; 0.4 to 2.0 x 10(9) erythrocytes/mL from diabetic uremic patients with secondary hypertension and from normal subjects . Suspensions from diabetic uremic patients with secondary hypertension 0.42 +/- 0.06 to 2.05 +/- 0.28; normal subjects showed a percentage uptake of 22Na+ of 0.27 +/- 0.05 to 1.28 +/- 0.22 . The uptake of 22Na+ in 2.0 x 10(9) cells/mL was 60% more (P less than .05) in diabetic uremic patients than in the controls . These studies indicate that 22Na+ influx determinations may be used to distinguish secondary hypertensive patients from normal subjects.

Br J Pharmacol, 1990 Oct, 101(2), 253 - 6
Inhibition of human platelet activation by polymorphonuclear leukocytes; Schattner MA et al.; 1 . The effect of unstimulated human polymorphonuclear leukocytes (PMNs) on platelet activation was examined . 2 . Human platelet aggregation and adenosine 5'-triphosphate (ATP) release induced by collagen (1-2 micrograms ml-1); thrombin (0.01-0.02 u ml-1) or arachidonic acid (AA) (0.1-0.2 mM) were markedly inhibited when conducted in the presence of unstimulated PMNs . 3 . Platelet inhibition induced by PMNs was dependent on the number of PMNs and on the incubation time of the mixed cell suspension . 4 . Platelet inhibition was not reversed in time when PMNs were depleted from the mixed-cell suspension . 5 . PMN-mediated platelet-inhibition was not mediated by AA metabolites, oxygen reactive intermediates, nitric oxide or proteases . 6 . The factor(s) accounting for the platelet inhibition mediated by PMNs are not yet characterized.

Cryobiology, 1990 Oct, 27(5), 576 - 84
A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration; Koebe HG et al.; Current methods of cryopreservation of hepatocytes in single cell suspensions result in low overall yields of hepatocytes, demonstrating long-term preservation of hepatocellular functions . A novel culture method has recently been developed to culture liver cells in a sandwich configuration of collagen layers in order to stabilize the phenotypic expression of these cells in vitro (J . C . Y . Dunn, M . L . Yarmush, H . G . Koebe, and R . G . Tompkins, FASEB J . 3, 174, 1989) . Using this culture system, rat hepatocytes were frozen with 15% (v/v) Me2SO to -70 degrees C, and stored at approximately -100 degrees C . Following rapid thawing, long-term function was assessed by measuring albumin secretion in culture for 7-14 days postfreezing . Comparison was made with cryopreservation of liver cells in single cell suspensions . Cryopreservation of liver cells in suspension resulted in only a 2% yield of cells which could be successfully cultured; albumin secretion rates in these cultured cells over 48 hr were 26-30% of secretion rates for nonfrozen hepatocytes . Freezing cultured liver cells in the sandwich configuration after 3, 7, and 11 days in culture maintained 0, 26, and 19% of the secretion rates of nonfrozen hepatocytes, respectively . Morphology of the cryopreserved cells appeared grossly similar to cells without freezing; however, this morphological result was patchy and represented approximately 30% of the cells in culture . These results represent the first demonstration of any quantitative long-term preservation of hepatocellular function by cryopreservation, suggesting that cultured hepatocytes can survive freezing and maintain function.

In Vitro Cell Dev Biol, 1990 Oct, 26(10), 1004 - 10
Culture of endocrine pancreatic cells in protein-free, chemically defined media; Kinard F et al.; Cell suspensions prepared by collagenase digestion of pancreata obtained from 21.5-d-old rat fetuses were preincubated in RPMI medium containing 10% fetal bovine serum (FBS), to ensure cell adhesion . Twenty hours later, this medium was replaced by a chemically defined medium . Dulbecco's modified Eagle's (DME)-F12 was used alone or supplemented with various combinations of transferrin, sodium selenite, or Ultroser G . The evolution of the culture and the islet ultrastructure were similar in defined and serum-containing media . However, in the defined medium, the neoformed islets seemed less numerous, and the fibroblast layer less dense, when compared to the RPMI + 10% FBS control medium . At Day 7, in defined media, the total insulin content per dish was half that of control cultures . None of the tested additives improved the yield of the cultures . The fractional insulin release per day was elevated in defined media . In subsequent incubations, glucose and leucine stimulated insulin release in a way characteristic of these cells of fetal origin . The labeling index of islet cells cultured in DME-F12 reached 10.7%, which is not far from that observed in RPMI + 10% FBS . Such a defined medium is useful to study B cell physiology, avoiding the possible interaction of serum components with substances to be tested.

Strahlenther Onkol, 1990 Oct, 166(10), 663 - 8
{Oxygen consumption during photodynamic therapy in vitro}; Wandl EO et al.; The consumption of dissolved molecular oxygen was measured during photodynamic therapy in vitro . Aqueous solutions of hematoporphyrin derivative (HpD) containing different concentrations of serum or numbers of cells were prepared . Decrease in oxygen in the solution and cell suspensions was monitored during light-irradiation (at a wavelength of 610 to 640 nm) . The rate of oxygen consumption increased with increasing concentrations of HpD, serum levels or numbers of cells . Upon repeated irradiation of the same solutions (after restitution of the initial pO2 of 100 mm Hg), the rate of oxygen consumption decreased indicating a disaggregation process of HpD . The rate of oxygen content of the solutions decreased independently of the actual oxygen partial pressure . Even at a low pO2 below 10 mm Hg, oxygen consumption was unimpeded until the solutions were free of oxygen . No O2-consumption was noted in aqueous solutions of HpD (up to 40 g/ml) without serum or cells during irradiation.

Am J Physiol, 1990 Oct, 259(4 Pt 1), L328 - 34
Endogenous xanthine oxidase-derived O2 metabolites inhibit surfactant metabolism; Baker RR et al.; The ability of xanthine oxidase (XO)-derived, partially reduced O2 species (PROS) to inhibit surfactant production was examined in freshly isolated alveolar type II (ATII) pneumocytes from New Zealand White rabbits . {Methyl-3H}choline chloride and {1-14C}palmitate incorporation into phosphatidylcholine (PC) decreased in a dose-dependent manner, whereas peak media hydrogen peroxide (H2O2) concentration increased, when 1, 5, or 10 mU/ml XO were added to cell suspensions containing 500 microM xanthine . Addition of 100 microM allopurinol inhibited H2O2 production and abolished the decrease in choline and palmitate incorporation into PC . ATII cells incubated with 500 microM xanthine alone incorporated choline and palmitate at 90 and 80% of control levels, respectively . However, 100 microM allopurinol restored precursor incorporation to control values . To identify a possible intracellular source of PROS, ATII cell xanthine dehydrogenase (XDH) and XO activities were measured . Both total activity (XDH + XO; 45 +/- 7 microU/mg protein) and the percentage activity in the oxidase form (%XO; 30 +/- 4%) remained unchanged in ATII cells incubated in media only (control) for 2 h . In contrast, incubation of ATII cells with 500 microM xanthine resulted in a 50% loss of XDH + XO activity and a 21% increase in %XO within 10 min . After 2 h there was no measurable XDH + XO activity in xanthine-treated cells . Total XDH + XO activity in cells incubated with 500 microM xanthine and 100 microM allopurinol was less than 6% of control values throughout the incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Hum Pathol, 1990 Oct, 21(10), 1036 - 40
Multilobated lymphoma of B cell type: a multiparameter investigation; Westermann CD et al.; Multilobated lymphomas were originally described as T-cell neoplasms, but many of B-cell type have subsequently been reported . A case of B-cell origin is reported in which both immunophenotypic and genotypic studies performed on a cell suspension of the lymphoma gave inconclusive and potentially misleading information, while paraffin and frozen section immunohistologic studies, as well as genotypic studies performed on DNA obtained from snap-frozen tissue, were definitive . Thus, this case illustrates some of the problems that may be encountered using cell suspensions as a source for immunophenotypic, and even the much more sensitive genotypic, studies.

Am J Respir Cell Mol Biol, 1990 Oct, 3(4), 377 - 91
Airway intra-luminal macrophages: evidence of origin and comparisons to alveolar macrophages; Lehnert BE et al.; Airway intra-luminal macrophages (AI-LM) are a little-studied subpopulation of pulmonary macrophages that are located on the surfaces of the conducting airways of the lower respiratory tract . In this study, we: (1) developed a flow cytometric approach by which AI-LM can be viably isolated in high purity from cell suspensions obtained by airway washings; (2) comparatively examined various functional, biochemical, biophysical, and morphologic features of the rat's AI-LM and alveolar macrophage (AM) phenotypes, and (3) investigated the origin of the AI-LM in the rat . Airway cells were harvested from the tracheas of adult Fischer-344 rats, and AM were obtained from the lungs by conventional bronchopulmonary lavage or via prosthetic airway circuits that supplanted the removed tracheas . Flow cytometric analyses of lavaged airway cells revealed that the AI-LM fell within the range of the electro-optical phenotype of AM, and subsequent cell-sorting experiments demonstrated that virtually all viable AI-LM could be sorted from contaminating airway epithelial cells in greater than 95% purity based on their electro-optical characteristics, e.g., electronic volume, axial light loss, 90 degrees light scatter, and blue and green autofluorescence signals . In Fc gamma receptor-mediated phagocytic studies, approximately 90% of AM engulfed opsonized erythrocytes (EIgG) whereas only 60% of the AI-LM were able to do so . Comparisons of the numbers of EIgG in phagocytic AM and in phagocytic AI-LM indicated the AI-LM were less phagocytic . Densitometric analyses of sorted AI-LM and of sorted AM stained for acid phosphatase, nonspecific esterase, and beta-glucuronidase indicated that the activities of these enzymes were generally less in the AI-LM than in the AM . Morphometric comparisons of sorted AM and of sorted AI-LM showed that the AI-LM were generally larger than the AM and that the surfaces and nuclei of the AI-LM were more regular than those of the AM . The AI-LM were found to strongly label with the monoclonal antibody ED1, which recognizes an antigen on the surfaces of rat AM, but the AI-LM did not label with the monoclonal antibody ED2, which recognizes an antigen on the surfaces of rat peribronchial and pulmonary perivascular macrophages . Over the course of alveolar phase clearance of a lung burden of polystyrene microspheres, the frequency distributions of the particles in AI-LM and in AM were found to be virtually identical.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatogastroenterology, 1990 Oct, 37(5), 474 - 9
Specificity of androgen receptors of hepatocellular carcinoma and liver in humans; Nagasue N et al.; The specificity of androgen receptor in hepatocellular carcinoma and the liver was investigated using auto-radiographic techniques . Partial hepatectomy was carried out on 11 patients with hepatocellular carcinoma and associated parenchymal disease of the liver . Androgen receptors were assayed biochemically for hepatocellular carcinoma and the surrounding liver in all cases . Estrogen receptor was also measured in five patients . In eight patients, fresh resected specimens as thick as 3 mm were first incubated for 15 minutes in a medium containing estradiol and hydrocortisone, and then radio-labeled testosterones were added to the medium . After another 60 minutes incubation, macro-autoradiographic studies were carried out . With the same medium and chemicals, and using the same principle, micro-autoradiographic studies were performed using fresh hepatocellular carcinoma and liver cell suspensions in six cases . The radio-labeled testosterones were incorporated into hepatocellular carcinoma and the liver to a parallel extent with the androgen receptor titers biochemically assayed . The current results seem to indicate that androgen receptors present in hepatocellular carcinoma and the liver of humans specifically bind androgens . Further studies are needed to elucidate the role of AR in hepatocarcinogenesis in humans.

J Immunol, 1990 Oct 1, 145(7), 2115 - 22
Thymic B cells from myasthenia gravis patients are activated B cells . Phenotypic and functional analysis; Leprince C et al.; Thymic cell populations from 12 patients displaying myasthenia gravis were submitted to a phenotypic and functional study . Immunofluorescence analysis of thymic sections revealed the presence in germinal centers of B lymphocytes expressing the B cell markers--CD19, CD21, IgD, or IgM . After T cell and macrophage depletion of thymic single cell suspensions, B cell-enriched populations were isolated . Enriched B cells expressed at variable levels activation markers such as CD71, 4F2, CD23, and B8.7, indicating that a marked proportion of them are activated . Moreover, addition of B cell growth factor 12kDa and to a lesser extent of rIL-2 induced a spontaneous proliferation of these B cell populations . These functional and phenotypic signs of activation may reveal the first steps of an autoimmune response against acetylcholine receptor as enriched B cell populations have the capacity to spontaneously secrete anti-acetylcholine receptor antibody.

J Appl Physiol, 1990 Oct, 69(4), 1270 - 5
Stiffened erythrocytes augment the pulmonary hemodynamic response to hypoxia; Doyle MP et al.; Isolated rat lungs were perfused with suspensions containing normal and stiffened erythrocytes (RBCs) during normoxic and hypoxic ventilation to assess the effect of reduced RBC deformability on the hypoxic pressor response . RBC suspensions were prepared with cells previously incubated in isotonic phosphate-buffered saline with or without 0.0125% glutaraldehyde . The washed RBCs were resuspended in isotonic bicarbonate-buffered saline (with 4% albumin) to hematocrits of approximately 35% . The lungs were perfused with control and experimental cell suspensions in succession while pulmonary arterial pressure was measured during normoxic (21% O2) and hypoxic (3% O2) ventilation . On the attainment of a peak hypoxic pressor response, flow rate was changed so that pressure-flow curves could be constructed for each suspension . RBC deformability was quantified by a filtration technique using 4.7-microns-pore filters . Glutaraldehyde treatment produced a 10% decrease in RBC deformability (P less than 0.05) . Over the range of flow rates, Ppa was increased by 15-17% (P less than 0.05) and 26-31% (P less than 0.05) during normoxic and hypoxic ventilation, respectively, when stiffened cells were suspended in the perfusate . The magnitude of the hypoxic pressor response was 50-54% greater with stiffened cells over the three flow rates . In a separate set of experiments, normoxic and hypoxic arterial blood samples from conscious unrestrained rats were used to investigate the effects of acute hypoxia on RBC deformability . Deformability was measured with the same filtration technique . There was no difference in the deformability of hypoxic compared with normoxic RBCs . We conclude that the presence of stiffened RBCs enhances the hemodynamic response to hypoxia but acute hypoxia does not affect RBC deformability.

Br J Cancer, 1990 Oct, 62(4), 607 - 13
Serine protease-induced enhancement of blood-borne metastasis of rat ascites tumour cells and its prevention with deoxyribonuclease; Sugihara S et al.; Serine proteases, such as alpha-chymotrypsin or elastase, caused an aggregation of rat ascites tumour cell lines, AH-130, AH-109A and YS, in a protein free medium which preserved the cell viability . This aggregation, which was monitored spectrophotometrically, was dependent upon the protease activities and was resistant to treatment with either a calcium chelating reagent (EDTA) or neuraminidase . However, the tumour cell aggregates were redispersed by treatment with deoxyribonuclease I (DNase I) . This dispersal effect was dependent upon the DNase activity . A possible relationship between the tumour cell aggregation and development of blood-borne metastasis was studied . An intravenous inoculation in rats of tumour cell aggregates performed by the alpha-chymotrypsin treatment resulted in significantly higher numbers of lung metastatic foci than an injection of single cells . When the re-separated single cells, prepared in vitro by treatment with DNase I following alpha-chymotrypsin treatment, were injected instead of the aggregates, the enhancement of metastasis was reversed . These enhancement and reversal effects were mimicked in vivo by intravenous injections of protease and nuclease following inoculation of a single cell suspension . That is, the number of metastatic foci caused by single cell inoculation followed by an intravenous alpha-chymotrypsin injection, was higher than that in a control group receiving PBS instead of alpha-chymotrypsin . Again, this augmentation was reversed by an injection of DNase I following alpha-chymotrypsin injection . Furthermore, an injection of DNase I alone itself reduced the starting number of metastases resulting from injection of the single tumour cell suspension . These data suggest that the metastatic behaviour of tumour cells may be increased by protease inducible DNA dependent cell aggregation should it occur in the blood stream.

Am J Physiol, 1990 Oct, 259(4 Pt 1), C660 - 7
Glycolytic component of rat spermatid energy and acid-base metabolism; Reyes JG et al.; The impact of glycolysis on rat spermatid energy metabolism is made apparent by the simultaneous occurrence of the following three events upon glucose addition to the extracellular medium of a rat spermatid cell suspension: decrease in ATP content, exit of acid equivalents, and increased lactate production and efflux . In this work, we have studied the interrelations between these three phenomena . By measuring ATP content, net acid transport, lactate exit, oxygen consumption, intracellular pH, CO2 production, and glycolytic intermediates in the presence of glucose and glucose analogues, we conclude that 1) lactate production, decrease in ATP content, and acid equivalent exit are dependent on the metabolism of glucose up to different stages in glycolysis . 2) The decrease in ATP content is not directly related to the exit of acid equivalents from rat spermatids . 3) Glucose metabolism is a net ATP-consuming process at high intracellular ATP content but is a net ATP-producing process at low intracellular ATP concentration in rat spermatids . 4) Acid equivalent production arises from the metabolism of glucose beyond glyceraldehyde 3-phosphate dehydrogenase . 5) Lactic acid diffusion and/or lactate transport and CO2 production and exit could account for the glucose-dependent acid equivalent efflux in rat spermatids.

Endocrinol Jpn, 1990 Oct, 37(5), 649 - 63
Morphologically and functionally distinct subpopulations of steroidogenic cells in corpora lutea during pregnancy in rats; Miyauchi F et al.; On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CL) were dissected and dissociated into single cell suspensions by enzyme treatments . The suspended luteal cells were allowed to sediment in a BSA gradient at 4 degrees C for 3.5 hours . Five fractions were collected from the top (Fraction (Fr.) 1) to the bottom (Fr . 5) of the gradient . Cells were incubated in serum-free DME-F12 for 20 hours with or without hCG (100 ng/ml) to test them functionally, and the accumulation of progesterone and testosterone was determined by radioimmunoassay . To assess 3,3-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used . Cells were examined by light microscopy, and the percentage of cells containing dark blue formazan deposits and their diameters were determined in at least 40 microscopic fields . The number of cells staining for 3 beta-HSD did not vary by day 15 but decreased from 141.6 +/- 16.5 X 10(3) cells/CL on day 15 to 113.8 +/- 13.2 X 10(3) cells/CL on day 20 of pregnancy . However, 3 beta-HSD-positive cells maintained the same levels of progesterone secretion until the advent of luteolysis, then they increased in size progressively throughout pregnancy . In BSA gradients, the relatively larger 3 beta-HSD-positive cells migrated faster than the smaller 3 beta-HSD-positive cells on each day of pregnancy . The diameters of 3 beta-HSD-positive cells differed significantly in Frs . 2, 3, 4 and 5 on days 15 and 20 of pregnancy . On day 15 of pregnancy, less progesterone accumulated in wells containing 3 beta-HSD-positive cells from Fr . 2 (mean diameter; 24.96 microns) than from Fr . 3, Fr . 4 and Fr . 5 (mean diameters; 27.20, 30.79 and 31.28 microns, respectively) but the Fr . 2 cells responded more to hCG stimulation . Fr . 2 also showed a higher ratio of testosterone accumulation to progesterone accumulation than the other fractions . The response to hCG stimulation of cells in Fr . 2 tended to be higher than that in Fr . 3 on day 20 of pregnancy . These data suggest that the steroidogenic rat luteal cells are comprised of morphologically and functionally different cell types after day 15 of pregnancy . No stimulating nor inhibiting effects were observed in co-incubation of cells from Fr . 2 with cells from Fr . 3 or Fr . 4 on days 15 and 20 of pregnancy.

NMR Biomed, 1990 Oct, 3(5), 227 - 32
Proton magnetic resonance spectroscopy of small regions (1 mL) localized inside superficial human tumors . A clinical feasibility study; van Zijl PC et al.; It is demonstrated that the stimulated echo technique for proton magnetic resonance spectroscopy (MRS) can be used to study metabolites in volumes of interest (VOIs) as small as 1 mL localized within superficial human tumors . Access to these small VOIs is important for characterization of tissue regions within a tumor, before, during and after treatment . Spectral appearance resembles that from studies on extracts, and cell suspensions and perfused cells of several tumor types . For the first time proton MRS was used to study cancer treatment in vivo in humans, for a case of radiation treatment of squamous cell carcinoma . No spectral evidence of changed metabolism prior to reduction in tumor size was found . However, after the first period of radiation (39 Gy, 4 weeks), complete disappearance of the metabolite resonances from the spectrum was observed, while a considerable mass still remained, suggesting effective cell destruction upon treatment . Needle aspiration cytology of this mass showed absence of malignant cells, supporting this result.

J Immunol, 1990 Sep 15, 145(6), 1659 - 63
Differences in turnover between thymic medullary dendritic cells and a subset of cortical macrophages; Kampinga J et al.; To investigate the turnover of thymic accessory cells, we performed vascular thymus transplantation in RT7 congenic rats . mAb specific for one of the two allelic variants of the RT7 molecule, as well as mAb specific for either medullary interdigitating cells or a subset of cortical macrophages (M phi), were used on cryostat sections and cell suspensions prepared from grafted thymuses to monitor the turnover of these two cell types . In contrast to the complete turnover of interdigitating cells within 3 wk after transplantation, ED2-labeled cortical M phi showed a very slow turnover . Seventy-six days after transplantation, more than 30% of these M phi were found to be still of donor origin . The different turnover rates of these thymic accessory cells could reflect their function in T cell development.

Biochemistry, 1990 Sep 11, 29(36), 8313 - 8
External electric fields stimulate the electrogenic calcium/sodium exchange in plant protoplasts; Graziana A et al.; External electric fields of low intensity stimulated calcium influx in protoplasts isolated from carrot cell suspension cultures in field intensity dependent and frequency-dependent ways . The field-induced calcium uptake involved a temperature-dependent system that was saturable by external calcium . The induction process appeared mainly cumulative as long as the morphology of the protoplasts did not change (up to 10 min) . The stimulation elicited by the electric fields was effective even after switching the field off; the influx increased for 5 min and then slowed down to its initial value 15 min later . During electrostimulation, an additional amount of ATP was accumulated; on removal of the stimulatory field, the extra amount of ATP was consumed, whereas the plasma membrane was hyperpolarized and sodium ions were expelled from the protoplasts . Inhibition of either ATP accumulation or consumption results in the inhibition of both calcium influx and sodium efflux, demonstrating that these processes are coupled . From the data obtained in this work, it may be concluded that the electric field stimulates an ATP synthase like activity; the consumption of the ATP thus formed elicits an electric potential (probably due to the efflux of cations and more specifically sodium) that drives the influx of calcium.

Mol Cell Biochem, 1990 Sep 3, 97(1), 17 - 33
Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum; Brickey DA et al.; We have recently reported the existence of two forms of glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate-alpha-glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum . During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased . The total phosphorylase activity remained constant . The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme . In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK) . We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5'AMP and a 104 kd peptide appears . We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions . The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK . We found that relatively low levels, 100 microM, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity . The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK . Two other analogs, Bt2-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 microM) for 'b' to 'a' conversion . cDNAs to three cAMP-regulated genes--PL3, D11, and D3--were used as controls in the above experiments . In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture--a medium in which adenylate cyclase is inhibited . Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP . The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.

Anal Cell Pathol, 1990 Sep, 2(5), 287 - 95
DNA flow cytometry on breast carcinomas: comparison of a detergent and an enzyme-detergent preparation method; Risberg B et al.; In this study we have compared two different preparation methods for DNA flow cytometry on breast cancer . Tumour cell suspensions from 49 breast cancers were analysed on a Facscan flow cytometer . In seven of 49 cases, additional aneuploid peaks were found after enzyme/detergent treatment (E/D), not seen after the detergent (D) preparation . S-Phase fractions were significantly higher after D than after E/D preparation (mean values, 15 and 8%, respectively), although the correlation was high between the two methods . S-Phase fraction estimated after background correction diminished the differences between the two methods (mean values, 8 and 6%) . Furthermore, the fraction of G2/M cells were generally greater with the D method . These differences can be explained by increased number of cell doublets and nuclear fragments after D compared to E/D preparation . This clearly shows that the preparation method influences the result of DNA flow cytometry on human breast cancers.

Trop Med Parasitol, 1990 Sep, 41(3), 234 - 40
Experimental ocular onchocerciasis: local and systemic antibody and cell-mediated immune responses; Haldar JP et al.; Hartley guinea pigs injected subconjunctivally with Onchocerca lienalis (OL) microfilariae (Mf) develop punctate corneal opacities resembling the punctate keratitis of human onchocerciasis . Antibody production and antigen-induced proliferative responses were studied in conjunctival-associated lymphoid tissues (CALT), spleens (SL) and peripheral blood lymphocytes (PBL) from experimentally infected guinea pigs . Cultured single cell suspensions of CALT, SL and PBL were assayed for IgG1, IgG2, IgA and IgE antibody production . IgG1, IgG2, and IgA Onchocerca-specific antibodies were found in culture supernatants of CALT, SL and PBL . When initiated 10 days after a challenge injection of OL, CALT cultures produced antibody levels equal to or less than those produced by the corresponding SL cultures . When initiated 66 days after the last injection of Mf, CALT cultures produced significantly more antibody than the corresponding SL cultures . Blastogenic responses to OL Mf antigen were observed in peripheral and splenic lymphocytes of OL-infected guinea pigs . Animals given subconjunctival injections of Mf followed by treatment with a microfilaricide had greater responses to OL antigen than those given Mf alone, while responses to phytomitogens were similar in drug-treated and non-treated animals . The CALT was locally immunologically responsive against the subconjunctivally injected OL Mf, with the capacity for localized memory responses . The local immunologic responses to conjunctival Onchocerca microfilariae may play a significant role in the immunopathological reactions of ocular onchocerciasis.

J Lipid Res, 1990 Sep, 31(9), 1683 - 91
ELISA measurement of LDL receptors; May K et al.; An enzyme-linked immunosorbent assay was developed for measurement of low density lipoprotein (LDL) receptors . A monospecific polyclonal antibody to LDL receptor purified from rat liver that reacted with rat, mouse, canine, and human LDL receptor was used . With this assay, LDL receptors could be measured on 2-4 x 10(5) adherent cells and 1.0 x 10(5) cells in suspension, although results were more variable with cell suspensions . Membranes from a variety of receptor-rich and receptor-poor tissues could be assayed directly after adherence of the membranes to the ELISA plate by an overnight incubation . In some instances, the quality of the assay was improved by first solubilizing the membranes . The sensitivity of the assay is such that between 0.15 and 2 micrograms of membrane protein is required . This could be obtained from leukocytes in a modest (20-30 ml) quantity of human blood . The assay was used to demonstrate the rapid down-regulation of LDL receptors in human mononuclear leukocytes in response to a cholesterol-containing meal . Overall, the results support the use of ELISA technology to measure LDL receptors, particularly for physiologic studies.

Anticancer Res, 1990 Sep-Oct, 10(5A), 1303 - 6
Factors in prostate cancer metastasis; Geldof AA et al.; Death related to prostate cancer is invariably due to the tumor metastasis (to lungs, skeleton and lymph nodes) . Tumor cell metastatic behaviour is still poorly understood . The R3327 prostate tumor model in the male Copenhagen rat offers an opportunity to investigate the different aspects of metastatic processes in vivo and to evaluate effects of current and new treatment approaches . Lymphatic spread of cancer cells can be measured by determining volume of local load in successive draining lymph node stations . Surgical removal of primary tumor and inguinal lymph node shows that lymphatic metastasis in the R3327-MATLyLu tumor variant is a continuous progressive phenomenon, which starts already in early stages of tumor growth after subcutaneous transplantation . Spread to the lungs can be quantified by enumeration of macroscopically visible pleural lung colonies . Metastatic spread to the lungs can be mimicked by intravenous injection of monodispersed tumor cell suspension . Within 10 days pleural tumor colonies become visible . Effects of different agents and treatments like chemotherapeutic drugs, heparin, surgery and high energy shock wave (HESW) treatment have been described using these methods . Recently, metastasis to the vertebral column was induced by temporal occlusion of venous blood flow through the caval veins while injecting tumor cells in the lateral tail vein . The resulting osteoblastic metastatic lesions in the lumbar vertebrae and the concomitant spinal cord compression led in time to the loss of motoric and sensory function of the hind legs . These observations permit investigation of the mechanisms of bone metastasis based on biological functions, i.e . sensory and motoric function of the hind legs . Finally, it can be said that the various variants of the R3327 rat prostate tumor offer an appropriate model to study various aspects of prostate cancer and metastasis.

Scand J Clin Lab Invest, 1990 Sep, 50(5), 497 - 507
A review of 23Na nuclear magnetic resonance spectroscopy for the in vitro study of cellular sodium metabolism; Nissen H et al.; Changes in intracellular sodium have been associated with a number of different diseases . Consequently, various methods have been used to quantify the level of intracellular sodium concentrations . Traditional methods like flame photometry and ion-selective electrodes are destructive or invasive, thereby potentially altering the intracellular sodium levels . There has been an increasing interest in evaluating the method of 23Na nuclear magnetic resonance in recent years, since this method allows for non-invasive continuous monitoring of intracellular sodium in cell suspensions and tissues . A phenomenological approach to basic theory, review of methodology, applications to the in vitro study of cellular sodium metabolism, and difficulties of interpretation of this analytical modality is presented.

Int J Cell Cloning, 1990 Sep, 8(5), 377 - 84
The effect of carbamylcholine on CFU-s differentiation; Hu XT et al.; The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types . Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively . Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays . The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively . The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.

J Biomed Mater Res, 1990 Sep, 24(9), 1241 - 62
Microencapsulation of mammalian cells in a HEMA-MMA copolymer: effects on capsule morphology and permeability; Crooks CA et al.; A new process for preparing uniform microcapsules with a hydroxyethyl methacrylate-methyl methacrylate copolymer (HEMA-MMA) has been devised . Capsule diameters were 900-1000 microns in diameter, (+/- 10-20 microns, +/- SD) depending on the precipitation conditions . The process involved the coextrusion of polymer solution (in PEG 200) and the mammalian cell suspension (here erythrocytes) through a needle assembly which is submerged in a layer of hexadecane which is in turn sitting above a stirred isotonic aqueous solution in a volumetric flask . The needle is repeatedly withdrawn from the hexadecane overlayer shearing a droplet from the needle tip which falls into the water, where the solvent is extracted to precipitate the polymer around the cells to yield the capsules . The morphology of the capsule wall was altered by changing the precipitation bath from phosphate buffered saline (PBS) to 0.3 M glycerol . This resulted in greater macroporosity in the wall, presumably because of the faster precipitation due to the higher solvent/precipitant compatibility with 0.3 M glycerol . The permeability to a series of test solutes (glucose, inulin, albumin, and alcohol dehydrogenase, ADH) increased by a factor of approximately 2, presumably because of the increased macroporosity . Addition of 15% water to the polymer solvent enhanced the macroporosity, presumably by bringing the system closer to the cloud point; however, there was no corresponding increase in permeability . There was a significant decrease in permeability between that of albumin (approximately 69,000 D) and ADH (approximately 150,000 D) suggesting that the molecular weight cut-off of these capsules was on the order of 100,000 D as desired . This process is now being evaluated for the encapsulation of pancreatic islets and other cells of potential clinical interest.

