A purified capsular polysaccharide markedly inhibits inflammatory response during endotoxic shock.

Capsular material of the opportunistic fungus Cryptococcus neoformans is composed mainly of a polysaccharide named glucuronoxylomannan (GXM). In this study, the effects of GXM were analyzed in an in vivo experimental system of lipopolysaccharide (LPS)-induced shock. Endotoxic shock was induced in mice by a single intraperitoneal injection of LPS from Escherichia coli. GXM treatment reduced the mortality of mice at early stages. Mice treated with LPS alone showed markedly increased plasma levels of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-6, whereas mice that were also treated with GXM showed significantly lower plasma levels of these cytokines. This effect was related to a marked suppression of Akt and IκBα activation. Importantly, the inhibitory effect of GXM on proinflammatory cytokine secretion was reproduced by treatment with wortmannin, an inhibitor of the Akt transcription pathway. Our results indicate that GXM has a beneficial effect on endotoxic shock, resulting in a significant increase in the rate of survival by dampening the hyperinflammatory response.

Association of pregnancy and Candida vaginal colonization in women with or without symptoms of vulvovaginitis.

AIM: Candida infection is one of the main causes of vulvovaginitis. The experience of symptoms of vulvovaginitis during pregnancy changes in relation to clinical, behavioral, and demographic factors. Candidiasis is associated with an increased risk of delivery complications. In some studies pregnant women are found more symptomatic than non-pregnant women, but in others a higher prevalence of asymptomatic infections is described during pregnancy. The aims of this study were to evaluate the prevalence of Candida vaginal colonization in pregnant women, and investigate if the occurrence of symptoms is influenced by pregnancy, in a population of Italian native and immigrant women. METHODS: A total of 344 outpatients, who visited the laboratory for routine genital examination, independently of pregnancy or presence or absence of symptoms of vulvovaginitis, were evaluated. RESULTS: Colonization by Candida spp. was significantly higher in pregnant than non-pregnant patients (31.4% vs. 19.9%; χ2=5.59; P=0.018), nevertheless pregnant women were significantly more often asymptomatic compared to non-pregnant (46.5% vs. 16%; χ2=42.31; P<0.0001). In the sub-group of women colonized by Candida spp., pregnancy resulted significantly associated to asymptomatic infection (58.1% vs. 30.8%; χ2 =6.18; P=0.013). A binary logistic regression analysis showed pregnancy or lactobacilli colonization independently associated to a lower probability of experiencing symptoms of vulvovaginitis (respectively: P<0.0001 and P=0.008). CONCLUSION: Pregnancy seems to be independently associated to Candida spp. asymptomatic vaginal infection. Given that candidiasis has been associated with possible delivery complications, these results suggest to screen for Candida spp. vaginal colonization asymptomatic women during pregnancy.

A critical role for FcgammaRIIB in up-regulation of Fas ligand induced by a microbial polysaccharide.

The microbial capsular polysaccharide glucuronoxylomannan (GXM) from the opportunistic fungus Cryptoccocus neoformans is able to alter the innate and adaptive immune response through multi-faceted mechanisms of immunosuppression. The ability of GXM to dampen the immune response involves the induction of T cell apoptosis, which is dependent on GXM-induced up-regulation of Fas ligand (FasL) on antigen-presenting cells. In this study we elucidate the mechanism exploited by GXM to induce up-regulation of FasL. We demonstrate that (i) the activation of FasL is dependent on GXM interaction with FcgammaRIIB (FcγRIIB); (ii) GXM induces activation of c-Jun NH(2) -terminal kinase (JNK) and p38 signal transduction pathways via FcγRIIB; (iii) this leads to downstream activation of c-Jun; (iv) JNK and p38 are simultaneously, but independently, activated; (v) FasL up-regulation occurs via JNK and p38 activation; and (vi) apoptosis occurs via FcγRIIB engagement with consequent JNK and p38 activation. Our results highlight a fast track to FasL up-regulation via FcγRIIB, and assign to this receptor a novel anti-inflammatory role that also accounts for induced peripheral tolerance. These results contribute to our understanding of the mechanism of immunosuppression that accompanies cryptococcosis.

Neutralizing antibody to interleukin 4 induces systemic protection and T helper type 1-associated immunity in murine candidiasis.

