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Background: Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis., Objective: We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections., Methods: PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated., Results: We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ-induced gene expression, but we found impaired responses to IFN-γ restimulation., Conclusion: Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ-mediated inflammation., (Published by Mosby, Inc.)

Galectin-3 (gal3) is known for its immunoregulatory functions in infectious, autoimmune, and inflammatory diseases. However, little is known about its regulatory role in the host's IL-17A response to infection. Using a mouse model of histoplasmosis in which both Th1 and Th17 responses contribute to fungal clearance, we investigated how gal3 regulates IL-17A responses. Our study showed that Histoplasma infection induced gal3(-/-) dendritic cells to produce significantly higher levels of IL-23, TGF-β1, and IL-1β than did gal3(+/+) cells. Infected by the same inoculum of Histoplasma, gal3(-/-) mice had lower fungal burden and produced higher levels of IL-23/IL-17-axis cytokines and lower levels of IL-12 and IFN-γ. Additionally, there was an increase in Th17 cells and a reduction in Th1 cells in infected gal3(-/-) mice. In vitro Th1/Th17-skewing experiments excluded the intrinsic effect of gal3 on Th cell differentiation. Although neutrophils from both gal3(+/+) and gal3(-/-) mice produced IL-17A upon IL-23 stimulation, their contribution to IL-17A production was greater in gal3(-/-) mice than in gal3(+/+) mice. Compared with gal3(+/+) dendritic cells, adoptive transfer of gal3(-/-) dendritic cells resulted in production of significantly higher levels of IL-17-axis cytokines and reduced fungal burden. It appears that reduced fungal burden and preferential IL-17A response in gal3(-/-) mice by both Th17 cells and neutrophils were the result of preferential production of IL-23/IL-17-axis cytokines by dendritic cells. Our study showed that gal3 negatively regulates IL-17A responses through inhibition of IL-23/IL-17-axis cytokine production by dendritic cells.

Histoplasmosis has attained increasing relevance in the past 3 decades because of the appearance of the human immunodeficiency virus (HIV). In most immunocompetent persons, the infection is asymptomatic or can produce a respiratory condition with symptoms and radiological images similar to those observed in pulmonary tuberculosis; in non-HIV+ immunocompromised patients, it can cause respiratory symptoms or evolve into a disseminated infection. The same can occur in acquired immunodeficiency syndrome (AIDS) patients. We have observed a series of HIV+ patients with AIDS who presented with cutaneous histoplasmosis and in whom the clinical and histopathological features were highly unusual, including variable mucocutaneous lesions that were difficult to diagnose clinically. These patients displayed unusual, previously undescribed, histological patterns, including lichenoid pattern, nodular pseudomyxoid pattern, pyogenic granuloma-like pattern, perifollicular pattern, and superficial (S), mid (M), and deep perivascular dermatitis; and more commonly encountered patterns, such as histiocytic lobular panniculitis and focal nodular dermatitis. The novel histopathological patterns of cutaneous involvement by histoplasmosis seen in these patients resembled other common inflammatory and infectious conditions and required a high level of suspicion and the application of special stains for organisms for confirmation. These new, clinical, and histological findings do not seem to be commonly encountered in HIV- patients infected with the fungus but seem to be displayed most prominently in HIV+ patients with AIDS.

