Paracoccidioides spp. and Histoplasma capsulatum: Current and New Perspectives for Diagnosis and Treatment.

The thermally-dimorphic systemic fungal group includes several important human pathogens: Blastomyces dermatitides, Coccidioides immitis and C. posadasii, Histoplasma capsulatum, Paracoccidioides brasiliensis, P. lutzii, and Talaromyces (Penicillium) marneffei. They usually are geographically restricted and have natural habitats in soil or in plants, and when fungal propagules invade mammalian host by inhalation, they initiate an inflammatory reaction that can result in self-resolution of the infection or cause an acute or chronic disease. In the setting of the AIDS pandemic and the developments in modern medicine, such as immunosuppressive therapy in cancer surgery patients and in transplantation and autoimmune diseases, the incidence of endemic mycoses has progressively increased. Another important factor of the increased incidence of systemic mycoses in certain regions is the progressive devastation of tropical and subtropical forests. In this review, we focus on two of the most important systemic mycoses: paracoccidioidomycosis and histoplasmosis, and their major characteristics in epidemiology, clinical aspects and laboratorial diagnosis.

Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

Fungal extracellular vesicles: modulating host-pathogen interactions by both the fungus and the host.

The secretion of biomolecules by fungal cells occurs via the conventional export of signal peptide-coupled soluble molecules, but it also results from transport within extracellular vesicles (EV). During the last ten years since the description of this non-conventional secretion pathway, varied, interesting biological roles have been associated with EV release by fungi. The various organic molecules carried by these structures are involved in pathogenesis and immune evasion, and may be associated with cell-cell communication. In regards to host-pathogen interactions, EV roles are diverse and organism-specific, although some features seem to be conserved among the pathogenic fungal organisms studied to date. This review aims to highlight our current understanding of the biologically relevant findings regarding EV released by the pathogenic fungal organisms and describes our knowledge of the roles of EV in host-pathogen interactions.

Butyricimonas virosa bacteraemia and bowel disease: case report and review.

Only two cases of human infection with the anaerobic Gram-negative bacillus Butyricimonas virosa have been previously reported. We describe the case of a 69-year-old man with B. virosa and diverticulitis, further supporting an association of bacteraemia with this pathogen to bowel disease. We also summarize the characteristics of the previously described cases.

A novel inhibition ELISA for the detection and monitoring of Penicillium marneffei antigen in human serum.

The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 µg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment.

PARACOCCIDIOIDOMYCOSIS: CHALLENGES IN THE DEVELOPMENT OF A VACCINE AGAINST AN ENDEMIC MYCOSIS IN THE AMERICAS.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

Paracoccidioidoyycosis: challenges in the develpment of a vaccine against an eendemic mycosis in the americas / Paracoccidioidomicose: desafios no desenvolvimento de uma vacina contra micose endêmica nas Américas

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

RESUMOA paracoccidioidomicose (PCM), causada por Paracoccidioides spp, é importante micose endêmica na América Latina. Com base em diferenças filogenéticas, existem duas espécies reconhecidas de Paracoccidioides, P. brasiliensis e P. lutzii, no entanto, a patogênese e as manifestações clínicas de ambas são indistinguíveis atualmente. Aproximadamente 1853 (~51,2%) de 3583 mortes confirmadas, atribuídas a micoses sistêmicas de 1996-2006, no Brasil foram causadas por PCM. Tratamento antifúngico é necessário para pacientes com PCM. O tratamento inicial dura de dois a seis meses e derivados de sulfa, anfotericina B, azóis e terbinafina são utilizados na prática clínica; no entanto, apesar da terapêutica prolongada, as recaídas ainda são um problema. Uma resposta imune celular eficaz, tendendo a Th1, é essencial para controlar a doença que pode ser induzida por antígenos exógenos, ou moduladas por vacinas profiláticas ou terapêuticas. A estimulação de células B ou a transferência passiva de anticorpos monoclonais também são meios importantes que podem ser utilizados para melhorar a eficácia do tratamento da paracoccidioidomicose no futuro. Esta revisão detalha criticamente os principais desafios que o desenvolvimento de uma vacina para combater a PCM enfrenta.