An interleukin 4 (IL-4)-specific monoclonal antibody (mAb) was administered to mice infected systemically with the yeast Candida albicans, and the animals were monitored for mortality, development of delayed-type hypersensitivity, production of antibodies of different isotypes, release of IL-2, IL-4, IL-6, and interferon gamma (IFN-gamma) in vitro by splenic CD4+ lymphocytes, and levels of IL-4 and IFN-gamma mRNA in these cells. Neutralization of IL-4 by three weekly injections of mAb in several independent experiments resulted in an overall cure rate of 81% versus 0% of controls. Cure was associated with efficient clearance of the yeast from infected organs and histologic evidence of disease resolution, detection of strong T helper type 1 (Th1) responses, and establishment of long-lasting protective immunity. Soon after infection, and as a result of the first or second injection of mAb, there was a decrease in IL-4 mRNA in CD4+ cells, which was accompanied by an increase in the levels of IFN-gamma-specific transcripts. Our data thus indicate that the production of IL-4 by Th2 cells may limit Th1-associated protective immunity in murine candidiasis.

Impaired neutrophil response and CD4+ T helper cell 1 development in interleukin 6-deficient mice infected with Candida albicans.

To define the role of interleukin (IL)6 in Candida albicans infection, IL-6 deficient mice were assessed for susceptibility to systemic or gastrointestinal infection, as well as for parameters of elicited T helper cell (Th) immunity. IL-6-deficient mice were more susceptible than wild-type mice to either type of infection caused by virulent C. albicans. In response to systemic challenge with a live vaccine strain of yeast, IL-6-deficient mice failed to mount Th1-associated protective immunity, but the resulting Th2-biased response could be redirected to the Th1 phenotype by IL-10 neutralization. Severe impairment of the macrophage and neutrophil response to infection was observed in IL-6-deficient mice, but administration of IL-6 would increase both neutrophil response and resistance to infection. IL-6 seems to oppose the Th2-promoting role of IL-10 in candidiasis, its early regulatory activity involving effects on neutrophil function.

Retroperitoneal abscess: an uncommon localization of tubercular infection.

We describe a rare case of a 29-year-old immunocompetent Nigerian male affected by an abdominal abscess due to Mycobacterium tuberculosis infection. Diagnosis was achieved with cultures from surgical drainage. No pulmonary, renal, or gastrointestinal involvement was identified. The patient was successfully treated with standard four-drug antitubercular therapy.

Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii.

The micronemal protein 2 (MIC2) of Toxoplasma gondii shares sequence and structural similarities with a series of adhesive molecules of different apicomplexan parasites. These molecules accumulate, through a yet unknown mechanism, in secretory vesicles (micronemes), which together with tubular and membrane structures form the locomotion and invasion machinery of apicomplexan parasites. Our findings indicated that two conserved motifs placed within the cytoplasmic domain of MIC2 are both necessary and sufficient for targeting proteins to T. gondii micronemes. The first motif is based around the amino acid sequence SYHYY. Database analysis revealed that a similar sequence is present in the cytoplasmic tail of all transmembrane micronemal proteins identified so far in different apicomplexan species. The second signal consists of a stretch of acidic residues, EIEYE. The creation of an artificial tail containing only the two motifs SYHYY and EIEYE in a preserved spacing configuration is sufficient to target the surface protein SAG1 to the micronemes of T. gondii. These findings shed new light on the molecular mechanisms that control the formation of the microneme content and the functional relationship that links these organelles with the endoplasmic reticulum of the parasite.

Relationships among tumor load, route of tumor inoculation, and response to immunochemotherapy in a murine lymphoma model.

The combined effects of nonspecific immunostimulation with Candida albicans (CA) and chemotherapy were studied in (BALB/cCr X DBA/2Cr)F1 and (C57BL/6Cr X DBA/2Cr)F1 mice bearing virus-induced LSTRA lymphomas. Paradoxically, animals treated with a relatively high number of tumor cells responded better to therapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) than those challenged with a low number of tumor cells. However, the majority of mice subjected to low initial tumor load were cured when they were treated with chemotherapy or chemotherapy plus booster injection of CA at a relatively "late" stage of the disease, i.e., when high tumor load was present in tumor-bearing hosts. It has been shown that this phenomenon, provisionally called high tumor load protection, occurs when the animals are challenged ip but not when they are challenged iv with the tumor and is abolished by total-body gamma-irradiation. Moreover, marked host protection can be attained when immunostimulated mice, inoculated iv with lymphoma cells, are subjected to simultaneous challenge with high inocula of the same tumor ip, followed by BCNU administration. These data stress the importance of the peritoneal cavity for successful CA plus drug treatment and suggest that optimal tumor "antigen load" should be present at the time of CA and/or BCNU administration.

