Cocultures effects

Tyrosine phosphorylation plays an important role in synaptic transmission and plasticity. Evidence for this role is that modulators of PTKs and PTPs have been shown to be intimately involved in these synaptic functions. Among the various modulators of PTKs, neuro-trophins have been extensively studied in this regard and will be our focus in the following discussion (for details of growth factors, see Ch. 27). BDNF and NT-3 have been shown to potentiate both the spontaneous miniature synaptic response and evoked synaptic transmission in Xenopus nerve-muscle cocultures. Neurotrophins have also been reported to augment excitatory synaptic transmission in central synapses. These effects of neurotrophins in the neuromuscular and central synapses are dependent on tyrosine kinase activities since they are inhibited by a tyrosine kinase inhibitor, K-252a. Many effects of neurotrophins on synaptic functions have been attributed to the enhancement of neurotransmitter release BDNF-induced increase in neurotransmitter release is a result of induced elevation in presynaptic cytosolic calcium. Accordingly, a presynaptic calcium-depen-dent phenomenon - paired pulse facilitation - is impaired in mice deficient in BDNF. 

The effect of NK cell subsets on IgE regulation was examined in cocultures of in vitro differentiated NK cells with peripheral blood mononuclear cells or B cells. NKl cells significantly inhibited... 

Figure 6 Immunopotentiating reconstituted influenza virosomes (IRIV) adjuvant effects in the induction of tumor associated antigen-specific cytotoxic T cell. CD14-negative cells from a healthy donor peripheral blood mononuclear cells were cocultured with autologous immature dendritic cells (iDC) in the presence of Melan-A/Mart-l27-35, alone (A) or supplemented with either control liposomes (B) or IRIV (1 50, C). On day 7, culture cells were restimulated with Melan-A/MART-127-35 pulsed iDC and cultured for six further days [see Materials and Methods ]. On day 7 after restimulation cells were stained with fluorescein isothiocyanate-conjugated anti-CD8 and phosphatidylethanolamine-conjugated HL A-A0201 /Melan-A/MART -127-3 5 tetramers. Source From Ref. 6.

Whatever the nature of the receptor tyrosine kinase subclass 111 ligand isoforms involved, it is presently unclear whether they induce different signaling effects in early hematopoietic cells upon binding to its receptor. To determine the function of the SCF/CSF-1 isoforms we used an embryonic epithelial cell line to express ectopically each membrane-bound and soluble ligand. In cocultures with human and murine early progenitor cells we determined their proliferative and developmental responses. 

Within one week the total number of hematopoietic cells increased on stimulation with S1+MS5 stroma 5-fold (Fig. 4a). Membrane-bound SCF was slightly more effective (3.5-fold) than soluble SI SCF (2.8-fold) in enhancing the total number of hematopoietic cells at this time point. A proliferating cell population was also initially detected in coculture with untransduced epithelial cells, although the proliferation capacity declined within two weeks (Fig. 4a). After four weeks in coculture the proliferation potential of SI SCF induced CB cells was 6-fold decreased, that of mb SCF stimulated cells 1.5-fold. Thus, proliferating cells were maintained significantly longer in cultures supported by membrane-bound SCF than by Sf SCF. 

We have evaluated this assumption by utilizing CB CD34+ cells in coculture experiments with epithelial cells expressing either membrane-bound or SI SCF. Cycling of primary stem cells have been reported to be correlated with a sequential loss of the proliferation capacity. Our results have shown that the reduction of proliferation is less when membrane-bound SCF stimulates the CD34+ cells as opposed to soluble SI SCF (Fig. 4). Consequently, membrane-bound SCF stimulated CB cells show higher proliferation potential than cells stimulated by soluble SCF. Thus, the overall effect of the SCF isoforms is similar on primary as well as on immortalized TFl cells. 

Different susceptibility among species to reproductive effects seems also related to the metabolism of 1,3-DNB. Rats were much more susceptible to adverse reproductive effects of 1,3-DNB than hamsters (McEuen and Miller 1991 Obasaju et al. 1991). This was correlated with the fact that blood levels of 1,3-DNB in the hamster reached only half those found in the rat and that blood levels of the metabolite 1,3-nitroaniline were higher in the rat (McEuen and Miller 1991). Furthermore, rats excreted more unconjugated and less phenolic metabolites than hamsters. Results from studies with rat Sertoli/germ cell cocultures suggest that reactive metabolic intermediates such as nitrosonitrobenzene and nitrophenylhydroxylamine may be responsible for the testicular toxicity of... 

Rotating culture vessels such as simulated microgravity systems are primarily used to study 3-D tumor growth and differentiation. However, mixed cell populations combined with matrix proteins can be used to generate a complex microenvironment in which cell-cell interactions and invasion can be measured (95). A similar system has also been described for the coculture of endothelial cells, myofibroblasts, and tumor cell clusters embedded in Matrigel . Differential labeling of the cell populations enables their invasion and the effects of inhibitors to be measured (96). 

Immunological Effects. The effects of carbon tetrachloride on the immune system have not been evaluated in humans. Immune responses were not affected in rats orally exposed to carbon tetrachloride (Smialowicz et al. 1991). Parenteral exposure of animals to carbon tetrachloride has been reported to impair the immune system (Kaminski et al. 1989 Muro et al. 1990 Tajima et al. 1985), and oral exposure caused depletion of lymphocytes, hemorrhage, and hemosiderin deposition in the pancreaticoduodenal lymph node (Doi et al. 1991). These findings are supported by in vitro studies in which the IgM antibody formation response of isolated mouse splenocytes to sheep erythrocytes was inhibited in a dose-dependent manner when the splenocytes were exposed to carbon tetrachloride for 1-3 hours in the presence of cocultured hepatocytes (Kaminski and Stevens 1992). No effects were observed in the absence of cocultured hepatocytes. Mice appear to be more sensitive than rats to carbon tetrachloride-induced immunosuppression, but the biological significance to humans of these reported effects are yet ascertainable from the available data. 

The dramatic effects observed when nitric oxide ( N=0) synthase is induced in cocultures of macrophages and tumor cells (Hibbs et al., 1987 Stuehr and Nathan, 1989) as well as Kupffer cells and hepatocytes (Billiar et al., 1989 ... 

V. EFFECT OF NITRIC OXIDE SYNTHESIS IN DEFINED MACROPHAGE-LYMPHOCYTE COCULTURES... 

Previous studies on the effects of N=0 production on the alloimmune response utilized bulk populations of responder and stimulator cells. In order to more clearly define the circumstances that induce -N=0 synthesis in allogeneic macrophage-lymphocyte cocultures, mouse splenocyte populations were depleted of accessory cells (>90% Thy 1.2+) and cultured with mitomycin-C-treated macrophage cell lines, as the alloantigen presenting cells. The P388D1 (H2 ) and RAW 264.7 (H2 ) macrophage lines were selected because... 

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