Reduced Smad3 protein expression and altered transforming growth factor-beta1-mediated signaling in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)-beta1, it was our goal to examine components of TGF-beta1-mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of protein levels of the TGF-beta1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF-beta1-stimulated levels of luciferase expression using the TGF-beta1-responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF-beta1-stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF-beta1-mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 protein expression in CF epithelium may also influence inflammatory responses. Our data demonstrate that both CF-phenotype cells lacking Smad3 expression, and A549 cells expressing a dominant-negative Smad3, are unable to support TGF-beta1-mediated inhibition of either the interleukin (IL)-8 or the NOS2 promoter. We conclude that a CF-related reduction in Smad3 protein expression selectively alters TGF- beta1-mediated signaling in CF epithelium, potentially contributing to aggressive inflammatory responses.

Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides undergo normal isotype switching after immunization with polysaccharide-protein conjugate vaccines.

Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the gamma 3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3(-/-) mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3(-/-) mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3(-/-) mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream gamma 3 heavy-chain gene.

Effects of exposure to polychlorinated biphenyl (PCB) from conception on growth, and development of endocrine, neurochemical, and cognitive measures in 60 day old rats.

Industrially employed PCB has caused wide-spread environmental contamination through improper disposal and has been associated with detrimental physiological states in exposed organisms, including depressed body weight, food consumption, and circulating levels of T4 and T3. Previously, the activity of choline acetyltransferase (ChAT) in the basal forebrain and hippocampus was shown to be depressed in young rats exposed to the PCB diet from the time of conception. The present study measured the neurochemical effects of similar PCB exposure in older (i.e., 60 day old) rats, and examined possible restoration of PCB-induced deficits by removing PCB at weaning (28 days). Possible PCB-induced impairment of memory was also evaluated with a radial arm maze. Findings included a significant depression of circulating levels of T4 in all treatment groups with the most profound depression seen in rats continuously fed PCB. Also, T3 levels and relative thyroid weights were not found to be severely depressed. The ChAT activity in both the basal forebrain and hippocampus was not different from control in all treatment groups. It appears that the effect of PCB on thyroxine is persistent, but its influence on ChAT activity is not. However, modest memory deficits were observed despite normal ChAT activity. Average number of working memory errors per test session in the maze increased in a dose-dependent manner across treatment groups.