Glucuronoxylomannan (GXM) is the major component of Cryptococcus capsular polysaccharide, which represents an essential virulence factor for this yeast. Cryptococcus neoformans infections in immunocompetent rats are associated with inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by macrophages. This study demonstrates in vitro and in vivo that GXM promotes iNOS expression with NO production in rat macrophages. GXM also induced macrophage apoptosis after 48 h of culture, with this phenomenon being prevented by the iNOS inhibitor, aminoguanidine. The NO-induced macrophage apoptosis triggered by GXM was dependent on interactions with CD18, Fcgamma receptor II and protein kinase C activation, without participation of tyrosine kinases or mitogen-activated protein kinases. Furthermore, this study reveals that GXM down-regulates the macrophage caspase-3 activity, induces a caspase-independent cell death and promotes depolarization of mitochondria membrane potential with increased cytosolic expression of the apoptosis-inducing factor. Taken together, this study describes the pathways and mechanisms involved in the macrophage apoptosis promoted by GXM through NO generation. These findings indicate new mechanisms of immunomodulation for the main capsular polysaccharide of C. neoformans.

Summary Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, revealing a strong granulomatous response and a low susceptibility to dissemination. Moreover, it has been shown that eosinophils are components of the inflammatory response to C. neoformans infections. In this in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, and that the phenomenon involves the engagement of FcγRII and CD18. Moreover, our results showed that the phagocytosis of opsonized C. neoformans triggers eosinophil activation, as indicated by (i) the up‐regulation of major histocompatibility complex (MHC) class I, MHC class II and costimulatory molecules, and (ii) an increase in interleukin (IL)‐12, tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) production. However, nitric oxide (NO) and hydrogen peroxide (H2O2) synthesis by eosinophils was down‐regulated after interaction with C. neoformans. Furthermore, this work demonstrated that CD4+ and CD8+ T lymphocytes isolated from spleens of infected rats and cultured with C. neoformans‐pulsed eosinophils proliferate in an MHC class II‐ and class I‐dependent manner, respectively, and produce important amounts of T‐helper 1 (Th1) type cytokines, such as TNF‐α and IFN‐γ, in the absence of T‐helper 2 (Th2) cytokine synthesis. In summary, the present study demonstrates that eosinophils act as fungal antigen‐presenting cells and suggests that C. neoformans‐loaded eosinophils might participate in the adaptive immune response.

SUMMARYGlucuronoxylomannan (GXM) is the major Cryptococcus neoformans capsular polysaccharide and represents the main virulence factor of this fungus. In in vitro studies we have demonstrated previously that this acidic and high-molecular-weight polysaccharide suppresses lymphoproliferation, modulates cytokine production and promotes apoptosis in spleen mononuclear (Spm) cells from rats. In this study we demonstrate that these phenomena also occur in vivo after the intracardiac inoculation of GXM into normal Wistar rats. The results of this study show suppression of the proliferative response Spm cells to concanavalin A (Con A) or heat-killed C. neoformans (HKCn) in the first 2 weeks after polysaccharide administration. In addition, increased levels of interleukin (IL)-10 were produced by Con A-stimulated Spm cells, coinciding with immunohistochemical GXM detection in the white pulp of spleen. In particular, high production of IL-10 with diminution of IL-2, interferon (IFN)-c and tumour necrosis factor (TNF)-a synthesis were detected 14 days after GXM administration. In situ cell death detection by TdT-mediated biotin-dUTP nick-end labelling (TUNEL) reaction in sections of spleen, lung and liver demonstrates apoptosis in tissues with deposits of GXM. These data demonstrate the in vivo ability of GXM to modify cytokine synthesis by Spm cells and to promote host cell apoptosis.

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The present study was designed to determine the relationships among biofilm formation, cellular stress and release of Shiga toxin (Stx) by three different clinical Shiga toxin-producing Escherichia coli (STEC) strains. The biofilm formation was determined using crystal violet stain in tryptic soy broth or thioglycollate medium with the addition of sugars (glucose or mannose) or hydrogen peroxide. The reactive oxygen species (ROSs) were detected by the reduction of nitro blue tetrazolium and reactive nitrogen intermediates (RNI) determined by the Griess assay. In addition, the activities of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied. For the cytotoxicity studies, Vero cells were cultured with Stx released of STEC biofilms. The addition of sugars in both culture mediums resulted in an increase in biofilm biomass, with a decrease in ROS and RNI production, low levels of SOD and CAT activity, and minimal cytotoxic effects. However, under stressful conditions, an important increase in the antioxidant enzyme activity and high level of Stx production were observed. The disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx evaluated under different conditions of biofilm formation may contribute to a better understanding of the relevance of biofilms in the pathogenesis of STEC infection.