Jpn J Cancer Res, 1990 Sep, 81(9), 962 - 6
Mechanism of cell damage by ultrasound in combination with hematoporphyrin; Umemura S et al.; The mechanism of cell damage by ultrasound in combination with hematoporphyrin was studied . Mouse sarcoma 180 cell suspensions were exposed to ultrasound for up to 60 s in the presence and absence of hematoporphyrin, with and without active oxygen scavengers . The cell damage enhancement by hematoporphyrin was suppressed by adding histidine but not by mannitol . The enhancement was doubled in rate by substitution of deuterium oxide medium for normal water . Sonoluminescence was produced in a saline solution under similar acoustic conditions and observed to have spectral components that can excite hematoporphyrin molecules . These results suggest that cell damage enhancement is probably mediated via singlet oxygen generated by ultrasonically activated hematoporphyrin.

J Dent Res, 1990 Sep, 69(9), 1602 - 6
Mechanism of endotoxin inhibition of human gingival fibroblast attachment to type I collagen; Pitaru S et al.; Bacterial endotoxin inhibits the attachment of human gingival fibroblasts to collagen . The present study attempted to elucidate the possible mechanism of this inhibition . Two mechanisms were considered: direct toxicity to the cells and steric interference . Collagen substrates were prepared by rat type I collagen being air-dried in the wells of 24 multi-well plates . Experimental collagen substrates were treated with 50 micrograms of endotoxin/well, while untreated collagen substrates served as controls . Two mL of cell suspension (10(4) cells/mL) was added to each well, and these were incubated at 37 degrees C for two h . The average cell number/mm2 attached to experimental and control substrates was determined . Cell attachment to endotoxin-treated collagen was inhibited by 78%, compared with that to untreated collagen . The washing of the endotoxin-treated collagen for two h did not affect the inhibition of cell attachment, whereas after 24 h of washing, cell attachment was inhibited by 54%, compared with that to untreated collagen . Pre-incubation of the cells in endotoxin for two h did not affect their attachment to collagen . The addition of fetal calf serum (15%) to the experimental system completely reversed the inhibition of fibroblast attachment to endotoxin-treated collagen . These findings suggest that endotoxin interferes with fibroblast attachment to collagen through a steric phenomenon, possibly by blocking the binding sites on the collagen molecule recognized by the membrane receptor for collagen.

Radiat Res, 1990 Sep, 123(3), 325 - 30
Response of human squamous cell carcinoma xenografts of different sizes to irradiation: relationship of clonogenic cells, cellular radiation sensitivity in vivo, and tumor rescuing units; Baumann M et al.; The relationship of clonogenic cells, cellular radiation sensitivity at tumor control does in vivo, and tumor rescuing units at different tumor sizes was investigated in the human squamous cell carcinoma FaDu growing in NCr/Sed nude mice . The composition of the tumors was determined in single cell suspensions and compared to tumor control data after single-dose irradiation . To avoid the influence of varying oxygen concentrations in the tumors, all irradiations were performed under clamp hypoxia . Nude mice and animals further immunosuppressed by 6-Gy whole-body irradiation were used to assess the immunological effects . The numbers of total cells, cells excluding trypan blue, host cells, and colony-forming cells increased linearly with the weight of FaDu tumors . Comparable results were obtained for cell suspensions prepared from tumors growing in nude of pretreated nude mice . The radiation dose required to control 50% of tumors (TCD50) of different sizes between 36 and 470 mm3 increased from 52.1 to 60.1 Gy when the tumors were maintained in normal nude mice and from 50.8 to 61.3 Gy in whole-body-irradiated mice . The D0 of FaDu cells in vivo was calculated by regression analysis of TCD50 vs the logarithm of the clonogenic cell number, assuming an oxygen enhancement ratio of 3.0 . The resultant D0S of 1.1 and 1.2 Gy in vivo correspond well to the radiosensitivity of FaDu cells in vitro determined previously . Assuming the single-hit multitarget model of cell killing and extrapolation numbers between 2 and 20, the mean number of tumor rescuing units would be 10(5) to 10(6) for a 100-mm3 tumor growing in whole-body-irradiated nude mice . Comparison of the number of tumor rescuing units to the estimated number of clonogenic cells does not conflict with the assumption that every surviving clonogenic cell is able to repopulate FaDu tumors after irradiation; however, it seems more likely that more than one clonogenic cells is necessary . The proportion of tumor rescuing units in the clonogenic cell population is independent of tumor size.

Tokai J Exp Clin Med, 1990 Sep, 15(5), 363 - 7
Contribution of growth factors to heat production by cultured rat fibroblasts; Okuda A et al.; The heat released from rat 3Y1 fibroblasts was measured by the stopped-flow method using a flow microcalorimeter . The heat output from a monolayer culture is defined as the maximum heat output from the cell suspension, after dispersion of the monolayer . The heat output per cell was not significantly changed when exponentially proliferating cells ceased dividing, with a G1 DNA content, at confluent cell density . When the growth-arrested (resting) cells were exposed to fresh medium containing various combinations of growth factors, heat output increased in parallel with the fraction of the cells that entered S phase of the cell cycle . The increase in heat output was correlated with the increase in cell volume . We infer from these findings that the resumption of cell proliferation stimulated by growth factors involves an increase in cell volume and a concomitant increase in heat production, but that entry into the resting state does not involve abrupt decreases in cell volume and heat production.

Arch Ophthalmol, 1990 Sep, 108(9), 1323 - 5
Ascorbic acid is cytotoxic to dividing human Tenon's capsule fibroblasts . A possible contributing factor in glaucoma filtration surgery success; Jampel HD; Successful glaucoma filtration surgery depends on the incomplete healing of the surgical wound, with formation of a filtration bleb . In most other tissues, however, complete healing is the rule . I have explored the possibility that the high concentration of ascorbic acid normally present in aqueous humor inhibits wound healing after filtration surgery . At the concentration normally present in aqueous humor (1.1 mmol/L), ascorbic acid decreased the plating efficiency of cell suspensions of human Tenon's capsule fibroblasts by a mean (+/- SD) of 40% +/- 10% . When added to low-density monolayer cultures of fibroblasts, ascorbic acid decreased the cell number by 90% +/- 5%, an effect that was completely prevented by catalase . When added to confluent cultures, the cell number was decreased by only 14% +/- 2% . If ascorbic acid has similar effects on fibroblasts in vivo, it may contribute to the incomplete wound healing that characterizes successful glaucoma surgery.

Acta Virol, 1990 Sep, 34(5), 449 - 56
Influenza virus detection in clinical specimens; Havlickova M et al.; The authors compared the results of influenza A (H1N1) and influenza A (H3N2) virus detection in nasopharyngeal swabs from flu patients by molecular hybridization (MH), ELISA, virus isolation and seroconversion . Using the immunofluorescence (IF) technique influenza virus was detected in cell suspensions from the first chick embryo passage . Altogether 63 swabs from various epidemic seasons were separated into 3 groups according to specimen sampling and storage . It was shown that influenza virus RNA could be found in 16 out of 22 swab specimens (72%) stored at -70 degrees C without thawing and that ELISA revealed the influenza virus antigen in 19 cases (86%); in contrast, IF was positive in 6 (27%) and virus isolation in 5 (22%) cases only . However, the positive rate of MH decreased to 9% in 21 swab specimens repeatedly thawed and stored at -20 degrees C and was completely negative after prolonged storage of repeatedly thawed samples . Despite these conditions, ELISA was still successful in both latter sample groups (71-80%) . For specificity control, 29 samples coming from patients with influenza B virus and other respiratory virus diseases (adeno- and respiratory syncytial virus) were used.

J Immunol, 1990 Sep 1, 145(5), 1615 - 20
Long-term survival of adoptively transferred tumor-infiltrating lymphocytes in mice; Alexander RB et al.; We examined the long term survival of adoptively transferred, cultured tumor-infiltrating lymphocytes (TIL) in sublethally irradiated nontumor-bearing mice . TIL were produced by culturing Thy-1-enriched single cell suspensions from a variety of murine tumors in exogenous IL-2 along with irradiated tumor cells and splenocytes . TIL were adoptively transferred i.v . to mice and such animals demonstrated a statistically significant degree of protection from a subsequent i.v . tumor challenge . This protection lasted up to 6 wk and occurred in the absence of exogenous IL-2 administration in vivo . Using congenic B6.PL Thy-1 alpha/CY mice we demonstrated directly that TIL can survive in vivo for up to 6 wk and that the adoptively transferred TIL were the source of the relative immunity to subsequent tumor challenge . We conclude that TIL contain cells with substantial functional longevity in vivo even in the absence of exogenous IL-2 . These studies are relevant to ongoing clinical trials of TIL as a therapy for patients with advanced cancer.

In Vivo, 1990 Sep-Oct, 4(5), 309 - 15
Nude mouse xenografts as in vivo models for lung carcinomas; Bepler G et al.; Eighty-eight primary and secondary lung tumor specimens were subcutaneously transplanted into athymic nude mice . One third of all carcinoma specimens yielded tumor growth . Success rates were highest if fresh tumor pieces or fresh or frozen cell suspensions were implanted or injected . More than half of squamous cell and adenocarcinoma xenografts showed a lower degree of differentiation than the original tumor . The degree of dedifferentiation led to the diagnosis of large cell carcinoma by light microscopical criteria in 4 of these cases . All small cell carcinoma xenografts showed intermediate-cell type morphology irrespective of the cell type of the original tumor, and all large cell carcinoma xenografts showed features similar to the original tumor . Tumor latent periods were approximately twice as long during the first nude mouse passage than subsequent passages, and tumor doubling times remained stable during serial passages . We conclude that the large cell lung carcinoma subtype is a mixed bag of tumors and includes highly undifferentiated squamous cell and adenocarcinomas, that the small cell carcinomas remain in that histologic subtype during xenotransplantation, and that lung carcinoma xenografts display stable morphologic and kinetic features during serial xenotransplantation . Nude mouse xenografts may serve an in vivo model to study the biologic relationship of non-small cell lung carcinomas.

Development, 1990 Sep, 110(1), 221 - 7
Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig; Corps AN et al.; 125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15-19 of pregnancy . Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days . The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones . Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types . The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors . Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000 . The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst.

Cell Tissue Kinet, 1990 Sep, 23(5), 391 - 400
The changes of BPA level in 31 cases of children with aplastic anaemia and its clinical significance; Wang W et al.; Burst-promoting activity (BPA) was measured in the sera from 31 children with aplastic anaemia (AA) . BPA levels were elevated in most of the children with AA (65.2%), the mean value (137.7 +/- 18.4%) being significantly higher than that in normal children (69.6 +/- 9.4%), in children in the recovery period and in children with non-aplastic anaemia . There was a negative relationship between the BPA level in children with AA and the peripheral haemoglobin concentration . The BPA level was higher in those whose duration of illness was shorter than 1 year . In three cases of AA caused by chloramphenicol and benzene hexachloride and one case of congenital pure red cell AA, the BPA level was not elevated . Eleven patients received fetal liver cell suspensions intravenously (FLI) . After FLI the BPA level in their sera was significantly reduced . According to these results, it appears that the elevation of BPA level is a special phenomenon of AA . The measurement of BPA in serum is helpful for differentiation between AA and other kinds of anaemia . The elevation of the BPA level in serum is a biological compensation for the haematopoietic disorder, and the measurement of BPA in the serum of patients with AA may be helpful in evaluating the haematopoietic condition.

Endocrinology, 1990 Sep, 127(3), 1487 - 94
Neuropeptide-Y enhances luteinizing hormone (LH)-releasing hormone-induced LH release and elevations in cytosolic Ca2+ in rat anterior pituitary cells: evidence for involvement of extracellular Ca2+ influx through voltage-sensitive channels; Crowley WR et al.; The present studies were designed to investigate the mechanism by which neuropeptide-Y (NPY) augments the effect of LHRH to stimulate the release of LH from cultured rat anterior pituitary cells . Anterior pituitary cells from ovariectomized rats were enzymatically dispersed, cultured for 3 days, and then exposed to various secretagogues during 3-h incubations . As reported by this laboratory previously, NPY alone (100 nM) did not affect LH release, but significantly enhanced the LH response to 1 nM LHRH . This facilitatory action of NPY was mimicked by the dihydropyridine Ca2+ channel agonist Bay K 8644 (1 microM), and the enhancement of LHRH-induced LH release by either NPY or Bay K 8644 was prevented by the dihydropyridine antagonist nitrendipine (1 microM) . Nitrendipine alone reduced the response to LHRH by approximately 25%, but did not affect basal LH release . In contrast, NPY failed to amplify the release of PRL in response to TRH, another Ca2(+)-mobilizing hormone . To test whether NPY also enhances the increase in cytosolic Ca2+ induced by LHRH, anterior pituitary cells were acutely dispersed into single cell suspensions, loaded with the fluorescent Ca2+ probe Indo-1 AM, and analyzed with a UV laser in an EPICS-753 flow cytometer at a rate of 500 cells/sec for 200 sec . The ratio of intracellular fluorescence resulting from Ca2+ bound to the Indo-1 to the fluorescence from Indo-1 alone (Indo-1 ratio), which is an index of the concentration of free cytosolic Ca2+, was determined for each cell . Approximately 7% of anterior pituitary cells responded to LHRH (1 or 10 nM) with significant increases in Indo-1 ratios, indicative of an increase in the concentration of free cytosolic Ca2+ . EGTA (2.5 mM) reduced the basal Indo-1 ratios and attenuated, but did not abolish, the initial increase in response to LHRH, consistent with the initial extracellular Ca2+ influx-independent phase of the response to LHRH . NPY alone (100 nM) did not affect the Indo-1 ratios in anterior pituitary cells, but pretreatment with the peptide for 10 min before the scans significantly augmented the Indo-1 ratio response to 10 nM LHRH . This effect of NPY was also blocked by EGTA . Taken together, these biochemical and pharmacological studies suggest that NPY enhances the release of LH stimulated by LHRH by increasing extracellular Ca2+ entry, possibly by selectively affecting that component of the response involving dihydropyridine-sensitive L-type voltage-sensitive Ca2+ channels during the initial stages of the cellular response to LHRH.

J Invest Dermatol, 1990 Sep, 95(3), 359 - 62
Granulocyte macrophage--colony-stimulating factor (GM-CSF) decreases CD1a expression by human Langerhans cells and increases proliferation in the mixed epidermal cell-lymphocyte reaction (MELR); Kolenik S et al.; Langerhans cells (LC) undergo a variety of phenotypic and functional changes in vitro . To determine the effects of granulocyte macrophage--colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-alpha (IL-1) on LC phenotype in vitro, epidermal cell suspensions were enriched for LC by density-gradient centrifugation and cultured in the presence of 10 ng/ml of these cytokines . The percentage of cells expressing the surface protein CD1a was determined by flow cytometry . This percentage typically dropped after 48 h culture in both control and cytokine-treated medium to less than half that of the starting value . By the fifth day, the percentage of cells expressing CD1a in TNF-alpha and IL-1--treated cultures was still near half of the starting value, slightly above that of control cultures . Treatment with GM-CSF caused large and consistent decreases in the percentage of epidermal cells expressing CD1a . Cell viability in each of the three cytokine-treated cultures was identical to the control cultures, with essentially all cells having died by the sixth day after isolation . To determine the functional effects of these cytokines, the cytokine-containing medium was replaced after 72 h with medium containing purified allogeneic T cells and proliferation measured . Preliminary experiments showed no increased proliferation induced by IL-1 or TNF-alpha--treated epidermal cells . GM-CSF-treated epidermal cells induced 2-3 times more T-cell proliferation than epidermal cells cultured without additional cytokines . We conclude that GM-CSF, a cytokine known to be produced by keratinocytes in vitro, decreases CD1a expression by human LC and increases their ability to stimulate proliferation by allogeneic T cells.

Enzyme Microb Technol, 1990 Sep, 12(9), 653 - 5
Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii; Trevors JT et al.; Azotobacter vinelandii cells were transformed via high-voltage electroporation, with the broad host-range plasmid pRK2501 . The number of transformants was dependent on the applied voltage, capacitance, and recovery procedure after electroporation . For example, Log, 4.44 transformants microgram-1 DNA were recovered in the A . vinelandii cell suspension electroporated at 1500 V and 25 microF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 microgram/ml-1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance) . Electroporation at 2500 V and capacitance settings of 25 and 3 microF did not produce any transformants . Cell survival was also poor at high voltages . A . vinelandii transformants were not recovered on N-free agar medium . In addition, no viable cells were recovered on N-free agar after electroporation at 2500 V, 25 microF; 2500 V, 3 microF; and 1500 V, 25 microF . Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.

Brain Res, 1990 Aug 13, 525(1), 45 - 58
Dispersed cell suspensions of fetal SCN restore circadian rhythmicity in SCN-lesioned adult hamsters; Silver R et al.; Overt circadian rhythms are permanently disrupted following lesions of the suprachiasmatic nucleus (SCN) in hamsters . It has previously been demonstrated that whole tissue grafts which include the fetal SCN restore circadian locomotor rhythms to hamsters previously made arrhythmic by SCN lesions . In the present study, we ask whether the intrinsic peptidergic organization of the SCN is a prerequisite for functional recovery of circadian rhythms of locomotor activity . To this end, dispersed cell suspensions of {3H}thymidine-labelled fetal anterior hypothalamic tissue which contains the SCN, were injected stereotaxically into the brain of adult hamsters . Dispersed cell suspensions restored free-running locomotor rhythms, but not entrainment or gonadal regression . The period of the restored free-running rhythms following injections of SCN cell suspensions was shorter than 24 h, in contrast to intact hamsters and SCN-lesioned hamsters whose rhythms are restored by whole tissue grafts . In animals with restored rhythms, a majority of {3H}thymidine-labelled cells were located within nuclei of the midline thalamus and zona incerta . In a few individuals, donor cells were also deposited along the injection tract as far ventrally as the medial hypothalamus . Restoration of free-running locomotor rhythmicity was correlated with the presence of small numbers of isolated VIP cells along with small plexuses of VIP fibers . In animals which did not recover locomotor rhythmicity, grafts were identical in location and size to those in recovered hamsters, but did not contain peptidergic cells characteristic of the SCN . The results suggest that structural integrity of the fetal SCN is not necessary for restoration of rhythmicity after grafting.

Brain Res, 1990 Aug 13, 525(1), 155 - 9
Intra-accumbens implants of embryonic dopaminergic neurons reverse the behavioral supersensitivity to opiates evoked by lesion of the mesolimbic dopaminergic pathway; Abrous DN et al.; The ascending mesotelencephalic dopaminergic systems of rat pups of 3 days of age were bilaterally lesioned using 6-hydroxydopamine injected at the level of the lateral hypothalamus . A sub-group of lesioned pups received, 5 days after the lesion, a dopamine neuron-rich cell suspension graft implanted bilaterally into the striatum and nucleus accumbens . Behavioral tests were conducted 6 months later . The lesion induced an increase in the locomotor activation induced by D-Ala2-Met5-enkephalinamide injected into the nucleus accumbens (2.5 micrograms/side) as compared to the activation observed in control animals . Locomotor activation by systemic apomorphine (0.1 mg/kg s.c.) was also increased while that induced by amphetamine (1.5 mg/kg i.p.) was abolished . The presence of DA neuron implants reversed each of these post-lesion modifications.

J Electron Microsc Tech, 1990 Aug, 15(4), 414 - 5
Easy handling of cell suspensions for electron microscopy; Pihakaski-Maunsbach K et al.; We present here a new, simple agar encapsulating technique, which is helpful when preparing a small quantity of isolated fragile cells, e.g . protoplasts, for electron microscopy.

Br J Cancer, 1990 Aug, 62(2), 189 - 94
A feasibility study of the MTT assay for chemosensitivity testing in ovarian malignancy; Wilson JK et al.; We assess the feasibility of using the MTT assay as a measure of cell viability in chemosensitivity testing in ovarian malignancy . The assay utilises the conversion of the tetrazolium salt MTT to formazan by dehydrogenase enzymes in living cells . We show that the optical density of the formazan produced from MTT is directly proportional to the number of live cells tested . Optimum MTT conversion occurred after 4 h incubation and dimethyl sulphoxide was found to be the most suitable solvent for the formazan . Seventy-five samples of ascitic fluid and/or solid tumour were collected from 56 patients with FIGO stage III-IV ovarian adenocarcinoma . Malignant cell suspensions with a viability greater than 75% were prepared from 95% of ascitic fluid and 75% of biopsy samples by simple techniques . The effect of cytotoxic drugs was assessed in 91% of patients included in the study . Variation in drug effect between patients was evident following a 48 h incubation period and was reproducible . Overall platinum and anthraquinone analogues produced the greater effect but resistance did occur . Our results mirrored reported clinical response rates . Only one sample tested against chlorambucil showed any drug effect . As this assay produces results in a high percentage of tests and is rapid and simple it appears suitable for prospective clinical trials to correlate the in vitro results with in vivo response.

Exp Neurol, 1990 Aug, 109(2), 191 - 9
Embryonic mesencephalic and striatal co-grafts: development of grafted dopamine neurons and functional recovery; Yurek DM et al.; Previous neural grafting studies have shown that embryonic dopamine neurons survive transplantation into the parenchyma of the brain; however, fiber outgrowth from those cells is often limited to the immediate vicinity of the graft . More extensive outgrowth is desirable for promoting and maintaining functional recovery of damaged neural systems in animal models as well as human neurodegenerative disorders . The present study examined the possibility of stimulating fiber outgrowth of grafted neurons by simultaneously grafting dopamine neurons with their embryonic target cells . Subsequent functional recovery was evaluated in concert with morphological characteristics of these grafts . Co-grafts of embryonic mesencephalic and striatal cells were implanted into the DA-denervated striatum of rats previously given unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway . Two types of co-grafts were implanted into the DA-denervated striatum: mixed or separate cell suspensions . Tyrosine hydroxylase immunocytochemical analysis of brain sections containing co-grafts revealed extensive arborization of TH-positive neurons in both types of co-grafts . When mesencephalic and striatal nerve cells were implanted into separate sites, TH-positive neurons extended projections that appeared to preferentially reach regions occupied by embryonic striatal neurons . Moreover, the average size of TH-positive cell bodies found in mixed or separate co-grafts was significantly larger than the size of those found in single mesencephalic grafts . Amphetamine-induced rotational behavior was used to assess the degree of functional recovery . In the majority of co-grafted animals, rotational behavior was attenuated by 3 weeks and reversed (amphetamine-induced contralateral rotation) by 5 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Exp Hematol, 1990 Aug, 18(7), 789 - 93
Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry; Hegyi E et al.; Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation . Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes . The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes . We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS . A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes . The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules . During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled . Polyploidization also affected the proportion of labeled AGR-SVS . Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules . These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.

J Histochem Cytochem, 1990 Aug, 38(8), 1215 - 21
Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies; Otsuki Y et al.; We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method . Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study . Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy . The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy . Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles . However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles . Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph . Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.

Cancer, 1990 Aug 1, 66(3), 480 - 90
Lymphomas in dogs . A morphologic, immunologic, and clinical study; Greenlee PG et al.; One hundred seventy-six canine lymphomas were classified morphologically using four of the major human lymphoma classification schemes (Rappaport, Lukes-Collins, Kiel, and the Working Formulation) . All 176 dogs received the same chemotherapeutic protocol . Sixty-two of these lymphomas had their immunophenotypes established by examination of cell surface markers by automated cytofluorography . Several different morphologic types of canine lymphoma were identified and these were comparable to morphologic categories in human classification schemes . Follicular and low grade lymphomas were rare . The two most common morphologic types were diffuse large cell (centroblastic) and immunoblastic . The Kiel classification appeared to be the most useful human scheme for classifying the canine lymphomas . Cytofluorographic analysis was generally straightforward, and 60 of the 62 lymphomas were placed into one of three immunophenotypic categories: 27 pan-T(LQ1)+SIg+, 21 pan-T(LQ1)-SIg+, and 12 pan-T(LQ1)+SIg- . Two of the lymphomas could not be characterized immunologically because a pre-existing or reactive non-neoplastic population of lymphocytes made interpretation of single cell suspension analysis difficult . The authors identified correlations between morphology and survival and disease-free remission; dogs with high-grade tumors generally survived the longest and had the longest remissions . No correlations were identified between high concentrations of serum lactate dehydrogenase, age, sex, or stage of disease, and morphology, immunophenotype, remission, or survival times . A significant correlation between clinical illness and survival time was documented . The median age of the dogs was nine years, no significant effect of sex on prevalence was observed, and some breeds were significantly overrepresented . Significant morphologic-immunophenotypic correlations included shorter remission and survival times for T-cell tumors than B-cell tumors, and a highly significant correlation between the pan-T(LQ1)+SIg-"T cell" phenotype and hypercalcemia.

Analyst, 1990 Aug, 115(8), 1145 - 6
Rapid simple cell suspension enzyme-linked immunosorbent assay to demonstrate and measure antibody binding; O'Kennedy RJ et al.; A simple cell suspension enzyme-linked immunosorbent assay system, which can be used to test the reactivity of antibodies with cells, is described . Such a system is of particular use with cells that are difficult to fix to microtitre plates or when measuring antigens which are labile to fixation techniques.

Radiother Oncol, 1990 Aug, 18(4), 349 - 56
Radiosensitivity testing of primary cervical carcinoma: evaluation of intra- and inter-tumour heterogeneity; Davidson SE et al.; Biopsies from 89 patients with cervical carcinoma were studied using a clonogenic assay to obtain values for the surviving fraction at 2 Gy (SF2) . Heterogeneity in intrinsic radiosensitivity was investigated by independently processing multiple biopsies from 18 tumors . No significant differences between intra-tumour SF2 values were demonstrated (p = 0.30) . The results have shown that intra-tumour heterogeneity is not a limitation to radiosensitivity testing using the Courtenay-Mills assay . A wide range of values (0.13-0.97) for SF2 was obtained with a mean value of 0.47 +/- 0.18 (+/- 1 S.D., CV = 38%) for 52 squamous cell carcinomas and 0.59 +/- 0.27 for four adenocarcinomas . There were statistically significant differences between the individual tumours (p less than 0.001) . From the analysis-of-variance of all the SF2 results it appears to be the surviving fractions below about 0.40 and those above about 0.7 which show significant differences in radiosensitivity between pairs of tumours (p = 0.05) . Also 36% of the values of SF2 show significant differences from the mean SF2 of all tumours . The storage of tumour cell suspensions in liquid nitrogen improved the colony-forming efficiency (CFE) but it did not alter the radiosensitivity.

Mol Biochem Parasitol, 1990 Aug, 42(1), 109 - 17
A 31P nuclear magnetic resonance study of Crithidia luciliae; Grote R et al.; 31P NMR has been used to observe phosphorus-containing compounds in both perchloric acid and KOH extracts and in whole cell suspensions of Crithidia luciliae . Cells were grown in Bone and Steinert medium, or in a modified RPMI culture medium and harvested after about 72 h in mid- to late log phase . 31P NMR spectra of the perchloric acid extracts indicated that 3-phosphoglycerate, which is normally at low concentrations in most cells, was the dominant phosphorus-containing compound in the sugar phosphate region . 3-Phosphoglycerate is the end product of glycosomal glycolysis and our finding is consistent with previous observations of the failure to detect prior glycolytic intermediates . Other metabolites observed were ATP, ADP, NAD(P)+, phosphoenolpyruvate and low molecular weight polyphosphates (PPn, n less than 20) . The presence of high-molecular-weight polyphosphates was established by spectra recorded on extracts obtained through subsequent treatment of the insoluble fraction with KOH . 31P NMR experiments on whole cells indicated that the average main internal pH of cells in late-log growth phase was approx . pH 7.2 +/- 0.1, using the orthophosphate resonance as an indicator . The cells responded to the addition of glucose (final concentration approx . 35 mM) with a decrease in pH, both internal (delta pH = -0.9 (55 min)-1) and external (delta pH = -1.3 (15 min)-1) . Polyphosphates and ATP could not be observed in whole cell experiments, although perchloric acid extracts of identically treated cells showed no significant depletion of these compounds.

J Biomech Eng, 1990 Aug, 112(3), 257 - 62
Effects of cell lysis on the rheological behavior of red blood cell suspensions; Tran-Son-Tay R et al.; Rheological studies of lysed cell suspensions are performed with a magneto acoustic ball microrheometer . Two methods for lysing the cells are developed in order to provide cell volume concentrations identical to control intact cell suspensions . The first uses a freeze-thaw technique and the second uses sonication . It is found that cell suspensions disrupted by sonication have a lower viscosity than intact suspensions, whereas cell suspensions lysed by the freeze-thaw method exhibit a higher viscosity . Sonication is discovered to have a detrimental impact on the cell membrane, and to cause complete destruction of the cell membrane structure . Measurements of the steady state viscosity show that indeed the presence of the membrane is not detected, and that what is measured is mainly the viscosity of the hemoglobin solution . On the other hand, freeze-thaw results indicate that at least two phenomena occur . The first phenomenon, occurring during the first freeze-thaw cycle, produces an increase in viscosity and in viscoelasticity . The second one, taking place after subsequent freeze-thaw cycles, induces a decrease in the bulk rheological properties . Several possible mechanisms are presented to explain the observed phenomena.

Hybridoma, 1990 Aug, 9(4), 389 - 95
Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method; Pancino GF et al.; This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology . Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma . A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast . The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections . These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.

J Periodontol, 1990 Aug, 61(8), 491 - 6
The effects of scaling titanium implant surfaces with metal and plastic instruments on cell attachment; Dmytryk JJ et al.; This study examined the ability of tissue culture fibroblasts to attach and colonize on the surface of pure titanium dental implants following instrumentation of the implant surface with curettes of dissimilar composition . Pure titanium dental implants were scaled with a plastic, titanium-alloy, or stainless steel curette and then immersed in a cell suspension of 3T3 fibroblasts . Counts of attached cells were made at 24 and 72 hours; the implants were then processed for scanning electron microscopy (SEM) . At 24 hours, only surfaces scaled with a stainless steel curette showed a significant reduction in number of attached cells relative to untreated control surfaces . At 72 hours, both stainless steel and titanium-alloy curette instrumented surfaces showed significantly fewer attached cells than untreated control surfaces, with the greatest reduction in cell attachment observed on the stainless steel curette instrumented surfaces . SEM observations showed that fibroblasts on stainless steel instrumented surfaces tended to show a somewhat rounded morphology and a relatively reduced degree of spreading: while fibroblasts on untreated control, plastic, or titanium-alloy instrumented surfaces showed a well-spread, polygonal morphology, more typical of fibroblasts in favorable culture conditions . To the extent that such observations of cell attachment and morphology are indicative of in vivo biocompatibility, these findings could have clinical implications for the proper maintenance of titanium dental implants.