Indoleamine 2,3 dioxygenase (IDO) plays an important role in immunoregulation as it is involved in downregulating immune responses to infections. We sought to characterize IDO activity in histoplasmosis and to do so, C57Bl6 mice were infected intranasally with Histoplasma capsulatum. After infection, lung and spleen IDO activity was assessed by HPLC and IDO expression by qRT-PCR. The distribution of IDO was determined by immunohistochemical staining. Cytokine levels were measured in lung and spleen homogenates using cytokine bead array. Fungal burden was quantified by culture. Subcutaneous pellets containing methyltryptophane (1-MT) were employed to inhibit IDO in vivo. Histoplasma infection strongly induced functional lung IDO, with activity at its highest at weeks 1 and 2 and then decreased thereafter as the mice cleared the infection. Lung IDO activity positively correlated with the fungal burden (Rho = 0.845), interferon-γ (Rho = 0.839) and tumor necrosis factor-α (Rho = 0.791) levels, P < 0.001. In contrast, spleen IDO activity was not induced despite high infection burden and cytokine levels. IDO expressing cells were predominately located at the ring edge of Histoplasma-induced granulomas. IDO inhibition prior to infection reduced fungal burdens and inflammation in lungs and spleen. Histoplasma preferentially induces lung IDO, as early as one week after infection. IDO appears to modulate the immune response to Histoplasma infection.

Background: Histoplasmosis is a common opportunistic fungal infection that is observed almost exclusively in immunodeficient patients, especially those with AIDS. Immunocompetent individuals that suffer from histoplasmosis are rarely reported, especially those with disseminated lesions, such as disseminated histoplasmosis. The observation of disseminated histoplasmosis with prominent gastrointestinal involvement, no respiratory symptoms (which is presumed to be the portal of infection), gastrointestinal pathological changes, and minor digestive system disorders make this case study exceedingly rare., Case Presentation: We report the case of a 33-year-old immunocompetent male who presented with fever and weight loss. Based on investigations, the patient showed pancytopenia, hepatosplenomegaly, bone marrow involvement and marked colonic involvement. Finally, disseminated histoplasmosis was diagnosed and confirmed by stained smears of fine needle aspirates and biopsy from lesions in the bone marrow and colon. The patient showed appreciable regression of lesions following prompt treatment with amphotericin B deoxycholate, and was treated thereafter with oral itraconazole following discharge from hospital., Conclusion: Disseminated histoplasmosis could be underestimated in immunocompetent patients. A high degree of clinical suspicion is essential in both immunocompromised and immunocompetent patients, regardless of pulmonary symptoms, and whether in endemic or non-endemic areas. Early and accurate diagnosis is extremely important for the appropriate treatment of infection and to improve disease outcome.

Background: Infection of local bone with Histoplasma capsulatum is rare and difficult to diagnosis, and occurs particularly in immunocompetent subjects, who are more likely to be affected by a wide range of organisms., Case Presentation: An 11-year-old boy presented with localized histoplasmosis osteomyelitis in the left fibula without any evidence of abnormal immunological function or systemic disease. After surgical clearance of the lesion and homologous cancellous bone, the patient was treated orally with voriconazole for 6 months. The patient completely recovered with full function of his left leg during the 5-year follow-up., Conclusions: Histoplasmosis osteomyelitis can occasionally occur in immunocompetent individuals and can be complete cured by surgical clearance of the lesion and antibiotic treatment.

History and Clinical Findings: A 24-year-old woman (student of biology) was part of a study group in Uganda. She developed fever and headache, which was empirically treated as malaria. After she had returned to Switzerland, a chest x-ray showed bilateral miliary nodular infiltrates. In assumption of an atypical pneumonia, she was treated with levofloxacin, although without success. On admission, she was in a bad general condition and was markedly dyspneic. Rales were heard over both lungs., Investigations and Diagnosis: CRP, liver enzymes and LDH were elevated. A lung function test revealed a marked impairment of the diffusion capacity. The chest x-ray showed a progression of the lung infiltrates. The informal medical data exchange among the group members by a virtual social network abbreviated our diagnostic workup substantially, since we heard that histoplasmosis had been assumed in another group member. It turned out that the affected persons had visited a colony of bats living in a cave inside the trunk of a tree. Antibodies against Histoplasma capsulatum were positive., Treatment and Course: We began a treatment with itraconazole. The condition improved gradually; chest x-ray and lung function normalized after the 8 week treatment., Conclusion: Histoplasmosis with such a severe course is rare in immunocompetent humans, which indicates that the inoculum must have been very high. Soil contaminated with bats guano favours the proliferation of Histoplasma capsulatum. Our case is also an illustration of how the widespread use of electronic media can sometimes facilitate our work., (© Georg Thieme Verlag KG Stuttgart · New York.)