The effect of methamphetamine on an animal model of erectile function.

In the US methamphetamine is considered a first-line treatment for attention-deficit hyperactivity disorder. It is also a common drug of abuse. Reports in patients and abusers suggest its use results in impotence. The efficacy of phosphodiesterase-5 inhibitors (PDE5i) to restore erectile function in these patient groups also has not been determined. In these studies, we determined if the rat is a suitable animal model for the physiological effects of methamphetamine on erectile function, and if a PDE5i (tadalafil) has an effect on erectile function following methamphetamine treatment. In acute phase studies, erectile function was measured in male Sprague-Dawley rats, before and after administration of 10 mg/kg methamphetamine i.p. Chronically treated animals received escalating doses of methamphetamine [2.5 mg/kg (1st week), 5 mg/kg (2nd week), and 10 mg/kg (3rd week)] i.p. daily for 3 weeks and erectile function compared with untreated controls. The effect of co-administration of tadalafil was also investigated in rats acutely and chronically treated with methamphetamine. Erectile function was determined by measuring the intracorporal pressure/blood pressure ratio (ICP/BP) following cavernous nerve stimulation. In both acute and chronic phase studies, we observed a significant increase in the rates of spontaneous erections after methamphetamine administration. In addition, following stimulation of the cavernous nerve at 4 and 6 mA, there was a significant decrease in the ICP/BP ratio (approximately 50%), indicative of impaired erectile function. Tadalafil treatment reversed this effect. In chronically treated animals, the ICP/BP ratio following 4 and 6 mA stimulation decreased by approximately 50% compared with untreated animals and erectile dysfunction (ED) was also reversed by tadalafil. Overall, our data suggest that the rat is a suitable animal model to study the physiological effect of methamphetamine on erectile function. Our work also provides a rationale for treating patients that report ED associated with therapeutics-containing methamphetamine or amphetamine with PDE5i.

Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Prophylactic and therapeutic vaccination using dendritic cells primed with peptide 10 derived from the 43-kilodalton glycoprotein of Paracoccidioides brasiliensis.

Vaccination with peptide 10 (P10), derived from the Paracoccidioides brasiliensis glycoprotein 43 (gp43), induces a Th1 response that protects mice in an intratracheal P. brasiliensis infection model. Combining P10 with complete Freund's adjuvant (CFA) or other adjuvants further increases the peptide's antifungal effect. Since dendritic cells (DCs) are up to 1,000-fold more efficient at activating T cells than CFA, we examined the impact of P10-primed bone-marrow-derived DC vaccination in mice. Splenocytes from mice immunized with P10 were stimulated in vitro with P10 or P10-primed DCs. T cell proliferation was significantly increased in the presence of P10-primed DCs compared to the peptide. The protective efficacy of P10-primed DCs was studied in an intratracheal P. brasiliensis model in BALB/c mice. Administration of P10-primed DCs prior to (via subcutaneous vaccination) or weeks after (via either subcutaneous or intravenous injection) P. brasiliensis infection decreased pulmonary damage and significantly reduced fungal burdens. The protective response mediated by the injection of primed DCs was characterized mainly by an increased production of gamma interferon (IFN-γ) and interleukin 12 (IL-12) and a reduction in IL-10 and IL-4 compared to those of infected mice that received saline or unprimed DCs. Hence, our data demonstrate the potential of P10-primed DCs as a vaccine capable of both the rapid protection against the development of serious paracoccidioidomycosis or the treatment of established P. brasiliensis disease.

Isolation of Histoplasma capsulatum from bats in the urban area of São Paulo State, Brazil.