Hepatology, 1990 Aug, 12(2), 295 - 300
Liver tumor promoters stimulate growth of transplanted hepatocellular carcinomas; Seglen PO et al.; Cell suspensions or tissue fragments from primary hepatocellular carcinomas and benign neoplastic nodules, induced by treating rats with chemical carcinogens, were transplanted by intraportal injection or subcapsular implantation in the livers of syngeneic host rats . Both nodule and carcinoma transplants produced high numbers of hepatocellular carcinomas in the hosts 2 to 5 mo after transplantation . Treatment of the host rats with liver tumor promoters (phenobarbital or 2-acetylaminofluorene) greatly stimulated tumor outgrowth, demonstrating that even established carcinoma cells can be promoter-sensitive . Tumor outgrowth was also stimulated by partial hepatectomy of the hosts, the regenerative stimulus interacting synergistically with the tumor promoters.

Fertil Steril, 1990 Aug, 54(2), 356 - 9
Fluorescent detection of rabbit endometrial implants resulting from monodispersed viable cell suspensions; Manyak MJ et al.; A model for early stage endometriosis was prepared in rabbits by intraperitoneal injection of monodispersed viable endometrial cells . With this model, we have found that DHE fluorescence increases the sensitivity of detection of endometrial tissue . The potential experimental and clinical significance of ectopic endometrial detection by porphyrin fluorescence is enhanced by the recent report of endometrial transplant destruction by PDT . Although caution must be exercised for extrapolation from animal experiments to human conditions, these results encourage further evaluation of photosensitization for the study and potential treatment of disorders involving endometrial tissue.

Biochem J, 1990 Aug 1, 269(3), 671 - 7
Metabolism of saturated and polyunsaturated very-long-chain fatty acids in fibroblasts from patients with defects in peroxisomal beta-oxidation; Street JM et al.; The metabolism of {1-14C}lignoceric acid (C24:0) and {1-14C}tetracosatetraenoic acid (C24:4, n-6) was studied in normal skin fibroblast cultures and in cultures from patients with defects in peroxisomal beta-oxidation (but normal peroxisomal numbers) . Cells from X-linked adrenoleukodystrophy (ALD) patients with a presumed defect in a peroxisomal acyl-CoA synthetase, specific for fatty acids of carbon chain lengths greater than 22 (very-long-chain fatty acids; VLCFA), showed a relatively normal production of radiolabelled CO2 and water-soluble metabolites from {1-14C}C24:0 . However, the products of synthesis from acetate de novo (released by beta-oxidation), i.e . C16 and C18 fatty acids, were decreased, and carbon chain elongation of the fatty acid was increased . In contrast, cell lines from two patients with an unidentified lesion in peroxisomal beta-oxidation (peroxisomal disease, PD) showed a marked deficiency in CO2 and water-soluble metabolite production, a decreased synthesis of C16 and C18 fatty acids and an increase in carbon chain elongation . The relatively normal beta-oxidation activity of ALD cells appears to be related to low uptake of substrate, as a defect in beta-oxidation is apparent when measurements are performed on cell suspensions under high uptake conditions . Oxidation of {1-14C}C24:4 was relatively normal in ALD cells and in the cells from one PD patient but abnormal in those from the other . Our data suggest that, despite the deficiency in VLCFA CoA synthetase, ALD cells retain a near normal ability to oxidize both saturated and polyunsaturated VLCFA under some culture conditions . However, acetate released by beta-oxidation of the saturated VLCFA and, to a much lesser degree, the polyunsaturated VLCFA, appears to be used preferentially for the production of CO2 and water-soluble products, and acetate availability for fatty acid synthesis in other subcellular compartments is markedly decreased . It is likely that the increased carbon chain elongation of the saturated VLCFA which is also observed reflects the increased availability of substrate (C24:0) and/or an increase in microsomal elongation activity in ALD cells.

Endocrinology, 1990 Aug, 127(2), 613 - 20
Calcitonin inhibits thyrotropin-releasing hormone-induced increases in cytosolic Ca2+ in isolated rat anterior pituitary cells; Shah GV et al.; Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells . Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect . Secretogogue-induced elevations of cytosolic free Ca2+ ({Ca2+}i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry . AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min . Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of {Ca2+}i, was determined for each cell . Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio . Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in {Ca2+}i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA . In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH . In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced {Ca2+}i increase was also abolished by prior exposure to sCT . These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in {Ca2+}i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.

Biull Eksp Biol Med, 1990 Aug, 110(8), 196 - 8
{Fast direct method of obtaining metaphase and prometaphase chromosomes from chorion biopsy cells and human embryos during the lst semester of pregnancy}; Baranov VS et al.; Samples of chorionic villi and embryonic tissues (brain, brain--sheaths) are thoroughly washed with Hank's solution, immediately subjected to hypotonic treatment (0.9% sodium citrate plus few drops of 0.01% colchicine) 37 degrees C, 30 min, prefixed 20 min with equal amount of standard fixative mixture, twice fixed in standard fixative solution (1 hour, -10 degrees C), hydrated with equal volume of distilled water (5-10 min), dried, macerated directly on the slide with 60% acetic acid . The cell suspension is then evenly spread on the slide surface, dried, postfixed and stained . The method provides sufficient amount of metaphase and prometaphase mitotic plates suitable for differentiating staining in 1.5-2 hours after sampling and might be recommended for routine chromosomal analysis in prenatal diagnosis of inherited diseases during early pregnancy.

J Neurosci Methods, 1990 Aug, 33(2-3), 93 - 100
Primary culture of astrocytic glial cells from rainbow trout, Salmo gairdneri L., brain; Tocher DR et al.; A method is described for the preparation and maintenance of rainbow trout brain astrocytes in primary culture . A dissociated cell suspension was prepared from brains from young fish by mechanical sieving through nylon guazes, and the cells cultured in polylysine-treated plastic tissue culture flasks at 22 degrees C . The resultant cultures were characterised by specific immunofluorescent staining using glial fibrillary acidic protein (GFAP) for astrocytes and Thy 1 for fibroblastic cells . The cultures were greater than 95% astrocytes with fibroblasts the only other cell type present . These highly enriched astrocyte cultures provide a unique system for various neurochemical, neurophysiological and biochemical studies of fish neural cells . Polyunsaturated fatty acid metabolism will be an area of particular interest.

Int J Hyperthermia, 1990 Jul-Aug, 6(4), 793 - 800
Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice; Wierenga PK et al.; Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting . There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e . the amount of stem cells which will lodge in the spleen . The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice . Furthermore, we investigated the seeding efficiency before and after heat treatment . Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions . The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen . Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Br J Radiol, 1990 Jul, 63(751), 542 - 6
The effect of ultrasound on the cytotoxicity of adriamycin; Loverock P et al.; The effect of continuous wave ultrasound exposures on the cytotoxicity of adriamycin has been studied . It has been found that 2.6 MHz, 2.3 Wcm-2 (spatial average) ultrasound can enhance the cell killing potential of adriamycin both in suspensions of single V79 chinese hamster fibroblast cells and in spheroids formed from these cells . The ratio of the slopes of the survival curves for single cell suspensions is 1.5 . For spheroids, the growth delay is increased by 1.3 days by simultaneous ultrasound exposure . Flow cytometric studies of the intracellular concentration of adriamycin following ultrasound exposure reveals that this is increased when compared with that measured when the cells are only exposed to adriamycin . Evidence is presented to suggest that this is a non-thermal effect of ultrasound.

Res Vet Sci, 1990 Jul, 49(1), 39 - 45
Separation of equine bronchopulmonary lavage cells by density gradient centrifugation and expression of procoagulant activity in unpurified cells and cell subpopulations; Grunig G et al.; Bronchopulmonary lavage was performed in 10 healthy horses and in 39 horses with chronic pulmonary disease . The predominant cell types were macrophages in healthy horses and neutrophils in severely diseased horses . Procoagulant activity (PCA) was detected in all 32 cell-free supernatants examined and in all 49 unpurified cell suspensions . Cells were separated by centrifugation on discontinuous gradients prepared either with Percoll or with Metrizamide . Macrophages were enriched in subpopulations of low density . Neutrophils could not be purified by density gradient centrifugation using either gradient medium . PCAs of cell subpopulations were plotted against their respective macrophage, neutrophil, and lymphocyte content . PCA was positively correlated with macrophage content (P less than 0.001) and negatively correlated with neutrophil (P less than 0.02) and lymphocyte (P less than 0.001) content . Therefore, PCA of equine lung cells most likely originates from macrophages as shown in other species . The density shift of lung neutrophils requires further investigation.

Int J Radiat Oncol Biol Phys, 1990 Jul, 19(1), 85 - 7
Intra-cerebral ventricular infusion of 5-iodo-2-deoxyuridine (IUDR) as a radiosensitizer in the treatment of a rat glioma; Deutsch M et al.; The efficacy of 5-iodo-2-deoxyuridine (IUDR) as a radiosensitizer when administered by continuous infusion into the cerebral spinal fluid (CSF) of the lateral cerebral ventricle was evaluated in a 9L gliosarcoma rat brain tumor model . Stereotactic implantation of a 5 x 10(4) tumor cell suspension into the left caudate nucleus was carried out in four groups of 10 rats each . Control animals had a median survival of 16.9 days (range 16-21 days) . IUDR, 8.4 mg over 7 days administered by continuous infusion into the left lateral ventricle produced a slight survival advantage (median survival 21.5 days, range 12-56) . Irradiation of the entire brain, 8 Gy on days 4, 6 and 7 after tumor cell implantation also produced a slight improvement in survival (median 19.5 days, range 17-34) . The combination of radiation and IUDR infusion into the CSF produced a marked survival advantage (median 30.5, range 22-54) compared to the control and single modality treatment groups . This is the first demonstration of the effectiveness of IUDR as a radiosensitizer when administered into the lateral cerebral ventricle in the treatment of an intraparenchymal brain tumor.

Magn Reson Med, 1990 Jul, 15(1), 58 - 69
Determination of extracellular/intracellular fluid ratios from magnetic resonance images: accuracy, feasibility, and implementation; Martin MA et al.; This study determines the accuracy and feasibility of using localized spin-lattice (T1) relaxation time measurements from magnetic resonance (MR) images to follow changes in extracellular/intracellular fluid ratios in defined subvolumes of living tissue . A red blood cell suspension was used as a test system and a simple two-compartment model incorporating fast exchange was found to suffice for the conversion of T1 values to volume ratios . The technique requires the addition of gadolinium-DTPA to the model system to selectively enhance relaxation in the extracellular fluid space . No detectable amount of gadolinium-DTPA was found to enter the intracellular fluid space, and all magnetization decay plots obtained from both intracellular constituents and complete RBC suspensions consisted of a single exponential . Both of these results are compatible with assumptions underlying our physical model . The NMR-determined fluid ratio values were compared to those measured via the microhematocrit technique . Partial saturation image-mode determinations are strongly correlated to microhematocrit data (R2 = 0.945) and indicate that localized cell volume changes may be followed with a sensitivity of +/- 2.2% . These values compare favorably with those produced when nonimaging inversion-recovery techniques are used to determine the MR hematocrit (R2 = 0.962, sensitivity = +/- 1.1%) . This technique, with modification, should be applicable to the comparison of ratios of extracellular/intracellular fluid volumes in structurally complex tissues where small subvolumes of homogeneous cell structure could be examined.

Endocrinology, 1990 Jul, 127(1), 500 - 2
Expression of steroid metabolizing enzymes by aggregating fetal brain cells in culture: a model for developmental regulation of the progesterone 5 alpha-reductase pathway; Barnea A et al.; In this study, we established an in vitro model system for the study of developmental regulation of steroid enzyme expression in the perinatal brain . Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation under constant rotation . On day 0, 3, 6, or 12 of culture, aggregates were incubated for 4 or 20 h with 3H-progesterone (P4) and the profile of 3H-steroids in the medium analyzed . Five major metabolites were formed from 3H-P4: 5 alpha-pregnan-3, 20-dione (DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP), and two unidentified polar substances designated A and B . Progressively with aggregate-age in culture, there was a decrease in the relative amounts of 3H-P4 recovered in the medium and a sequential increase in DHP, 3 beta-OH-DHP, 3 alpha-OH-DHP, A and B . Aggregates maintained in a chemically defined, serum-free medium metabolized P4 at an accelerated rate compared to those maintained in serum . An inhibitor of the enzyme 5 alpha-reductase completely inhibited P4 metabolism, indicating that 5 alpha-reduction is the primary step in this pathway . Thus, the aggregates express three key enzymes in P4 metabolism: 5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductases and the operation of this pathway is proposed: P4----DHP----3 alpha-OH-DHP + 3 beta-OH-DHP; A and B are derived from one or more of the latter three . Hence, this culture system can serve as a model to study regulatory processes in the developing steroidogenic brain.

J Leukoc Biol, 1990 Jul, 48(1), 27 - 37
Development, differentiation, and maturation of macrophages in the fetal mouse liver; Naito M et al.; Primitive macrophages emerged in the sinusoidal lumen of the fetal mouse liver at 10 days of gestation before the initiation of hepatic hematopoiesis and matured into fetal macrophages . In the culture of cell suspensions from the fetal liver with LP3-conditioned medium, monocyte colonies were formed, but monocytopoiesis was poor in the early stage of hepatic hematopoiesis in vivo . In the culture of cell suspensions obtained from the fetal liver at 10 days of gestation on the monolayer of a mouse bone marrow stromal cell line, ST2, primitive/fetal macrophage colonies were formed before the development of monocyte/macrophage colonies and showed differentiation of primitive macrophages into fetal macrophages without passing through the stage of promonocytes and monocytes . At this time, the fetal cardiovascular system was connected with the vitelline vein just before the formation of the liver . With the progress of gestation, a monocytic cell series was observed to develop and form a monocyte/macrophage population . This was confirmed by in vitro studies with an LP3-conditioned medium and on a monolayer of ST2 . Thus, it appears that there exist two different macrophage populations, a primitive/fetal macrophage population and a monocyte/macrophage population in hepatic hematopoiesis . It also appears that fetal macrophages are differentiated from primitive macrophages which are colonized into the fetal liver from the yolk sac or which develop in loco, presumably from hematopoietic stem cells.

Placenta, 1990 Jul-Aug, 11(4), 349 - 67
Human trophoblast-endometrial interactions in an in vitro suspension culture system; Kliman HJ et al.; We developed an in vitro suspension co-culture system to examine the interaction of 1st, 2nd and 3rd trimester purified cytotrophoblasts with human endometrium . Endometrium explants were added to cytotrophoblast cell suspensions and placed on an angled gyrating platform in a 37 degrees C incubator . When endometrium was cultured alone it was able to remain viable for up to 3 days . When trophoblasts were cultured alone, they formed small and large aggregates, and occasionally spherical shells with hollow centers . When trophoblasts and endometrium were cultured together, the trophoblasts adhered to the exposed stromal surfaces of the tissue fragments . The surface epithelium was not receptive to trophoblast attachment except in one experiment when day 19 endometrium was used for the co-incubation, suggesting that surface attachment is usually restricted . A common finding was the presence of an acellular zone in the endometrium only adjacent to the attached trophoblasts . We speculate that this zone may be caused by proteolysis and resynthesis of ECM proteins by the trophoblasts . Based on our results, this in vitro suspension should prove useful for examining those factors which: (1) induce endometrial permissiveness, (2) promote paracrine effects on the endometrium, and (3) facilitate human trophoblast invasion.

Obstet Gynecol, 1990 Jul, 76(1), 139 - 46
The safety of obstetric ultrasonography: concern for the fetus; Reece EA et al.; It has been estimated that more than half of all pregnant women in the United States undergo diagnostic ultrasound during their pregnancies . In light of this, the question of safety is of fundamental importance . Nondiagnostic ultrasound has been shown to produce biologic effects by thermal and cavitational activities . However, diagnostic ultrasound uses much lower intensities, and no evidence exists to suggest that it is associated with adverse effects . Numerous studies have examined the biologic effects of diagnostic ultrasound in insects, plants, cell suspensions, and even small mammals . The data from these experiments are confusing when attempting to relate these findings to the human . Epidemiologic data in humans, used to evaluate the potential adverse effects of exposure to diagnostic ultrasound, have revealed no ill effects from such exposure . Current data indicate that there are no confirmed biologic effects on patients and their fetuses from the use of diagnostic ultrasound and that the benefits to patients exposed to prudent use of diagnostic ultrasound outweigh the risks, if any . This review discusses the available information on the safety of obstetric ultrasonography.

Am J Physiol, 1990 Jul, 259(1 Pt 2), F95 - 103
ADH modulates plasma membrane lipid order of living MDCK cells via a cAMP-dependent process; Giocondi MC et al.; Using 1-{4-(trimethylamino)phenyl}-6-phenyl-hexa-1,3,5-triene, a fluorescent probe that specifically labels the external leaflet of the plasma membrane of living cells, we examined the effects of antidiuretic hormone (ADH) and various agents known to raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on the physical state of the plasma membrane of Madin-Darby canine kidney (MDCK) cells . In polarized cells grown as a monolayer, {desamino-Cys1, DArg8}-vasopressin (V2-agonist) elicited a biphasic decrease in the lipid order as estimated from the decrease in fluorescence anisotropy (from r = 0.317 to r = 0.304, 37 degrees C) of the apical domain of the plasma membrane, equivalent at the peak response (t = 5 min) to that produced by an upward shift in temperature of 5-6 degrees C . A similar response was obtained by adding dibutyryl cAMP to the monolayers . Experiments on cell suspensions further indicated that the biphasic decrease in lipid order could also be evoked by forskolin, prostaglandin E2, and bradykinin but not by bradykinin plus indomethacin and was inhibitable by the protein kinases inhibitor compound H7 . These data demonstrate that the lipid order of the plasma membrane of MDCK cells can be modulated in situ by cAMP-dependent processes probably involving protein kinase A activity, i.e., that membrane "fluidity" might act in the regulation of the cellular function of living epithelial cells . They provide a rationale for the changes in lipophilic solute permeability that accompany the increase in water permeability of target cells on ADH administration.

Dev Biol, 1990 Jul, 140(1), 189 - 95
New roles for DIF? Effects on early development in Dictyostelium; Wurster B et al.; The DIFs are unusual, chlorinated molecules which induce stalk cell differentiation during the later, multicellular phase of Dictyostelium development . Here we provide evidence that one or more DIFs have a role during early development, when small amounts are known to be made . Initial indications came from an optical technique which detects changes in shape or cohesion of cells in suspension (Gerisch and Hess, PNAS 71, 2118, 1974) . After a period of optical inactivity at the start of development, cell suspensions normally produce spontaneous spike-shaped light-scattering oscillations synchronised by oscillations in extracellular cAMP levels, followed by sinusoidal oscillations where the synchroniser is not known . DIFs 1 and 2 produce optical responses from cells at all these early stages of development . The phase of both spiked and sinusoidal oscillations can be shifted, indicating an effect on the oscillator in each case . We find further: (1) cAMP oscillations and cAMP relay during spiked oscillations are transiently inhibited by DIF-1 . (2) DIF-1 causes a transient decrease in cellular cGMP levels in cells taken before oscillations commence and likewise inhibits the cGMP response to a cAMP stimulus in cells taken later in development . Cytoskeletal organization and hence cell shape might be affected by DIF-1 by this indirect route . (3) The effects of DIF-1 are transient, even though it is essentially stable in the cell suspension . Cells somehow adapt to DIF-1 . (4) The effects are chemically specific: DIF-1 and DIF-2 are active at 10(-7) to 10(-8) M, with DIF-2 being the more active, whereas related compounds have little or no activity at 10(-6) M . These results indicate that cells are responsive to DIFs 1 and 2 from the start of development and suggest a wider role for the DIFs . This role might involve effects on cAMP signalling and on intracellular second messengers.

Res Immunol, 1990 Jul-Aug, 141(6), 491 - 504
Relationship between the synthesis of autoimmune antibodies and the formation of clusters of B, T and APC cells during the syngeneic mixed lymphocyte reaction in BALB/c and NZB mice: a technique for isolation of the spleen autoimmune compartment of non-immunized pathogen-free mice; Bruyns C et al.; Cells from the spleens of non-immunized mice were cultured in horizontal tubes, rotating very slowly around their long axis . Under these conditions, the flux speed gradient of the cell suspension near the tube walls greatly increased the chances of cells coming into contact with one another . Mixed clusters of B, T and APC cells were soon found adhering firmly to the walls of the tube; cluster formation leveled off after about 3 h . The clustered cells were easily separated from those remaining in suspension and constituted a particular cell compartment comprising a maximum of 20-30% of the total . B cells from this compartment, cultured in complete medium for 48 h, almost exclusively produced IgM antibodies . Antibodies reacting with self antigens were so numerous in the culture medium that it is likely all IgM were self antibodies . That the clusters obtained under these conditions constituted a compartment of autoimmune cells is supported by previous work which showed that 20-30% of spleen cells secrete IgM antibodies almost exclusively . Cluster formation as a function of age was compared in NZB mice which are used as a model of lupus erythematosus, and in BALB/c mice which never manifest self-immune pathology . The number of cells found in clusters per whole spleen increased exponentially with age in NZB mice and linearly in BALB/c mice . The production of autoimmune antibodies as a function of age also increased exponentially for NZB mice and linearly in BALB/c mice, which provides further striking support for the hypothesis that the clusters formed constitute the autoimmune comportment.

Immunology, 1990 Jul, 70(3), 351 - 6
Patterns of membrane TcR alpha beta and TcR gamma delta chain expression by normal blood CD4+CD8-, CD4-CD8+, CD4-CD8dim+ and CD4-CD8- lymphocytes; Scott CS et al.; Enriched CD4+CD8-/CD4-CD8-, CD4-CD8+/CD4-CD8- and CD4-CD8- cell suspensions were prepared from normal peripheral blood by selective immunomagnetic depletion of monoclonal antibody-defined lymphocyte populations . Subsequent examination of these modified cell fractions by two-colour flow cytometry provided a means of determining the expression of membrane T-cell receptor (TcR)alpha beta and TcR gamma delta chains by both major (CD4+ and CD8+) and minor (CD3+CD4-CD8dim+ and CD3+CD4-CD8-) lymphocyte subpopulations . Normal CD4+CD8- lymphocytes were almost invariably (greater than 99%) TcR alpha beta+, whereas lymphocytes expressing membrane CD8, which could be further subdivided according to differences in fluorescent staining intensity into CD3+CD4-CD8+, CD3+CD4-CD8dim+ and CD3-CD4-CD8dim+ components, were characterized by distinct differences in patterns of TcR chain expression . In contrast to CD3+CD4-CD8+ cells, which were predominantly (99%) TcR alpha beta+, CD3+CD4-CD8dim+ lymphocytes showed a significant proportion (33%) of TcR gamma delta+ cells (natural killer-associated CD3-CD4-CD8dim+ cells were uniformly TcR-) . The highest proportion (62%) of TcR gamma delta+ cells was associated with the CD3+CD4-CD8- fraction, but these studies also revealed that a significant minority of this population was TcR alpha beta+ . Despite some evidence for normal inter-individual variation, further analysis of membrane CD8 fluorescent intensities confirmed clear differential relationships for TcR alpha beta and TcR gamma delta chain expression.

Surgery, 1990 Jul, 108(1), 56 - 62
In vitro assessment of parathyroid immunogenicity: the effect of cryopreservation; Saxe AW et al.; Post-parathyroidectomy hypoparathyroidism, although fortunately uncommon, is a disorder of major inconvenience and potential morbidity . Attempts at modifying parathyroid tissue to facilitate allotransplantation without host immunosuppression are warranted . Cryopreservation has been reported to improve survival of canine parathyroid allografts . We employed a modification of the mixed lymphocyte culture to study the effect in vitro of cryopreservation on human parathyroid tissue . Dispersed parathyroid cells from fresh and previously cryopreserved tissue from 10 patients were incubated with unrelated mononuclear cells for 6 days, and incorporation of tritiated thymidine was measured after a 1-day pulse . Studies with irradiated mononuclear cells and parathyroid cells confirmed the model as a one-way test in which mononuclear cells respond to parathyroid cells but not vice versa . An antigenicity index was computed to express mononuclear cell tritiated thymidine incorporation for similar numbers of viable parathyroid cells . Although absolute values of the antigenicity index varied from patient to patient, there were no consistent differences in the antigenicity index of patients' fresh compared with cryopreserved tissue . In an attempt to identify the cells responsible for immunogenicity, we incubated cytocentrifuged parathyroid cell suspensions with antiserum directed at leukocyte common antigen, a marker of lymphoid tissue . Cell suspensions of parathyroid tissue demonstrated leukocyte common antigen-positive cells (median, 2.7% positive; range, 0% to 16%) . There were no consistent differences in the number of leukocyte common antigen-positive cells in fresh compared with cryopreserved tissue, and the number of leukocyte common antigen-positive cells did not correlate with the antigenicity index.

Endocrinology, 1990 Jul, 127(1), 200 - 10
Endocytosis and degradation of ovine prolactin by Nb2 lymphoma cells: characterization and effects of agents known to alter prolactin-induced mitogenesis; Augustine EC et al.; Rat Nb2 node lymphoma cells proliferate in response to lactogens, but the signal transduction mechanism involved remains unclear . Specific binding, internalization, and degradation of ovine PRL (oPRL) were examined under a variety of experimental conditions to characterize the metabolism of receptor-bound hormone by these cells . Stationary-phase cells were incubated with {125I}oPRL in Fischer's medium containing horse serum . Cell suspensions were centrifuged, and the cell pellets were assayed to determine specific cell-associated radioactivity . Internalized ligand was measured by exposing the cells to an acidic buffer before centrifugation to dissociate hormone from plasma membrane receptors, and cell-surface ligand was calculated by subtracting internalized hormone from the total {125I}oPRL bound by the cells . Hormone degradation was assessed by measuring the radioactivity in an acid-soluble fraction prepared from the incubation medium . Endocytosis of {125I}oPRL was observed within 30 min at 37 C, and the internalized component accounted for approximately 50% of the bound hormone under steady-state conditions . Hormone degradation was detectable within 1 h at 37 C and continued at a relatively linear rate thereafter; by 4 h, 8% of the added {125I}oPRL was acid soluble . Chloroquine (0.2 mM), methylamine (20 mM) and monensin (20 microM) prevented {125I}oPRL degradation and elevated both cell-surface and intracellular hormone 2-fold during a 4-h incubation . Leupeptin (0.2 mM) decreased degradation by only 15% under the same conditions . Phorbol 12-myristate 13-acetate (PMA; 20 nM), a comitogen for lactogen-stimulated Nb2 cells, increased cell-surface hormone by 20% and decreased intracellular hormone by a corresponding amount 1 h after administration . Calcium ionophore A23187 (1 microM) produced similar changes, and a synergistic effect was noted when cells were exposed to both agents for 4 h . Amiloride (125 microM), an inhibitor of Nb2 cell mitogenesis, decreased {125I}oPRL degradation by 25% during a 4-h incubation . This response was abolished when the cells were exposed simultaneously to PMA . These experiments demonstrate that receptor-bound oPRL is rapidly internalized and extensively degraded via the endosome-lysosome pathway when Nb2 cells are maintained at 37 C . The inhibitory effect of PMA on oPRL internalization may help to explain the comitogenic action of this phorbol on Nb2 cells . Since amiloride also produced major changes in oPRL metabolism, post-binding events in lactogen processing by target cells could play an important role in the mitogenic response elicited by such hormones.

Res Immunol, 1990 Jul-Aug, 141(6), 461 - 75
Tissue distribution and cytofluorometric analysis of oral mucosal T cells in the BALB/c mouse; Bolduc C et al.; The incidence and distribution of Thy-1.2+, Lyt-2.2+ and L3T4+ cells in the murine oral mucosa were investigated using qualitative and quantitative approaches . From immunostaining of frozen tissue sections, it appeared that the majority of oral T cells are located either in the epithelium or within the minor salivary gland network . The occurrence of Thy-1.2+, L3T4+ and Lyt-2.2+ cells at these sites points to two strategic lines of defence in the event of mucosal infections or aggression . A quantitative analysis of oral T-cell subsets was made possible by optimizing an enzymatic digestion procedure which preserves all three T-cell surface markers . Flow cytometric analysis of oral mucosal cells demonstrated that the helper phenotype is about twice as numerous as the cytotoxic/suppressor phenotype in the mucosa . Furthermore, in single cell suspensions, virtually all Thy-1+ cells were either L3T4+ or Lyt-2.2+ in the mucosa and in the spleen . From this frequency analysis and our previous studies, we conclude that T cells are a major component of the oral immune system, being 2-3 times as numerous as B cells or macrophages . Present data on the spatial distribution and characteristic ratio of T-cell subsets assess the basal activity of the local T-cell populations in healthy animals and lay the basis for comparative studies of both qualitative and quantitative variations occurring during mucosal infections or autoimmune reactions.

Br J Haematol, 1990 Jul, 75(3), 325 - 32
Immunohistocytochemical correlation of DAP IV-CD 26 reactivity with immunologic markers of lymphocyte activation in human lymphoid tissues; Cozzi M et al.; The topographic distribution of the dipeptidylaminopeptidase IV (DAP IV-CD 26) and II (DAP II) positive T-cell population in six reactive lymph nodes and seven follicular B-cell non-Hodgkin's lymphomas (NHL) was analysed with regard to the distribution of activated T-cells, as visualized by a panel of monoclonal antibodies including Tac-CD 25, HLA-DR, OKT 9-CD 71, ICAM-1-CD 54, LFA-1-CD 11a . For comparative studies serial frozen sections of the lymph nodes were tested by enzyme histochemistry and immunohistochemistry . In addition, cell suspensions obtained from 10 B-NHL and interleukin-2 (IL-2) activated T-cells were investigated by a combined cytochemical and immunological method for simultaneous visualization of DAP IV-CD 26 cytoplasmic activity and surface immunostaining for markers of lymphocyte activation . Both in reactive and lymphomatous lymph nodes the topographic distribution of DAP IV-CD 26+ and DAP II+ lymphocytes was rather similar to that of Tac-CD 25+ lymphocytes . On the contrary, the DAP IV-CD 26 and DAP II distribution pattern substantially differed from that of the other immunologic markers . In a cell suspension of IL-2 activated T-cells, more than 80% of the cells with a blastic morphology were DAP IV-CD 26+; DAP IV-CD 26+ cells coexpressing Tac-CD 25, OKT 9-CD 71, HLA-DR positivity, relative to the total number of DAP IV-CD 26 positive cells, were 90.5%, 70.5% and 87% respectively . Only small (not activated) lymphocytes expressed a focal cytoplasmic DAP II positivity . In cell suspensions from 10 cases of B-NHL the mean percentage of DAP IV-CD 26+ Tac-CD 25+ cells was 75.8 . Only a small number of DAP IV-CD 26+ cells coexpressed HLA-DR, the mean percentage being 9.6 . The results support the view that DAP IV-CD 26 may be considered as a marker of lymphocyte activation; this marker seems to be restricted to T lymphocytes that reside in the T dependent areas of reactive lymph nodes and to non malignant T-cells surrounding neoplastic follicles of follicular NHL.