We report the detection of high-titre anti-Histoplasma capsulatum IgM in the serum of three young adult males occupationally exposed to bat guano. Multidrug treatment with trimethoprim- sulfamethoxazole was started, followed by ciprofloxacin, clarithromycin, metamizole sodium, rifampicin/isoniazid/pyrazinamide, moxifloxacin and lastly amphotericin B and ceftriaxone. Despite treatment the condition of one patient deteriorated, and he died 23 days after exposure. The other two patients recovered after receiving similar therapy with the addition of voriconazole. They are currently being treated with itraconazole for a 1-year period.

Histoplasmosis has emerged as an important opportunistic fungal infection in immunocompromised patients. Histoplasma is a dimorphic fungus that primarily involves lung and the environmental reservoir is soil. Although several cases of histoplasmosis have been reported in India but cytological diagnosis was made in a few cases. We are presenting two cases of histoplasmosis diagnosed on fine-needle aspiration cytology. In the first case, pulmonary histoplasmosis was diagnosed on transbronchial needle aspiration of lung in a 41-year-old immunocompetent male, while second case was of disseminated histoplasmosis in 40-year-old immunocompromised female diagnosed on cytology of cervical lymph node. FNAC is a simple, safe, and rapid technique to establish the initial diagnosis, thus promoting early treatment and favorable outcome especially in the immunocompromised patients., (Copyright © 2011 Wiley Periodicals, Inc.)

Disseminated histoplasmosis is an emerging infection in patients with cellular immune deficiency in non-endemic countries, caused by the migration from endemic regions and the development of travels. Diagnosis can be challenging in this context because rapid diagnostic tools such as Histoplasma antigen detection or appropriate molecular tools are generally unavailable, serology is often negative in immunosuppressed patients, and isolation of the fungus from cultures often takes several weeks. Here, we report the contribution of galactomannan serum detection for the management of an HIV-infected patient with disseminated histoplasmosis.

Histoplasma in patients with impaired cellular immunity can disseminate to various organs and is known as progressive disseminated histoplasmosis. Similarly cytomegalovirus (CMV) is the most common opportunistic pathogen in an immunocompromised host. The authors report an older male with symptoms and radiological evidence of subacute intestinal obstruction. The patient had concerns for compromised immune system as he was on chronic prednisone and methotrexate therapy. Follow-up colonoscopy revealed a stricture in the proximal ascending colon. Biopsy of the stricture revealed ulcer with granulomatous inflammation including well-formed granulomas and an infiltrate of histiocytes within the lamina propria. Special stains on the specimen showed fungal structures consistent with Histoplasma capsulatum. Immunochemistry showed presence of CMV in the tissue. The patient had good response to antimicrobial therapy and did not have progression of intestinal obstruction. This case highlights the need to consider infectious pathology in immunocompromised patients presenting with obstructive symptoms.

Histoplasma capsulatum is a prevalent fungal pathogen in the United States, infecting approximately 500,000 individuals each year. Host protection requires an intact cell-mediated immune response. In this review, we will discuss how cytokines and chemokines influence protective immunity in H. capsulatum infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In murine histoplasmosis, tumor necrosis factor α (TNF-α) antagonism increases the number of regulatory T cells (Tregs) in lungs, and these cells profoundly hinder protective immunity. Because CCR5 mediates Treg homing and proliferation, we determined the outcome of antagonizing TNF-α in CCR5(-/)(-) mice infected with Histoplasma capsulatum. The absence of CCR5 attenuated the severity of infection associated with TNF-α neutralization. Infected controls given anti-TNF-α had a 10-fold increase in the number of Tregs in lungs compared with a <2-fold increase in CCR5(-/)(-) lungs. This difference was partially attributable to impaired homing in the absence of CCR5. Neutralization of TNF-α-enhanced CCR5 ligands in wild-type lungs thus promotes a gradient between lungs and the thymus. This study elucidates the interplay between TNF-α and CCR5 in histoplasmosis. The data suggest that targeting CCR5 may improve host immunity in individuals receiving TNF-α antagonists during infection.

