Fungal burden, dimorphic transition and candidalysin: Role in Candida albicans-induced vaginal cell damage and mitochondrial activation in vitro.

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans.

Lactobacillus acidophilus, L. plantarum, L. rhamnosus, and L. reuteri Cell-Free Supernatants Inhibit Candida parapsilosis Pathogenic Potential upon Infection of Vaginal Epithelial Cells Monolayer and in a Transwell Coculture System In Vitro.

Vulvovaginal candidiasis (VVC) is a common clinical condition with symptoms and signs of vaginal inflammation in the presence of Candida species. At least one episode of VVC is experienced in up to 75% of women in the reproductive age group during their lifetime, and 5% to 8% of such women suffer from the chronic form. Most cases of VVC are still caused by C. albicans. However, the incidence of VVC cases by non-albicans Candida (NAC) species, such as C. parapsilosis, is continuously increasing. Despite the prevalence of VVC from NAC, little is known about these species and almost nothing about the mechanisms that trigger the VVC. Lactobacillus spp. are the most widely before represented microorganisms in the vaginal microbiota of healthy women. Here, cell-free supernatants (CFS) obtained from L. acidophilus, L. plantarum, L. rhamnosus, and L. reuteri were assessed for their effect on C. parapsilosis virulence traits. Moreover, we assessed if such an effect persisted even after the removal of the CFS (CFS preincubation effect). Moreover, a transwell coculture system was employed by which the relevant antifungal effect was shown to be attributable to the compounds released by lactobacilli. Our results suggest that lactobacilli can work (i) by reducing C. parapsilosis virulence traits, as indicated by the reduced fungal proliferation, viability, and metabolic activity, and (ii) by improving epithelial resistance to the fungus. Overall, these data suggest that, in the context of the vaginal microbiota, the lactobacilli may play a role in preventing the onset of mucosal C. parapsilosis infection. IMPORTANCE The incidence of VVC by non-albicans Candida (NAC) species, such as C. parapsilosis, is increasing. Treatment failure is common in NAC-VVC because some species are resistant or poorly susceptible to the antifungal agents normally employed. Research on C. parapsilosis's pathogenic mechanisms and alternative treatments are still lacking. C. albicans triggers the VVC by producing hyphae, which favor the loss of epithelial tolerance. Differently, C. parapsilosis only produces pseudohyphae. Hence, different virulence factors may trigger the VVC. Likewise, the therapeutic options could also involve different fungal targets. Substantial in vitro and in vivo studies on the pathogenicity mechanisms of C. parapsilosis are lacking. The data presented here ascribe a novel beneficial role to different Lactobacillus spp., whose CFS provides a postbiotic-like activity against C. parapsilosis. Further studies are needed to unravel the mechanisms involved in the bioactivities of such compounds, to better understand the role of single postbiotics in the CFS.

Prevention and treatment of autoimmune diseases with plant virus nanoparticles.

Plant viruses are natural, self-assembling nanostructures with versatile and genetically programmable shells, making them useful in diverse applications ranging from the development of new materials to diagnostics and therapeutics. Here, we describe the design and synthesis of plant virus nanoparticles displaying peptides associated with two different autoimmune diseases. Using animal models, we show that the recombinant nanoparticles can prevent autoimmune diabetes and ameliorate rheumatoid arthritis. In both cases, this effect is based on a strictly peptide-related mechanism in which the virus nanoparticle acts both as a peptide scaffold and as an adjuvant, showing an overlapping mechanism of action. This successful preclinical testing could pave the way for the development of plant viruses for the clinical treatment of human autoimmune diseases.

In vitro effects of commercial mouthwashes on several virulence traits of Candida albicans, viridans streptococci and Enterococcus faecalis colonizing the oral cavity.