Leukemia, 1990 Jul, 4(7), 508 - 16
Detection of minimal residual disease in acute myeloid leukemia; Gerhartz HH et al.; The distinction of clonogenic leukemic cells (CFU-L) and normal myeloid progenitors (GM-CFU) is a problem because both types of cells respond to the same growth factors and their clones resemble each other morphologically in culture . We investigated by means of an indirect enzyme-immunoassay the expression of "early" and "late" differentiation markers on bone marrow cell suspensions, as well as on agar clones in 18 cases of newly diagnosed acute myeloid leukemia (AML) as compared with 13 normal controls . Uncultured AML cells carried only low amounts of "late" myeloid differentiation antigen (CD15) but expressed nearly normal levels when cultured in agar with colony-stimulating factor (CSF) . In contrast to normal bone marrow, AML cells were strongly reactive with "early" differentiation markers (CD10, CD20, CD34) and remained so during culture . Normal and leukemic agar clones could be specifically distinguished by CD20- and CD34 antibodies . By means of a double marker technique, it could be shown that "late" myeloid differentiation markers (CD15) and "early" markers (CD10, CD20, CD34) were coexpressed on the same cells only in AML but not in normal bone marrow . Leukemic clones were identified by phenotyping of agar clones in 17 of 19 cases investigated during complete clinical remission (CR) of the disease . A formal proof of the leukemic origin of CD20/CD34 positive clones grown in CR was made possible in four cases either by Southern blot analysis or by a cytogenetic marker . These results demonstrate that AML cells can partially differentiate in vitro in the presence of CSF . A distinction of AML from normal clones, however, is possible by their reactivity with "early" differentiation markers, because this is maintained under the differentiating influence of CSF . The technique described here identifies residual leukemic clones in the majority of AML in CR, which persist at a constant rate and increase 6 months before cytological relapse.

J Invest Dermatol, 1990 Jul, 95(1), 60 - 4
Expression of integrins in junctional and dystrophic epidermolysis bullosa; Nazzaro V et al.; Recently, monoclonal antibodies (MoAb) have been raised against a family of adhesive membrane receptors (R) for extracellular matrix molecules known as integrins . In order to ascertain whether these adhesive proteins are normally expressed in inherited epidermolysis bullosa (EB) dermal epidermal junction, we studied the reactivity of MoAb recognizing receptors for VLA-1 (R for unknown ligand), VLA-2 (R for collagen), VLA-3 (R for collagen, laminin, fibronectin), VLA-4 (R for unknown ligand), VLA-5 (R for fibronectin), VLA-6 (R for laminin), VNR alpha, and VNR beta (R for vitronectin) on cryostat skin sections from EB patients and normal controls and on cytospins of normal epidermal cell suspensions with indirect immunohistochemical methods . Two cases of junctional EB (EBj) (lethal and non-lethal), three cases of dominant dystrophic EB (EBdd), two cases of recessive dystrophic EB (EBdr), and two normal controls skin sections and cell suspensions entered the study . No significant modification of the distribution of these adhesive receptors was observed in junctional and dystrophic EB skin . Both in normal and EB specimens MoAb against VLA-2, VLA-3, and VNR alpha determinants showed reactivity with the total cytoplasmic membrane of basal keratinocytes and basement membrane zone . Interestingly, anti-VLA-6 MoAb was characterized by an intense linear staining of the dermal-epidermal junction with the same localization on the roof of the blisters in EBj, EBdd, and EBdr as bullous pemphigoid (BP) serum . On the basis of these results we suggest that anti-VLA-6 MoAb could be used instead of BP serum for immunohistochemical detection of the cleavage of blisters in EB.

Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 1185 - 90
Intracellular protein phosphorylation in oat (Avena sativa L.) protoplasts by phytochrome action: involvement of protein kinase C; Park MH et al.; Phosphorylations of two proteins (27 KDa, 32 KDa) in oat cells were dependent on phytochrome action . To determine which kinase system(s) for the phosphorylation of these two proteins are controlled by the phytochrome, involvement of the Ca2+/DG dependent protein kinase (protein kinase C) was first investigated . When a protein kinase C inhibitor (1-(5-isoquinoline sulfonyl)-2-methylpiperazine:H-7) or the inositol phospholipid metabolic blocker Li+ was added into the cell suspension, respectively, the phosphorylations of these two proteins were substantially reduced . On the other hand, an addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG:activator of protein kinase C) or phorbol 12-myristate 13-acetate (TPA: tumor promoting phorbol ester) enhanced the phosphorylations of these proteins . These results suggest that phytochrome action is certainly connected with the protein phosphorylation via the activation of protein kinase C or a similar molecule with protein kinase C.

Biochim Biophys Acta, 1990 Jun 27, 1025(2), 173 - 8
Membrane fluidity of non-activated and activated human blood platelets; Feijge MA et al.; The steady-state fluorescence anisotropy of membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or its 4'-trimethylammonio derivative, TMA-DPH, is generally considered a measure for the lipid order and, hence, inversely related to membrane fluidity . We now report that anisotropy values of DPH- and TMA-DPH-labeled human platelets are considerably influenced by experimental conditions like the platelet concentration, which do not affect membrane fluidity . Activation of platelets with thrombin increases, but activation with ionomycin decreases anisotropy values with both labels . Such anisotropy changes are not detected in platelet membranes or platelet lipids, when isolated after activation of the intact platelets . We present evidence that the anisotropy changes of intact platelets are not a consequence of modified lipid composition (e.g., as would be induced by phospholipase A2 activity) but are, at least partially, caused by changed optical properties of the cell suspension . Measurement of membrane fluidity of platelets by fluorescence polarization is severely hindered by a high turbidity of the platelet suspension and also by changes in the turbidity and platelet morphology during the activation process.

Cancer Res, 1990 Jun 15, 50(12), 3681 - 90
Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity; Heo DS et al.; A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors . With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01) . Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release . In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay . Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients . This colorimetric assay is especially well suited to adherent tumor cell targets . The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets . Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

FEBS Lett, 1990 Jun 4, 265(1-2), 104 - 6
Biologically active ether lipids . Biotransformation of rac-1(3)-O-alkylglycerols in cell suspension cultures of rape and semisynthesis of 1-O-alkyl-2-palmitoyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolamines, potent antitumor agents; Apte SS et al.; Biotransformation of rac-1(3)-O-hexadecylglycerol by photomixotrophic rape (Brassica napus) cells in suspension culture leads to 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines and small proportions of other ether lipids, e.g . 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamines . Reaction of the hexadecylacyl-glycerophosphocholines with ethanolamine in the presence of phospholipase D from Streptomyces chromofuscus yields additional hexadecylacylglycerophosphoethanolamines . Partial hydrolysis of the combined hexadecylacylglycerophosphoethanolamines followed by reacylation of the resulting lyso compound with palmitic anhydride gives 1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phospho-(N-palmitoyl) ethanolamine, a nontoxic ether glycerophospholipid with antitumor activity . The corresponding 1-O-tetradecyl,1-O-octadecyl, and 1-O-{(Z)-9'-octadecnyl} derivatives are prepared similarly.

Gematol Transfuziol, 1990 Jun, 35(6), 9 - 12
{Isolation of hematopoietic stem cells from peripheral blood of patients with hemoblastoses}; Aleksanian MZh et al.; To isolate peripheral blood stem cells, a fraction of the mononuclear blood cell suspension, 51 leukapheresis procedures were conducted in 8 patients with hemoblastosis using the continuous-flow blood cell separator Fenwal CS-3000 . The blood volume processed in a procedure was 10 l at a flow rate of 40-60 ml/min, the centrifuge speed was 1600 rpm . The standard computer program of the Separator was modified depending on the whole blood rate and the patient's hematocrit . The mean yield of mononuclear cells per run was 6.3 X 10(9) . Leukapheresis has not significantly affected the patient's blood cell concentration . The mean content of granulocytes in the leucocyte suspension comprised 1.89% . No complications or side effects were observed in the patients during leukapheresis procedures.

J Endocrinol, 1990 Jun, 125(3), 397 - 402
Luteal steroidogenesis and regression in the rat: progesterone secretion and lipid peroxidation induced in luteal cells by human chorionic gonadotrophin, phospholipase A2 and prostaglandin F2 alpha; Greenhalgh EA; Mid-luteal phase (control) and mid-luteal phase luteal cell suspensions from rats given prostaglandin F2 alpha (PGF2 alpha) to induce luteal regression and killed 24 h later (P24-regressed) were incubated at 37 degrees C in serum-free medium . Progesterone secretion and lipid peroxidation induced by treatment with human chorionic gonadotrophin (hCG) alone or hCG plus phospholipase A2 and PGF2 alpha were measured . Differences were demonstrated in progesterone secretion and lipid peroxide measurements between control and P24-regressed cell suspensions . The experiments suggested that malondialdehyde formation may be a useful indicator of luteal regression and cell death.

J Periodontol, 1990 Jun, 61(6), 328 - 33
Human gingival Langerhans cells stimulate allogeneic lymphocytes: requirement for MHC class II antigens; Walsh LJ et al.; Langerhans cells (LC) are antigen-presenting cells which express high levels of Class II MHC antigens on their plasma membranes . While the expression of these antigens on gingival LC has been documented, their functional significance is unclear . In this study, the mixed epithelial cell-lymphocyte culture reaction (MECLR) between stimulator cells (LC) and allogenic lymphocytes was used as an in vitro model for investigating the role of the MHC Class II antigens HLA-DR, -DQ, and -DP in alloantigen presentation by gingival LC . In epithelial cell suspensions prepared from human gingiva, MHC Class II antigen expression (HLA-DR, -DP, -DQ) was confined to CD1a-positive LC . Depletion of Class II antigen-bearing LC from epithelial cells using monoclonal antibodies (L243, B7/21, and SK10) and complement inhibited the ability of epithelial cells to stimulate proliferation in the MECLR . Pre-treatment of epithelial cell suspensions with the same monoclonal antibodies suppressed proliferation in the MECLR, as did direct addition of these antibodies to co-cultures of epithelial cells and lymphocytes . These results indicate that HLA-DQ and -DP, together with DR antigens on gingival LC, are involved in LC-lymphocyte interactions . Since LC are potent antigen presenting cells, alterations in the expression of MHC Class II antigens on the surface of these cells will influence their ability to stimulate lymphocytes during the initiation of the cellular immune response to the accumulation of dental plaque.

J Bacteriol, 1990 Jun, 172(6), 3221 - 8
Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei; Southam G et al.; In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella . However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell . Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm . Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing . Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol) . The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000 . However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1 . This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins . On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated . This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels . Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.

Zhonghua Nei Ke Za Zhi, 1990 Jun, 29(6), 347 - 9, 382
{Clinical and experimental study of treating aplastic anemia with fetal liver cell suspension and fetal liver cell-free suspension}; Han JR et al.; Fresh fetal liver obtained from 3- to 6-month fetus was prepared . Fetal liver cell suspension (FLC) or fetal liver cell-free suspension (FLCF) were then transfused into two groups of patient of aplastic anemia . 15 of 21 patients of aplastic anemia treated with FLC showed reconstitution of haemopoietic function or improvement of peripheral blood pictures, while 27 of 30 patients treated with FLCF showed reconstitution or improvement . It is verified that there is a stimulating factor for CFU-CM, BFU-E, and CFU-E and also a immunologic stimulant for improving the nonspecific immunologic function of the organism as shown by clinical analysis and experimental study . It is obvious that the therapeutic effect of FLCF is much better than that of the FLC.

Immunol Cell Biol, 1990 Jun, 68 ( Pt 3), 147 - 53
Biochemical manifestations of a rat mammary adenocarcinoma-producing cachexia: in vivo and in vitro studies; Coyle P et al.; The physical and metabolic characteristics of a Dark Agouti rat mammary adenocarcinoma and its effects on host metabolism are described . The tumour was characterized by a lack of glandular differentiation, tetraploidy, a rapid mitotic index and a high rate of glycolysis . The adenocarcinoma was readily maintained in tissue culture and could be passaged through the host by inoculating either cell suspensions or tissue explants . In the rat, tumour growth resulted in a loss of adipose tissue at a tumour mass of less than 5% body weight indicating that increased energy expenditure was already present at that stage . In addition the tumour caused anaemia, hypercalcaemia and hypoglycaemia . Hyperketonaemia was also observed in fasted tumour-bearing rats . Methotrexate arrested tumour growth in vivo . These aspects of the tumour model make it useful for investigations into host-tumour competition and mechanisms of cachexia.

J Cell Physiol, 1990 Jun, 143(3), 494 - 500
Insulin-like growth factor-1 (IGF-1), insulin, and epidermal growth factor (EGF) are survival factors for density-inhibited, quiescent Balb/c-3T3 murine fibroblasts; Tamm I et al.; The great majority of murine Balb/c-3T3 fibroblasts in density-inhibited, quiescent cultures disintegrate and die rapidly when cells are deprived of serum in the medium . Platelet-derived growth factor (PDGF, 5 ng/ml) used alone and insulin-like growth factor (IGF-1, 40 ng/ml) + epidermal growth factor (EGF, 10 ng/ml) prevent most of this cell death and all three factors used together protect close to all cells in the confluent monolayer as determined by counting trypsinized cell suspensions in a Coulter counter . IGF-1 used alone affords a high level of protection during the first 5 hours of incubation in serum-free medium but the protective effect declines subsequently unless EGF is also present . EGF alone has little protective activity . The survival-promoting activity of PDGF used alone or of PDGF + EGF + IGF-1 is not significantly decreased by selective inhibition of messenger precursor RNA transcription with 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB, 20 or 40 microM), which prevents G1 traverse of the cells mediated by the combination of the three growth factors . DRB also does not interfere with the early protective effect of IGF-1 + EGF, but decreases the late protective effect of this growth factor combination . DRB by itself decreases cell viability in the absence of growth factors or serum . In these experiments viability was assayed by neutral red uptake by using an automated microplate reader . Inhibition of protein synthesis with cycloheximide (CHX, 1 or 5 micrograms/ml) over a 20-hour period was associated with decreased survival of cells protected by IGF-1 + EGF or PDGF + EGF + IGF, but also with decreased survival of cells incubated in the absence of growth factors or serum . The decrease in survival was somewhat more marked when IGF + EGF was present than when PDGF + EGF + IGF-1 was present . Insulin (1,500 ng/ml) mimics the action of IGF-1 (40 ng/ml) . The cell survival-enhancing activities of growth factors are concentration dependent . The evidence presented indicates that PDGF, EGF, and IGF-1 (or insulin) act through distinctive mechanisms in affording protection of cells against death . The short-term protective effects of the growth factors are independent of gene expression and may be mediated via metabolic events . Long-term protection may be dependent on gene expression, especially in the case of IGF-1 + EGF.

Am Rev Respir Dis, 1990 Jun, 141(6), 1453 - 8
Identification of lipoxin A4 and its relationship to the sulfidopeptide leukotrienes C4, D4, and E4 in the bronchoalveolar lavage fluids obtained from patients with selected pulmonary diseases; Lee TH et al.; Lipoxins are biologically active trihydroxytetraene containing products derived from arachidonic acid that are formed by interactions between lipoxygenases . Although the lipoxins have been generated from mixed cell suspensions in vitro, it has not been established whether these products are synthesized in vivo . We have performed bronchoalveolar lavage (BAL) in 12 patients with lung disease (sarcoid, six; pneumonia, two; asthma, two; carcinoma, one; alveolitis of unknown cause, one) and in six normal control subjects . The BAL fluid was analyzed for lipoxin A4 (LXA4) using gas chromatography mass spectrometry with selective ion monitoring, and the levels of the sulfidopeptide leukotrienes were determined using reverse-phase high-performance liquid chromatography and radioimmunoassay . LXA4 was detected in BAL fluid from nine of the 12 patients studied . The levels of LXA4 ranged from 0.4 to 2.8 ng/ml . LXA4 was not detected in any of the six normal subjects . Sulfidopeptide leukotrienes were detected in all the BAL samples, ranging from 0.04 to 0.7 ng/ml, and there was no significant difference between the patients and the normal subjects . In patients with detectable LXA4 in BAL fluid, the ratio of the concentrations of LXA4 to those of the sulfidopeptide leukotrienes ranged from 1.9 to 62 (mean, 19.0) . This is the first demonstration of the presence of LXA4 in disease.

J Photochem Photobiol B, 1990 Jun, 6(1-2), 39 - 48
Time-gated fluorescence spectroscopy of porphyrin derivatives incorporated into cells; Cubeddu R et al.; Time-gated fluorescence spectroscopy was performed on the tumour-localizing fraction (TLF) of haematoporphyrin derivative (HPD) incorporated into cells . Three different cell lines were incubated with 20 and 5 micrograms ml-1 of TLF for various time periods; they were then washed and resuspended in buffer . Fluorescence decay measurements and time-integrated and time-gated spectra were then obtained from the cell suspensions . Similar experiments were repeated using HPD containing 60% of the active material . The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF . In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a consequence of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to monomers and unfolded polymeric chains . The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is more pronounced for HPD . The results obtained seem to indicate that both the composition of the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the incorporated sensitizer.

J Gen Physiol, 1990 Jun, 95(6), 1061 - 75
Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++; White RL et al.; Gap junctional conductance (gj) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with CO2 when extracellular calcium activity is near physiological levels (1.0 mM CaCl2 added; 470 microM Ca++) . Intracellular calcium concentration (Cai), measured by fura-2 fluorescence in cell suspensions, was 148 +/- 39 nM (+/- SEM, n = 6) and intracellular pH (pHi), measured with intracellular ion-selective microelectrodes, was 7.05 +/- 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air . Cai increased to 515 +/- 12 nM (n = 6) and pHi decreased to 5.9-6.0 in medium equilibrated with 100% CO2 . In air-equilibrated low-calcium medium (no added CaCl2; 2-5 microM Ca++), Cai was 61 +/- 9 nM (n = 13) at pHi 7.1 . Cai increased to only 243 +/- 42 nM (n = 9) at pHi 6.0 in CO2-equilibrated low-calcium medium . Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pHi 5.9-6.0 in low-calcium medium . Cell pairs could still be electrically uncoupled reversibly by the addition of 100 microM octanol, an agent which does not significantly affect Cai . In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO2 increased Cai to 425 +/- 35 nM (n = 11) at pHi 5.9-6.0 and gj was reduced to near zero . Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187 . The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification . In contrast, acidification did not substantially reduce gj when intracellular calcium was low . Increasing intracellular calcium did not appreciably reduce gj at pHi 7.0 . These results suggest that, although other factors may play a role, H+ and Ca++ act synergistically to decrease gj.

Oral Microbiol Immunol, 1990 Jun, 5(3), 137 - 42
Production of volatile sulfur compounds by various Fusobacterium species; Claesson R et al.; In 12 species of Fusobacterium the following characteristics were studied; the desulfhydration of L-cysteine and L-methionine by resting cell suspensions, the formation of alpha-keto-acids from L-cysteine, D-cysteine and L-methionine by cell extracts, and the formation of hydrogen sulfide from L-cysteine, D-cysteine and L-cysteine by cell extracts separated by polyacrylamide gel electrophoresis . Multiple forms of L-cysteine desulfhydrase activity were found in most of the species . In some of them also D-cysteine desulfhydrase activity was demonstrated . Seven of the species had high L-methionine gamma-lyase activity . L-cysteine activity was present in 5 of the species.

Thymus, 1990 Jun, 15(4), 213 - 21
The effect of trypsin on CD1a molecule of human thymocytes; Dezutter-Dambuyant C et al.; The cortical thymocytes expressed at least three distinct cell-surface differentiation antigens . CD1a (Mr 49,000), CD1b (Mr 45,000) and CD1c (Mr 43,000) which are non-covalently attached to beta 2-microglobulin . In the present study, we confirm the presence of two out of the three CD1 molecules on epidermal Langerhans cells by biochemical analysis . Furthermore some CD1a monoclonal antibodies immunoprecipitated an additional molecule with an apparent relative mass of 27,000 from Langerhans cell-enriched epidermal cell lysates and not from fresh iodinated thymocyte lysates . From trypsin-treated thymocyte lysates, this low molecular weight protein was considered as a cleavage product of Mr 49,000 molecule (CD1a molecule) by this enzyme which is used to obtain epidermal cell suspensions . This Mr 27,000 was found to content one N-linked oligosaccharide residue by endoglycosidase F treatment . On CD1-expressing cells (thymocytes and Langerhans cells) it would be tempting to take advantage of the sensitivity of CD1a molecule to trypsin in order to precise the structure/function relationship of CD1a antigen.

Brain Res, 1990 May 7, 515(1-2), 193 - 206
Amelioration of spatial memory impairment by intrahippocampal grafts of mixed septal and raphe tissue in rats with combined cholinergic and serotonergic denervation of the forebrain; Nilsson OG et al.; Previous studies in the rat have shown that a serotonergic depletion greatly potentiates the learning and memory impairments produced by pharmacological or lesion-induced cholinergic blockade in the forebrain . The impairment produced by combined serotonergic-cholinergic lesions is reminiscent of that seen in memory-impaired aged rats . In the present experiment, we investigated whether grafts of cholinergic septal tissue and serotonergic mesencephalic raphe tissue, placed in the hippocampus, could reverse the severe memory impairment produced by combined cholinergic-serotonergic lesions . Adult rats were given an intraventricular injection of 5,7-dihydroxytryptamine followed by a radiofrequency lesion of the septum 1-2 weeks later . Three weeks after lesion surgery, the rats were given bilateral intrahippocampal cell suspension grafts of either fetal septal or mesencephalic raphe tissue, or both . The rats were tested for spatial learning and memory in the Morris water maze task at 4 and 10 months after grafting . At 4 months, lesioned and grafted groups were all impaired compared to the normal controls in their swim time and distance swum to find the platform, and they did not show any spatially focussed search strategy in the spatial probe trial when the platform was removed from the tank . At 10 months, the rats with mixed cholinergic and serotonergic grafts were no longer impaired compared to normals in their swim time and distance to find the platform, and they were significantly improved compared to the other grafted groups . Moreover, in the spatial probe trial, the rats with mixed cholinergic and serotonergic grafts displayed a spatially focussed search behaviour over the previous platform site, which was not seen in the lesioned control rats or in the other graft groups . Morphological analysis of the hippocampus revealed that the septal grafts produced an acetylcholinesterase-positive innervation but were totally devoid of serotonin innervation . The raphe grafts produced mainly a serotonin innervation, of both acetylcholinesterase- and serotonin-positive fibres . The results suggest that a mixture of septal and raphe tissue is required when grafted to the hippocampal formation in order to ameliorate the severe spatial learning and memory impairments produced by a combined cholinergic and serotonergic denervation, and that each of these graft types separately are not sufficient to ameliorate such deficits.

Int J Hyperthermia, 1990 May-Jun, 6(3), 571 - 80
Combined effects of hyperthermia (to 45 degrees C) and ultrasound irradiation on the surface ultrastructure of HeLa cells; Shammari MA et al.; Hyperthermic treatment of HeLa cells in suspension combined with ultrasound irradiation produced alterations to the cell surfaces . The changes induced were related to ultrasound intensity in the standing wave and to heat treatments between 37 and 45 degrees C . Two transducers were used, driven at resonant frequencies of 0.75 and 1.5 MHz, and producing peak intensities up to 7 W/cm2 . These intensities produced a negligible rise in temperature of the cell suspension medium . Ultrastructural damage in standing wave fields, as seen by scanning electron microscopy, progressed through stages . The first stage was characterized by the loss of microvilli and smooth appearance of the cell surface, e.g . after insonation at 41.5 degrees C for 10 min; damage increased to a final stage where the surface appeared heavily pitted and porous, with the cells showing signs of disintegration, e.g . after insonation at 45 degrees C for 10 min . The monitoring of ultrasound-induced cavitation suggested that damage was caused by bubble oscillations, not collapse cavitation . Shearing stresses accentuated by hyperthermia were considered the probable cause of such damage . Coulter counter studies of cell size distribution showed that the extent of cell damage depended on the geometry of the vessel in which insonation was carried out.

Int J Hyperthermia, 1990 May-Jun, 6(3), 563 - 70
The effect of cis-diamminedichloroplatinum(II) treatment at elevated temperatures on murine fibrosarcoma, FSa-II; Urano M et al.; The effect of cis-diamminedichloroplatinum(II) (cis-DDP) on the murine fibrosarcoma cells was investigated in vitro and in vivo . For in vitro experiments tumour cell suspensions containing a given amount of cis-DDP were treated in water bath maintained at a desired temperature, and cell survival was determined by the lung colony assay . The D0 or the time to reduce survival from 1.0 to 0.37 on the exponential portion of the survival curve was determined and 1/D0 was plotted as a function of 1/T, where T stands for the absolute temperature . The slope of this Arrhenius plot indicated that the activation energy for chemical reaction of cis-DDP was 44 kcal/M between the temperature range from 37 to 41 degrees C . For in vivo experiments tumours were transplanted into the foot and treated by immersing the animal foot into a water bath when each tumour reached an average diameter of 4 mm (35 mm3) . The drug was injected i.p . immediately before hyperthermia . The tumour growth (TG) time or the time required for a tumour to reach 1000 mm3 from the treatment day was determined, and the median TG time was obtained by logit analysis . Dose-response curves between the TG time and drug dose indicated that the cytotoxic effect of cis-DDP was enhanced at elevated temperatures . This enhancement increased with increasing temperature from room temperature to 43.5 degrees C . Because of short plasma half-time of cis-DDP, continuous infusion and pulse injections were attempted.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Res Dev Brain Res, 1990 May 1, 53(2), 186 - 93
Embryonic and early postnatal hippocampal cells respond to nanomolar concentrations of muscimol; Fiszman ML et al.; Embryonic and early postnatal tissue taken from rat hippocampi were papain digested in order to obtain cell suspensions suitable for analysis in a fluorescence-activated cell sorter (FACS) . Cell suspensions consisted of two major peaks of forward-angle light scatter (FALS) . FACS analysis showed that the population which stained intensely with the vital dye Acridine orange (AO) scattered significant levels of light (high FALS) and amounted to 85% of the total events collected in embryonic cell suspensions and 65% in postnatal (PN) samples . Two minor populations were weakly stained with AO and scattered little light . Oxonol, a voltage-sensitive indicator dye, was used to detect membrane polarization changes . The AO and oxonol staining patterns were very similar . All the events exposed to media containing 50 mM KCl were depolarized (increase in intensity of oxonol fluorescence) . The depolarizing effect of veratridine, a sodium channel activator, was more pronounced in the high FALS subpopulation . In embryonic hippocampal cell suspensions nanomolar concentrations of GABAA agonists depolarized the high FALS subpopulation in a dose-dependent manner . This effect was prevented by preincubation with bicuculline or picrotoxin . In hippocampal cell suspensions obtained from 5-7-day-old rat pups (PN5-7), GABAA agonists depolarized one cell subpopulation and hyperpolarized another . Our results indicate that physiological responses can be resolved in subpopulations of hippocampal cell suspensions by FACS analysis . This technique seems to be a sensitive assay to measure physiological responses (changes in membrane potential) as a parameter of receptor expression . GABAA agonists induced pure depolarizing responses in embryonic and early postnatal hippocampus when active neurogenesis is taking place . The response become hyperpolarizing-depolarizing ones after inhibitory synapses appear.

Int J Radiat Oncol Biol Phys, 1990 May, 18(5), 1061 - 7
Evaluation of cell subpopulations isolated from human tumor xenografts by centrifugal elutriation; Keng PC et al.; Human epidermoid tumor cells (Coll2, ME180, A431, HEp3) grown as xenografts in nude mice, were dissociated into single cell suspensions using an enzyme cocktail containing 0.025% collagenase, 0.05% pronase, and 0.04% DNase . The dissociated cell suspensions were separated by centrifugal elutriation into fractions containing homogeneous cell subpopulations primarily based on the differences in the rates of sedimentation . The quality of separation was evaluated by several techniques including flow cytometry, cell volume distributions, in vitro colony forming assay and morphological examination of Wright-Giemsa stained cells . In each separated fractions, the host to neoplastic cell ratio, the DNA ploidy, the plating efficiency and the cell cycle distribution were determined . After an initial separation of non-neoplastic host cells from malignant cells, a purity of greater than 95% host cells was obtained from the four xenografts studied . DNA analysis of tumor suspensions showed that neoplastic cells of different xenografts contained aneuploid cells with a DNA index of 1.51 to 1.95 . The neoplastic cells were further separated into fractions according to their positions in the cell cycle . Fractions containing greater than 95% G1, 65% S, and 72% G2M cells were obtained from HEp3 xenografts . Less efficient separation with respect to cell cycle was attained with cells derived from Coll2, ME180, and A431 xenografts . Colony forming abilities of the neoplastic cells were determined at different phases of the cell cycle and found to be similar to those of the unseparated cell suspensions after corrections for non-neoplastic host cells were made . These investigations indicate that centrifugal elutriation is an effective technique of obtaining homogeneous subpopulations of cells from human tumor xenografts for various tumor biology and cell kinetics studies.