A TH1-TH2-like switch in candidiasis: new perspectives for therapy.

An imbalance in TH1-type and TH2-type responses may allow Candida albicans to modify the host response to favor its own persistence. This hypothesis has important consequences for allergy, autoimmunity and co-infection, and also highlights a potential role for cytokine and anti-cytokine therapy in Candida-related pathology.

Glycosaminoglycan profile in macrophages exposed to Candida albicans and interleukins.

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity.

Normalization of anti-cryptococcal activity and interleukin-12 production after highly active antiretroviral therapy.

OBJECTIVE: To investigate the effect of highly active antiretroviral treatment (HAART) on antifungal and secretory functions of polymorphonuclear leukocytes (PMNL) from HIV-infected patients with high viral load. DESIGN: Antifungal activity, oxygen-dependent mechanisms and interleukin (IL)-12 secretion were evaluated in PMNL from HIV-infected patients before and 3 months after commencing HAART. METHODS: PMNL antifungal activity was evaluated by effects on fungal colony-forming units. Superoxide anion (O2-) production was determined by superoxide dismutase reduction and IL-12 was determined by enzyme-linked immunosorbent assay in supernatant fluids of PMNL cultured for 18 h. RESULTS: PMNL from HIV-infected patients showed dysregulation of antimicrobial and secretory functions. A selective defect in antimicrobial activity against encapsulated Cryptococcus neoformans correlated with baseline O2- overproduction, which drastically decreased upon microbial stimulation. Similarly, constitutive secretion of IL-12 was blocked by exposure to microbial products. PMNL analysed after 3 months of HAART showed restoration of antimicrobial activity against encapsulated C. neoformans, reduction in O2- formation by unstimulated cells and restoration of oxidative burst after appropriate stimulation, and reduction of IL-12 hypersecretion. CONCLUSIONS: PMNL from HIV-infected patients with high viral load have impaired function; HAART normalizes antimicrobial and secretory activities. The effects of HAART on innate immunity provide new prospects for reduction of HAART-mediated opportunistic infections.

Granulocyte-macrophage colony-stimulating factor and fluconazole enhance anti-cryptococcal activity of monocytes from AIDS patients.

OBJECTIVE: To investigate the effect of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and fluconazole on anti-cryptococcal activity of monocytes from AIDS patients and normal subjects. DESIGN: The effect of GM-CSF and fluconazole on fungistatic and fungicidal activity of monocytes was studied in an in vitro system. METHODS: Monocytes were treated in vitro with hrGM-CSF and fluconazole or either agent alone for 24 or 48 h, and fungistatic and fungicidal activity was evaluated in a colony-forming unit inhibition assay. CD11b/CD18 expression in monocytes was measured by flow cytometry analysis. Superoxide anion generation by peripheral blood monocytes was measured in the presence of pre-opsonized zymosan. RESULTS: Defective antifungal capacity of monocytes from AIDS patients was observed. GM-CSF treatment of monocytes from AIDS patients increased fungistatic activity, and the combination of hrGM-CSF and fluconazole resulted in fungicidal activity. The mechanisms involved in the GM-CSF-mediated effect appeared to be mediated by (i) enhancement of phagocytic activity, (ii) increase of superoxide anion generation, and (iii) upregulation of CD11b/CD18 expression on the monocyte surface. CONCLUSIONS: Our data highlight the effect of GM-CSF on anti-cryptococcal activity of human monocytes and show a synergistic effect of GM-CSF with fluconazole, suggesting a new therapeutic strategy in the treatment of cryptococcosis.

Antibody to capsular polysaccharide enhances the function of neutrophils from patients with AIDS against Cryptococcus neoformans.

