ABSTRACT:A new poly(ester amide) derived from L-alanine has been synthesized and characterized. The polymer has good fiber-and film-forming properties, as well as other characteristics like thermal stability and solubility in chloroform, which enhance its processing facilities. Degradation studies show that both pH and temperature influence in the hydrolisis rate that takes mainly place through the ester linkages. Degradation was also studied by using different enzymes. Results indicated that papain was the most efficient of these, and that the hydrolysis to water-soluble products could be attained in a few days. Basal cytotoxicity was assayed using a mouse L929 fibroblast permanent cell line. The MTT viability test was performed with liquid extract of the material (50 days, 37ЊC). An attachment and proliferation screening study with intact material was also carried out. No cytotoxic responses were detected, in either assay, after a 24-and 48-h incubation period with the cells. After 72 h a slight cytotoxicity was detected in the polymer material, while a more significant one was detected in the material extract.
Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin. C D14 is a glycosyl-phosphatidyl-inositol anchored membrane protein (mCD14) expressed on mature monocytes (1, 2). It functions as a coreceptor for bacterial lipopolysaccharide (LPS) and triggers the induction of inflammatory responses (3). One consequence of LPS-mediated monocyte activation is the release of soluble CD14 (sCD14) (4), and increased levels of circulating sCD14 correlate with infection and autoimmunity (5-8).sCD14 has been postulated to desensitize monocytes through blunting their production of inflammatory cytokines in response to endotoxin (9). However, this role remains contentious, as humans respond immediately and briskly to endotoxin despite containing 1,000-fold molar excess of serum sCD14 relative to the concentration of LPS observed during sepsis (10). The present study establishes a previously unrecognized function of sCD14 and demonstrates its broader spectrum of biological activities. Materials and MethodsCell Preparation, Activation, and Inhibition. All splenic B cell cultures were done in serum-free medium with high buoyant density cells isolated from C57BL͞6 mice as described (11). Cells (1.5 ϫ 10 5 ) were cultured in 0.2 ml in the presence of sCD14 or LPS derived from Salmonella typhosa 0901. Cultures were pulsed with 1 Ci of )]F 1 pups were determined by ELISA using mAb b-7-6 (14) as the capture antibody followed by biotinylated anti-mouse IgM a as the developing antibody. TEPC 183 (mouse IgM a , ) was used as standard. Signals were revealed by using horseradish peroxidase (HRP)-conjugated streptavidin.Human B cells were isolated from suspensions of tonsil leukocytes. Cells were labeled with biotinylated mAb specific for CD3 followed by avidin-conjugated ''microbeads'' and passed through MACS (Becton Dickinson). The effluent population contained Ͻ1% T cells and Ͼ98% B cells as assessed by immunofluorescence. B cells were cultured as described above in the presence or absence of submitogenic concentrations of plate-bound mAbs (coated at 1:1) specific for human Ig (mAb LO-HK-3, ref. Induction of membrane Ig (mIg) expression by ...
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