Hum Pathol, 1990 May, 21(5), 551 - 8
Flow-cytometric DNA analysis of hematopoietic and lymphoid proliferations: a comparison of fresh, formalin-fixed and B5-fixed tissues; Pelstring RJ et al.; Flow-cytometric DNA analysis of human tumors using paraffin-embedded tissue samples is becoming an increasingly popular method of determining ploidy and proliferative rate . Particularly with hematopoietic/lymphoid proliferations, little is known about how these data compare with data from fresh suspension studies, or how B5-fixed tissues compare with those fixed in formalin . For these reasons, flow-cytometric DNA ploidy and cell cycle analysis was performed on 16 hyperplastic tonsils and 28 lymphoid/hematopoietic neoplasms using fresh (or fresh-frozen) cell suspensions (FR), B5-fixed paraffin-embedded (B5), and formalin-fixed paraffin-embedded (FO) tissue . Ploidy analysis showed all tonsil specimens to be diploid regardless of preparative method; however, for the neoplastic cases the FO and B5 preparations agreed with the FR preparations in 50% and 61% of the cases, respectively . Complete agreement between all three tissue preparation methods was present in only 36% of the neoplastic cases . Percent S or S + G2M phase fractions showed relatively poor correlation between the three preparative methods, with the best correlations found between the FR and B5 samples (S phase, r = 0.44; S + G2M, r = 0.43) . In conclusion, while potentially useful data can be obtained from flow-cytometric DNA analysis of fixed, paraffin-embedded lymphoid/hematopoietic tissues, the specific limitations of such analyses must be recognized . Ploidy and proliferative phase data from fixed tissue preparations are not equivalent to information obtained from fresh suspension studies . B5-fixed tissue provides an acceptable alternative to formalin-fixed tissue for such analyses.

J Leukoc Biol, 1990 May, 47(5), 449 - 56
Inhibition of neutrophil and eosinophil chemotactic responses to PAF by the PAF-antagonists WEB-2086, L-652,731, and SRI-63441; Hakansson L et al.; The influence of the three PAF-antagonists WEB-2086, L-652,731, and SRI-63441 on the chemotactic response of neutrophil and eosinophil granulocytes to PAF was investigated . When the PAF-antagonists were added to the cell suspension that was exposed to a gradient of PAF, WEB-2086 and SRI-63441 at the concentration of 10(-6) mol/litre inhibited (P less than .01) the neutrophil and eosinophil chemotactic response to 10(-8) and 10(-9) mol PAF per litre; at the concentration of 5 x 10(-6) mol/litre, WEB-2086 and SRI-63441 also inhibited (P less than .02) the response to 10(-7) mol PAF per litre . Under the same conditions L-652,731 at the concentration of 5 x 10(-6) mol/litre inhibited (P less than .01) the eosinophil chemotactic response to 10(-8) and 10(-9) mol PAF per litre . The inhibition of the chemotactic response to PAF by the three PAF-antagonists was specific, since the chemotactic response to C5f, f-MLP, and LTB4 was not affected by WEB-2086, L-652,731, or SRI-63441, neither was the chemokinetic migration induced by albumin.

Am J Clin Pathol, 1990 May, 93(5), 615 - 20
Deoxyribonucleic acid ploidy and cell cycle analysis of colorectal carcinoma by flow cytometry . A prospective study of 137 cases using fresh whole cell suspensions; Hood DL et al.; Several retrospective studies suggest that abnormal deoxyribonucleic acid (DNA) content in colorectal carcinoma correlates with adverse clinical outcome . Many of these studies have used naked nuclei retrieved from formalin-fixed, paraffin-embedded tissues for flow cytometry . The purpose of this study was to prospectively analyze 137 colorectal carcinomas using fresh whole-cell suspensions for flow cytometry and to determine whether abnormal DNA content (DNA aneuploidy or tumors with high proliferative activity) correlates with Dukes' stage, histologic grade, lymphocytic infiltration of the tumor, tumor fibrosis, extramural venous spread, or tumor size . Cell suspensions for flow cytometry were prepared by enzyme disaggregation with collagenase XI, DNase, and trypsin . Satisfactory DNA histograms were obtained from 132 of the 137 samples . The mean coefficients of variance for the G1/G0 of the external 2C control, internal 2C populations, and aneuploid populations were 2.5, 3.5, and 3.5, respectively . The mean percentage of viable cells was 97% . Of 132 cases, 102 (77%) demonstrated abnormal DNA histograms, of which 77 (58%) showed DNA aneuploidy . Abnormal DNA histograms of DNA aneuploidy did not correlate with Dukes' stage . Tumors of higher histologic grade were more likely to demonstrate DNA aneuploidy, however, these differences did not reach statistical significance . The authors conclude that (1) satisfactory DNA histograms can be obtained with the use of a fresh, whole-cell technique; (2) abnormal DNA histograms did not statistically correlate with standard clinical, grading, or staging parameters; and (3) carcinomas of high histologic grade showed an increased proportion of aneuploid DNA histograms, but this trend did not reach statistical significance.

Exp Hematol, 1990 May, 18(4), 304 - 10
Early B-lymphocyte precursor cells in mouse bone marrow: subosteal localization of B220+ cells during postirradiation regeneration; Jacobsen K et al.; The localization of early B-lymphocyte precursor cells in the bone marrow of young mice has been studied during recovery from sublethal whole body gamma-irradiation (150 rad) . Initial studies by double immunofluorescence labeling of the B-lineage-associated cell surface glycoprotein, B220, and of mu heavy chains in bone marrow cell suspensions, demonstrated a sequential wave of regeneration of early B precursor cells, pre-B cells, and B cells . Early B precursor cells expressing B220 but not mu chains were enriched at 1-3 days following irradiation . After in vivo administration of 125I-labeled monoclonal antibody 14.8 to detect B220+ cells in situ, light and electron microscope radioautography of femoral bone marrow sections revealed concentrations of labeled B220+ cells located peripherally near the cortical bone at 1-3 days following irradiation, increasing in numbers in more central areas by 5-7 days . Proliferative B220+ precursor cells were found within layers of bone-lining cells and in a subosteal area characterized by a prominent electron-dense extracellular matrix, often associated with stromal reticular cells . The results demonstrate that the precursor cells that are active in the bone marrow early in the recovery of B lymphopoiesis after gamma-irradiation are located both within and near the endosteum of the surrounding bone . The distinctive extracellular matrix and stromal cell associations noted in this region may contribute to a supportive local microenvironment for early hemopoietic progenitor cells.

J Nat Prod, 1990 May-Jun, 53(3), 579 - 86
New hydroxylated benzo{c}phenanthridine alkaloids from Eschscholtzia californica cell suspension cultures; Tanahashi T et al.; From cell cultures of Eschscholtzia californica and their spent medium, three new benzo{c}phenanthridine alkaloids--namely 10-hydroxysanguinarine {2a}, 12-hydroxychelirubine {4a}, and 10-hydroxychelerythrine {7a}--and two new dihydrobenzo{c}phenanthridine alkaloids--10-hydroxydihydrosanguinarine {2b} and 12-hydroxydihydrochelirubine {4b}--together with the known constituents sanguinarine {1a}, chelirubine {3a}, macarpine {5a}, dihydrosanguinarine {1b}, dihydrochelirubine {3b}, and dihydromacarpine {5b}, were isolated and characterized . Structure elucidations were done by 1H nmr, decoupling experiments, and NOESY spectra . Isolated microsomes from E . californica, the site of hydroxylation activity within the cells, contained the whole set 1b to 8b of 5,6-dihydrobenzo{c}phenanthridines . A scheme for the biosynthesis of macarpine {5a} from protopine {9} via dihydrosanguinarine {1b} is presented.

Development, 1990 May, 109(1), 37 - 40
A study of meiosis in chimeric mouse fetal gonads; Dolci S et al.; The influence of somatic environment on the onset and progression of meiosis in fetal germ cells was studied in chimeric gonads produced in vitro by dissociation-reaggregation experiments . Germ cells isolated from testes or ovaries of 11.5-13.5 days post coitum (dpc) CD-1 mouse embryos were loaded with the fluorescent supravital dye 5-6 carboxyfluorescein diacetate succinimyl ester (CFSE) and mixed with a cell suspension obtained by trypsin-EDTA treatment of gonads of various ages and of the same or opposite sex . Whereas 11.5 dpc donor germ cells appeared unable to survive in the chimeric gonads obtained, about 76% of the CFSE-labeled female germ cells obtained from 12.5 dpc donor embryos (premeiotic germ cells) found viable within host ovarian tissues showed a meiotic nucleus . In contrast, a smaller number (about 19%) were in meiosis in chimeric testes . None or very few of donor male germ cells entered meiosis in testes or ovarian host tissues . Aggregation of meiotic 13.5 dpc female germ cells with testis tissues from 13.5 to 14.5 dpc embryos resulted in inhibition of meiotic progression and pyknosis in most donor germ cells . These results support the existence of a meiosis-preventing substance or a factor causing oocyte degeneration in the fetal mouse testis, but not of a meiosis-inducing substance in the fetal ovary.

Hear Res, 1990 May, 45(3), 237 - 46
Fetal tectum grafted as a cell suspension into the adult rat inferior colliculus; Zrull MC et al.; The known structural and functional features of the inferior colliculus provided an advantageous substrate for examining the outcome of neural transplantation . In the present study, tissue from the midbrain tectum of Sprague-Dawley rat fetuses was removed at 15, 16 and 19 days of gestation (E15, E16 and E19), incubated as a dissociated cell suspension for 12 to 15 h in a medium containing True Blue for prelabeling, and injected into the central nucleus of the inferior colliculus (CNIC) of 28 normal male adult conspecifics . Viable tectal grafts were found in 57.5% of host animals with 65.2% located in CNIC . Grafts of E16 tissue had a survival rate of 84.2%, which was twice that of E15 or E19 donors, and there was no difference in graft survival rate in hosts sacrificed up to 6 months after the implantation procedure . Neurons prelabeled with True Blue and counterstained with thionin were identified as uniquely green stained cells in brightfield illumination and brilliant opaque cells in darkfield microscopy . Grafts were organized in tightly packed clusters of cells including large rounded polygonal and smaller cells similar in size to normal CNIC large and small multipolar neurons . Cell morphology of the larger ovoid and fusiform grafted neurons most closely resembled that of typical adult CNIC cell populations; other implanted cell types appeared immature even when host post-implant survival was extended . At longer post-implant intervals graft and host cells further intermingled, and both cellular and fibrous bridges were observed at the interface between host and graft tissue . The present work demonstrates the viability and development of homotypic cells grafted into a central auditory structure utilizing a simple method of prelabeling donor tissue with a fluorescent dye.

Int J Microcirc Clin Exp, 1990 May, 9(2), 141 - 61
Is the laser Doppler flow signal a measure of microcirculatory cell flux?
Driessen G, Rutten W, Inhoffen W, Scheidt H, Heidtmann H.
To assess oxygen transport as a function of hematocrit, microcirculatory red blood cell flux (microflux) was measured by laser Doppler flowmetry (LDF) in the isolated rat mesentery as well as in cat sartorius muscle . The hematocrit value was varied from 0.5 to 50% while the perfusion pressure ranged from about 2.7 to 13 kPa . Simultaneously macroflux which is the volume flow times the systemic hematocrit value was determined . The curves LDF versus pressure showed saturation at several hematocrit values conveying the impression of autoregulation of microflux . The macroflux curves, on the other hand, curved upward . To clarify whether this discrepancy had a physiological cause, pressure-microflux as well as pressure-macroflux curves were collected in a flow chamber model where regulatory effects are absent . In the model the same discrepancies between micro- and macroflux as in vivo were observed . These discrepancies are explained by maximum values in velocity of about 3 mm/s and in hematocrit value of less than or equal to 20% which the LDF can discern . The limit in velocity is explained by the low pass filter of 12 kHz of the LDF instrument, the limit in hematocrit measurement is probably caused by the light scattering properties of red blood cell suspensions . A complicated combination of effects appears to be responsible for the linearity of the LDF signal versus macroflux at normal hematocrit value.

In Vitro Cell Dev Biol, 1990 May, 26(5), 493 - 501
Density separation of rat adrenocortical cells: morphology, steroidogenesis, and P-450scc expression in primary culture; Roskelley CD et al.; We have developed a method that separates rat adrenocortical cells by density into populations which retain zone specific properties in primary culture . Two different parenchymal populations were obtained and designated 2FASC (1.034 g/ml, 18.0 microns cell diameter) and 7GLOM (1.069 g/ml, 11.7 microns cell diameter) . In freshly isolated cell suspensions the physical characteristics and differential steroidogenic responses to adrenocorticotropin and angiotensin II suggested that 2FASC cells originated predominantly from the zona fasciculata and 7GLOM cells from the zona glomerulosa . In primary culture (Dulbecco's Modified Eagle's Medium-F12 medium with 15% horse serum and 2.5% fetal bovine serum) the two populations exhibited different morphologies . 2FASC cells retained lipid and formed cohesive epithelial monolayers that remained stationary for 3 wk . 7GLOM cells were initially epithelial but rapidly lost lipid, spread, and assumed fibroblastic shapes . Both cell types were positive for the cholesterol side-chain cleavage cytochrome P-450 by immunofluorescence . Therefore, the morphologic changes seen in 7GLOM cultures were due to modulation, not fibroblastic overgrowth . This phenotypic plasticity may reflect the mesodermal origin of the adrenal cortex, and the subcapsular location of 7GLOM cells in vivo . In contrast, cells such as 2FASC which are located deeper in the cortex seem to have a more restricted, fully committed parenchymal phenotype.

Ultrasonics, 1990 May, 28(3), 155 - 8
Positive and negative effects of diagnostic intensities of ultrasound on erythrocyte blood group markers; Rosenfeld E et al.; Human erythrocytes were exposed in vitro to diagnostic intensities of ultrasound . Various antigenic tests were conducted to see if the different blood group markers had been changed . No change was observed following sonication for Ss, M, K1, ABO, and the rhesus factors . However a marked reduction was found for the N antigen when assayed using the lectin isolated from Vicia graminea . Several experiments were performed to ensure that this effect was not an artefact . If the cells were not resuspended in fresh medium immediately following sonicating then the magnitude of the effect rapidly decreased . We were unable to detect free N antigen in the supernatants from the sonicated cell suspensions . We were also unable to demonstrate changes in the level of the N antigen following sonication if we used anti-N sera from rabbits . A surprising observation was that different batches of the lectin preparation from the same manufacturer could eliminate the effect.

Plant Mol Biol, 1990 May, 14(5), 669 - 85
Yeast RAS2 affects cell viability, mitotic division and transient gene expression in Nicotiana species; Hilson P et al.; Overexpression of the budding yeast RAS2 gene in Nicotiana plumbaginifolia cells revealed that RAS2 acted as 'suicide' gene in freshly isolated protoplasts from leaves and blocked cell proliferation in cell suspension-derived protoplasts . Among a series of genes tested (such as npt II, CDC35, PDE2), RAS2 was the only one to block the expression of the cat gene, as measured in a transient gene expression assay . Another ras gene, v-Ha-ras, had similar effects . Furthermore, the RAS2 effect was species-specific and depended on the modulation of hormonal metabolism in the transfected cells, while no differences were noticed between the normal and the activated val19 gene . Transfected plant cells are shown to synthesize a RAS2 protein of the same electrophoretic mobility as the yeast RAS2 product . The results are discussed in the broader context of the evolutionarily conserved ras genes involved in vital cellular functions.

In Vivo, 1990 May-Jun, 4(3), 201 - 4
Isolation of viable intestinal epithelial cells and their use for in vitro toxicity studies; Kralovanszky J et al.; The application of the collagenase portal vein perfusion technique for the isolation of intestinal cells resulted in the preparation of highly viable enterocytes . Cell viability was found to be greater than 90% as tested by LDH release and Trypan blue exclusion techniques . According to the results of marker enzyme determinations, collected cells were mostly of matured villus type, characterized by high disaccharidase and very low thymidine kinase activity . In vitro treatment of the isolated cells with the anticancer agent cis-diamminedichloroplatinum (II) caused decrease of the metabolic processes, i.e . glucose oxidation and protein synthesis, demonstrating that beyond the production of DNA-crosslinks other mechanisms may play a role in the cytotoxic effect of the drug . It should be stressed, however, that prolonged incubation of the cell suspension over 30 min at physiological temperature may itself lead to gradual decrease of the viability and to disturbance of the metabolic activity of the cells.

Cell Growth Differ, 1990 May, 1(5), 225 - 31
Association of hst gene expression with metastatic phenotype in mouse mammary tumors; Murakami A et al.; From the pregnancy-dependent mouse mammary tumor TPDMT-4, four autonomous sublines were established after its independent progression under different conditions . Despite their similar growth rates in inguinal fat pads, three sublines formed lung metastases, and one did not when they were injected i.v . into mice as a single cell suspension . The TPDMT-4 tumor and the nonmetastatic subline expressed mRNA for the orf gene of mouse mammary tumor virus, whereas all metastatic sublines did not . This suggested that the loss of its expression may have been a prerequisite for the progression toward metastatic ability . To identify the gene(s) participating in the generation and the progression of TPDMT-4, the expression of 23 different oncogenes was analyzed . The expression of int-2 was detected in TPDMT-4 and in all sublines, indicating that TPDMT-4 was generated by activation of this gene, whereas hst expression occurred only in the metastatic sublines . These results demonstrated that the hst gene may contribute to tumor progression from a nonmetastatic to a metastatic phenotype in the mouse mammary tumor system.

Gematol Transfuziol, 1990 May, 35(5), 23 - 5
{Production of transfusion media on the base of erythrocytes and plasma substitute solutions}; Selivanov EA et al.; A review of investigations has been made conducted in the USSR and abroad on the production of combined polyfunctional hemotransfusion media--red blood cell suspensions in plasma-substituting solutions possessing preservative properties and hemodynamic action . The advantages of the hemotransfusion therapy with the use of these media have been shown associated with the diminution of the risk of posttransfusion complications and reactions (especially under extreme conditions) and depending on economic aspects . The properties of red blood cell suspension in the modified deionization gelatin solution have been considered in detail.

J Biol Chem, 1990 Apr 15, 265(11), 6360 - 8
Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures; Dietrich A et al.; Treatment of suspension-cultured parsley (Petroselinum crispum) cells with fungal elicitor triggers rapid, transient and sequential phosphorylation of a number of proteins, as shown by electrophoretic analysis on two-dimensional gels . This response is rapidly reversed by removal of the elicitor from the medium and appears to be specific . It is not observed in cells exposed to other environmental stress factors, such as heat shock, UV irradiation or treatment with mercuric chloride . Pronase digestion of the elicitor has the same negative effect on protein phosphorylation as its previously demonstrated effect on the activation of some pathogen defense-related genes, suggesting a link between these two phenomena . Some of the changes in protein phosphorylation are among the earliest known events following elicitation . The phosphorylation of a neutral 45-kDa protein, which is found in both the microsomal and cytoplasmic fractions, can be observed as early as 1 min after the onset of elicitor treatment . The phosphorylation of a 26-kDa nuclear protein also starts increasing very early . The changes in protein phosphorylation in response to the elicitor are dependent on the presence of Ca2+ in the medium . Our data are compatible with the hypothesis that protein phosphorylation is involved in the signal transduction processes following elicitor recognition by parsley cells.

Environ Health Perspect, 1990 Apr, 85, 107 - 12
DNA synthesis in alveolar macrophages and other changes in lavaged cells following exposure of CBA/H mice to cigarette smoke; Hornby SB et al.; Traditional methods to determine the proportion of cells in S-phase use radiolabeled precursors of DNA, such as 3H-thymidine, which become incorporated into DNA during its synthesis and are visualized either in tissue sections or in cell preparations by autoradiography . At the Harwell Laboratory the effects of inhaled alpha-emitting actinides on the pulmonary alveolar macrophage population of the rodent lung are being studied . For this research the use of an autoradiographic technique to determine the proportion of cells in S-phase is inappropriate, because of the possible presence of competing sources of radioactivity in the cells under investigation . Consequently, an alternative method has been developed . In this method, 5-bromodeoxyuridine (BrdU), an analogue of thymidine, is incorporated into cells undergoing DNA synthesis . Fluorescein-conjugated monoclonal antibodies, highly specific for BrdU substituted DNA, are available commercially and may be used as a probe for BrdU-labeled cells . This technique for identifying cells in S-phase has been described previously for the flow cytometric analysis of cell suspensions and for cells in tissue sections . An adaptation of this technique for use on cytocentrifuge preparations of cells recovered from mouse lung by bronchoalveolar lavage has been developed and its use is described . Some preliminary results of a short-term experiment with CBA/H mice to determine the effects of exposure to cigarette smoke on the DNA synthesis of alveolar macrophages are also included.

Nippon Kyobu Geka Gakkai Zasshi, 1990 Apr, 38(4), 625 - 9
{Hemolysis and red cell deformability during cardiopulmonary bypass--the effect of prostaglandin E1 for prevention of hemolysis}; Yamaguchi H et al.; Prevention of hemolysis which is related to renal failure during cardiopulmonary bypass (CPB) is very important . We discussed the relationship between hemolysis and red cell deformability during CPB, and moreover the effect of PGE1 to reduce hemolysis . PGE1 was given during CPB (10 approximately 20 ng/kg/min) . Red cell deformability was measured using 20% Ht red cell suspension and Nucleopore Microfilter (pore size: 5 mu) . Red cell filtration rate (RFR: microliter/sec) was calculated as a value of red cell deformability . Red cell deformability (RFR: microliter/sec) was reduced during CPB in almost all patients . Secondly, plasma Hb (mg/dl) was measured in the controls (n = 8) and PGE1 group (n = 12) . The mean pre-bypass level were 20.6 in the control group and 23.8 in the PGE1 group . The mean values of plasma Hb at 30, 60, 90 and 120 min of CPB were 37.8, 52.4, 52.3 and 61.2 in the control group and 24.1, 25.0, 26.3 and 29.0 in the PGE1 group . Our study showed conclusively that CPB has a detrimental effect on red cell deformability and that this effect is accentuated by prolongation of CPB time . PGE1 lessened the decrease in red cell deformability during CPB and was shown to be very effective for prevention of hemolysis.

Cell Struct Funct, 1990 Apr, 15(2), 79 - 84
Effects of gangliosides on cell maturation of murine megakaryocytes in a liquid culture system; Watanabe Y et al.; Formation of platelet-producing megakaryocytes, the cytoplasm of which showed the terminal stage of cell maturation, heavy granulation and platelet-fields delineated with demarcation membranes, was observed in a short-term culture system, using megakaryocyte-enriched bone marrow cell suspension . Approximately 6-8% of the megakaryocytes changed to the platelet-producing megakaryocytes during 12-hour incubation . In the presence of inhibitors of energy metabolism, formation of the platelet-producing megakaryocytes was inhibited, suggesting that the process is dependent on energy producing systems . Ganglioside GD1a increased both the number of total megakaryocytes and the ratio of the platelet-producing megakaryocytes to total megakaryocytes, while GM1 did not influence the number of total megakaryocytes, but increased the ratio . Gangliosides GM2, GM3 and GD1b showed little effect on either the number of total megakaryocytes or the ratio . The results suggest that ganglioside GD1a stimulates at least two steps of megakaryocyte maturation, the change of megakaryocytic progenitors to megakaryocytes and the subsequent maturation of megakaryocytes to the platelet-producing megakaryocytes, while GM1 stimulates only the latter step of the maturation.

J Pharmacokinet Biopharm, 1990 Apr, 18(2), 121 - 35
Kinetic assessment of apparent facilitation by albumin of cellular uptake of unbound ligands; Morgan DJ et al.; Previous studies of the effect of albumin on initial uptake of ligands by isolated cell suspensions or cultures found that the apparent uptake for unbound ligand appeared larger in the presence of binding to the albumin than when albumin was absent . Furthermore, when ligand and albumin were increased in a fixed molar ratio, uptake appeared to be competitively inhibited by the excess albumin . We examined the kinetics underlying this apparent facilitation phenomenon by incorporating unbound fraction of ligand in the medium (fu1) into the general model for diffusion between two compartments . The analysis showed that even in the absence of facilitation by albumin, the apparent rate constant for uptake of unbound ligand (k/fu1) increases as albumin concentration increases but the uptake clearance of unbound ligand remains constant . This theoretical analysis was verified experimentally by measuring the effect of albumin on uptake rates of 14C-taurocholate (12, 24, 48, 60, and 96 microM, with and without 0.87 mM albumin) in a nonphysiological system consisting of two solutions separated by a cellulose membrane . Moreover, when the taurocholate and albumin concentrations were increased in a fixed molar ratio of 0.06 (taurocholate 12-96 microM, albumin 0.2-1.6 mM), the initial uptake rate exhibited the same nonlinear pattern as the previous studies that used living cells . This pattern was due not to saturation of a putative albumin receptor but simply to the concomitant decrease in fu1 which tended to offset the increase in uptake rate due to the increasing total taurocholate concentration . The model was also used to evaluate published data describing the effect of albumin on the uptake of iopanoic acid by cultured hepatocytes . In accordance with the model, k1/fu1 increased as albumin concentration increased, but uptake clearance was independent of albumin concentration . Therefore, the kinetic pattern found in this and other studies with isolated cell suspensions or cultures argues against a special role for albumin in facilitating cellular ligand uptake.

Eur J Immunol, 1990 Apr, 20(4), 913 - 7
Nurse cells of the bursa of Fabricius: do they exist?
Wuorela M, Jalkanen S, Pelliniemi LJ, Toivanen P.
Cell-cell interactions in B lymphocyte development have so far been incompletely characterized, mostly due to lack of a special organ for B cell maturation in the mammalian species . Certain well-known lymphostromal interactions in the thymus have raised the question whether similar interactions with nurse cells would also operate in the development of B cells . We have tested this hypothesis in the chicken bursa of Fabricius, an organ specific for the B cell maturation . To identify possible nurse cells, with viable lymphocytes enclosed, the cells in the bursa of Fabricius were dispersed with collagenase and trypsin . Light and electron microscopic examination of bursa cell suspensions showed four types of aggregates, identified by low magnification light microscopy as potential nurse cell-like complexes . Electron microscopy revealed that all aggregates consisted of epithelial cells, and complexes of epithelial cells with lymphocytes enclosed were not observed . These findings indicate that interactions similar to those seen in the avian and mammalian thymus between epithelial nurse cells and T lymphocytes are not a part of the avian B cell differentiation process.

Br J Oral Maxillofac Surg, 1990 Apr, 28(2), 85 - 8
Differences in in vitro growth of epithelium from inflammatory and developmental odontogenic cysts; Hume WJ et al.; Ninety-three odontogenic cysts, 42 of inflammatory and 51 of developmental origin, were grown in vitro from explants and/or cell suspensions . There was little difference in the success rate of culturing epithelium from explants of dentigerous cysts (N = 28) or odontogenic keratocysts (N = 23) (approximately 75% and 87%, respectively) and the dentigerous cyst grew particularly well from suspensions (N = 11) (91%) compared with the keratocyst (N = 19) (58%) . Epithelium from developmental odontogenic cysts grew much better in vitro than did cysts of inflammatory origin (56 to 58% from explants and 19 to 25% from suspension) . From this work there is little evidence to support previous statements that the dentigerous cyst cannot be grown from explants, or that the odontogenic keratocyst has 'aggressive' growth characteristics.

J Neurosci, 1990 Apr, 10(4), 1276 - 85
Neuron-glia interactions of rat hippocampal cells in vitro: glial-guided neuronal migration and neuronal regulation of glial differentiation; Gasser UE et al.; To examine neuron-glia interactions of hippocampal cells, including glial-guided neuronal migration, glial organization of neuronal positioning and neuronal regulation of astroglial differentiation, rat hippocampal tissue, harvested between embryonic day 16 (E16) and postnatal day 3 (P3), was dissociated into a single cell suspension and plated in glass coverslip microcultures (Hatten and Liem, 1981; Hatten et al., 1984) . Immunostaining the cells with antibodies against the glial filament protein (AbGFP) revealed developmental stage-specific changes in the number and extent of morphological differentiation of hippocampal astroglial cells . At E16-E18, fewer than 5% of the cells were AbGFP-positive; stained cells were immature, bearing very short processes . By E19-E20, the number of stained cells increased to 15% of the total cell population . Three forms of differentiated glial cells predominated, a bipolar form bearing processes 30-50 microns, an elongated form which resembled the radial glia of hippocampus, bearing processes 120 microns in length, and a stellate form with 3 or more processes 30-50 microns in length . At P0-P3, glial morphological differentiation varied with the culture substratum; differentiated forms resembling those seen at E20 occurred on Matrigel, but not on polylysine . Quantitation of the distribution of neurons relative to AbGFP-stained glial processes revealed developmental stage-specific changes in glial organization of neuronal positioning in the cultures . In cultures of E16-E18 hippocampal cells, the neurons did not preferentially associate with astroglial cells . By E19-E20, extensive neuron-glia interactions occurred, with 80-90% of the neurons being located within 5-10 microns of a glial process . In addition to their organization of neuronal positioning, E20 hippocampal astroglial cells supported extensive neuronal migration . Migrating hippocampal neurons displayed a cytology and neuron-glia cell apposition identical to that described for migrating cerebellar granule cells in vitro (Edmondson and Hatten, 1987), closely apposing their cell soma against the hippocampal glial process and moving along the glial arm by extending a thickened, leading process . Migration was seen only along highly elongated glial profiles resembling radial glial seen in vivo . The morphological differentiation of hippocampal glial cells in vitro was dependent on cell-cell interactions with neurons . In the absence of neurons, purified hippocampal astroglia had flat, undifferentiated profiles and proliferated rapidly . The addition of hippocampal neurons rapidly arrested glial growth and induced glial process extension.

Biochem J, 1990 Apr 1, 267(1), 141 - 7
Characterization of peptide fluxes into human erythrocytes . A proton-n.m.r . study; Odoom JE et al.; A new protocol for measuring cellular uptake of dipeptides was developed in which the problem of peptide hydrolysis is obviated by introduction into the cell suspension of a membrane-permeant peptidase inhibitor . The uptake of unlabelled dipeptide is readily monitored so long as some analytical technique is available for measuring the intracellular peptide concentration; in this study we used n.m.r . spectroscopy . Using this protocol, we demonstrated that dipeptide uptake by human erythrocytes occurs by simple diffusion through the lipid bilayer and not via a high-capacity protein-mediated transport system . Substantiating evidence includes demonstration that: (a) the fluxes are slow compared with known protein-mediated transport processes in human erythrocytes; (b) the uptake is not stereospecific; (c) the uptake does not display saturation kinetics; (d) the fluxes are significantly enhanced by butanol; (e) a distinct correlation exists between the size-corrected permeability coefficients of the dipeptides and their calculated n-octanol/water partition coefficients . It is calculated that under normal physiological conditions the diffusive fluxes of circulating plasma peptides into human erythrocytes are too small for these cells to play a significant role in dipeptide catabolism.