Summary Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, because as in healthy humans, rats can effectively contain cryptococcal infection. Moreover, it has been shown that eosinophils are components of the immune response to C. neoformans infections. In a previous in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, thereby triggering their activation, as indicated by the up‐regulation of MHC and co‐stimulatory molecules and the increase in interleukin‐12, tumour necrosis factor‐α and interferon‐γ production. Furthermore, this work demonstrated that C. neoformans‐specific CD4+ and CD8+ T lymphocytes cultured with these activated C. neoformans‐pulsed eosinophils proliferated, and produced important amounts of T helper type 1 (Th1) cytokines in the absence of Th2 cytokine synthesis. In the present in vivo study, we have shown that C. neoformans‐pulsed eosinophils are also able to migrate into lymphoid organs to present C. neoformans antigens, thereby priming naive and re‐stimulating infected rats to induce T‐cell and B‐cell responses against infection with the fungus. Furthermore, the antigen‐specific immune response induced by C. neoformans‐pulsed eosinophils, which is characterized by the development of a Th1 microenvironment with increased levels of NO synthesis and C. neoformans‐specific immunoglobulin production, was demonstrated to be able to protect rats against subsequent infection with fungus. In summary, the present work demonstrates that eosinophils act as antigen‐presenting cells for the fungal antigen, hence initiating and modulating a C. neoformans‐specific immune response. Finally, we suggest that C. neoformans‐loaded eosinophils might participate in the protective immune response against these fungi.

Shiga toxin-producing Escherichia coli are important food-borne pathogens. The main factor conferring virulence on this bacterium is its capacity to secrete Shiga toxins (Stxs), which have been reported to induce apoptosis in several cell types. However, the mechanisms of this apoptosis have not yet been fully elucidated. In addition, Stxs have been shown to stimulate macrophages to produce nitric oxide (NO), a well-known apoptosis inductor.The aim of this study was to investigate the participation of NO in apoptosis of rat peritoneal macrophages induced by culture supernatants or Stx2 from E. coli. Peritoneal macrophages incubated in the presence of E. coli supernatants showed an increase in the amounts of apoptosis and NO production. Furthermore, inhibition of NO synthesis induced by addition of aminoguanidine (AG) was correlated with a reduction in the percentage of apoptotic cells, indicating participation of this metabolite in the apoptotic process. Similarly, treatment of cells with Stx2 induced an increase in NO production and amount of apoptosis, these changes being reversed by addition of AG. In summary, these data show that treatment with E. coli supernatants or Stx2 induces NO-mediated apoptosis of macrophages. Key words Escherichia coli, hemolytic uremic syndrome, Shiga toxin 2.Shiga toxin-producing Escherichia coli infections are responsible for widespread disease, including HUS, which is characterized by thrombocytopenia, microangiopathic hemolytic anemia and renal failure (1-3). During intestinal infection, E. coli secretes different products into the intestinal lumen, including LPS and its main virulence factor, Stx (2). The latter is a holotoxin composed of an enzymatic A subunit (StxA) in association with five B subunits (StxB) (4). This toxin translocates across the intestinal epithelial cell layer into the circulation, allowing it to act on different cell populations at distant sites, such as the kidney and the brain (5). Numerous effects on different cellular populations have been attributed to Stx, including cytokine production, secretion of ROI such as List of Abbreviations: AG, aminoguanidine; CHOP, C/EBP homologous protein; E coli, Escherichia coli; ER, endoplasmic reticulum; HUS, hemolytic uremic syndrome; iNOS, inducible nitric oxide synthase; JNK, Jun N-terminal kinase; LPS, lipopolysaccharide; NO, nitric oxide; PI, propidium iodide; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates;rpm, revolutions per minute; SEM, standard error of the means; Stx2, Shiga toxin 2; TSB, tryptic soy broth the superoxide anion (O 2 − ), liberation of RNI such as NO, and apoptosis (6-9).Apoptosis (programmed cell death) is a phenomenon that has been demonstrated under both physiological and pathological conditions. This process is characterized by changes in cell morphology, chromatin condensation, nuclear DNA fragmentation, and apoptotic body formation (10). Induction of apoptosis occurs through two main apoptotic pathways: the extrinsic, or death receptor, pathway and the...

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