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.

Saccharomyces cerevisiae-based probiotic as novel anti-fungal and anti-inflammatory agent for therapy of vaginal candidiasis.

Previously we demonstrated that the treatment with live Saccharomyces cerevisiae exerts beneficial therapeutic effects against vaginal candidiasis. Here, we address potential mechanisms particularly examining the probiotic capacity to modulate both fungus and host-related factors. We show that the S. cerevisiae-based probiotic markedly affects the expression of virulence traits of Candida albicans such as aspartyl proteinases (SAPs) as well as hyphae-associated proteins Hwp1 and Ece1 in the vaginal cavity. On the host side, the probiotic suppression of the influx of neutrophils caused by the fungus into the vaginas of the mice is likely related to: (1) lower production of interleukin-8; and (2) inhibition of SAPs expression. However, these neutrophils displayed reactive oxygen species hyperproduction and increased killing activity as compared to the neutrophils of placebo-treated mice. There was no evidence of any cytotoxic effect by the probiotic, either when used in vivo on vaginal epithelial cell and organ architecture, or in in vitro in human vaginal epithelium. Inactivated yeast cells did not affect any of the factors above. In summary, the data suggest that the beneficial effect exerted by this S. cerevisiae-based probiotic is the result of its interference with the expression of fungus virulence factors coupled with the modulation of the inflammatory response of the host.

NO synthesis in human saliva.

Human saliva contains nitrate that is converted into nitrite by the activity of facultative, anaerobic bacteria of the oral cavity. Nitrite can be reduced to NO in the acidic gastric milieu; some NO may also form in the mouth at acidic pH values. In this paper, we show that bacteria (S. salivarius, S. mitis and S. bovis) isolated from saliva, may contribute to NO production in human saliva. NO formation by bacteria occurs at neutral pH values and may contribute to the antibacterial activity of saliva.

Involvement of host macrophages in the immunoadjuvant activity of amphotericin B in a mouse fungal infection model.

We have recently reported the in vivo augmentation of resistance to experimental Candida albicans injection by amphotericin B in mice and have shown that this event is concurrent with the appearance in the spleen of a highly candidacidal cell population reactive in vitro against 51Cr-labeled yeast cells. In the present study we characterize these in vitro fungicidal effectors as macrophages and describe the conditions of amphotericin B treatment most suitable for inducing candidacidal activity. We also report that macrophages from intact mice can be activated in vitro to become cytotoxic against Candida. The possible mechanisms through which the amphotericin B activated macrophages exert their increased anti-Candida activity are also investigated.

[The role of Streptococcus mutans in human caries]. / Ruolo dello Streptococcus mutans nella carie umana.

Definite proof has been found that S. mutans, and probably S. sobrinus and lactobacilli, are responsible for caries in man. The aciduria of S. mutans encourages the selection inside the plaque and is directly responsible for its cariogenicity. Other aciduric species, as S. sobrinus, cause caries on the smooth surface of the tooth, where the principal causes of the rampant caries reside. If during the eruption of the tooth the fissures become colonized in depth by S. mutans, the development of caries becomes a highly probable event. Instead, if the colonization of the tooth by S. mutans takes place after the fissure depth have been occupied by other microbic species not cariogenic, it is probable that the caries will not manifest or will appear in a less severe form. In conclusion, the knowledge already acquired on the ecology of S. mutans and the mechanism of cariogenesis clearly indicate that all factors that interfere with the colonization of the tooth by S. mutans can greatly reduce the incidence of caries in man.

[Use of BCG as an immunoadjuvant in combination with antitumor agents in virus-induced leukemia]. / L'uso del BCG come immunoadiuvante in combinazione con farmaci antitumorali in unal leucemia virus indotta.