Oral microbiota consists of hundreds of different species of bacteria, fungi, protozoa and archaea, important for oral health. Oral mycoses, mostly affecting mucosae, are mainly caused by the opportunistic pathogen Candida albicans. They become relevant in denture-wearers elderly people, in diabetic patients, and in immunocompromised individuals. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis and periodontitis, which affect even immune-competent individuals. An appropriate oral hygiene can avoid (or at least ameliorate) such pathologies: the regular and correct use of toothbrush, toothpaste and mouthwash helps prevent oral infections. Interestingly, little or no information is available on the effects (if any) of mouthwashes on the composition of oral microbiota in healthy individuals. Therefore, by means of in vitro models, we assessed the effects of alcohol-free commercial mouthwashes, with different composition (4 with chlorhexidine digluconate, 1 with fluoride, 1 with essential oils, 1 with cetylpyridinium chloride and 1 with triclosan), on several virulence traits of C. albicans, and a group of viridans streptococci, commonly colonizing the oral cavity. For the study here described, a reference strain of C. albicans and of streptococci isolates from pharyngeal swabs were used. Chlorhexidine digluconate- and cetylpyridinium chloride-containing mouthwashes were the most effective in impairing C. albicans capacity to adhere to both abiotic and biotic surfaces, to elicit proinflammatory cytokine secretion by oral epithelial cells and to escape intracellular killing by phagocytes. In addition, these same mouthwashes were effective in impairing biofilm formation by a group of viridans streptococci that, notoriously, cooperate with the cariogenic S. mutans, facilitating the establishment of biofilm by the latter. Differently, these mouthwashes were ineffective against other viridans streptococci that are natural competitors of S. mutans. Finally, by an in vitro model of mixed biofilm, we showed that mouthwashes-treated S. salivarius overall failed to impair C. albicans capacity to form a biofilm. In conclusion, the results described here suggest that chlorhexidine- and cetylpyridinium-containing mouthwashes may be effective in regulating microbial homeostasis of the oral cavity, by providing a positive balance for oral health. On the other side, chlorhexidine has several side effects that must be considered when prescribing mouthwashes containing this molecule.

The synthetic killer peptide KP impairs Candida albicans biofilm in vitro.

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.

Role of the glucocorticoid-induced leucine zipper gene in dexamethasone-induced inhibition of mouse neutrophil migration via control of annexin A1 expression.

The glucocorticoid-induced leucine zipper (GILZ) gene is a pivotal mediator of the anti-inflammatory effects of glucocorticoids (GCs) that are known to regulate the function of both adaptive and innate immunity cells. Our aim was to investigate the role of GILZ in GC-induced inhibition of neutrophil migration, as this role has not been investigated before. We found that GILZ expression was induced by dexamethasone (DEX), a synthetic GC, in neutrophils, and that it regulated migration of these cells into inflamed tissues under DEX treatment. Of note, inhibition of neutrophil migration was not observed in GILZ-knockout mice with peritonitis that were treated by DEX. This was because DEX was unable to up-regulate annexin A1 (Anxa1) expression in the absence of GILZ. Furthermore, we showed that GILZ mediates Anxa1 induction by GCs by transactivating Anxa1 expression at the promoter level via binding with the transcription factor, PU.1. The present findings shed light on the role of GILZ in the mechanism of induction of Anxa1 by GCs. As Anxa1 is an important protein for the resolution of inflammatory response, GILZ may represent a new pharmacologic target for treatment of inflammatory diseases.-Ricci, E., Ronchetti, S., Pericolini, E., Gabrielli, E., Cari, L., Gentili, M., Roselletti, E., Migliorati, G., Vecchiarelli, A., Riccardi, C. Role of the glucocorticoid-induced leucine zipper gene in dexamethasone-induced inhibition of mouse neutrophil migration via control of annexin A1 expression.

Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis.

Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection.

In vivo induction of neutrophil chemotaxis by secretory aspartyl proteinases of Candida albicans.

Secretory aspartyl proteinases (Saps) of Candida albicans are key virulence traits which cause inflammasome-dependent, aseptic inflammation in a mouse model of vaginitis. In this paper, neutrophil migration in response to Sap2, Sap6 and chemo-attractive products released from Sap-treated vaginal epithelium was measured in vitro, ex vivo and in vivo. Our results show that Sap2 and Sap6 induce neutrophil migration and production of potent chemoattractive chemokines such as IL-8 and MIP-2 by vaginal epithelial cells. Our data suggest that at least part of MIP-2 production depends upon IL-1ß activity. The vaginal fluid of Candida-infected mice contained a heat-labile inhibitor of neutrophil candidacidal activity that was absent from the vaginal fluid of Sap-treated mice. Overall, our data provide additional information on the capacity of C. albicans Saps to cause aseptic vaginal inflammation and highlight the potential role of some chemokines released from vaginal epithelial cells in this phenomenon.

Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice.

UNLABELLED: Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C. albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophil influx and accumulation of the inflammasome-dependent interleukin-1ß (IL-1ß) but not of inflammasome-independent tumor necrosis factor alpha. Sap2 could be replaced by other Sap, while no inflammation was induced by the vaccine antigen, the N-terminal-truncated, enzymatically inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library, inhibited or abolished the inflammatory response, provided the antibodies were able, like the Sap inhibitor Pepstatin A, to inhibit Sap enzyme activity. The same antibodies and Pepstatin A also inhibited neutrophil influx and cytokine production stimulated by C. albicans intravaginal injection, and a mutant strain lacking SAP1, SAP2, and SAP3 was unable to cause vaginal inflammation. Sap2 induced expression of activated caspase-1 in murine and human vaginal epithelial cells. Caspase-1 inhibition downregulated IL-1ß and IL-18 production by vaginal epithelial cells, and blockade of the IL-1ß receptor strongly reduced neutrophil influx. Overall, the data suggest that some Sap, particularly Sap2, are proinflammatory proteins in vivo and can mediate the inflammasome-dependent, acute inflammatory response of vaginal epithelial cells to C. albicans. These findings support the notion that vaccine-induced or passively administered anti-Sap antibodies could contribute to control vaginitis. IMPORTANCE: Candidal vaginitis is an acute inflammatory disease that affects many women of fertile age, with no definitive cure and, in its recurrent forms, causing true devastation of quality of life. Unraveling the fungal factors causing inflammation is important to be able to devise novel tools to fight the disease. In an experimental murine model, we have discovered that aspartyl proteinases, particularly Sap2, may cause the same inflammatory signs of vaginitis caused by the fungus and that anti-Sap antibodies and the protease inhibitor Pepstatin A almost equally inhibit Sap- and C. albicans-induced inflammation. Sap-induced vaginitis is an early event during vaginal infection, is uncoupled from fungal growth, and requires Sap and caspase-1 enzymatic activities to occur, suggesting that Sap or products of Sap activity activate an inflammasome sensor of epithelial cells. Our data support the notion that anti-Sap antibodies could help control the essence of candidal vaginitis, i.e., the inflammatory response.

Induction of caspase-11 by aspartyl proteinases of Candida albicans and implication in promoting inflammatory response.

We recently demonstrated that the secreted aspartyl proteinases (Saps), Sap2 and Sap6, of Candida albicans have the potential to induce the canonical activation of the NLRP3 inflammasome, leading to the secretion of interleukin-1ß (IL-1ß) and IL-18 via caspase-1 activation. We also observed that the activation of caspase-1 is partially independent from the NLRP3 activation pathway. In this study, we examined whether Sap2 and Sap6 are also able to activate the noncanonical inflammasome pathway in murine macrophages. Our data show that both Sap2 and Sap6 can activate caspase-11 through type I interferon (IFN) production. Caspase-11 cooperates to activate caspase-1, with a subsequent increase of IL-1ß secretion. Endocytosis and internalization of Saps are required for the induction of type I IFN production, which is essential for induction of noncanonical inflammasome activation. Our study indicates a sophisticated interplay between caspase-1 and caspase-11 that connects the canonical and noncanonical pathways of inflammasome activation in response to C. albicans Saps.

Mouse strain-dependent differences in estrogen sensitivity during vaginal candidiasis.