Am J Clin Pathol, 1990 Apr, 93(4), 569 - 71
Plasmacytoid T-cells in a reactive lymph node . Detection by flow cytometry?
Brubaker R, Swerdlow SH.
Plasmacytoid T-cells (PTCs) are a histologically recognized component of some reactive nodes . To the best of the authors' knowledge, plasmacytoid T-cells have never been recognized as a distinct cell population in a reactive lymph node using cell suspension flow cytometric studies . The authors here report a case of reactive lymph node hyperplasia in which prominent aggregates of PTCs were present and a distinct cell population with an immunophenotype characteristic of PTCs was identified by flow cytometry . Aside from the recognition of PTCs, this observation is important so that cell suspensions showing a distinct population of PTCs with an "aberrant" T-cell phenotype are not misinterpreted as providing evidence for a T-cell neoplasm.

Am J Clin Pathol, 1990 Apr, 93(4), 545 - 8
Immunophenotypic analysis of cells isolated from bone marrow biopsies in patients with failed bone marrow aspiration ('dry tap'); Pihan GA et al.; A method for the immunophenotypic analysis of bone marrow cells in cases of failed bone marrow aspiration is described . Cell suspensions are obtained by mechanical disaggregation of bone marrow core biopsies . The isolated cells are stained with the appropriate antibodies and analyzed by flow cytometry . The usefulness of the method is illustrated by presenting immunophenotypic data obtained in eight consecutive cases accessioned by the authors' laboratory . The method is simple and reproducible . It allows for parallel morphologic examination with Romanovski-type stains and is capable of generating multivariate, quantitative, immunophenotypic data useful in the diagnosis of leukemia and lymphoma.

Circ Res, 1990 Apr, 66(4), 1143 - 55
Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes; Capogrossi MC et al.; We used left ventricular myocytes from adult rats to investigate the effect of 4 beta-phorbol 12-myristate 13-acetate (PMA) and of sn-1,2-dioctanoylglycerol (DiC-8) on the membrane association of protein kinase C (PKC), cytosolic {Ca2+}, (Cai) homeostasis, and the contractile properties of single cardiac cells . Because PKC activity is known to be highly Ca2+ sensitive, the K+ concentration of the bathing medium was raised from 5 to 30 mM in some experiments, a perturbation known to depolarize the cell and increase Cai . In cell suspensions both PMA (3 x 10(-10) and 3 x 10(-7) M) and DiC-8 (10(-5) and 10(-4) M) increased membrane association of PKC . The effect of PMA (10(-7) M) on PKC translocation was enhanced in 30 mM KCl compared with 5 mM KCl . During steady field stimulation at 1 Hz in 1 mM bathing {Ca2+}, both PMA (10(-7) M) and DiC-8 (10(-5) M) decreased twitch amplitude to approximately 60% of control in 5 mM KCl, and the negative inotropic effect of either drug was more pronounced in 30 mM KCl than in 5 mM KCl . In single cardiac myocytes loaded with the Ca2+ indicator indo-1 and bathed in 5 mM KCl, we simultaneously measured cell length and Cai . The myofilament responsiveness to Ca2+ was assessed by the relation between contraction amplitude and the peak of the Cai transient . The negative inotropic effect of both PMA and DiC-8 was related to a diminished amplitude of the Cai transient and not to a decreased myofilament responsiveness to Ca2+ . In the absence of electrical stimulation, PMA (10(-7) M) and DiC-8 (10(-5) M) decreased the frequency of contractile waves due to spontaneous Ca2+ release from the sarcoplasmic reticulum, and DiC-8 also decreased resting Cai . Thus, activation of PKC, which is thought to occur as part of the response of cardiac muscle to alpha 1-adrenergic stimulation, is associated with a negative inotropic action due to a smaller Cai transient rather than to a decrease in the myofilament responsiveness to Ca2+ . These effects on the membrane association of PKC and on contractility are enhanced by cell depolarization achieved by raising {KCl} in the bathing medium.

Anal Cell Pathol, 1990 Apr, 2(3), 139 - 48
The resolution of aneuploid DNA stem lines by flow cytometry: limitations imposed by the coefficient of variation and the percentage of aneuploid nuclei; Cusick EL et al.; Factors important in the resolution of cell sub-populations with differing DNA contents were investigated using an EPICS C flow cytometer . Software is available for the EPICS C which permits data from any two histograms to be superimposed or added together before display . Samples of fresh and archival thyroid tissue, stained with propidium iodide, were analysed on the flow cytometer and the peak channel number noted . The photomultiplier (PMT) voltage was increased and the sample analysed again producing a second histogram with a higher peak channel number . The two histograms were added together to simulate a cell suspension with two sub-populations with a different DNA content . By systematically altering the PMT voltage and the number of nuclei included in each analysis, it was possible to examine the importance of DNA index and the percentage of tumor cells with an aneuploid DNA content for both fresh and paraffin-embedded thyroid nuclei . The crucial importance of achieving a low coefficient of variation (CV) was demonstrated and consequently the reservations that pertain when archival material is studied, particularly in tumours where DNA aneuploidy is frequently expressed with a low DNA index.

J Exp Med, 1990 Apr 1, 171(4), 997 - 1013
Human epidermal T cells predominantly belong to the lineage expressing alpha/beta T cell receptor; Foster CA et al.; The epidermis of clinically normal-appearing human skin harbors a phenotypically heterogeneous population of T lymphocytes (TCs), the majority of which are CD2+/CD3+/CD5+ "memory" cells, but in an unactivated state, and express the TCR-alpha/beta . In contrast to murine skin, only a very minor subpopulation of CD3+ cells in the human epidermis bears the TCR-gamma/delta . Epidermal TCs primarily are distributed along the rete ridges in the basal keratinocyte layer and are often in close apposition to Langerhans cells (LCs) . These TCs were propagated from epidermal cell suspensions after stimulation with TC activating agents (Con A, rIL-1, rIL-2), then evaluated for phenotypic features and TCR diversity . Similar to the in situ situation, most were CD4-/CD8+/TCR-alpha/beta+ . In addition, two cultures contained TCR-gamma/delta+ cells; one of these determined to be an adherent CD4-/CD8+ population . Epidermal TCs were significantly (p less than 0.0001) more abundant in the sole than in the other body regions examined (i.e., 40 vs . 7 CD3+ cells/linear centimeter of epidermis) and seemed to have a particular affinity for the acrosyringial epithelium of eccrine sweat ducts . Moreover, the sole usually contained a greater number of CD8+ relative to CD4+ TCs, whereas the epidermal CD4/CD8 ratio in the trunk and extremities was quite variable, although the trend also was towards a slightly larger percentage of CD8+ cells . Collectively, our data suggest that the volar epidermis has a unique microenvironment which is responsible for both the higher density of TCs, preferentially CD8+, and lower number of LCs . This study has not only provided evidence for significant regional variability in the human epidermal TC population of normal skin, but also strengthens the concept for skin-associated lymphoid tissues (SALT), whereby memory TCs recirculate back to the epidermis and interact with resident antigen-presenting cells (i.e., LC).

Gynecol Oncol, 1990 Apr, 37(1), 24 - 8
Ovarian cancer-associated antibodies recovered from ascites: their use for the isolation of ovarian cancer-associated antigen to produce monoclonal antibodies; Giancotti FR et al.; Immune complexes (ICs) were recovered from the ascites of a patient with stage IV endometrioid ovarian cancer by sequential precipitation with 33% saturated ammonium sulfate and 2.5% polyethylene glycol 6000 (PEG 6000), followed by affinity chromatography on protein A-Sepharose CL-4B . The IgG-containing ICs were dissociated using 8 M urea, separated by ion-exchange chromatography on Sephadex QAE-50, and subsequently analyzed for purity by immunoelectrophoresis (IEP) and radial immunodiffusion (RID) . Recovered antibody was tested for reactivity by immunohistologic techniques against paraffin-embedded tumor tissue and acetone-fixed cell suspensions of epithelial tumors . The antibody which demonstrated ovarian cancer-associated activity was absorbed with antigen extracts of breast, colon, and lung cancers as well as keratin to reduce cross-reactivity . The absorbed endometrioid ovarian cancer-associated antibody (OCAAb) was used to produce an immunoadsorbent column for the recovery of tumor-associated antigens . A mouse monoclonal antibody designated FEN-1 was produced using this antigen-containing fraction, and preliminary screening has demonstrated ovarian tumor-associated reactivity . The use of autologous ICs as reagents for preparing tumor antigen-rich immunogens may provide a valuable tool in the search for tumor-associated antigens.

Dev Biol, 1990 Apr, 138(2), 400 - 9
Granule cell induction of 9-O-acetyl gangliosides on cerebellar glia in microcultures; Mendez-Otero R et al.; In previous studies we have shown that the expression of acetylated gangliosides recognized by the JONES monoclonal antibody is correlated with regions of cell migration in the developing rat nervous system . In this study we have investigated the expression of these gangliosides in two different types of cultures prepared from dissociated postnatal rat cerebella . In the first type, cells are plated after dissociation under conditions where most of the glial cells develop a stellate morphology that anchors neurons but does not support their migration . In the second type of culture, cells are plated in a ratio of four neurons to one glial cell and under these conditions the predominant form of astroglia is an elongate form that supports the migration of granule neurons . Granule neurons express JONES antigens in dissociated cell suspensions and in cultures in which cells are plated either after dissociation or in a 4:1 neuron:glia ratio . On the other hand, glial cells grown in the absence of neurons are JONES negative . In addition, the expression of JONES gangliosides by glial cells is different in the two types of culture . In cultures where the astroglial cells display the stellate morphology only a small proportion show JONES staining . Cultures in which the glial cells assume the elongate morphology have a significantly higher number of JONES-positive astroglia.

J Mol Endocrinol, 1990 Apr, 4(2), 177 - 85
Transmembrane Na+/H+ exchange in the rat thyroid cell strain FRTL-5: a possible role in insulin-like growth factor-I-mediated proliferation; Woods DJ et al.; Using the fluorescent pH indicator 2'7'-bis(2-carboxyethyl)-5'-(6')-carboxyfluorescein to monitor intracellular pH (pHi), we have investigated whether transmembrane Na+/H+ exchange, as measured by experimental changes in pHi under bicarbonate-free incubation conditions, may be involved in the early growth-promoting actions of insulin-like growth factor-I (IGF-I) on the rat thyroid cell stain FRTL-5 . In initial studies to characterize Na+/H+ exchange in FRTL-5 cell suspensions, the recovery of a resting pHi in acid-loaded cells was shown to be dependent upon the presence of extracellular Na+, was enhanced by the presence of the sodium ionophore monensin and was abolished by amiloride, an antagonist of Na+/H+ antiport activity . Unlike TSH, which was without effect on the pHi of FRTL-5 cells for up to 15 min after addition, IGF-I (1000 micrograms/l) caused a rapid and sustained increase within 3 min, which was abolished in medium in which Na+ had been replaced with an iso-osmotic level of choline chloride . The change in pHi in response to IGF-I was mimicked by phorbol 12-myristate 13-acetate (PMA; 100 nmol/l), an activator of thyroid cell proliferation . In the presence of TSH, exposure of cells to IGF-I or PMA had no additional effect on the cytoplasmic alkalinization induced by either of these two agonists alone . However, blockade of transmembrane Na+/H+ exchange with amiloride inhibited both the individual actions of IGF-I and PMA on {methyl-3H}thymidine incorporation, and the synergistic interaction between TSH and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomed Sci, 1990 Apr, 1(4), 407 - 13
Oxidative stress, disturbance of energy balance, and death of ascites tumour cells under menadione (vitamin K3) action; Gabai VL et al.; Resting ascites tumour cells (Ehrlich and EL-4 thymoma) treated with menadione (50 microM) died (up to 80% cell death over 2 h) without dividing (i.e . interphase) . Glucose (25 mM) added to the cell suspensions partially protected these cells from menadione action . During incubation of the cells with menadione, the rates of free oxidation and lipid peroxidation were elevated, cellular ATP and nonprotein SH-group levels were much decreased, and {Ca2+}i was moderately increased . From a comparison of these effects and cell survival rates with those seen with rotenone, KCN (both inhibitors of oxidative phosphorylation), and H2O2 (an inducer of oxidative stress), it is concluded that ATP depletion is the main factor leading to the death of cells treated with menadione . The level of cellular ATP dropped to less than 10% of its initial value after 1 h incubation with menadione and may have resulted in irreversible damage to cytoskeletal structures, bleb formation, and changes in plasma membrane permeability that are incompatible with cell viability.

J Vet Diagn Invest, 1990 Apr, 2(2), 120 - 2
Identification of Brucella abortus strain 19 by decreased ability to utilize erythritol as determined by gas liquid chromatography; Ewalt DR et al.; A method to identify Brucella abortus strain 19 by erythritol utilization using gas liquid chromatography (GLC) was developed . A total of 69 strains of B . abortus (41 virulent field strain isolates and 28 strain 19 isolates) were tested . Following incubation of the isolate with a standard amount of erythritol, the erythritol present in the cell suspension was acetylated and measured by GLC . Field strains of B . abortus utilized an average of 90.9% of the erythritol, whereas vaccine strains utilized an average of 42.4% . This difference in erythritol utilization will allow a more rapid identification of B . abortus strain 19.

J Photochem Photobiol B, 1990 Apr 1, 5(1), 69 - 84
Experimental tests of the feasibility of singlet oxygen luminescence monitoring in vivo during photodynamic therapy; Patterson MS et al.; Singlet oxygen (1O2) is thought to be the cytotoxic agent in photodynamic therapy (PDT) with current photosensitizers . Direct monitoring of 1O2 concentration in vivo would be a valuable tool in studying biological response . Attempts were made to measure 1O2 IR luminescence during PDT of cell suspensions and two murine tumour models using the photosensitizers Photofrin II and aluminium chlorosulphonated phthalocyanine . Instrumentation was virtually identical to that devised by Parker in the one positive report of in vivo luminescence detection in the literature . Despite the fact that our treatments caused cell killing and tissue necrosis, we were unable to observe 1O2 emission under any conditions . We attribute this negative result to a reduction in 1O2 lifetime in the cellular environment . Quantitative calibration of our system allowed us to estimate that the singlet oxygen lifetime in tissue is less than 0.5 microsecond . Some technical improvements are suggested which would improve detector performance and perhaps make such measurements feasible.

Proc Natl Acad Sci U S A, 1990 Apr, 87(7), 2647 - 51
Acetylcholine release from intrahippocampal septal grafts is under control of the host brain; Nilsson OG et al.; The activity of intrahippocampal transplants of cholinergic neurons was monitored by microdialysis in awake, freely moving rats . Fetal septal-diagonal band tissue was implanted into rats with a complete transection of the fimbria-fornix cholinergic pathway either as a cell suspension injected into the hippocampus or as a solid graft implanted in the lesion cavity . The grafts restored baseline acetylcholine release in the graft-reinnervated hippocampus to normal or supranormal levels . The graft-derived acetylcholine release was dependent on intact axonal impulse flow, and it was markedly increased during behavioral activation by sensory stimulation or by electrical stimulation of the lateral habenula . The results demonstrate that the septal grafts, despite their ectopic location, can become functionally integrated with the host brain and that the activity of the transplanted cholinergic neurons can be modulated from the host brain during ongoing behavior . Anatomical observations, using immunohistochemistry and retrograde tracing, indicate that direct or indirect brainstem afferents to the graft could mediate this functional integration . Host afferent control of the graft may thus play a role in the recovery of lesion-induced functional deficits seen with these types of transplants.

Biol Trace Elem Res, 1990 Apr, 25(1), 39 - 45
Aluminum transfer as glutamate complex through blood-brain barrier . Possible implication in dialysis encephalopathy; Deloncle R et al.; In vitro distribution of aluminium between plasma and erythrocytes has been studied in the presence of variable amounts of sodium L-glutamate . With a red blood cell suspension in isotonic sodium chloride, aluminium remains confined in erythrocytes even when the sodium L-glutamate concentration increases in the medium . Aluminium initially present in plasma penetrates red blood cells when sodium L-glutamate increases in whole blood, showing that this metal is able in vitro to cross the erythrocyte membrane as glutamate complex . In vivo experiments with male Wistar rats prove that aluminium is also able to pass the blood--brain barrier as glutamate complex and deposit in the brain cortex.

Agents Actions, 1990 Apr, 30(1-2), 294 - 6
Cytokine-induced release of histamine from basophil leukocytes from AIDS patients; Pedersen M et al.; Cytokine-induced histamine release from basophil leukocytes was examined in cell suspension from AIDS patients and compared with healthy controls . Cells from approximately half of the AIDS patients, in contrast to none from the control group, showed histamine release after stimulation with interleukin-4 (IL-4), tumor necrosis factor alpha (TNF alpha), lymphotoxin (LT) and interferon gamma (IFN gamma) . These cytokines seem to induce histamine release from cells from AIDS patients by interaction with the cell surface immunoglobulins, since removal of the immunoglobulins prior to the exposure of the cytokines completely abolished the response to the cytokines . IL-1 alpha, IL-1 beta, IL-3, colony stimulating factor (CSF) and granulocyte-macrophage-CSF (GM-CSF) caused significant histamine release from cells from a similar number of AIDS patients and controls.

Biophys J, 1990 Apr, 57(4), 835 - 49
Two mechanisms by which fluorescent oxonols indicate membrane potential in human red blood cells; Pratap PR et al.; Optical potentiometric indicators have been used to monitor the transmembrane electrical potential (Em) of many cells and organelles . A better understanding of the mechanisms of dye response is needed for the design of dyes with improved responses and for unambiguous interpretation of experimental results . This paper describes the responses to delta Em of 20 impermeant oxonols in human red blood cells . Most of the oxonols interacted with valinomycin, but not with gramicidin . The fluorescence of 15 oxonols decreased with hyperpolarization, consistent with an "on-off" mechanism, whereas five oxonols unexpectedly showed potential-dependent increases in fluorescence at less than 2 microM {dye} . Binding curves were determined for two dyes (WW781, negative response and RGA451, positive response) at 1 mM {K}o (membrane hyperpolarized with gramicidin) and at 90 mM {K}o (delta Em = 0 with gramicidin) . Both dyes showed potential-dependent decreases in binding . Changes in the fluorescence of cell suspensions correlated with changes in {dye}bound for WW781, in accordance with the "on-off" mechanism, but not for RGA451 . Large positive fluorescence changes (greater than 30%) dependent on Em were observed between 0.1 and 1.0 microM RGA451 . A model is suggested in which RGA451 moves between two states of different quantum efficiencies within the membrane.

Immunology, 1990 Apr, 69(4), 622 - 5
Subsets of keratinocytes and Langerhans' cells express epitopes associated with suppressor-inducer capabilities in resting normal human epidermis; De Panfilis G et al.; In recent years two cell populations with down-regulatory immune capabilities have been identified in murine epidermis . The present report demonstrates that even in human epidermis at least two populations of cells expressing suppressor-inducer phenotypes (i.e . CD45R-positive) exist, namely small subsets of keratinocytes and Langerhans' cells, respectively . Highly specific and sensitive 5-nm colloidal gold-immunoelectronmicroscopic techniques were carried out using anti-CD45R monoclonal antibodies, on freshly isolated crude epidermal cell suspensions, and 4000 cells were scrutinized in the electron microscope . Over 2% of the total epidermal cell population was CD45R+ . Subpopulation analysis revealed that approximately 2% of keratinocytes and about 5% of the total Langerhans' cell population showed strong gold-plasma membrane staining, whilst the remaining epidermal cells were absolutely negative . Heterogeneity of staining together with this somehow surprising distribution of CD45R positivity on non-lymphoid epidermal cells was confirmed by the negative controls . These CD45R+ Langerhans' cells and keratinocytes are clearly candidates for the cells which have been functionally demonstrated as being capable of inducing down-regulation responsiveness in the human epidermis . However, functional investigations are needed to clarify the roles of the CD45R+ keratinocyte and Langerhans' cell subsets in the modulation of cutaneous immune responses.

Biotechnology (N Y), 1990 Apr, 8(4), 333 - 7
Gel microdroplets and flow cytometry: rapid determination of antibody secretion by individual cells within a cell population; Powell KT et al.; We report a new method capable of rapidly determining the secretion of biologically important macromolecules from each of many individual cells within a large population . This method combines flow cytometry with gel microdroplets (GMDs), which in this study were agarose particles ranging from about 53 to 88 mu in diameter . The GMDs were formed from a liquid 2.5% agarose suspension with cells at a concentration which yielded mostly zero or one cell per GMD . A large number of extracellular binding sites were also provided within each GMD, allowing the capture of secreted molecules, and their subsequent measurement by solid phase, fluorescence immunoassay . The method was explored using a model system of mouse hybridoma (secreting) and mouse masticytoma (non-secreting) cells . The method was able to determine subpopulations of individual cells that secreted antibody in less than fifteen hours after receipt of a conventional cell suspension.

FEBS Lett, 1990 Mar 26, 262(2), 228 - 30
Reversal by EGTA of the enhanced secretory responsiveness of mast cells due to treatment with ouabain; Johansen T et al.; The effect of EGTA on the enhancement by ouabain of compound 48/80-induced secretion from mast cells was compared with the effect on the Na(+)-K+ pump activity . The time-dependent secretory enhancement by ouabain was blocked by addition of EGTA to the cell suspension concomitantly with the addition of ouabain, and EGTA caused a large increase in the pump activity . Addition of 10 microM EGTA to ouabain-treated cells stopped but did not reverse the enhancement . The experiments show that the effect of ouabain was due to changes in a calcium pool utilized in compound 48/80-induced secretion following changes in the Na+,K+ pump activity.

J Immunol Methods, 1990 Mar 9, 127(2), 207 - 14
A flow cytometric rosetting assay for the analysis of IgG-Fc receptor interactions; Tuijnman WB et al.; We have developed a sensitive and flexible method for the qualitative evaluation of IgG-Fc receptor interactions in cell suspensions . The assay is based on the flow cytometric quantitation of antibody-coated erythrocyte (EA) rosetting using fluorescein-labelled indicator erythrocytes (E) . The number of IgG molecules on indicator E, an important parameter in EA rosetting, was estimated by calibrated flow cytometry . EA binding quantitated by this method was correlated with microscopically evaluated rosette formation . Besides automated quantitation of EA binding, this method offers the additional advantage of simultaneously using a second fluorescence parameter, permitting analysis of FcR activity in subpopulations of cells . As an example of the applicability of this approach the binding characteristics of E sensitized with a series of murine heavy chain isotype switch variant monoclonal antibodies against glycophorin A, to the low affinity receptor on K562 cells were determined . Remarkably, the results suggest a comparable affinity of Fc gamma RII on these cells for immunoglobulins of the murine IgG1, IgG2a and IgG2b isotypes.

J Invest Dermatol, 1990 Mar, 94(3), 267 - 72
Cutaneous dermal Ia+ cells are capable of initiating delayed type hypersensitivity responses; Tse Y et al.; The presence of Langerhans cells (LC) within the epidermis has been shown to be critical for inducing T-cell-mediated immune responses in the skin . The purpose of this study was to assess whether cells in the dermis can initiate T-cell-mediated delayed-type hypersensitivity responses in vivo . Initially, back skins from C3H mice were trypsinized to remove the epidermis . The dermis was enzymatically dispersed and filtered to obtain a cell suspension . However, dermal cells exposed to trypsin were contaminated with numerous disaggregated hair follicles . These hair follicles contained Ia+ cells (presumably LC), and upon haptenation in vitro with trinitrophenyl, initiated contact hypersensitivity reactions in vivo . We therefore used dispase in place of trypsin to prevent follicular disaggregation and to allow preparation of dermal cell suspensions free of hair follicles . These hair follicle-free dermal cells were haptenated with trinitrophenyl and injected intradermally . Elicitation of contact hypersensitivity by epicutaneous painting 6 d later revealed the mean +/- SEM incremental ear-swelling response to be 53 +/- 8 mm X 10(-3) . In contrast, mice sensitized by injection with dermal cells depleted of Ia+ cells demonstrated only 10 +/- 1 mm X 10(-3) of ear swelling . Thus, like dendritic LC of the epidermis, perivascular dendritic Ia+ cells of the dermis are capable of initiating T-cell-mediated contact hypersensitivity in vivo and may be highly relevant for presentation of antigen to T cells trafficking through the dermis.

Boll Soc Ital Biol Sper, 1990 Mar, 66(3), 295 - 302
Neoplastic cell spreading in rodent transplantable tumor models . II . Santamaria tumor (syngeneic keratinizing squamous cell carcinoma); Roveta G; A syngeneic transplantable tumor was obtained in our laboratory by inducing a skin squamous cell carcinoma in BALB/c mice treated with benzo(a) pyrene and UVA . Single tumor cell suspensions obtained by finely disrupted tumor masses were either i.p . or intramuscularly injected and developed (100% takes) invasive tumors maintaining in subsequent analogue serial transplantations identical histopathological aspects . May Grunwald Giemsa stained organ imprints of tumor bearing mice showed disseminated tumor cells as well as a number of infiltrating host defense cells (principally neutrophils) despite the mice were in a terminal status . May Grunwald Giemsa stained cryostat sections showed numerous mast cells lining the invasive tumor front and allowed to detect in the liver tumor cells migrated from primary tumor (localized in femoral muscle) adhering to endothelial cells may be to perform extravasation.

J Helminthol, 1990 Mar, 64(1), 35 - 45
Early lymphocytic responses to Heligmosomoides polygyrus infections in mice; Parker SJ et al.; Responses to parasite antigens were studied in three strains of mice, BALB/c, CBA and NIH, during the initial phases of a primary infection with the intestinal nematode Heligmosomoides polygyrus . Changes in the rate of in vivo cell division were analysed in mesenteric lymph nodes and spleens during the phases of larval maturation and adult establishment, and related to changes in organ size and cellularity . The nature of the proliferating cell populations was also investigated by flow cytometry, carried out on cell suspensions prepared at the time when larval development was complete . The variation in the ability of the strains of mice to become resistant to a challenge infection was manifest as only slight differences in their initial responses to infection . All three strains showed an increase in 125I-iododeoxyuridine incorporation in their mesenteric lymph nodes and spleen, and an increase in B cell frequency over that of T cells in the draining lymph nodes . Although lymph node weight in NIH mice continued to rise over a 4 week period, the majority of responses measured were short lived, peaking 10 to 14 days after infection . The low responder status of CBA mice was thus reflected in a transient and relatively small enlargement of lymphoid tissues, but their early proliferative responses to antigen were similar in scale to those of responder strains.

In Vitro Cell Dev Biol, 1990 Mar, 26(3 Pt 1), 237 - 49
Isolated trout liver cells: establishing short-term primary cultures exhibiting cell-to-cell interactions; Blair JB et al.; Composition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated . Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages . A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120 X g for 1 min . Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA . When in culture, significant changes in and among the cells were observed . Initial preparations were rounded, single cells . Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d . Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates . Transmission EM indicated that these projections represent biliary ductules forming in vitro . Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver . After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate . Analysis showed the dislodged cells were viable but vacuolated . The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver.

Am J Physiol, 1990 Mar, 258(3 Pt 2), H880 - 6
Heat production in isolated heart myocytes: differences among species; Ponce-Hornos JE et al.; A new calorimetry method has been developed to measure heat production from heart cell suspensions under continuous perfusion . The method is technically independent of the temperature at which the measurements are made, allows full control of the perfusion media, and is suitable for various biological preparations such as cells from diverse tissues, membrane vesicles, or skinned cells . The resting heat rate (Hr) measured at 18.5 degrees C in three different species (19.2 +/- 0.43, 12.8 +/- 0.56, and 9.4 +/- 0.52 mW/g dry wt for rat, guinea pig, and rabbit ventricular myocytes, respectively) agrees with that obtained with other methodologies such as oxygen consumption, thermopiles, and whole heart calorimetry . The Hr measurements showed an excellent correlation with the percentage of rod-shaped cells, indicating that rounded cells are metabolically inactive . Although the time course of the effect of increasing extracellular {K} was dependent on the species, the new steady level of Hr observed under higher extracellular {K} was significantly higher in all three species (+8.3 +/- 1.2, +9.5 +/- 4.0, and +9.3 +/- 2.7 mW/g dry wt for rat, guinea pig, and rabbit ventricular cells, respectively) . This indicates that the commonly used "arrested-heart" preparation (with high extracellular {K}) for evaluation of basal metabolism most probably overestimates the real resting values . The present results also show that the wide range of resting metabolism reported in whole tissue is not due to cellular heterogeneity nor to myocyte interaction and supports the idea of an inverse relationship between resting metabolism and body weight or animal size across species.

Radiat Res, 1990 Mar, 121(3), 274 - 81
Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells; Joshi DS et al.; The induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) were measured in the spermatogenic cells of mice using the alkaline elution technique . The animals were injected with {3H}thymidine and sacrificed on subsequent days to examine selectively cohorts of radiolabeled cells in the successive stages of maturation . A significantly increased frequency of SSB was observed in the unirradiated early spermatocytes and late spermatids, associated with genetic recombination and chromatin compaction, respectively . The frequency of SSBs induced by irradiation of animals in vivo remained constant from the early spermatocyte through mid-spermatid stages and decreased significantly only after the cells matured to the late spermatid stage . The frequency of SSBs after in vitro irradiation of testicular cell suspensions also decreased as round spermatids matured to late spermatids . Such decreases for both modes of irradiation may result from maturation-dependent alterations in chromatin in late spermatids, such as condensation and replacement of histones with protamines, rather than from changes in oxygen tension . Rejoining of SSBs in vivo was efficient in the spermatocytes and early spermatids but declined in late spermatids . Possible reasons for the discrepancy between the greater number of unrepaired lesions and lower susceptibility to mutation induction in late spermatids than in round spermatids are discussed.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1825 - 9
Modulation of constitutive cytochrome P-450 expression in vivo and in vitro in murine keratinocytes as a function of differentiation and extracellular Ca2+ concentration; Reiners JJ Jr et al.; A procedure was developed for the per cell estimation of cytochrome P-450-dependent monooxygenase activities in cultures and whole cell suspensions of murine epidermal keratinocytes (MEKs) . Murine keratinocytes cultured in medium containing less than or equal to 0.04 mM Ca2+ can be induced to differentiate by raising medium Ca2+ concentrations to 1.2 mM . The per cell activities of the monooxygenases 7-ethoxyresorufin O-deethylase (7-ER) and 7-ethoxycoumarin O-deethylase (7-EC) were elevated greater than or equal to 2090% and approximately 460%, respectively, within 13-24 hr of Ca2+ shift . These increases could be completely suppressed by supplementation of culture medium with actinomycin D or cycloheximide immediately prior to Ca2+ shift . After prolonged culture in low Ca2+ medium, some MEKs detached from the monolayer . These detached cells had the characteristics of differentiating MEKs but did not have elevated 7-EC or 7-ER activities . Percoll gradient centrifugation of freshly isolated dorsal skin MEKs was used to prepare four subpopulations that differed in their stages of terminal differentiation . 7-EC and 7-ER activities varied among these subpopulations and correlated with the degree of MEK differentiation . Specifically, the lowest and highest per cell activities (greater than 7-fold difference) were in the basal and most differentiated spinous cell populations, respectively . Collectively, the current studies demonstrate that in vivo P-450 activities are markedly different in proliferating and differentiating MEKs and suggest that constitutive P-450 expression may be modulated as a function of changes in Ca2+ concentration that occur during keratinocyte terminal differentiation.