The presence of bats in caves, attics, ceilings, and roofs is important epidemiologically as they can increase the chance of human acquisition of pathogens, including Histoplasma capsulatum. Brazilian urban areas contain many species of bats, especially insectivorous bats, that are attracted by a wide range of readily available food and shelter. From August 2003 to December 2008, we analysed 2427 bats in the São Paulo State region. Homogenates of the livers and spleens of the bats were plated on specific medium to identify animals infected with H. capsulatum. The fungus was isolated from 87 bats (3·6%). The infected bats were identified as Molossus molossus (74), Nyctinomops macrotis (10), Tadarida brasiliensis (1), Molossus rufus (1) and Eumops glaucinus (1), all insectivorous species. The data presented are a relevant contribution to the epidemiology of H. capsulatum in densely populated urban areas such as in São Paulo State, especially since histoplasmosis is not included in the mandatory disease notification system.

The monoclonal antibody against the major diagnostic antigen of Paracoccidioides brasiliensis mediates immune protection in infected BALB/c mice challenged intratracheally with the fungus.

The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

Protective antibodies and endemic dimorphic fungi.

The host response to infection is the outcome of a complex interaction between a microbe and a host's innate and adaptive immune system. In this context, the role of antibody in the endemic mycoses is relatively poorly understood. Recently, a monoclonal antibody to a cell surface protein has been shown to be protective in a murine histoplasmosis model. The findings with Histoplasma capsulatum may provide a paradigm for antibody protection against endemic fungi. This paper reviews the recent data on protective antibody in histoplasmosis and previous data supporting a role for antibody in protective responses in other dimorphic fungi.

Scytalidium dimidiatum causing recalcitrant subcutaneous lesions produces melanin.

Scytalidium dimidiatum is a pigmented dematiaceous coelomycete that typically causes chronic superficial skin diseases and onychomycosis, as well as deeper infections, such as subcutaneous abscesses, mycetoma, and even fungemia in immunocompromised patients. A second species, Scytalidium hyalinum, has hyaline hyphae and arthroconidia and is considered by some authors to be an albino mutant of S. dimidiatum. This study aimed to confirm the presence of melanin or melanin-like compounds (which have been previously implicated in the virulence of other fungal pathogens) in S. dimidiatum from a patient with multiple subcutaneous nodules. Treatment of the hyphae and arthroconidia with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles, which were stable free radicals, consistent with their identification as melanins. Extracted melanin particles from S. dimidiatum cultures were labeled by melanin-binding monoclonal antibodies (MAbs) from Sporothrix schenckii, Aspergillus fumigatus, and Cryptococcus neoformans. Lesional skin from the patient infected with S. dimidiatum contained fungal cells that were labeled by melanin-binding MAbs, and digestion of the tissue yielded dark particles that were also reactive. S. hyalinum was also subjected to the melanin extraction protocol, but no dark particles were yielded.

Feasibility of radioimmunotherapy of experimental pneumococcal infection.

Streptococcus pneumoniae is an important cause of community-acquired pneumonia, meningitis, and bacteremia. The problem of pneumococcal disease is exacerbated by increasing drug resistance. Furthermore, patients with impaired immunity are at high risk for invasive pneumococcal infections. Thus, there is an urgent need for new approaches to antimicrobial therapy. Antibody therapies take advantage of the specificity and high affinity of the antigen-antibody interaction to deliver antibacterial compounds to a site of infection in the form of naked or conjugated antibodies. We have recently established that radioimmunotherapy (RIT) can be used to treat experimental fungal infections in mice. In the present study, we investigated the feasibility of applying a RIT approach to the treatment of S. pneumoniae infection by evaluating the susceptibility of S. pneumoniae to radiolabeled antibody in vitro and in an animal infection model. For the specific antibody carrier, we used human monoclonal antibody D11, which binds to pneumococcal capsular polysaccharide 8. We have selected the alpha particle emitter (213)Bi as the radionuclide for conjugation to the antibody. Incubation of serotype 8 S. pneumoniae with (213)Bi-D11 resulted in dose-dependent killing of bacteria. RIT of S. pneumoniae infection in C57BL/6 mice showed that 60% more mice survived in the (213)Bi-D11-treated group (80 micro Ci) than in the untreated group (P < 0.01). The treatment did not cause hematological toxicity, as demonstrated by platelet counts. This feasibility study establishes that RIT can be applied to the treatment of bacterial infections.