The animal models available for studying the immune response to genital tract infection require induction of a pseudo estrous state, usually achieved by administration of 17-ß-estradiol. In our experimental model of vaginal candidiasis, under pseudo estrus, different strains of mice were used. We observed major differences in the clearance of Candida albicans infection among the different strains, ascribable to differing susceptibility to estradiol treatment. In the early phase of infection CD1, BALB/c, C57BL/6 albino and C57BL/6 mice were colonized to similar levels, while in the late phase of infection, BALB/c mice, which are considered genetically resistant to C. albicans infection, exhibited greater susceptibility to vaginal candidiasis than CD1 and C57BL/6 albino strains of mice. This was because estradiol induced "per se" enlarged and fluid-filled uteri, more pronounced in infected mice and consistently more evident in BALB/c and C57BL/6 mice than in CD1 mice. Unlike CD1, BALB/c and C57BL/6 mice showed a heavy fungal colonization of the uterus, even though C57BL/6 mice apparently cleared C. albicans from the vagina. The presence of C. albicans in the vagina and uterus was accompanied by a heavy bacterial load. Collectively these observations prompted us to carry out a careful analysis of estradiol effects in a mouse model of vaginal infection.

Peptides of the constant region of antibodies display fungicidal activity.

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Cryptococcus neoformans galactoxylomannan is a potent negative immunomodulator, inspiring new approaches in anti-inflammatory immunotherapy.

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised individuals and occasionally in those with no known immune impairment. The fungus is endowed with several virulence factors, including capsular polysaccharides that play a key role in virulence. The capsule is composed of 90-95% glucuronoxylomannan (GXM), 5-8% galactoxylomannan (GalXM) and <1% mannoproteins. Capsular polysaccharides are shed into tissue where they produce many deleterious effects. Since GalXM has a smaller molecular mass, the molar concentration of GalXM in polysaccharide that is shed could exceed that of GXM in C. neoformans exopolysaccharides. Moreover, GalXM exhibits a number of unusual biologic properties both in vitro and in vivo. Here, we summarize the principal immunomodulatory effects of GalXM described during the last 20 years, particularly the mechanisms leading to induction of apoptosis in T lymphocytes, B lymphocytes and macrophages. Since the capacity of GalXM to induce widespread immune suppression is believed to contribute to the virulence of C. neoformans, this property might be exploited therapeutically to dampen the aberrant activation of immune cells during autoimmune disorders.

Role of CD45 signaling pathway in galactoxylomannan-induced T cell damage.

Previously, we reported that Galactoxylomannan (GalXM) activates the extrinsic and intrinsic apoptotic pathways through an interaction with the glycoreceptors on T cells. In this study we establish the role of the glycoreceptor CD45 in GalXM-induced T cell apoptosis, using CD45(+/+) and CD45(-/-) cell lines, derived from BW5147 murine T cell lymphoma. Our results show that whereas CD45 expression is not required for GalXM association by the cells, it is essential for apoptosis induction. In CD45(+/+) cells, CD45 triggering by GalXM reduces the activation of Lck, ZAP70 and Erk1/2. Conversely, in CD45(-/-) cells, Lck was hyperphosphorylated and did not show any modulation after GalXM stimulation. On the whole, our findings provide evidence that the negative regulation of Lck activation occurs via CD45 engagement. This appears to be related to the capacity of GalXM to antagonize T cell activation and induce T cell death. Overall this mechanism may be responsible for the immune paralysis that follows GalXM administration and could explain the powerful immunosuppression that accompanies cryptococcosis.

Antibody complementarity-determining regions (CDRs): a bridge between adaptive and innate immunity.

BACKGROUND: It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities. METHODOLOGY/PRINCIPAL FINDINGS: In this study we demonstrate that a synthetic peptide with sequence identical to V(H)CDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, V(H)CDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. CONCLUSIONS/SIGNIFICANCE: These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

A microbial polysaccharide reduces the severity of rheumatoid arthritis by influencing Th17 differentiation and proinflammatory cytokines production.

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease characterized by chronic joint inflammation with subsequent cartilage and bone destruction. RA is emerging as a model of IL-17-driven autoimmune inflammatory disease. IL-17 is a marker for Th17 cells, with its master regulator being the retinoic acid receptor-related orphan receptor (RORgammat) regulated by STAT3 signaling. Glucuronoxylomannan (GXM), a polysaccharide representing the main component of the capsular material of the opportunistic yeast Cryptococcus neoformans, exhibits potent immunosuppressive properties both in vitro and in vivo. The present study investigates the effects of GXM treatment on the progression of collagen-induced arthritis. GXM suppressed clinical signs of collagen-induced arthritis and blocked joint erosion progression. This effect was mediated by down-regulation of key cytokines involved in the pathogenesis of RA such as TNF-alpha and IL-1beta, and up-regulation of the inhibitory cytokine IL-10. Moreover, a reduction of IL-6 and TGF-beta, which inhibit Th17 differentiation with consequent decreased IL-17 production at the local and systemic level, was observed. The effect of GXM on Th17 differentiation mirrored the reduction in STAT3 activation and inhibition of RORgammat synthesis. Consequently, this work highlights the beneficial properties of an efficacious compound that could eventually be destined to the clinic.