Introduction: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction., Methods: Mice were infected with 2.2 x 10⁴ H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection., Results: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components., Conclusions: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

5-Lipoxygenase-derived products have been implicated in both the inhibition and promotion of chronic infection. Here, we sought to investigate the roles of endogenous 5-lipoxygenase products and exogenous leukotrienes during Histoplasma capsulatum infection in vivo and in vitro. 5-LO deficiency led to increased lung CFU, decreased nitric oxide production and a deficient primary immune response during active fungal infection. Moreover, H. capsulatum-infected 5-LO(-/-) mice showed an intense influx of neutrophils and an impaired ability to generate and recruit effector T cells to the lung. The fungal susceptibility of 5-LO(-/-) mice correlated with a lower rate of macrophage ingestion of IgG-H. capsulatum relative to WT macrophages. Conversely, exogenous LTB4 and LTC4 restored macrophage phagocytosis in 5-LO deficient mice. Our results demonstrate that leukotrienes are required to control chronic fungal infection by amplifying both the innate and adaptive immune response during histoplasmosis.

Apoptosis of leukocytes is known to strongly influence the immunopathogenesis of infection. In this study, we dissected the death pathways of murine macrophages (MΦs) infected with the intracellular pathogen Histoplasma capsulatum. Yeast cells caused apoptosis of MΦs at a wide range of multiplicity of infection, but smaller inocula resulted in delayed detection of apoptosis. Upon infection, caspases 3 and 1 were activated, and both contributed to cell death; however, only the former was involved in apoptosis. The principal driving force for apoptosis involved the extrinsic pathway via engagement of TNFR1 by TNF-α. Infected MΦs produced IL-10 that dampened apoptosis. The chronology of TNF-α and IL-10 release differed in vitro. The former was detected by 2 h postinfection, and the latter was not detected until 8 h postinfection. In vivo, the lungs of TNFR1(-/-) mice infected for 1 d contained fewer apoptotic MΦs than wild-type mice, whereas the lungs of IL-10(-/-) mice exhibited more. Blockade of apoptosis by a pan-caspase inhibitor or by simvastatin sharply reduced the release of TNF-α but enhanced IL-10. However, these treatments did not modify the fungal burden in vitro over 72 h. Thus, suppressing cell death modulated cytokine release but did not alter the fungal burden. These findings provide a framework for the early pathogenesis of histoplasmosis in which yeast cell invasion of lung MΦs engenders apoptosis, triggered in part in an autocrine TNF-α-dependent manner, followed by release of IL-10 that likely prevents apoptosis of newly infected neighboring phagocytes.

Vaccines may help reduce the growing incidence of fungal infections in immune-suppressed patients. We have found that, even in the absence of CD4(+) T-cell help, vaccine-induced CD8(+) T cells persist and confer resistance against Blastomyces dermatitidis and Histoplasma capsulatum. Type 1 cytokines contribute to that resistance, but they also are dispensable. Although the role of T helper 17 cells in immunity to fungi is debated, IL-17 producing CD8(+) T cells (Tc17 cells) have not been investigated. Here, we show that Tc17 cells are indispensable in antifungal vaccine immunity in hosts lacking CD4(+) T cells. Tc17 cells are induced upon vaccination, recruited to the lung on pulmonary infection, and act non-redundantly in mediating protection in a manner that requires neutrophils. Tc17 cells did not influence type I immunity, nor did the lack of IL-12 signaling augment Tc17 cells, indicating a distinct lineage and function. IL-6 was required for Tc17 differentiation and immunity, but IL-1R1 and Dectin-1 signaling was unexpectedly dispensable. Tc17 cells expressed surface CXCR3 and CCR6, but only the latter was essential in recruitment to the lung. Although IL-17 producing T cells are believed to be short-lived, effector Tc17 cells expressed low levels of KLRG1 and high levels of the transcription factor TCF-1, predicting their long-term survival and stem-cell like behavior. Our work has implications for designing vaccines against fungal infections in immune suppressed patients.