The effect of administration of BCG in association with chemotherapy in histocompatible CD2F1 mice challenged ip with Moloney-virus-induced lymphoma LSTRA of Balb/c origin was studied. All untreated mice died with comparable median survival time (MST). Immunochemotherapy experiments were performed in histocompatible mice using BCG according to various treatment schedules with respect to tumor challenge and 3 nitrosureas of clinical interest (i.e. BCNU, MeCCNU and CCNU) administration. If recipients were subjected to ip treatment with drugs alone or in association with the non specific immunoadjuvant (IA) given after tumor challenge (on day +1); no significant antitumor effect was detected. Synergistic antitumor effects were evidenced when the antineoplastic agents were associated with IA administered on the "-14+1" regimen with respect to the tumor. The results pointed out that the antilymphoma effects of chemotherapy could be amplified by IA only when the treatment schedule included adjuvants administration prior to tumor challenge.

[The use of BCG as immunoadjuvant in combination with antitumor drugs in a virus induced leukemia. II]. / L'uso del BCG come immunoadiuvante in combinazione con farmaci antitumorali in una leucemia virus indotta. II.

Various treatment schedules of BCG with respect to tumor challenge and drug administration were applied in a histocompatible tumor-host system. LSTRA, an ascitic lymphoma induced by Moloney leukemia virus in BALB/c mice, was inoculated ip in histocompatible CD2F1 mice. BCG was administered ip before and/or after (-14, +1, -14+1) the tumor challenge. The drugs used in our experiments: cyclophosphamide (CY), iphosphamide (IPHO), nitrogen mustard (NM), were given at graded doses on day +5. In our experimental system the BCG treatment schedule (-14+1) only showed synergistic antitumor effects at defined doses: only the association BCG-CY have no significant survival percentage increase. No synergistic antitumor activity was evidenced when the drugs were associated with BCG given 14 days before or 1 day after the tumor challenge. The degree of immunochemotherapy treatment efficacy was different according to various antineoplastic agents used. It was never found any treatment schedule was able to cure experimental mice with the best survival percentage increase.

Drug-induced modulation of IL-2 production in experimental murine trypanosomiasis.

In this study we evaluated the effects of N-acetyl-cysteine and indomethacin in restoring IL-2 producing ability in vitro of splenocytes from mice infected with Trypanosoma equiperdum. Spleen cells from these mice were found to produce significantly lower levels of interleukin-2 (IL-2) in response to mitogen stimulation than spleen cells from uninfected control mice. This was accompanied by considerable suppression of IL-2-receptor expression, which was not attributable to the elimination of a particular T-cell subset. Impairment of IL-2 production was not due to a primary defect in L3T4+ T-cells, but rather to the presence of both adherent and non-adherent suppressor cells that apparently acted via prostaglandin-independent and dependent mechanisms. In fact, the IL-2-producing ability of lymphocytes from infected mice could be efficiently restored by in vitro exposure to N-acetyl-cysteine or indomethacin.

Cytotoxic T lymphocyte antigen costimulation influences T-cell activation in response to Cryptococcus neoformans.

The kinetics of cytotoxic T lymphocyte antigen 4 (CTLA-4) expression on T cells responding to Cryptococcus neoformans and its role in regulating the T-cell response were examined. Using peripheral blood mononuclear cells stimulated with encapsulated or acapsular C. neoformans we showed that (i) the encapsulated strain augmented CTLA-4 expression on the T-cell surface while the acapsular strain was a weaker modulator, (ii) CTLA-4 molecules were rapidly up-regulated after the addition of encapsulated C. neoformans, (iii) CTLA-4 was up-regulated predominantly in CD4+ T cells responding to C. neoformans, and (iv) blockage of CTLA-4 with (Fab')2 of monoclonal antibody to CTLA-4 induced T-cell proliferation that paralleled the enhancement of interleukin-2 and gamma interferon production. These results suggest that capsular material, the major virulence factor of C. neoformans, promotes synthesis and expression of CTLA-4 molecules predominantly in CD4+ T cells. CTLA-4-mediated deactivation is due not to lack of costimulation but to specific recognition of CTLA-4 for B7 molecules. This appears to be a new mechanism by which C. neoformans may elude the host immune response.

[Pathogenicity of various species of Candida in an experimental murine system]. / Patogenicità di differenti specie di Candida in un sistema sperimentale murino.