The GITRL-GITR system alters TLR-4 expression on DC during fungal infection.

The glucocorticoid-induced TNFR-related (GITR) protein is a member of the tumor necrosis factor receptor superfamily influencing natural and acquired immune response. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting cells. Previously, we demonstrated that GITR plays a role in regulating immune response to Candida albicans. Here we analyzed whether GITRL-GITR interaction influences the recognition of C. albicans by regulating the expression of pattern recognition receptors on splenic dendritic cells. Our report demonstrates that under physiological conditions and during candidiasis the GITRL-GITR system affects TLR-2 and TLR-4 expression on DC. These changes correlate with decrease in: MyD88 activation; CD80 and CD40 expression on DC; T cell activation response, including CD28 expression, IL-2 and IFN-gamma production. Our results point out that, during fungal infection, GITRL-GITR interaction modulates TLR-4 and TLR-2 expression, thereby altering the antigen presentation process, and suggesting a role of GITRL-GITR interaction in resistance against infectious diseases.

Indinavir influences biological function of dendritic cells and stimulates antifungal immunity.

In this study, we analyzed the possibility that Indinavir (IDV), a well-known protease inhibitor (PI) used in highly active antiretroviral therapy, could affect immune response against the opportunistic fungus Cryptococcus neoformans. In particular, the quality of dendritic cell (DC) response was analyzed. The results reported here show that IDV treatment induces an expansion of DC with CD8alpha phenotype in spleens of infected hosts. Splenic CD11c+ DC expressed elevated costimulatory molecules such as CD40 and CD80, showed an increased expression of mRNA for proinflammatory cytokines, and secreted abundant IL-12. Integration of all aforementioned regulatory effects results in development of an efficient, T cell-protective response that reflects a consistent reduction in fungus colonization at a cerebral level. These results could help to elucidate the immunoregulatory activity of PI and point out the beneficial effects of IDV in regulating DC functions and antifungal activity. Therefore, although new PI are being introduced in the clinical setting, nevertheless, given its low cost and proven efficacy, IDV could still be considered a potential key compound in the treatment of HIV in resource-limited settings.

Functional defect of natural immune system in an apparent immunocompetent patient with pulmonary cryptococcosis.

We report a case of pulmonary cryptococcosis in a 21-year-old Italian female smoker with no apparent immune disorder. In this study we demonstrated that: (i) patient's neutrophils and monocytes manifested a significant reduction of killing activity against Cryptococcus neoformans as well as Candida albicans; (ii) the suppression was more pronounced in monocytes than in neutrophils; (iii) neutrophils and monocytes showed a significant impairment of TNF-alpha, IL-1beta, and nitric oxide production. These results suggest that the apparent immunocompetent host with pulmonary cryptococcosis could have specific defects in natural immune system mechanisms.

Microbial immune suppression mediated by direct engagement of inhibitory Fc receptor.

A microbial polysaccharide (glucuronoxylomannan (GXM)) exerts potent immunosuppression by direct engagement to immunoinhibitory receptor FcgammaRIIB. Activation of FcgammaRIIB by GXM leads to the recruitment and phosphorylation of SHIP that prevents IkappaBalpha activation. The FcgammaRIIB blockade inhibits GXM-induced IL-10 production and induces TNF-alpha secretion. GXM quenches LPS-induced TNF-alpha release via FcgammaRIIB. The addition of mAb to GXM reverses GXM-induced immunosuppression by shifting recognition from FcgammaRIIB to FcgammaRIIA. These findings indicate a novel mechanism by which microbial products can impair immune function through direct stimulation of an inhibitory receptor. Furthermore, our observations provide a new mechanism for the ability of specific Ab to reverse the immune inhibitory effects of certain microbial products.