Antibody responses to Cryptococcus neoformans in Indian patients with cryptococcosis.

An important element of the host response to cryptococcosis is humoral immunity. Specific antibody responses in patients with cryptococcosis however, have not been extensively studied. We analyzed the antibody responses of 22 Indian patients with cryptococcosis, including both HIV+ and HIV- individuals. Sera from 10 Indian patients with AIDS and without cryptococcosis were studied as controls. Antibody responses to cryptococcal proteins were detected by immunoblot, while antibodies to glucuronoxylomannan (GXM), the main component of the cryptococcal capsular polysaccharide were measured by ELISA. Our results indicate that cryptococcosis elicits antibodies to a specific pattern of cytoplasmic proteins. Further, we find that antibody responses to both cytoplasmic proteins and GXM are less robust in HIV+ patients when compared with HIV- patients.

Pulmonary cryptococcosis induces chitinase in the rat.

BACKGROUND: We previously demonstrated that chronic pulmonary infection with Cryptococcus neoformans results in enhanced allergic inflammation and airway hyperreactivity in a rat model. Because the cell wall of C. neoformans consists of chitin, and since acidic mammalian chitinase (AMCase) has recently been implicated as a novel mediator of asthma, we sought to determine whether such infection induces chitinase activity and expression of AMCase in the rat. METHODS: We utilized a previously-established model of chronic C. neoformans pulmonary infection in the rat to analyze the activity, expression and localization of AMCase. RESULTS: Our studies indicate that intratracheal inoculation of C. neoformans induces chitinase activity within the lung and bronchoalveolar lavage fluid of infected rats. Chitinase activity is also elicited by pulmonary infection with other fungi (e.g. C. albicans), but not by the inoculation of dead organisms. Enhanced chitinase activity reflects increased AMCase expression by airway epithelial cells and alveolar macrophages. Systemic cryptococcosis is not associated with increased pulmonary chitinase activity or AMCase expression. CONCLUSION: Our findings indicate a possible link between respiratory fungal infections, including C. neoformans, and asthma through the induction of AMCase.

[Mutation of arginines near the active site Cys124 of human dual-specificity phosphatase and its effect on the enzymatic activity].

To study the effect of three positively charged arginine residues near the active site Cys(124) of the human dual-specific phosphatase on the catalytic function, six VHR mutants R(125)L, R(130)L, R(130)K, R(130)L/S(131)A, R(158)K and R(158)L were obtained using QuikChange site-directed mutagenesis method. The recombinant plasmids containing mutant genes were transformed into the Escherichia coli strain BL21(DE3), and the expressed proteins were found to be water soluble after the induction of IPTG. The proteins with purity greater than 90% were obtained using Ni(2+) chelating affinity chromatography. The measurement of the steady-state kinetic parameters and arsenate inhibition constants K(i) of the enzyme and their mutants showed that the k(cat)/K(m) values of Arg(130) and Arg(158) mutants decreased, and K(i) values increased obviously compared with those of the wild enzyme. These results indicated that Arg(130) and Arg(158) were necessary for the enzymatic activity, and were probably related to the binding with the negatively charged phosphate group of the substrate. In addition, the slight difference for the k(cat) values between the R(130)L and R(130)L/S(131)A mutants suggested that Arg(130) mutation disrupted the hydrogen bond between Ser(131) and Cys(124). Furthermore, the arsenate binding affinity for R(125)L, R(130)L and R(158)L mutants was decreased, suggesting that positive charges in the side chains of these three arginine residues may be helpful for the binding of the enzyme to the substrate.