The systemic infection induced by Candida albicans, Candida krusei and Candida viswanathii was studied in an experimental murine system. Candida albicans is able to kill outbred CD1 mice within a few days and at a very low concentration; C. krusei is not pathogenic not even when inoculated at a higher concentration; C. viswanathii is able to kill animals only a a higher concentration. The different resistances do not seem to be under genic control, in as much as the different strains of mice used (hybrid CD2F1 and B6C3HF1, inbred Balb/c) show the same degree of resistance as the CD1 mice to the three species of Candida. The colony forming units (CFU) in the kidneys of CD1 mice inoculated intravenously with 10(5) cells of the three species of Candida, collected at various intervals showed a good correlation with the median survival times: a rapid moltiplication of the C. albicans is evident in the kidneys of the animals 24 hours after the inoculation, while the C. krusei and the C. viswanathii do not moltiply.

[Pathogenicity of various species of Candida in mice immunodepressed with cyclophosphamide]. / Patogenicita' di specie differenti di Candide in topi immunodepressi con ciclofosfamide.

The resistance of outbred CD1 mice immunodepressed with cyclophosphamide to the systemic infection induced by the three species of Candida: C. albicans, C. krusei and C. viswanathii was studied. The administration of cyclophosphamide, three days before the challange of the Candida species causes a degree of immunodepression in relation to the dose of drug: decrease of leucocytes of the peripheral blood and decrease of the weight and of the cellularity of the spleens. The CD1 mice were immunodepressed with cyclophosphamide (150mg/Kg) three days before the intravenous challange with graded doses of cells of the three species of Candida. They are much more susceptible, compared to normal mice, to the systemic infection induced by C. albicans and also by C. viswanathii but in this case only when inoculated at the highest concentration. They are resistant, like normal mice, towards the C. krusei even in the case in which the degree of immunodepression of the animal is furtherly increased (with stronger doses of the drug). The number of colony forming units (CFU) from the kidneys of the immunodepressed mice with cyclophosphamide correlates the median survival times.

A comparison of experimental pathogenicity of Candida species in cyclophosphamide-immunodepressed mice.

The experimental pathogenicity of Candida albicans, C. krusei, C. guilliermondii, C. parapsilosis, C. tropicalis and C. viswanathii was tested in normal and in cyclophosphamide-(Cy) immunodepressed mice. In unpretreated CD1 mice only C. albicans, C. tropicalis and C. viswanathii were pathogenic on intravenous challenge, with LD50 of 1.0 X 10(6), 4.8 X 10(6), 7.2 X 10(8) cells, respectively, per kg. Three days after a single intraperitoneal injection of Cy (150 mg kg-1) mice had a marked decrease in spleen weight and cellularity as well as reduced numbers of circulating leukocytes. Under these conditions, there was a significant, proportional increase in pathogenicity of C. albicans, C. tropicalis and C. viswanathii but the animals were still resistant to challenge with C. krusei, C. guilliermondii and C. parapsilosis. This pattern of susceptibility was not influenced by higher doses of Cy. Only C. albicans and C. tropicalis were capable of rapid and extensive multiplication in target organs such as kidney and brain in normal and Cy-treated mice and for both these species of Candida, there was a 'rebound' effect of increased resistance to experimental infection after 12 days from Cy administration. This study shows that the strong immunodepression provoked by Cy does not modify significantly the susceptibility of the animal to those species of Candida which were endowed with low or no pathogenicity for normal mice, but it greatly increases the susceptibility to those species of Candida that are already pathogenic for unmodified host.

Turkey red blood cell passive haemagglutination assay as guideline for specific prevention of tetanus in injured persons.