OBJECTIVE: To determine the contribution of anti-glucuronoxylomannan monoclonal antibody (MAb18B7) to the fungicidal capacity of polymorphonuclear leukocytes (PMNL) from HIV-infected patients towards Cryptococcus neoformans. DESIGN: Killing activity and superoxide anion generation were evaluated in the presence or absence of MAb18B7 in an in vitro system. METHODS: Killing activity was determined by colony forming unit inhibition assay. Superoxide generation was measured in the presence or absence of zymosan, C. neoformans, or Candida albicans. CD16, CD32, and CD64 molecules on PMNL were evaluated by cytofluorometric analysis. RESULTS: MAb18B7 strongly influenced the phagocytic and killing activities against encapsulated C. neoformans and consistently enhanced superoxide anion generation. Expression of CD16, and to a lesser extent CD64, on PMNL was required for MAb18B7-induced superoxide generation. By blocking CD16 and CD64 molecules with anti-CD16 and anti-CD64 MAb, a significant down-regulation of MAb18B7-induced fungicidal activity was observed. CONCLUSIONS: Our results demonstrate that MAb18B7 selectively enhances the killing mechanisms of PMNL from HIV-infected patients against encapsulated C. neoformans. The availability of CD16 and CD64 molecules on PMNL plays a critical role.

Inhibition of fungicidal activity of polymorphonuclear leukocytes from HIV-infected patients by interleukin (IL)-4 and IL-10.

OBJECTIVE: To investigate the effect of human recombinant interleukin (hrIL)-4 or hrIL-10 on the functional status of polymorphonuclear leukocytes (PMNL) from normal subjects and HIV-infected patients. DESIGN: In an in vitro system we studied the effect of hrIL-4 or hrIL-10 on phagocytosis, fungicidal activity and superoxide anion production by PMNL. METHODS: PMNL were treated in vitro with hrIL-4 or hrIL-10 or their combination for 6 h and then candidacidal activity was evaluated in a colony-forming unit inhibition assay. Superoxide anion generation by PMNL was measured in the presence or absence of preopsonized zymosan or Candida albicans. RESULTS: Treatment in vitro with hrIL-4 or hrIL-10 of PMNL for 6 h was able to impair candidacidal activity of neutrophils in both normal or HIV-infected patients. The inhibitory effect was time- and dose-dependent and was more evident in PMNL from HIV-infected subjects, and reflected in these latter cells a decrease of superoxide anion generation. The impairment of candidacidal activity in PMNL from HIV-infected patients was accompanied by survival of the yeasts shown by budding formation into phagosomic organelles of cytokine-treated PMNL. CONCLUSIONS: Our data highlight new biological effects of IL-4 and IL-10 evidenced by suppressed effector function of neutrophils; this phenomenon is emphasized in HIV-infected patients suggesting a role for these cytokines in mediating increased susceptibility to microbial infection during AIDS progression.

T helper cell dichotomy to Candida albicans: implications for pathology, therapy, and vaccine design.

Acquired immunity to Candida albicans is believed to prevent mucosal colonization of adult immunocompetent individuals from progressing to symptomatic infection. Resistance to disease appears to correlate with the detection of delayed-type hypersensitivity responses in vivo and a T helper type 1 (Th1) cytokine secretion profile in vitro. Cellular immunodeficiency, particularly HIV infection, greatly increases the risk of mucosal infection, confirming that CD(4+)-cell-directed immunity is effective locally in controlling infectivity of the yeast. While Th1-type CD4+ cell activation resulting in phagocyte-dependent immunity clearly represents an important mechanism of anticandidal resistance, clinical observations suggest that Th2-type CD4+ cell reactivity may be triggered by Candida antigens in several disease states, including symptomatic infections and immunopathology. This may imply that a Th1-type pattern of reactivity characterizes the saprophytic yeast carriage and resistance to disease by healthy humans, whereas Th2-type responses would be mostly associated with pathology. Moreover, Candida-specific T helper responses, namely humoral and cell-mediated immunity, appear to be reciprocally regulated, as typically occurs in experimental models of parasitic and retroviral infection, where the Th1/Th2 paradigm of acquired immunity has been best characterized. Recent studies, besides providing direct evidence for the occurrence of cross-regulatory Th1 and Th2 responses in mice with candidiasis, emphasize the potential of cytokine/anticytokine therapy for recruiting Candida-specific responses toward protective, Th1-type CD4+ cell reactivity. At the same time, these studies call attention to the possible consequences of C. albicans infection for immunopathology, allergy, and coinfection.

A radiolabel release microassay for phagocytic killing of Candida albicans.

The chromium release technique for quantifying intracellular killing of radiolabelled Candida albicans particles was exploited in a microassay in which murine and human phagocytes acted as effectors under peculiarly simple conditions. At appropriate effector: target ratios and with a 4 h incubation, up to 50% specific chromium release could be detected in the supernatant with no need for opsonization or lysis of phagocytes. This simple microassay permits easy-to-perform, simultaneous testing of a variety of different phagocytes even if only available in limited amounts, and provides an objective measurement of intracellular killing of Candida albicans.

