Comparison of cryptococcal meningitis in HIV-negative patients with and without lung infections.

OBJECTIVE: To investigate the clinical features and outcomes of cryptococcal meningitis (CM) in HIV-negative patients with and without lung infections. METHODS: We retrospectively reviewed the medical records of HIV-negative patients with CM admitted to two university hospitals in Southwest China over the past 5 years. RESULTS: Seventy-one patients were included, of whom 35 (49.3%) had lung disease. Compared with patients without lung infection, CM patients with lung infection tended to be male and younger (≤30 years), experienced more fever, less vomiting and fewer central nervous system symptoms; more often had low white blood cell (WBC) counts (<20 × 106/L), and fewer often had ethmoid sinusitis, maxillary sinusitis, paranasal sinusitis, and otitis media. Cryptococcus neoformans isolates from these patients were sensitive to itraconazole, voriconazole, fluconazole, and amphotericin B but resistant to flucytosine. CM patients with lung infection had higher mortality at discharge compared with patients without lung infection (8.6% vs. 0%). Multivariable analyses showed that a WBC count <20 × 106/L was significantly associated with poor treatment outcome (odds ratio 0.01, 95% confidence interval 0-0.83). CONCLUSION: HIV-negative CM patients with lung infections tended to be male and younger. Fever, fewer central nervous system symptoms, and WBC counts <20 × 106/L were characteristic of this patient group.

Favorable Evolution of Cryptococcal Meningitis in the Context of Flucytosine Resistance.

Cryptococcal meningitis is a critical illness affecting 0.2% to 5% solid-organ transplant recipients with a 40% to 50% mortality. We report the case of a 48-year-old lung transplant recipient, who, 15 months after a right lung graft, kept parakeets and developed meningitis due to Cryptococcus neoformans. Immunosuppressive treatment was based on a quadruple sequential immunosuppressive therapy that included induction therapy with thymoglobulin, followed by corticosteroids, calcineurin inhibitors, and mycophenolate mofetil. Antifungal susceptibility testing of Cryptococcus neoformans showed resistance to flucytosine and intermediate sensitivity to fluconazole. Initial treatment adhered to international guidelines; however, the patient could not tolerate an effective double-antifungal therapy during the first 2 months of treatment. Despite this delayed treatment for an aggressive infection in an immunocompromised patient, the patient survived without relapse and received maintenance treatment with fluconazole during the course of 3 years. Administration of calcineurin inhibitors as immunosuppressive treatment may partly explain this outcome, as this therapeutic class is known to protect from severe forms of cryptococcal meningitis.

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.

Synthesis and immunological studies of glycoconjugates of Cryptococcus neoformans capsular glucuronoxylomannan oligosaccharide structures.

Antibodies to the glucuronoxylomannan (GXM) component of the polysaccharide capsule of Cryptococcus neoformans are protective and GXM-protein conjugate vaccines can elicit protective immune responses. We report the synthesis of a heptasaccharide oligosaccharide representing the putative dominant motif of serotype A GXM and demonstrate that it is recognized by some monoclonal antibodies (mAbs) generated to GXM. Conjugation of the heptasaccharide to human serum albumin (HSA) resulted in an immunogenic compound that elicited high-titer IgG responses in mice when given with complete Freund's adjuvant. The antibody response elicited by the oligosaccharide conjugate vaccine had characteristics of a T-cell-dependent response. The availability of an immunogenic oligosaccharide representing a structural motif of GXM will prove useful in studies of antibody epitope specificity and represents a potential synthetic oligosaccharide vaccine against this fungal pathogen.

Effects of antifungal interventions on the outcome of experimental infections with phenotypic switch variants of Cryptococcus neoformans.