Evaluation of acute hematologic and long-term pulmonary toxicities of radioimmunotherapy of Cryptococcus neoformans infection in murine models.

We evaluated acute hematological and long-term pulmonary toxicity of radioimmunotherapy in murine models of Cryptococcus neoformans infection. Activities up to 250 microCi were well tolerated by healthy A/JCr mice for (213)Bi-18B7 and (188)Re-18B7 monoclonal antibodies. In infected mice, doses up to 150 microCi produced only transient toxicity. The lungs of treated mice had no evidence of radiation fibrosis.

Activation of the alternative complement pathway by fungal melanins.

Melanins are complex biological pigments formed by the oxidative polymerization of phenolic and/or indolic compounds. These pigments have been implicated in the pathogenesis of some microbial infections, malignancies, degenerative disorders, and autoimmune diseases. Recent studies have demonstrated that melanins have antigenic and anti-inflammatory properties. These findings led us to further explore the interaction of melanins with the immune system. Melanin particles ("ghosts") were isolated from in vitro-melanized Cryptococcus neoformans cells and Aspergillus niger conidia and then incubated in normal human serum containing (125)I-labeled complement C3. The results demonstrated deposition of C3 fragments onto the melanin ghosts as early as 1 min after incubation, with maximum deposition occurring after 12 min for C. neoformans-derived melanin ghosts and after 25 min for A. niger-derived melanin ghosts. The blocking of classical pathway activation did not affect the kinetics or total deposition of C3 onto the melanin ghosts, indicating that melanins activate complement through the alternative pathway. Immunofluorescence analysis of lungs from BALB/c mice injected intratracheally with C. neoformans-derived melanin ghosts demonstrated deposition of C3 fragments onto the ghosts. Small granulomas were also observed surrounding the ghosts. However, melanization of the C. neoformans cell wall did not alter the kinetics or total deposition of C3 fragments onto the fungal cells. The finding that melanin surfaces can activate the complement system suggests a potential mechanism for the pathogenesis of some degenerative and/or autoimmune processes that involve melanized cells as well as another potential role for melanin in the virulence of melanin-producing microorganisms.

Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection.

Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-dihydroxyphenylalanine (L-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in L-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

Passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal Cryptococcus neoformans infection.

Passive immunization with monoclonal antibodies (MAbs) to melanin prolonged the survival of and reduced the fungal burden in Cryptococcus neoformans-infected mice in comparison to controls. MAbs to melanin reduced the growth rate of in vitro-melanized C. neoformans cells, suggesting a new mechanism of antibody-mediated protection.

Glyphosate inhibits melanization of Cryptococcus neoformans and prolongs survival of mice after systemic infection.

Cryptococcus neoformans is a major fungal pathogen and is a relatively common cause of life-threatening meningoencephalitis. Glyphosate is a widely used herbicide that inhibits the synthesis of aromatic amino acids via the shikimate acid pathway. This study investigated the effects of glyphosate on C. neoformans growth, melanization, and murine infection. C. neoformans was relatively resistant to glyphosate, requiring concentrations >250 microg/mL for inhibition. Melanization of C. neoformans in the presence of L-dopa was inhibited by subinhibitory concentrations of glyphosate. Glyphosate inhibited autopolymerization of L-dopa and oxidation of L-epinephrine by cryptococcal cells, which is mediated by a laccase. Administration of glyphosate to mice infected with C. neoformans delayed melanization of yeast cells in vivo and prolonged average mouse survival. The results suggest that inhibition of melanization in vivo may facilitate control of C. neoformans infection.