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 vert