Exp Hematol, 1990 Mar, 18(3), 167 - 73
Human megakaryocytes . VII . Analysis of megakaryocytes for nuclear DNA content distribution in whole marrow cell suspensions by flow cytometry; Rabellino EM et al.; These studies were designed to quantitate and determine the DNA content distribution of human marrow megakaryocytes using whole bone marrow . Cellular DNA content within megakaryocytic cells was established by measuring propidium iodide staining in marrow cells expressing platelet glycoprotein IIb/IIIa (Gp IIb/IIIa) . These studies were based on the development of a method for rapid analysis of whole marrow cell preparations by a dual fluorescent system . DNA values ranging from 2 to 64 C were observed in all samples tested, with 16 C corresponding to the modal ploidy class representing almost one-half of the cells containing Gp IIb/IIIa . The second most frequent ploidy class corresponded to 32 C, followed by 8 C, with 20% and 15%, respectively . Virtually all high-ploidy megakaryocytes (greater than or equal to 8 C) were of low density (less than 1.050 g/cm3), whereas 2 and 4 C megakaryocytes were evenly distributed between less than or equal to 1.050 and greater than 1.050 g/cm3 marrow cells . These studies conclusively establish DNA content distribution of normal human marrow megakaryocytes and provide a basis for the study of states of altered megakaryocytopoiesis.

J Biochem (Tokyo), 1990 Mar, 107(3), 476 - 9
Glucagon- and dibutyryl cyclic AMP-produced inhibition of cholesterol ester hydrolase in isolated rat hepatocytes: role of calcium; Ruiz MB et al.; The regulation of neutral cytosolic cholesterol ester hydrolase was studied in isolated rat liver cells . Addition of glucagon to cell suspensions caused a decrease in the enzyme activity which was significant at 1 nM concentration . The cyclic nucleotide analogue bibutyryl cyclic AMP (10 and 100 microM) also inhibited the esterase activity . In the absence of calcium, glucagon did not produce any effect on the enzyme . To see if calcium was involved in a regulatory mechanism, cholesterol ester hydrolase activity was measured in cytosol from cells preincubated in a medium without calcium and containing EGTA . This treatment produced a marked reduction in cytosolic Ca2+ concentration with a concomitant threefold stimulation of the esterase activity . Readdition of calcium to Ca2(+)-deprived cells diminished the activation due to calcium deficiency . The present results suggest that 1) cholesterol ester hydrolase could be modulated by a cAMP-mediated mechanism elicited by glucagon in which Ca2+ appears to be involved and 2) the enzyme activity may also be regulated by changes in the intracellular calcium concentration.

Am J Clin Pathol, 1990 Mar, 93(3), 322 - 6
A prospective comparison of DNA quantitation by image and flow cytometry; Bauer TW et al.; Advances in computer and video technology suggest that image analysis may be practical method of measuring DNA that also allows visual confirmation of cell type . The purpose of this study was to prospectively compare DNA quantitation from 92 solid tumors in which DNA indices had been measured by image analysis of touch preparations (CAS 100) and flow cytometry of cell suspensions (FACScan) . For 81 cases, there was excellent correlation between the two methods . For nine cases, however, an aneuploid population, usually near tetraploid, was identified by image but not by flow cytometry . Three cases had aneuploid peaks by flow cytometry that were not identified by image . Although these methods show good correlation, rare populations may be missed by CAS, presumably because of sampling errors in the touch preparation . Aneuploid populations may also be missed by flow cytometry, either because of cell loss during processing or because visual identification by image can increase sensitivity.

J Immunol, 1990 Mar 1, 144(5), 1880 - 5
Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene; Hirata K et al.; The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages . Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM) . However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable . Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes . When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4 . These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity . These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4 . The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.

Mol Cell Biol, 1990 Mar, 10(3), 918 - 22
Regulation of DdrasG gene expression during Dictyostelium development; Khosla M et al.; DdrasG gene expression during the early development of Dictyostelium discoideum has been examined in detail . The amount of DdrasG-specific mRNA increased approximately twofold during the first 2 to 3 h of development and then declined rapidly, reaching negligible levels by the aggregation stage . The increase in mRNA levels that occurred during the first 2 to 3 h of development also occurred during differentiation in cell suspensions and was enhanced when cells were shaken rapidly . This initial increase was unaffected by cell density . When cells were set up to differentiate on filters, the addition of a glucose-amino acid mixture slightly delayed differentiation and had a similar effect on the expression of the gene . The decline in DdrasG expression during development did not occur when cells were treated with cycloheximide, suggesting that the expression of a developmentally regulated gene product is essential for the reduction of DdrasG gene mRNA . There was no decrease in DdrasG mRNA level during differentiation in shake suspension, but the decrease did occur upon application of pulses of cyclic AMP to shaking cultures . The application of a continuously high level of cyclic AMP delayed the increase in expression of the gene and did not result in the subsequent decline . These results suggest that the induction of a functional cyclic AMP relay system is important in reducing DdrasG gene mRNA levels.

Plant Cell, 1990 Mar, 2(3), 215 - 24
A DNA-binding protein factor recognizes two binding domains within the octopine synthase enhancer element; Tokuhisa JG et al.; A protein that binds to the enhancing element of the octopine synthase gene has been identified in nuclear extracts from maize cell suspension cultures . Two protein-DNA complexes are distinguishable by electrophoretic mobility in gel retardation assays . Footprint analyses of these low and high molecular weight complexes show, respectively, half and complete protection of the ocs-element DNA from cleavage by methidiumpropyl-EDTA.FE(II) . Two lines of evidence indicate that the element has two recognition sites, each of which can bind identical protein units . Elements that are mutated in one or the other half and form only the low molecular weight complex interfere with the formation of both the low and high molecular weight complexes by the wild-type element . Protein isolated from a complex with only one binding site occupied can bind to the wild-type ocs-element and generate complexes with protein occupying one or both binding sites . Occupation of both sites of the ocs-element is a prerequisite for transcriptional enhancement.

Biochem J, 1990 Mar 1, 266(2), 355 - 61
Regulation of the mitochondrial ATP synthase in intact rat cardiomyocytes; Das AM et al.; The ATP synthase capacity of rat heart myocytes can be measured in sonicated cell suspensions and in sonicated preparations of cultured cardiomyocytes . This procedure allows the rapid measurement of mitochondrial function in response to changes in the metabolic status of the cell . In cultured myocytes, transitions in ATP synthase capacity (with no detectable change in cellular ATP concentration) accompany a change to anoxia or electrically stimulated contraction (rise of 70%) . These changes are reversed on returning to the original conditions . Exposure of myocytes to low pH has little effect on basal ATP synthase capacity (down to values less than pH 6), but markedly affects cellular ATP levels and the response of the cells to anoxia and reoxygenation, possibly mimicking changes seen in ischaemic heart . Similar effects are seen in suspensions of freshly prepared myocytes, but these preparations are less stable and more pH-sensitive than are cells in culture . It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase, and that a regulator protein, the naturally occurring inhibitor protein from mitochondria, may be responsible for this regulation.

J Exp Med, 1990 Mar 1, 171(3), 775 - 86
Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils; Nusse O et al.; The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements . It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane . Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein . The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+ . Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.

Am J Physiol, 1990 Mar, 258(3 Pt 1), C495 - 503
Halothane-dependent release of intracellular Ca2+ in blood cells in malignant hyperthermia; Klip A et al.; The concentration of ionized cytosolic calcium {( Ca2+}i) was determined in peripheral blood mononuclear cells from normal and malignant hyperthermia (MH)-susceptible humans and pigs, using the fluorescent Ca2+ indicator indo-1 . {Ca2+}i was slightly but significantly elevated in cells from MH human cells relative to normal cells (198 +/- 18 nM, n = 15, and 146 +/- 14 nM, n = 11, respectively, P less than 0.05) . Anesthetic concentrations of halothane in the cell suspension resulted in a rapid increase in {Ca2+}i in cells from both normal and MH humans or pigs . The increases (delta) were more pronounced in cells from MH subjects than from normal individuals (delta at 5.7 mM halothane: 245 +/- 53 vs . 57 +/- 11 nM, respectively) and from MH than from normal pigs (delta of 241 +/- 63 vs . 53 +/- 27 nM, respectively) . Removal of extracellular Ca2+ obliterated the delta{Ca2+}i caused by halothane in cells from normal humans or pigs but only decreased by about half the delta{Ca2+}i in cells from MH humans or pigs . In 1,2-bis-(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-loaded cells, in the absence of extracellular Ca2+, halothane failed to increase {Ca2+}i . This suggests that buffering Cai2+ with BAPTA precludes detection of release of Ca2+ from intracellular stores, explaining the previous observations made with quin2, a highly chelating Ca2+ indicator . It is concluded that clinical concentrations of halothane allow influx of Ca2+ in cells from both normal and MH-susceptible individuals but release Ca2+ from intracellular stores selectively in cells from the latter group.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Physiol Scand, 1990 Mar, 138(3), 369 - 76
Binding of cholecystokinin and somatostatin to isolated porcine gastric mucosal cells and effects on aminopyrine uptake; Sjodin L et al.; Mucosal cells were prepared by enzymatic digestion of porcine gastric mucosa with pronase and collagenase . The resulting cell suspension contained 10-15% parietal cells, which responded to histamine stimulation by an up to 20-fold increase in {14C}aminopyrine accumulation over control levels . Cholecystokinin-8 (CCK-8) evoked a more moderate stimulation of {14C}aminopyrine accumulation, whereas somatostatin inhibited histamine-stimulated accumulation . Parietal cells were enriched by elutriation and isopycnic centrifugation on density gradients of Percoll . A fraction with 60% parietal cells bound approximately three times more iodinated CCK-8 than a fraction containing 70% non-parietal cells . Binding of {125I}BH-CCK-8 to preparations containing 30-60% parietal cells was specifically inhibited to about 50% by 10(-9) M unlabelled CCK-8 but not by bombesin . Cell fractions containing about 30% parietal cells also bound {125I}somatostatin . Unlabelled somatostatin at 10(-9) M inhibited tracer binding by about 50%, while CCK-8 did not affect somatostatin binding to such a preparation . The results suggest the existence of specific receptors for CCK and somatostatin on porcine parietal cells exerting a regulatory influence on acid secretion.

Infect Immun, 1990 Mar, 58(3), 726 - 31
Enrichment of suppressor T cells by means of binding to monophosphoryl lipid A; Baker PJ et al.; The binding and elution of spleen cells from plastic dishes coated with monophosphoryl lipid A (MPL) resulted in a greater than 1,000-fold enrichment of antigen-specific suppressor T-cell (TS) activity when spleen cells from mice 18 to 24 h after exposure to a low dose of type III pneumonococcal polysaccharide (SSS-III) were used . The removal of MPL-adherent TS cells resulted in an increase in the degree of amplifier T-cell (TA) activity present in the remaining MPL-nonadherent cell fraction; however, both TS and TA activities were found in the MPL-adherent cell fraction when spleen cells from mice 4 days after immunization with an optimal dose of SSS-III were examined . These findings, as well as others, suggest that both TS and TA, once activated, acquire a cell surface receptor that enables them to bind to MPL . Because of differences in the kinetics for the activation of TS and TA during the course of the antibody response and the fact that TS, but not TA, activity appears as early as 18 to 24 h after exposure to SSS-III, it is possible to use this experimental approach to obtain cell suspensions greatly enriched in TS activity.

AIDS Res Hum Retroviruses, 1990 Mar, 6(3), 287 - 98
In vitro studies of HIV-1 infection in thymic lymphocytes: a putative role of the thymus in AIDS pathogenesis; De Rossi A et al.; To ascertain whether thymic lymphocytes represent suitable targets for HIV-1 infection, we infected thymic cell suspensions from normal donors with HIV-1 (HTLV-IIIB strain) . We found that, in vitro, thymic lymphocytes are readily infected and highly permissive for HIV-1 replication . In addition, immature cells with the CD4+/CD8+ phenotype, most likely the precursors of mature circulating CD4+ and CD8+ lymphocytes, showed a marked susceptibility to viral infection and replication . These findings suggest that thymus infection may play a triggering role in the pathogenesis of AIDS, particularly in pediatric cases, and may partially explain the lack of restoration of peripheral CD4+ lymphocytes killed by HIV-1.

J Immunol, 1990 Feb 15, 144(4), 1161 - 8
Ontogeny of T cell receptors in the chicken thymus; Bucy RP et al.; A panel of murine mAb against chicken TCR and associated molecules was used to study the ontogeny of T cells . The intrathymic maturation of the TCR-gamma delta, (TCR-1) and TCR-alpha beta (TCR-2) sublineages was the focus of these studies employing immunoperoxidase staining of tissue sections and immunofluorescence analysis of cell suspensions . The first CD3+ cells appeared in the thymus on embryonic day 9 (E9) when the CD3 Ag was restricted to the cytoplasm . In tissue sections, both TCR-1+ and TCR-2+ cells were observed on E12, whereas only the TCR-1 cells were identifiable by surface immunofluorescence . On the next day, when a discrete thymic medullary region was first recognizable, the TCR-1 cells were present in both cortex and medulla . Two days later (E15), TCR-1 cells were found in the spleen . Surface TCR-2+ cells did not appear until E14, began to migrate in to the medulla on E17, and appeared in the spleen on E19 . The first TCR-1 cells thus move quickly through this maturational pathway, whereas TCR-2 cells undergo a prolonged developmental period in the cortex . While most TCR-1+ cells were CD4-CD8-, a minor subpopulation (5 to 15%) were CD4-CD8+, and less than 1% were CD4+CD8+ . In contrast, immature TCR-2+ thymocytes in the cortex were predominantly CD4+CD8+, whereas cells expressing a higher density of the CD3/TCR-2 complex were either CD4+CD8- or CD4-CD8+ and were localized in the thymic medulla . In the medulla of the mature thymus, the TCR-1+ cells preferentially occupy the cortico-medullary junction and form small aggregates around vessels . TCR-2+ cells were less frequent in these areas of TCR-1 accumulation . The thymic ontogeny and, by implication, the selection of the receptor repertoire thus differs substantially for these two TCR isotypes.

Biochem Biophys Res Commun, 1990 Feb 14, 166(3), 1390 - 7
Defluorination of 1,1,1,2-tetrafluoroethane (R-134a) by rat hepatocytes; Olson MJ et al.; As part of its toxicological evaluation we assessed the in vitro metabolism of 1,1,1,2-tetrafluoroethane (R-134a), a non-ozone-depleting chemical likely to replace dichlorodifluoromethane (R-12) as an air-conditioning refrigerant . Hepatocyte suspensions in sealed flasks produced increasing quantities of F- (detected in the liquid media) as the headspace concentration of R-134a increased from 1% to 50% (balance of atmosphere 95% O2-5% CO2); the kinetics of defluorination suggested substrate-saturation . Little F- was detected in cultures without R-134a or in cell suspensions heated prior to addition of R-134a . Halothane (1,1,1-trichloro-2-bromo-2-chloro-ethane), although not defluorinated by hepatocytes maintained with 95% O2, inhibited defluorination of R-134a . Hepatocytes from phenobarbital-treated rats dehalogenated high (greater than or equal to 25%) concentrations of R-134a at greater rates than cells from untreated rats . These findings are consistent with the hypothesis that oxidative metabolism of R-134a by cytochrome P-450 can occur in vivo.

J Immunol Methods, 1990 Feb 9, 126(2), 205 - 11
Purification and properties of peritoneal eosinophils from pediatric dialysis patients; Roberts RL et al.; Peritoneal eosinophilia frequently occurs in patients undergoing peritoneal dialysis . We have devised a method for isolating large numbers of these peritoneal eosinophils from pediatric patients on continuous peritoneal dialysis . Patients were selected on the basis of previous high peritoneal eosinophil counts and had an age range of 1.5-11 years . The unfractionated peritoneal fluid contained 7.9 +/- 3.7% neutrophils, 3.8 +/- 1.0% lymphocytes, 11.0 +/- 3.7% monocytes/macrophages, and 77.3 +/- 6.3% eosinophils (based on Wright stain) and up to 2 x 10(9) cells could be recovered from 1 liter of peritoneal dialysate . The cells were concentrated by centrifugation and the cell suspension then layered over a discontinuous Percoll gradient consisting of layers of 45%, 55%, 65%, and 75% Percoll . The gradients were centrifuged resulting in the formation of bands of cells at the interfaces of the layers . The densest band of cells (above 75% Percoll) contained 94.7 +/- 1.8% eosinophils (mean with median of 98%) and 4.3 +/- 16% neutrophils . The eosinophil counts were 72.2 +/- 7.1% above the 65% layer, 57.1 +/- 8.7 above 55%, and 40.9 +/- 10.9% above 45% . The monocyte/macrophage count increased from 0.1% above the 75% layer to 42.9% above the 45% . The denser eosinophils (above 75% and 65%) had the appearance of normal blood eosinophils and comparable function to blood eosinophils in cytotoxic and oxidative assays . This method provides a means of obtaining large numbers of very pure eosinophils for study of eosinophil function, eosinophil subpopulations, or eosinophil granule constituents.

J Biol Chem, 1990 Feb 5, 265(4), 2399 - 408
Toxic injury from mercuric chloride in rat hepatocytes; Nieminen AL et al.; The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2 . In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min . Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers . Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy . In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium . Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+ . Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM) . After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential . The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+ . HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.

Harefuah, 1990 Feb 1, 118(3), 146 - 8
{T-cell lymphoma}; Hurwitz N et al.; 8 patients with lymphoproliferative disorders of T-cell origin were diagnosed during the years 1985-1987 . They included 2 cases of so-called Lennert's lymphoma, 1 case of T-cell lymphoma simulating malignant histiocytosis and 1 case of T-cell lymphatic lymphoma with splenic T-cell lymphoma which survived 10 years . The other cases presented with peripheral T-cell lymphomas . Immunologic typing of malignant lymphomas with cell suspensions is of diagnostic value.

J Anat, 1990 Feb, 168, 209 - 16
Regulation of haematopoietic stem cell proliferation by stimulatory factors produced by murine fetal and adult liver; Dawood KA et al.; Haematopoietic stem cells in murine fetal liver are in a proliferative state unlike those in normal bone marrow which are quiescent . A regulatory activity is produced by cells in the fetal liver which will switch quiescent normal bone marrow haematopoietic stem cells into cell cycle in vitro . This regulator from Day 15 fetal liver cells is produced by adherent cells and by cells fractionated on a Percoll gradient in the 1.064 and 1.076 g per cm3 density bands but not in the 1.123 g per cm3 band . Colony-stimulating factor cannot be detected in the supernatants containing the stem cell regulatory activity . The stimulator can be detected in supernatants produced from cell suspensions of liver cells at Day 15 and Day 17 of gestation and 24 hours and 72 hours after birth . However by 1 week after birth the production of the stimulator decreases and is undetectable 3 and 10 weeks after birth . The total numbers of haematopoietic stem cells (CFU-S) in fetal liver decrease from Day 15 of gestation and only small numbers are present 1 week after birth . Thus the decline in the production of haematopoietic stem cell proliferation stimulator correlates with the decrease in haematopoietic stem cell numbers in the liver through gestation and after birth.

Ann Neurol, 1990 Feb, 27(2), 174 - 80
Increased thymocyte differentiation in myasthenia gravis: a dual-color immunofluorescence phenotypic analysis; Durelli L et al.; Thymocytes express multiple, different surface antigens according to their stage of maturation . Surface differentiation antigens have been studied with the technique of simultaneous dual-color, direct immunofluorescence in the thymuses of 20 patients with myasthenia gravis (MG) and 10 control subjects with cardiac diseases . Fluorescein isothiocyanate-conjugated and phycoerythrin-conjugated monoclonal antibodies were used to stain thymic cell suspensions . A significant decrease in the percentage of immature and common thymocyte phenotypes (CD1+,3+ and CD4+,8+) and a significant increase in the percentage of mature thymocyte phenotypes (CD1-,3+; CD4+,8-; and CD4-,8+) and of B cells (CD20+) were found in MG thymuses compared with controls . These data, indicating an increased availability of mature, fully immunocompetent T and B cells, indirectly suggest the occurrence of an active immune response in MG thymus.

Am J Respir Cell Mol Biol, 1990 Feb, 2(2), 171 - 81
Uptake of muramyl dipeptide fluorescent congeners by normal rabbit bronchoalveolar lavage cells: a study using flow cytometry; Richerson HB et al.; Muramyl dipeptide (MDP) is the minimal adjuvant-active structure of mycobacterial cell walls and is known to activate monocytes/macrophages, but mechanisms involved with uptake and activation of these cells have not been completely defined . Earlier studies addressing uptake of MDP and the question of receptors have utilized radioligands and murine peritoneal macrophages . We used fluorescent congeners of MDP and flow cytometry to explore kinetics and specificity of uptake by bronchoalveolar cells of normal rabbits . Both washed cells and cell suspensions from which the fluorescent congeners were not washed were used, and incubation was carried out primarily at 4 degrees C . Fluorescence microscopy consistently revealed intracellular but no visible membrane fluorescence of alveolar macrophages . Uptake was dose dependent but was not saturable up to concentration limits of fluoresceinated muramyl tripeptide (MTP-FITC) imposed by the system, and was partially inhibited by excess unlabeled MDP, consistent with specific inhibition . Alveolar macrophages, but not lymphocytes, demonstrated specific uptake at 4 degrees C, with rapid on- and off-times . Uptake was enhanced 7-fold at 37 degrees C . Uptake was greater by larger, more granular macrophages than by smaller, less granular macrophages, but no difference in uptake was found when cells of similar size but different densities were compared . The exact mechanism of the rapid uptake at 4 degrees C is uncertain but appears to be competed for by unlabeled MDP.

Am J Clin Oncol, 1990 Feb, 13(1), 70 - 4
Screening test for hormone sensitivity by the autoradiographic method using cell mats; Matsuoka H et al.; We developed an autoradiographic screening test for hormone sensitivity of single cell suspensions of tumor tissues on cell mats, which inhibited the growth of normal cells alone . We applied this method to our newly established KSE-1 line derived from esophageal carcinoma and compared this method with well-established cytoplasmic and nuclear assays . Our assay, though taking longer to implement and providing only qualitative information, requires significantly smaller specimens than the current biochemical assay and will predict the hormone sensitivity of only viable neoplastic cells.

Transplantation, 1990 Feb, 49(2), 453 - 8
Graft rejection by cytolytic T cells . Specificity of the effector mechanism in the rejection of allogeneic marrow; Nakamura H et al.; Cellular effector mechanisms of allograft rejection remain incompletely described . Characterizing the rejection of foreign-marrow allografts rather than solid-organ grafts has the advantage that the cellular composition of the marrow graft, as a single cell suspension, can be altered to include cellular components with differing antigen expression . Rejection of marrow grafts is sensitive to lethal doses of radiation in the mouse but resistant to sublethal levels of radiation . In an effort to identify cells mediating host resistance, lymphocytes were isolated and cloned from spleens of mice 7 days after sublethal TBI (650 cGy) and inoculation with allogeneic marrow . All clones isolated were cytolytic with specificity for MHC encoded gene products of the allogeneic marrow donor . When cloned cells were transferred in vivo into lethally irradiated (1025 cGy) recipients unable to reject allogeneic marrow, results utilizing splenic 125IUdR uptake indicated that these MHC-specific cytotoxic clones could suppress marrow proliferation . In order to characterize the effector mechanism and the ability of the clones to affect final engraftment, double donor chimeras were constructed so that 2 target cell populations differing at the MHC from each other and from the host were present in the same marrow allograft . Results directly demonstrated an ability of CTL of host MHC type to mediate graft rejection and characterized the effector mechanism as one with specificity for MHC gene products.

Transfusion, 1990 Feb, 30(2), 117 - 25
A new radioimmunoassay for the detection of small amounts of white cells and platelets in red cell concentrates: implications for blood transfusion; Webster MR et al.; In the procedure for quality control of red cell concentrates, made white cell (WBC)-poor by filtration, the particle-counting technique was found to be insufficiently sensitive in detecting the remaining WBCs and platelets . Therefore, direct radioimmunoassays were developed using murine monoclonal antibodies specific for platelets, granulocytes, and T lymphocytes . The sensitivity for platelets was 40 x 10(3) per mL, that for granulocytes was 10 x 10(3) per mL (starting from 0.2 mL of red cell filtrate), and that for T lymphocytes was 0.006 x 10(6) per mL (starting from 5 mL) and 0.0015 x 10(6) per mL (starting from 50 mL) . These direct assays were used in experiments on filtration with three types of filters: the Cellselect B-1005, B-1014 and the B-1013 (bedside filter) . After filtration of 1 unit of blood cell suspension through the B-1005, the number of remaining platelets was found to vary between less than or equal to 0.04 x 10(6) and greater than 15 x 10(6) per mL (n = 16); after filtration through the B-1013 filter, the remaining platelets were greater than 0.04 x 10(6) per mL . Upon filtration of a second unit of blood cell suspension through the B-1013 filter, the number of remaining platelets varied between less than or equal to 0.04 x 10(6) and 5 x 10(6) . In both filter types, the number of remaining granulocytes was always less than 0.01 x 10(6) per mL . A study of T-lymphocyte contamination revealed that, upon filtration of 1 unit of blood through the B-1005, T-lymphocyte numbers were less than or equal to 0.0015 x 10(6) to 0.15 x 10(6) per mL (starting from 5 and 50 mL); upon filtration through the B-1013 filter, the number of remaining T lymphocytes varied between less than or equal to 0.006 x 10(6) and 0.2 x 10(6) per mL (starting from 5 mL) . After filtration of a second unit of blood cell suspension through the B-1013 filter, the number of remaining T lymphocytes ranged from less than or equal to 0.006 x 10(6) to 0.1 to 0.5 x 10(6) per mL (starting from 5 mL) . The direct radioimmunoassay is an improvement over the present electronic particle-counting techniques with regard to both sensitivity and specificity and may therefore be useful in quality control procedures in blood transfusion as well as in the development of new filters.

Artif Organs, 1990 Feb, 14(1), 7 - 13
Concentration profiles of platelet-sized latex beads for conditions relevant to hollow-fiber hemodialyzers; Waters CM et al.; A freeze-capture method was used to obtain the concentration profiles of platelet-sized latex beads (2.5 microns diameter) in saline-based red cell suspensions flowing through tubes with inner diameters of 135 and 200 microns . Without red cells the concentration profile exhibited a slight deficit near the wall . For hematocrits from 15 through 52%, the concentration profile exhibited a near-wall excess of beads, from two to eight times the central concentration . Near-wall excesses were small or nonexistent in the first half centimeter of the tube . For hematocrits of 15 and 30%, the profile and near-wall excess were equal at stations 2 cm or more from the entrance . These results suggest that there is a flow-driven mechanism that moves platelet-sized beads toward, but not to, the wall.

J Invest Dermatol, 1990 Feb, 94(2), 247 - 53
Effect of aging on epidermal dendritic cell populations in C57BL/6J mice; Sprecher E et al.; The density and function of epidermal dendritic cell populations were investigated in aged C57BL/6J mice . The densities of both Langerhans cells (LC) and Thy-1+ dendritic epidermal cells were found to decrease with age . Epidermal cell suspensions from aged mice showed impaired immunologic function as assessed in vitro by the skin-lymphocyte reaction assay and by measuring the ability of epidermal cell suspensions to stimulate the proliferation of sensitized T cells in the presence of the sensitizing antigen . However, the capacity of LC to transport antigen from the skin to the draining lymph nodes was found in vivo to be comparable to that of young mice . Results of transplantation of bone marrow cells from young and old donors into irradiated recipients indicate that the decreased Langerhans cell density found in old mice may result from a deficiency in Langerhans cell bone marrow progenitors.

Exp Cell Res, 1990 Feb, 186(2), 279 - 87
How is the flagellar length of mature sperm determined? II . Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro; Uno S et al.; In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster . The average final length of flagella in Xenopus round spermatids was 35 mum, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 mum, almost half that of mature sperm . Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids . The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids . But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species . Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded . Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded . Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids . The period of flagellar elongation almost coincided with the period of tubulin synthesis . The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes . Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.

Cell Immunol, 1990 Feb, 125(2), 535 - 9
Protein kinase-C involvement in thymocyte apoptosis induced by hydrocortisone; Ojeda F et al.; The involvement of protein kinase-C in thymocytes death induced by hydrocortisone was studied . Thymus cells were incubated 6 hr or in the presence of hydrocortisone, labeled with Acridine orange, and the DNA content of each nuclei was estimated by cytofluorimetry . The results indicate that hydrocortisone-induced DNA fragmentation can be prevented by adding the protein kinase-C inhibitor H-7 to the cell suspension . Incubation of the H-A 1004, an inhibitor of c-AMP-dependent protein kinase, with low effect on on protein kinase-C, did not interfere with the cortisone-mediated DNA fragmentation . Therefore, it can be concluded that protein kinase-C plays an important role in the process of lympholysis mediated by corticoids.

J Dairy Sci, 1990 Feb, 73(2), 342 - 50
Preinfection functions of blood polymorphonuclear leukocytes and the outcome of experimental Escherichia coli mastitis in the cow; Lohuis JA et al.; The relationship between preinfection functions of blood neutrophils and outcome of experimental Escherichia coli mastitis was studied in 11 cows . Random migration, chemotaxis, phagocytosis, and chemiluminescence by neutrophils were determined in white blood cell suspensions, and in purified neutrophil suspensions . The course of E . coli mastitis (10(4) E . coli 0:157 in rear quarters) was monitored using clinical parameters, counts of E . coli in mastitic secretion, and milk production . Regressions were calculated for areas under curves of these parameters and preinfection activities of neutrophils . Chemiluminescence by nonstimulated neutrophils in white blood cell suspensions was negatively correlated with counts of E . coli in secretion and with losses in milk production . The chemotactic differential in white blood cell suspensions minus the chemotactic differential in purified suspensions of neutrophils referred to as delta varied from -.66 to +.50, indicating, respectively, inhibition and stimulation of chemotactic activity of neutrophils in white blood cell suspensions . Delta correlated negatively with counts of E . coli in mastitis secretion, inhibition of the amplitude of rumen contractions, and losses in milk production . We hypothesize that a factor in white blood cell suspensions may be involved in the down-regulation of the migratory response of neutrophils during E . coli mastitis.