Turkey red blood cell passive haemagglutination assays (TRBC-HA) were carried out on serum samples from 873 injured patients in order to compare individual prophylactic treatment against tetanus based on the anti-tetanus antibody levels with interventions based on anamnestic criteria. The results showed a great difference: according to the anamnesis 124 persons (14.2%) were protected, 253 (29%) were partially protected, and 496 (56.8%) were unprotected; according to the TRBC-HA assay, 479 (54.9%) were protected, 279 (32%) partially protected, and 115 (13.2%) unprotected.The efficiency of the prophylactic treatments given on the basis of the two criteria was also compared in a study of 129 injured patients who were divided in two groups: group 1 (50 patients) received 250 IU of human tetanus immunoglobulin (HTI) regardless of their tetanus immunity, and group II (79 patients) received appropriate or no treatment depending on the level of anti-tetanus antibodies determined by TRBC-HA assay. The results showed that prophylactic interventions based on the anti-tetanus antibody levels can give protection in 100% of injured patients at minimum cost and risk.

Immunomodulation by a low-virulence, agerminative variant of Candida albicans. Further evidence for macrophage activation as one of the effector mechanisms of nonspecific anti-infectious protection.

Systemic infection of mice with a Candida albicans strain (PCA-2) incapable of yeast-mycelial conversion is known to activate host macrophages and confer protection against subsequent challenge with highly pathogenic cells of the same species or by other micro-organisms. In an attempt to define the relative contributions of different immune components to the protection mediated by PCA-2, we evaluated the effect of manipulations known to selectively deplete immune functions. By means of cytostatic drug or silica induced toxicity, it was possible to demonstrate that no crucial role in protection is played by cytotoxic T lymphocytes or B cells, nor by PCA-2 induced granulocytosis alone. The cells responsible for this effect were dacarbazine-resistant silica-sensitive macrophages whose activity in vivo paralleled the in vitro expression of splenic candidacidal activity. Macrophage activation by PCA-2 and increased anti-Candida resistance did not result from an immunological response mediated by T-dependent effectors, as these effects could be reproduced in athymic mice.

Humoral response against Cryptococcus neoformans mannoprotein antigens in HIV-infected patients.

Twenty-four sera from healthy donors, 18 from HIV-positive patients (< 200 CD4+/mm3) and 18 sera collected before and during cryptococcosis from HIV-positive patients were analysed for the presence of humoral response to C. neoformans mannoproteins. Our results show that samples from healthy subjects and from HIV-positive patients had one of three antibody response profiles: (i) presence of reactive antibodies against both 105 and 80 kilodalton mannoproteins; (ii) presence of reactive antibodies against one of the two mannoproteins; or (iii) absence of reactive antibodies. Importantly the percentage of unreactive sera increased 6-fold in HIV-positive patients and more than 10-fold in patients with cryptococcosis. In addition, in the latter patients no variation of humoral response before and during cryptococcosis was observed. These results suggest that HIV-positive patients show a marked difficulty in mounting or maintaining antibody response to mannoprotein and this could contribute to predisposition to cryptococcosis.

In-vitro killing of Candida species by murine immunoeffectors and its relationship to the experimental pathogenicity.

Killing of yeast cells of several species of Candida by murine phagocytic cells was assessed in vitro by a radiolabel release microassay and measurement of colony forming units. The most effective candidacidal phagocytes, i.e. polymorphonuclear and bone marrow cells, were able to kill equally well cells of any species or isolate tested, given sufficient time (4 h) and an appropriate effector: target ratio. However, C. guilliermondii, C. krusei and C. parapsilosis were killed by polymorphonuclear and bone marrow cells much more promptly (1 h) and at a significantly lower effector:target ratio than C. albicans, C. tropicalis and C. viswanathii. Moreover, there were immune effectors such as peritoneal resident macrophages and, mostly, spleen cells which were practically ineffective against C. albicans and C. tropicalis but showed significant activity against C. guilliermondii, C. krusei and C. parapsilosis, even in mice immuno-depressed with cyclophosphamide. Three isolates of C. albicans, differing in the capacity to form germ tubes, also differed in mouse virulence: the germ-tube forming isolate was the most virulent. However, they showed an identical pattern of susceptibility to killing by mouse immunoeffectors, suggesting that virulence is probably not due to the resistance of hyphal cell to phagocytosis.