In cryptococcal infection, phenotypic switching from a smooth to a mucoid variant can occur in vivo, producing variants with enhanced virulence that are subsequently selected and affect the outcome of infection. Here, we demonstrate that antifungal treatment of the chronically infected host can promote this phenomenon. Amphotericin B treatment reduces fungal burden less effectively in mucoid variant-infected than in smooth variant-infected mice. Consequently, amphotericin B treatment resulted in a more pronounced prolongation of survival in smooth variant-infected than in mucoid variant-infected mice (20 versus 42 days; P < 0.05). Administration of anticapsular monoclonal antibody mediated better protection in smooth variant-infected than in mucoid variant-infected mice, although a protective effect was not consistently observed at all doses. Most interestingly, both antifungal drug therapy and administration of anticapsular monoclonal antibody promoted the selection of mucoid variants in smooth variant-infected mice, a phenomenon manifested by a statistically higher percentage of mucoid colonies in smooth variant-infected mice than in nontreated control mice. This finding suggests that both chemotherapeutic and immunological antifungal interventions may promote the selection of the more virulent mucoid variant, which could affect the outcome of infection in chronically infected hosts.

Preventative and therapeutic vaccines for fungal infections: from concept to implementation.

Many challenges confront the development of fungal vaccines for humans including differences in host susceptibility, varied pathogenic mechanisms employed by the different species of fungi and mechanisms of host resistance. Hence, no single antigen can be expected to serve as a pan fungal vaccine. Instead, it is likely that progress for fungal vaccines will have to be made at the level of each individual organism. In recent years, tremendous strides have been made in understanding the immunopathogenesis of medically important fungal infections and identifying putative vaccine candidates. Such discoveries will facilitate the introduction of fungal vaccines into the therapeutic armamentarium of clinicians. The fungi under discussion in this review include Candida spp., Aspergillus spp., Cryptococcus neoformans, Coccidioides spp., Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Pneumocystis jirovecii.

Preclinical efficacy of a glucuronoxylomannan-tetanus toxoid conjugate vaccine of Cryptococcus neoformans in a murine model.

The encapsulated yeast, Cryptococcus neoformans, causes life-threatening meningoencephalitis in immunocompromised humans, especially in AIDS patients. Fatality and relapse rates remain quite high despite aggressive therapy. A conjugate vaccine composed of the cryptococcal capsular glucuronoxylomannan covalently coupled to tetanus toxoid (GXM-TT) was constructed and evaluated. The vaccine elicited high levels of capsular antibodies in mice by active and passive immunizations and conferred 70-80% protection against a moderate challenge with 10(3) C, neoformans. Monitoring of serum GXM and anti-GXM antibody levels and of incidence of cryptococcal isolation from various organs of mice suggested that presence of vaccine-induced antibodies during the first 4-6 weeks of infection is critical for clearance of cryptococci from various organs, for limiting serum GXM titers from reaching immunosuppressive levels and ultimately for survival. GXM-TT is the first defined fungal vaccine to confer antibody-mediated protection against a systemic mycosis in an animal model. GXM-TT is being evaluated for safety and immunogenicity in healthy and HIV-infected human volunteers at the National Institutes of Health.

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans.

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.

Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response.

Previous studies with a murine model have shown that immunization with cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund adjuvant (CFA) induces two populations of anticryptococcal reactive CD4+ T cells. One population (TDH cells) transfers anticryptococcal delayed-type hypersensitivity (DTH), and the other population (Tamp cells) amplifies the anticryptococcal DTH response of given to recipient mice at the time of immunization of the recipient. Treatment of mice with cyclosporin A (CsA) ablates the induction of Tamp cells but not TDH cells. The present study focused on assessing the cytokines produced by spleen cells taken from CsA-treated and control (solvent-treated) mice at days 1, 2, 4, and 6 after immunization. Supernatants from the spleen cells cultured in vitro for 24 or 48 h in medium alone or with CneF, concanavalin A, or phorbol 12-myristate 13-acetate plus calcium ionophore were assessed for the presence of interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-4, IL-5, and tumor necrosis factor. Spleen cells from CneF-CFA-treated mice produced IL-2 and IFN-gamma, but not IL-4 or IL-5, constitutively and in response to CneF, indicating that CneF-CFA induces a Th1 response. Tumor necrosis factor was not produced. Anticryptococcal TDH cells developed in spleens in which there were low levels of IFN-gamma and IL-2 (CsA-treated, immunized mice), whereas anticryptococcal Tamp cells along with TDH cells matured in spleens in which production of IFN-gamma and IL-2 was high (solvent-treated, immunized mice). The data also suggest that IL-2 and IFN-gamma produced by Tamp cells early after adoptive transfer are influential in the development of the amplified anticryptococcal DTH response that has been observed in Tamp cell-recipient mice.