Pathol Res Pract, 1990 Feb, 186(1), 37 - 62
Cytophotometry in tumor pathology . A critical review of methods and applications, and some results of DNA analysis; Mellin W; In tumor pathology the quantitation of cellular substances can be of diagnostic value . Microscope cytophotometry and digital image analysis and, on the other hand, flow cytometry are supplementary methods for measuring, each with a typical spectrum of application . The methods are predominantly used for DNA analysis: Static and image cytophotometry are applicable to cytologic and histologic slides preferably for identifying stem lines in tumors of heterogenous morphology and in merely circumscribed lesions (e.g., precancerous lesions) . On the other hand, sampling errors due to preselection, and the often low number of cells actually measured, may preclude the possibility of exact cell cycle analysis . This is, in fact, an important additional option of flow cytometry resulting from the high resolution of DNA histograms, which is explained by the large number of cells that can be measured in a short period . Sampling errors in flow cytometry may result from the preparation of single cell suspensions which in certain tumor entities may suppress a varying amount of particularly fragile cells or nuclei . The prognostic significance of DNA ploidy, stem line heterogeneity and S-phase fraction is clearly described in quite a number of tumor entities . Independent of its prognostic value, the cytometric identification of stem lines might be particularly useful in the follow-up of tumor patients, where it may indicate the effectivity of systemic therapy . The development of therapeutic concepts is aptly supported by flow cytometric cell cycle analysis which helps to assess the in vitro effect of combined cytostatics on the proliferative process . Moreover, multiparameter analysis of biopsy samples may provide greater accuracy in characterising individual tumor stem lines and may furthermore help to develop improved protocols for the therapy of solid tumors.

Immunology, 1990 Feb, 69(2), 329 - 31
The rat FcR for monomeric IgG is preferentially expressed on red pulp macrophages in the spleen; Denham S et al.; Rat monoclonal IgG2b immunoglobulins labelled with 125I or biotin were localized in the red pulp areas but were not found in the marginal zones of the white pulp of the spleen 8 hr after their i.v . injection . In cell suspensions made from the spleen, 90% of red pulp macrophages (M phi) bound 125I-IgG2b monomeric proteins in vitro, whereas similar estimates for marginal zone M phi were 12-30% . Red pulp and marginal zone M phi were distinguished primarily by monoclonal antibodies (mAb) ED2 and ED3, but also by their ability to take up FITC-labelled Ficoll in vivo.

Exp Neurol, 1990 Feb, 107(2), 143 - 53
Importance of catecholamine release for the functional action of intrastriatal implants of adrenal medullary cells: pharmacological analysis and in vivo electrochemistry; Decombe R et al.; The aim of the present experiments was to test whether adrenal chromaffin cells implanted into the striatum of rats could exert a functional effect through a release of catecholamines . A cell suspension obtained from bovine adrenal medulla was implanted unilaterally into the striatum . The striatal dopaminergic input was extensively destroyed beforehand to preclude the possibility of reinnervation of the striatum by endogenous dopaminergic neurons . The functional influence of the implant was tested through the measurement of drug-induced rotation, while catecholamine release was measured subsequently in the same animals by in vivo electrochemistry . Transplant survival, as shown by the immunohistochemical analysis performed at the end of the in vivo experiments, was highly variable . Surviving chromaffin cells maintained their endocrine morphology and no reinnervation of the host striatum could be detected . Rotation of the animals evoked by apomorphine (0.1 mg/kg, sc) or amphetamine (5.0 mg/kg, ip) following the lesion was left uninfluenced following transplantation, even when a large transplant was recovered . On the other hand, nicotine (0.5 mg/kg, sc) evoked a strong contraversive rotational response in the transplant-bearing animals . This response could not be ascribed to the central effect of substances released peripherally and entering the nervous system through the blood-brain barrier opened by the implantation procedure, as it could not be found in animals bearing implants of other peripheral endocrine tissue, viz, pituitary . The effect of nicotine was not blocked by the pretreatment of the animals with either the opiate antagonist naloxone (2.5 mg/kg, 10 min) or the dopamine receptor blocker pimozide (0.5 mg/kg, 1 h), although the latter pretreatment blocked the amphetamine-evoked rotation . No spontaneous catecholamine release could be detected from the implanted chromaffin cells by in vivo electrochemistry, while treatment with amphetamine or nicotine did evoke a release . The results suggest that the functional effects of such intrastriatal grafts of chromaffin cells, reported in previous studies, cannot be explained by the secretion from the grafted cells of catecholamines into the denervated striatum . On the other hand the results obtained following the pharmacological stimulation of these cells indicate that adrenal grafts can, under suitable conditions, influence the functioning of the host nervous system.

Am J Vet Res, 1990 Feb, 51(2), 216 - 21
Evaluation of serologic and cellular immune responses of cattle to a nonlipopolysaccharide antigen from Brucella abortus; Hoffmann EM et al.; Cows naturally infected with Brucella abortus developed antibody (Ab) responses to a nonlipopolysaccharide antigen (NLA) purified from B abortus strain 1119-3 . Sera from strain 19-vaccinated cows did not have detectable amounts of Ab . Weak lymphoproliferative responses to NLA were observed in blood mononuclear cell suspensions obtained from infected cows . There was no evidence of NLA-specific lymphoproliferation in cell suspensions from healthy cows . Nonlipopolysaccharide antigen binding to bovine blood mononuclear cells was observed by antigen-consumption assays and direct binding of radiolabeled antigen . Cells from infected cows bound less NLA than did cells from healthy cows when assays were conducted with intact blood mononuclear cell preparations (monocytes plus lymphocytes) . Monocytes obtained from any group did not bind NLA . Purified B lymphocytes from infected and healthy vaccinated cows bound about 3 times more NLA than did T lymphocytes, but there were no apparent differences between the 2 groups in extent of binding . Results of the study indicate that bovine lymphocytes have binding sites for a NLA purified from B abortus strain 1119-3.

Gastroenterol Jpn, 1990 Feb, 25(1), 78 - 87
Effects of intraperitoneal transplantation of microcarrier-attached hepatocytes on D-galactosamine-induced acute liver failure in rats; Nagaki M et al.; A study was conducted to investigate morphologic as well as metabolic characteristics of microcarrier-attached hepatocytes in culture, and also to evaluate the effect of intraperitoneal transplantation of the microcarrier-attached hepatocytes on acute hepatic failure in rats induced by D-galactosamine (GalN) . Rat hepatocytes were isolated by collagenase perfusion, and cultured on collagen-coated microcarriers . Protein synthesis estimated by {14C} leucine incorporation was four-fold higher in microcarrier culture than in cell suspension . The rates of albumin, transthyretin and bile acid syntheses in hepatocytes cultured on microcarriers were similar to those in monolayer culture . When microcarrier-attached hepatocytes were intraperitoneally transplanted into rats with Galn-induced acute liver failure, a marked improvement in survival rate was observed as compared with control rats which received injections of microcarriers alone (80% vs 0% beyond 6 days of transplantation) . Mean serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), methionine and glucose levels were similar in both groups, while serum bilirubin and ammonia levels were lower (P less than 0.1, P less than 0.05) in rats transplanted with the microcarrier-attached hepatocytes . Immunohistochemical examinations revealed that the transplanted hepatocytes around microcarriers had albumin synthesis activity, whereas almost no albumin synthesis was demonstrated in recipient liver . In conclusion, intraperitoneal transplantation of the microcarrier-attached hepatocytes will provide sufficient metabolic support, representing detoxication of ammonia (and presumably bilirubin) and synthesis of albumin, to allow GalN-damaged liver function to restore . Microcarrier culture of isolated hepatocytes seems to be one of the most appropriate tools for an artificial liver support.

Behav Brain Res, 1990 Jan 22, 36(3), 229 - 49
The effects of ibotenic acid lesions of the nucleus basalis and cholinergic-rich neural transplants on win-stay/lose-shift and win-shift/lose-stay performance in the rat; Sinden JD et al.; Rats were trained to criterion performance in 2-lever operant conditional memory tasks that required them to follow either a Win-stay/Lose-shift, or else a Win-shift/Lose-stay response rule . Substantial impairments in performance of both pretrained conditional tasks were seen following ibotenic acid lesions of the nucleus basalis, but not of the globus pallidus . The deficit in both tasks was apparent at all inter-response retention intervals, indicating that nucleus basalis lesions produced a general impairment in the performance of the complex conditional operant tasks, and not a specific deficit in short-term memory . The nucleus basalis lesion rats were then divided into groups matched for equivalent performance . One group was given cell suspension grafts into neocortex of E15 cholinergic-rich forebrain tissue; a second group was given similar grafts of E17 hippocampal tissue; and a third group was given sham transplants . Testing beginning 3 months post-transplant showed that there was no evidence of recovery of performance on these tasks in the cholinergic-rich transplanted groups compared to the controls . However, the rats with cholinergic-rich transplants subsequently showed a significant improvement in retention of a step-through passive avoidance task . The results indicate that either cholinergic deafferentation of the neocortex is not critical for the observed deficits in the operant conditional tasks, or recovery of function following cholinergic-rich transplants is task-specific, in that more complex cognitive tasks may require different levels of graft-host neural integration.

J Biol Chem, 1990 Jan 5, 265(1), 96 - 102
Pathways of purine metabolism in human adipocytes . Further evidence against a role of adenosine as an endogenous regulator of human fat cell function; Kather H; Previous results demonstrated that the adenosine that accumulates in human fat cell suspensions is derived from extracellular sources (Kather, H . (1988) J . Biol . Chem . 263, 8803-8809) . To get insight into the mechanisms responsible for the lack of adenosine release, extracellular adenine nucleotide catabolism was minimized by 10 mmol/liter beta-glycerophosphate and 10 mumol/liter alpha,beta-methyleneadenosine 5'-diphosphate . Intracellular adenine nucleotide catabolism resulted in a release of inosine and hypoxanthine under these conditions that was increased markedly by isoproterenol . Experiments with inhibitors of adenosine deaminase and adenosine kinase indicated that the production of inosine and hypoxanthine proceeded via AMP deamination . Consistently, IMP levels were increased transiently in the presence of isoproterenol . In addition, the cells possessed a nucleotide phosphomonoesterase that was resistant to the inhibitory actions of ATP and alpha,beta-methyleneadenosine 5'-diphosphate and showed preference for IMP over AMP . Adenosine (approximately 1 nmol/10(6) cells/h) was also produced inside the cells . However, adenosine production was unrelated to ATP turnover via adenylate cyclase, and any adenosine formed was immediately reconverted to adenine nucleotides in the absence and presence of isoproterenol . It was concluded that adenosine is not released by intact human adipocytes, because the alternative routes of intracellular AMP catabolism are compartmentalized (at least in functional terms), and adenosine kinase is not saturated with substrate in the absence and presence of isoproterenol.

Am J Clin Pathol, 1990 Jan, 93(1), 104 - 8
An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry; Warzynski MJ et al.; A comparison was made between a manual and automated method for preparing single cell suspensions from various types of human biopsy samples . The automated method uses the shearing action of fluid movement generated by a reciprocating paddle system . When compared on 11 samples, the automated instrument always isolated cells in less time, usually requiring only 15-30 seconds in contrast to 10-15 minutes for the manual method . The time comparisons involved "set up" time of the required minor equipment as well as the time to make a complete single cell suspension from the portion of the biopsy sample sent to the Flow Cytometry Lab so extra cells could be used for other purposes and cryogenically stored for future reference . Cells isolated by the automated method from various B-lymphocytic and T-lymphocytic malignancies were still viable and could be successfully immunophenotyped by flow cytometry . The immunophenotyping results were compared for both cell isolation methods on seven of these samples, and the results were comparable . The automated technique has now been used to satisfactorily immunophenotype more than 50 biopsy samples . The automated method should represent a significant aid for clinical flow cytometry laboratories performing immunophenotyping tests by efficiently preparing single cell suspensions in a few seconds instead of minutes . Furthermore, the automated method uses a closed sterile bag system that helps minimize the exposure of personnel to potential infectious material present in biopsy samples and prevents external contamination of cell suspensions . The automated technique proven successful for immunophenotyping may also be helpful for related procedures involving hematopoietic malignancies such as DNA content analysis and cell functional assays by flow cytometry, as well as other assays such as tissue typing, gene probe, and in vitro chemosensitivity assays.

Exp Brain Res, 1990, 81(2), 426 - 32
Intracortical grafts of embryonic basal forebrain tissue restore low voltage fast activity in rats with basal forebrain lesions; Vanderwolf CH et al.; Unilateral injections of kainic acid into the basal forebrain in a series of rats resulted in an increase in large amplitude slow waves, a correlated burst-suppression pattern of multi-unit activity, and a decrease in acetylcholinesterase staining in the neocortex ipsilateral to the kainic acid injection . Subsequently, a cell suspension, prepared from rat embryonic basal forebrain tissue, was injected adjacent to the recording electrodes ipsilateral to the kainic acid injection . This produced a gradual recovery of low voltage fast activity (LVFA) and a correlated continuous discharge pattern of multi-unit activity in the neocortex ipsilateral to the kainic acid injection . LVFA recovered more slowly at neocortical recording sites that received an injection of a cell suspension of hippocampal primordial cells or no injection at all . Acetylcholinesterase-positive fibers from the basal forebrain tissue invaded host cortex; no comparable outrgrowths were demonstrable in the hippocampal primordium tissue grafts . Restoration of cholinergic electrocortical activation may play an important role in the improvements in behavioral performance produced by basal forebrain grafts in the cortex in animals with basal forebrain lesions.

Biomed Biochim Acta, 1990, 49(2-3), S242 - 6
Prognosis of hemolytic anemia in G6PD- subjects . Multifactorial cluster analysis of biochemical characteristics of red cell age groups; Yermakov NV et al.; Individual susceptibility of 10 G6PD- hemizygotes to oxidative hemolytic agents was tested on the basis of multifactorial cluster analysis of biochemical indices of erythrocyte populations; the indices related to G6PD activity and glucose metabolism were analyzed under physiological and oxidative stress conditions in very young, exactly adult and very old red cell suspensions . Biochemical images of G6PD- erythrocytes were obtained and compared with the donor (7 subjects) biochemical image on a IBM-PC computer according to a special "taxon" program . As a result, a stable subdivision of 10 Gd- biochemical images into 5 taxons was formed; each taxon included G6PD subjects with a certain form of clinical appearance of G6PD deficiency . Multifactorial cluster analysis of biochemical data on the erythrocyte population allows a clinical prognosis for G6PD- subjects.

Biomed Biochim Acta, 1990, 49(2-3), S172 - 7
Effects of phenylhydrazine hydrochloride on energy metabolism in rabbit erythrocytes and reticulocytes; Zivkovic RV et al.; The effects of 1, 5 and 20 mmol/l phenylhydrazine hydrochloride on energy metabolism of rabbit erythrocyte and reticulocyte were studied . Significant depression of glycolysis, accompanied by loss of adenine nucleotides, mainly due to an extensive decline of ATP, was found in erythrocytes . Energy metabolism of reticulocytes appears to be more sensitive to deleterious effects of this drug . The declines of ATP and sum of all adenine nucleotides, as well as the accumulation of hypoxanthine were twofold in reticulocyte-rich red cell suspensions compared with suspensions of mature erythrocytes . It can be concluded that these changes are the consequence of lower energy production due to phenylhydrazine hydrochloride-induced inhibition of oxidative phosphorylation.

Cell Mol Biol, 1990, 36(2), 213 - 24
An enzymatic method for the isolation of mouse Leydig cells; Merkel U et al.; Mouse Leydig cells were obtained by dispersion of testes of adult animals (aged 6-15 months) with a neutral protease from B . polymxa (dispase; EC 3.4.24.4) . The crude Leydig cell suspension was purified by centrifugation on a discontinuous Percoll gradient using a special centrifugation procedure similar to elutriation . The crude cell suspension obtained from 50 testes could be processed in one run . The combination of these two methods yielded 320000 +/- 53000 Leydig cells/testis (n = 554 testes) . The purity of the Leydig cell fraction was greater than or equal to 95% (nucleated cells) based on morphological and histochemical (staining for naphthyl esterase) identification . The purified Leydig cells showed an excellent ultrastructural appearance . More than 98% excluded trypan blue . In the presence of NADPH, testosterone biosynthesis was increased only 1.15 +/- 0.1-fold yielding a "quality factor" of 34.8 . Maximal hCG doses induced 10(6) purified Leydig cells to produce 5 nmol testosterone/hr . (40-fold stimulation in comparison to basal values) . The Leydig cells showed 43100 +/- 2500 LH/hCG receptors and an association constant of Ka = 1.95 x 10(9) M-1 . Due to the reproducibility of the method, to the yield as well as to the morphological and functional state of the purified Leydig cells at least 25% of laboratory animals could be saved.

Blood Cells, 1990, 16(1), 5 - 20; discussion 20-3
The salvaged blood syndrome: a sequel to mechanochemical activation of platelets and leukocytes?
Bull BS, Bull MH.
Disseminated intravascular coagulopathy (DIC) and/or increased vascular permeability in the lungs (ARDS) or systemic circulation (anasarca) has been seen in an occasional patient following the administration of washed autologous red cells . We have found that platelets and leukocytes, activated by the process of salvage, can contaminate such red cell suspensions . Apparently, activation begins with the mechanical deposition of platelets on the centrifuge bowl wall during the cell-concentration phase of blood salvage if there has been substantial prior dilution of the salvaged blood with saline . Electron microscopy of the deposit reveals activated, degranulated platelets lining the inner surface of the bowl . There is a preferential "homing" of specific leukocyte types to local regions of the deposit . On the basis of morphological evidence, we hypothesize that these mechanically activated platelets release leukoattractant substances, including arachidonate-rich phospholipids which trigger the oxidative burst enzymatic pathway in exposed phagocytic cells . These cells, when reinfused, cause increased vascular permeability . This presents clinically as ARDS or anasarca, whereas DIC results from reinfused platelet phospholipid plus accompanying cellular debris.

Folia Biol (Praha), 1990, 36(1), 65 - 70
Increase in the capacity of bone marrow exposed to He-Ne laser radiation for growth of GM-CFC colonies in vitro; Vacek A et al.; Single exposure of murine bone marrow cell suspension to He-Ne laser radiation elevates the potential of haemopoietic stem cells for growth of GM-CFC colonies in vitro . The stimulatory action of He-Ne laser persists for 60 min after short-term irradiation.

Neoplasma, 1990, 37(2), 139 - 47
Functional heterogeneity of Sarcoma I cells in transplantation experiments; Sobotkova E et al.; Cell suspensions prepared from Sarcoma I (SaI) allografts at various stages of development in C57BL10/ScSn (B10) mice immunosuppressed with xenogeneic antithymocyte serum (ATS) were adoptively transferred into secondary syngeneic (A/Ph) and allogeneic (B10) recipients . In the syngeneic recipients, a gradual decrease in the tumorigenic capacity of transferred suspensions was proved, while in the allogeneic recipients the tumorigenic activity was proved in early and late periods . The suspensions from the period of both permanently and temporarily regressing tumors showed a suppressed growth capacity in syngeneic and immunosuppressed allogeneic recipients . On the other hand, the suspensions from growing tumors produced in both types of secondary recipients a sharp and permanent growth . The data obtained suggest changes in functional properties of SaI cells during the course of their development in allogeneic recipients.

Dermatologica, 1990, 180(3), 141 - 5
Immunogold scanning electron microscopy applied to the study of Langerhans cells immunophenotype; Manara GC et al.; An immunogold technique in scanning electron microscopy was applied to the detection of Langerhans cell surface CD1a antigen in a heterogeneous human epidermal cell suspension . Scanning electron microscopy investigations performed in the secondary electron imaging mode and in the backscattered electron imaging mode allowed to define Langerhans cell surface morphology and the labeling degree, respectively . In particular, the backscattered electron imaging mode of the scanning electron microscope appeared very useful in identifying colloidal gold particles and, as a result, made it possible to make total counts of the labeled surface antigenic sites . Finally, the combination of secondary electron and backscattered electron signals generated mixed images with the Langerhans cell surface morphology well recognizable together with distinct gold particles . The immunogold labeling in scanning electron microscopy here described represents a highly sensitive method and yields a new improvement in the study of Langerhans cells immunophenotype.

Cytometry, 1990, 11(3), 395 - 405
Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry; Johnson NF et al.; Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells . Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2 degrees and 90 degrees light scatter signals . Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle . The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells . Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency than secretory cells (3.4%) . Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells . These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium . Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability . Because of these traits they may be a critical cell at risk from damage by environmental agents.

Urol Res, 1990, 18(2), 107 - 11
Flow cytometric evaluation of Thomsen-Friedenreich antigen on transitional cell cancer using monoclonal antibody; Oda H et al.; In 31 transitional cell cancer (TCC) tissues and 5 normal bladder mucosae (NBM), we compared the results of flow cytometry (FCM) and immunohistochemical examination in evaluating the expression of Thomsen-Friedenreich antigen (T-Ag) using a monoclonal antibody . On immunohistochemical examination, 14 (45%) cancer tissues showed T-Ag, while 7 (23%) cancer tissues and all NBM showed only cryptic T-Ag, which was detected only after neuraminidase treatment . Ten (32%) high grade cancer tissues showed neither T-Ag nor cryptic T-Ag . ON FCM the T-Ag positive cells (TPC) and the T-Ag positive cells after neuraminidase treatment (nTPC) were counted in fresh cell suspensions . FCM was more sensitive than immunohistochemical study in detecting T-Ag . Additionally, FCM revealed that some tumors had both T-Ag and cryptic T-Ag at the same time . The ratio of nTPC to TPC was well correlated with the stage or grade of the tumor and may be a more reliable marker of TCC than the expression of T-Ag assessed by immunohistochemical techniques.

J Reprod Fertil, 1990 Jan, 88(1), 223 - 9
Secretion of prostaglandins and progesterone by cells from corpora lutea of mares; Watson ED et al.; Corpora lutea (CL) were collected from mares during early (Day 4-5), mid- (Day 8-9), and late (Day 12-13) dioestrus . Dispersed cell suspensions were obtained by enzymic digestion of tissue . Two distinct luteal cell populations (large and small) were observed . The proportion of small luteal cells significantly increased as age of CL advanced . Cells (2 x 10(6)) from CL which were incubated for 24 h secreted prostaglandin (PG) F, PGE-2 and 6-keto-PGF-1 alpha (the stable metabolite of prostacyclin) . Higher concentrations of all PGs were produced by cells from CL at early dioestrus than from those at mid- or late dioestrus . The ratio of PGF:PGE-2 increased from 0.33 in CL of early dioestrus to 1.34 in CL of mid-dioestrus, whereas ratios of PGF:6-keto-PGF-1 alpha remained relatively constant (approximately 0.6) . The ratio of PGE-2:6-keto-PGF-1 alpha from CL decreased between early (3.27) and mid-dioestrus (0.43) . Addition of LH, dbcAMP, or ionophore to cell cultures did not consistently affect secretion of progesterone or PGs by luteal cells . It is suggested that prostaglandins produced by luteal cells of mares may contribute to control of luteal function and that the changing ratios of prostaglandins may be more important in controlling the lifespan of the CL than absolute concentrations of each.

Eur J Nucl Med, 1990, 16(2), 69 - 76
In vivo imaging of rat lymphocytes with an indium 111-labelled anti-T cell monoclonal antibody: a comparison with indium 111-labelled lymphocytes; Loutfi I et al.; An indium 111-labelled mouse anti-rat T cell monoclonal antibody, MRC OX-19, was injected intravenously into rats to establish the usefulness of radiolabelled anti-lymphocyte antibodies in imaging lymphoid tissues . Antibody binding in vivo, measured by immunofluorescence analysis of cell suspensions made from lymphoid tissues, was detectable on lymphocytes in blood, spleen and lymph nodes . The extent of binding was time and antibody-dose dependent . Doses of antibody above 80 micrograms/kg body weight resulted in modulation, i.e . loss of CD 5 (T1) molecules from the cell surface, although the cells remained in the circulation . Modulation was demonstrable within 2 h and for at least 24 h after a single injection of antibody . Intravenous injection of 111In-MRC OX-19 resulted in levels of in vivo binding comparable with those seen with unlabelled antibody . Scintillation imaging showed early splenic localisation persisting over 48 h, a more gradual localisation in the lymph nodes seen clearly at 24 h and a steady background . Comparison of the in vivo distribution of labelled antibody and 111In-tropolone-labelled lymphocytes showed that both could be used for external imaging of lymphocytes by scintillation camera.

Chemotherapy, 1990, 36(2), 147 - 54
In vitro synergistic activity of 5-fluorouracil with low-dose ozone against a chemoresistant tumor cell line and fresh human tumor cells; Zanker KS et al.; We followed the concept that the chemical reactivity of ozone depends upon its oxidative properties . Activated oxygen species are causally involved in toxicity of certain chemotherapeutic drugs . We have tested this prediction in human cell cultures either to overcome chemoresistance and/or to increase chemical cytotoxicity . Our results indicate that ozone in combination with 5-fluorouracil (5-FU) makes a 5-FU-resistant cell line susceptible for the combined treatment modality . Furthermore, ozone acts synergistically or at least additive to chemotherapy in different tumor cell suspensions, derived from the breast and the colon.

Invasion Metastasis, 1990, 10(2), 101 - 12
Efficient recovery of clonogenic stem cells from solid tumors and occult metastatic deposits; Miller BE et al.; We describe the use of enzymes combined with brief, sequential mechanical disruptions in a Tekmar Stomacher blender for the recovery of clonogenic neoplastic cells from solid tumors, lungs, and livers . The method has yielded 3 X 10(8) to 5 X 10(8) total cells and 1.2 X 10(6) to 17 X 10(6) clonogenic cells per gram of tissue from three different mouse mammary tumor subpopulations growing in the subcutis . The clonogenic cell yields represent a 4- to 13-fold increase over our previous best method of tumor disaggregation . The increase in total cells recovered, while not as dramatic (up to 3-fold), was statistically significant for two of the three tumor lines . We were also able to efficiently recover 125I-iododeoxyuridine labelled neoplastic cells from lungs and livers after injecting the cells intravenously . Over half of the total radiolabel present in these organs prior to disaggregation could be recovered in the cell suspensions obtained.

Int J Hyperthermia, 1990 Jan-Feb, 6(1), 203 - 11
Treatment of the human retinoblastoma cell line Y-79 growing in the athymic mouse eye with fractionated hyperthermia and/or radiation; Koole P et al.; Concentrated cell suspensions of the human retinoblastoma cell line Y-79 were injected into the eyes of athymic nude mice . Three to four weeks after implantation the eyes were treated with fractionated hyperthermia and/or radiation according to various protocols . Radiation was applied with 250 kV X-rays in fractions of 3 Gy, three fractions a week (3 x XT) . Hyperthermia was applied once a week for 30 min at 43-45 degrees C (1 x HT) . A coaxial microwave applicator (2450 MHz) has been developed for hyperthermia treatment of the mouse eye . The effect of the treatment was evaluated by visual observation of the eyes during a follow-up of several months, and by histopathological examination . No tumour regression was observed after hyperthermia alone (3 x HT) . Treatment with 3 x XT or 3 x XT + 1 x HT initially caused tumour regression followed by regrowth in most cases . A treatment protocol of 6 x XT resulted in regrowth in 10 out of 15 eyes . However, after 6 x XT + 2 x HT regrowth was observed in only 3 out of 13 eyes . The difference between these two groups is statistically significant (P less than 0.05) . Thus it is concluded that hyperthermia enhances the effect of radiation when the Y-79 cell line is transplanted to the athymic mouse eye, using the fractionation scheme presented here.

Br J Cancer, 1990 Jan, 61(1), 65 - 8
Flow cytometric measurement of glutathione content of human cancer biopsies; Hedley DW et al.; Rice et al . (1986) have described a flow cytometric method where the non-fluorescent probe monochlorobimane (mBCl) forms a fluorescent adduct with cellular glutathione (GSH) under the action of glutathione-S-transferase . We show here that for EMT6 carcinosarcoma cells there is a close correlation between mean cell fluorescence, expressed as a ratio to that of fluorescence calibration beads, and biochemically determined GSH content over the range 0.2-2.0 fmol cell-1 . Single cell suspensions from 14 human cancers were prepared by 23-gauge needle aspiration or mechanical disaggregation of surgical specimens, stained using mBCl and examined by flow cytometry . There was a wide range in individual cell fluorescence, which in contrast to EMT6 cells was not strongly correlated with Coulter volume . By comparing tumour cell fluorescence to that of calibration beads, and assuming that the relationship with GSH content for EMT6 holds for other cells, a mean GSH content of 0.95 fmol cell-1 was derived for nine carcinomas, and 0.21 fmol cell-1 for five non-Hodgkin's lymphomas . Although this semi-quantitation needs further validation, the method used here is rapid, gives an indication of heterogeneity of tumour cell GSH content, and can be applied to fine needle biopsy samples . It therefore shows promise as a means for studying prospectively the relationship of GSH content to clinical drug and radiation sensitivity, and for monitoring the effects of agents such as buthionine sulphoximine which are intended to improve treatment results through tumour cell GSH depletion.

J Invest Dermatol, 1990 Jan, 94(1), 58 - 64
Modulation of the binding and endocytosis of concanavalin A by guinea pig keratinocytes: reversible antagonistic effects of cholesterol and phospholipid-liposomes; Callaghan TM et al.; Alteration of guinea pig keratinocyte membrane microviscosities (eta) by liposomes of varying composition was determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene . Measurements performed either with whole cell suspensions or Percoll-separated cell subpopulations, indicate a similar membrane microviscosity (eta = 3.37 poise +/- 10%) compared to those microviscosities reported for other cell types . Our findings show that treatment of guinea pig keratinocytes with liposomes composed of phospholipids results in a decreased membrane microviscosity (1.95 poise), whereas treatment of the cells with an emulsion of cholesterol hemisuccinate, or liposomes composed of cerebrosides, causes an increase in membrane microviscosity (3.85 poise and 5.55 poise +/- 10%, respectively) . Changes in membrane fluidity had no significant effect on cell viability . A reduced membrane microviscosity resulted in a decrease in the binding of Concanavalin A to keratinocytes, whereas an increased microviscosity resulted in an increased binding of Concanavalin A . Furthermore, endocytosis of Concanavalin A bound to keratinocytes plasma membranes was not significantly affected by a reduced membrane microviscosity, whereas an increased membrane microviscosity completely blocked the endocytosis of Concanavalin A . Another novel observation was that membranes "fluidified" by phospholipid liposomes could be "rigidified" by treatment with cholesterol hemisuccinate and vice versa . Moreover, these alternate changes in membrane microviscosity resulted in simultaneous alternate changes in the binding of Concanavalin A to the keratinocyte surface.

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