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Fungal infections have been increasing and life-threatening in recent years, but host immune responses, especially the humoral immunity, to fungi have not been fully understood. In the present study, we report that natural antibodies from unimmunized mice bind to Candida albicans. We established a monoclonal natural antibody, 3B4, which recognized a surface antigen located at germ tubes of C. albicans. The 3B4 antibody protected mice from C. albicans-induced death in passive immunization, by mechanisms involving suppressing germ tube formation and modulating phagocytosis. Interestingly, 3B4 also bound to a self-antigen keratin. To further study the generation and anti-C. albicans activities of natural antibodies in vivo, we constructed a mu chain transgenic mouse (TgV(H)3B4) using the V(H) gene from 3B4. TgV(H)3B4 had elevated serum anti-keratin/C. albicans IgM, and were resistant to C. albicans infections. Analyses of B cell development showed that in TgV(H)3B4, B cells secreting the anti-keratin/C. albicans antibodies were enriched in the B1 B cell compartment. Our findings reveal an important role of keratin-reactive natural antibodies in anti-C. albicans immune responses, and suggest that keratin may function in selecting B cells into the B1 B cell compartment, where natural antibodies are made to fight fungal infections.

Aim: To express human anti-keratin Fab in Pichia pastoris secretively and optimize the expression condition., Methods: Genes of kappa chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/Fab were subcloned into vectors pPIC9K and pPICZalphaA respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/L and pPICZalphaA/Fd were transducted into the genome of GS115 Pichia pastoris using two-step integrating technology. Mut(+) multiple insert transformants were screened by G418 and Zeocin. The pH value and methanol concentration was adjusted to optimize the expression condition., Results: Under the optimized expression condition, the Fab of anti-keratin antibody was efficiently secreted into the medium. Western blot assay proved that the expressed protein had specific keratin binding activity., Conclusion: The successful expression of the anti-keratin Fab in Pichia pastoris has laid a solid foundation for its further application.

Aim: To analyze the reaction patterns of IgG class of anti-keratin autoantibodies (AK auto Abs) in normal human sera before and after purification, and to explore the effects of serum non-IgG components on the reaction patterns of IgG class of AK auto Abs., Methods: Titers of IgG AK auto Abs in healthy human sera before and after purification were measured by indirect ELISA, and the reaction patterns of the AK auto Abs to a group of keratins with different relative molecule mass were analysed by Western blot., Results: Titers of purified IgG class of AK auto Abs were higher than those of unpurified sera. Western blot analysis showed purified AK auto Abs recognized more keratins with stronger avidity than unpurified AK auto Abs. The reaction patterns of AK auto Abs from different individuals tended to be homogenous after purification., Conclusion: The reaction patterns of serum IgG class of AK auto Abs are influenced by some non-IgG components in sera. The homogeneity of reaction patterns of AK auto Abs from different individuals indicated that B cells producing IgG class of AK auto Abs may be selected by a set of conserved self-antigens, and this process may have no relationship with an individual's exposure to foreign antigens.

We found previously that ACh can significantly inhibit the proliferation of cultured human pituitary adenoma cells. In order to make a further investigation of the mechanism of the inhibitory effect of ACh on the proliferation of pituitary adenoma cells, we observed the levels of protein kinase C (PKC), [Ca(2+)](i) and cAMP/cGMP in cultured pituitary adenoma cells after treatment with ACh. The results demonstrate that (1) compared with control, PMA, a PKC activator, increased the activity of cytoplasm, membrane and total PKC in human pituitary adenoma cells. However, after a 15-min treatment with ACh (10 micromol/L), a significant reduction of the activity of cytoplasm, membrane and total PKC in human pituitary adenoma cells was observed, and the reduction effect could be blocked by atropine. (2) The level of [Ca(2+)](i) of single adenoma cells was found to decrease immediately on the addition of ACh (10 micromol/L), which could also be blocked by atropine. (3) ACh increased the amount of cAMP in the cytoplasm of human pituitary adenoma cells, but had no effect on that of cGMP. These data provide an important clue to explore the molecular mechanisms of the inhibitory effect of ACh on the proliferation of pituitary adenoma cells, and suggest that the modulating effect of ACh on the proliferation of pituitary adenoma cells results from the interactions of several cellular signaling pathways.

In order to elucidate the effect of acetylcholine (ACh) on the occurrence and development of human pituitary adenoma, it was firstly observed whether there exists choline acetyl transferase (ChAT) which is necessary for the synthesis of acetylcholine in the cells of human pituitary adenoma, and then MTT method, (3)H TdR incorporation, cell cycle analysis and TUNEL were employed to estimate the influence of ACh on the proliferation, DNA synthesis and apoptosis of three kinds of human pituitary adenoma (human prolactinoma, somatotropinoma and non-functional tumor) cells cultured in vitro. The results showed that (1) the positive staining of ChAT was obviously observed in the cells of the three kinds of human pituitary adenoma, however, it was lower than that in normal human pituitary gland; (2) ACh had a similar effect on the proliferation of the three kinds of human pituitary adenoma cells. ACh at 0.1-10 micromol/L decreased the (3)H TdR incorporation and the MTT A value in a dose-dependent manner. At the same time, ACh decreased the ratio of S or G(2) phase pituitary adenoma cells significantly, but increased the ratio of G(1) phase pituitary tumour cells markedly; (3) the effect of acetylcholine on the proliferation of human pituitary adenoma cells was inhibited by atropine, but not by tubocurarine; (4) ACh had no effect on the apoptosis of human pituitary adenoma cells cultured in vitro. These data suggest that ACh may have a significant modulating effect on the proliferation of pituitary adenoma cells by means of paracrine or autocrine, and the effect is mediated by muscarinic receptor.