A rapid objective immunofluorescence microassay. Application for detection of surface and intracellular antigens.

An indirect immunofluorescence microassay, which permits automated reading, has been employed for simple, rapid and objective detection of surface and intracellular antigens. Initially, the cells, spun in microplates, are fixed with glutaraldehyde (0.25% v/v in PBS). Following fixation, the cells can be stored at 4 degrees C for up to 2 weeks before being used in the immunofluorescence microassay. The fixed cells are then stained according to standard procedures using appropriate first and fluorescein-conjugated second antibodies. An automated and quantitative evaluation of the fluorescence intensity of the cell samples was achieved using the Titertek Fluoroskan II automatic reader. This microassay was shown to be suitable for the detection of the surface MAC1 antigen and intracellular v-myc protein in the GG2EE macrophage cell line.

Microglial cell-mediated anti-Candida activity: temperature, ions, protein kinase C as crucial elements.

An in vitro established microglial cell line, BV-2, constitutively exhibits high levels of anti-Candida activity. To elucidate the cascade of events leading to the accomplishment of such activity, we studied its dependence on temperature and ion availability. The role of protein kinases has also been studied by the specific inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and N-(2-guanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA 1004). We found that (a) the BV-2 cell/Candida conjugate formation is a discrete step, temperature-, ion- and protein kinase-independent; (b) the phagocytic event, which is protein kinase-independent, is significantly impaired by temperature decrease and ion deprivation; (c) the fulfillment of anti-Candida effects is strictly dependent upon temperature, ion availability and functional protein kinase. Functional protein kinase C, but not other kinases, is required for the accomplishment of anti-Candida activity, which, in fact, is selectively abrogated by H7 but not HA. Furthermore, protein kinase C activators, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1-oleoyl-2-acetyl glycerol (OAG), consistently potentiate BV-2 cell-mediated anti-Candida activity, the phenomena being dose-dependent. These results indicate that the multistep events leading a microglial cell to express anti-Candida activity can be dissected and differentiated for biochemical and biological demands, the latest along the cascade being the most demanding steps.

Pattern of cytokine gene expression in brains of mice protected by picolinic acid against lethal intracerebral infection with Candida albicans.

Recently, we demonstrated that intracerebral (i.c.) administration of picolinic acid (PLA) confers protection against a lethal local challenge with the opportunistic pathogen Candida albicans. By histopathological studies, we show here that mice receiving PLA treatment survive challenge and no evidence of fungal invasion is found within the brain compartment. In contrast, PLA-untreated mice succumb to infection within 7-10 days and show massive brain colonization with extensive granulomatous reaction. By PCR analysis, we show that, unlike naive brains, PLA-treated brains show transient activation of TNF alpha, IL-1 beta and IL-6 genes. C. albicans infection results in high levels of all cytokine transcripts, the phenomenon being long-lasting in PLA-untreated brains, while gradually declining in PLA-treated brains. The only exception is IL-1 beta, whose levels remain high at the latest time-points tested, also in PLA-treated brains. Finally, IL-1 alpha, constitutively detectable in naive brains, is slightly enhanced by C. albicans challenge, regardless of prior treatment. These findings, together with the knowledge that PLA is a potent co-stimulus for macrophages, suggest the involvement of cytokine circuits, likely of macrophage origin, in anti-Candida resistance established by PLA at the cerebral level.

Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus.

A murine cell line (BV-2) has been generated by infecting primary microglial cell cultures with a v-raf/v-myc oncogene carrying retrovirus (J2). BV-2 cells expressed nonspecific esterase activity, phagocytic ability and lacked peroxidase activity. Such cells secreted lysozyme and, following appropriate stimulation, also interleukin 1 and tumor necrosis factor. Furthermore, BV-2 cells exhibited spontaneous anti-Candida activity and acquired tumoricidal activity upon treatment with interferon-gamma. Phenotypically, BV-2 cells resulted positive for MAC1 and MAC2 antigens, and negative for MAC3, glial fibrillary acidic protein (GFAP) and galactocerebroside (GC) antigens. Since BV-2 cells retain most of the morphological, phenotypical and functional properties described for freshly isolated microglial cells, we can conclude that J2 virus infection has resulted in the immortalization of active microglial cells.