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.

We have previously revealed the protective role of CD8(+) T cells in host defense against Histoplasma capsulatum in animals with CD4(+) T cell deficiency and demonstrated that sensitized CD8(+) T cells are restimulated in vitro by dendritic cells that have ingested apoptotic macrophage-associated Histoplasma antigen. Here we show that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently activated functional CD8(+) T cells whose contribution was equal to that of CD4(+) T cells in protection against Histoplasma challenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8(+) T cell but not the CD4(+) T cell response to pulmonary Histoplasma infection. In mice subcutaneously immunized with viable Histoplasma yeasts whose CD8(+) T cells are protective against Histoplasma challenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killed Histoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8(+) T cell activation. Taken together, our results revealed that phagocyte apoptosis in the Histoplasma-infected host is associated with CD8(+) T cell activation and that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently evokes a protective CD8(+) T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8(+) T cell as well as CD4(+) T cell responses to Histoplasma infection.

Histoplasmosis, an infection caused by the fungus Histoplasma capsulatum, has been reported all over the world and is considered endemic in the American continent, including Cuba. This fungus grows on the soils contaminated with bird and bat excreta, where it produces a great number of microconidia that could cause the infection when they are inhaled. The clinical spectrum varies from asymptomatic infections to serious disseminated diseases involving one or many organ systems and affects mainly AIDS patients, patients with hematological neoplasias, transplant recipients or other immunosuppressed patients. The main risk groups include those individuals whose working activities make them be exposed to aerosols contaminated with H. capsulatum. Lab diagnosis is based on the microscopic observation, isolation and identification of the fungus in fluid or tissue samples of patients, and on specific antigen and antibodies detection. The molecular methods based on polymerase chain reaction have not been sufficiently defined, and they could be an important advance in the early diagnosis of this mycosis. Itraconazole is recommended for treatment of moderate, localized and chronic infection whereas amphotericin B is the drug of choice for disseminated and serious manifestations, particularly in its lipidic formulations. At present, histoplasmosis is considered one of the most important systemic mycoses in the Americas, and it is widely spread over all regions of Cuba.

A 27-year-old HIV-positive male patient with disseminated cutaneous histoplasmosis was treated with both HAART and amphotericin B (total accumulated dose of 0.5 g). Amphotericin B was later replaced with itraconazole (200mg/day). Two months after therapy had been started and the cutaneous lesions had healed, the patient interrupted both treatments voluntarily and his health deteriorated. HAART was then re-introduced and CD4+ cell count increased sharply at the same time as lymph node histoplasmosis was diagnosed. This paradoxical response? the relapse of histoplasmosis and concomitant increase in CD4+ cell count and undetectable viral load after resumption of HAART ? suggests that this was a case of immune reconstitution inflammatory syndrome (IRIS).

CCR5 is a potent mediator of regulatory T cell (Treg) chemotaxis. In murine histoplasmosis, mice lacking CCR5 or endogenous CCL4 have a reduced number of Tregs in the lungs, which results in accelerated resolution of infection. In this study, we demonstrate that CCR5 controls the outcome of Histoplasma capsulatum infection by dictating thymic and lymph node egress of Tregs. Mice lacking CCR5 or treated with a mAb to CCL4 had more Tregs in the thymus prior to and during infection. Thymic accumulation was associated with diminished transcription of the sphingosine 1-phosphate 1 receptor and Krüppel-like factor 2, both of which regulate thymic and lymph node emigration of T cells. The significance of CCR5 in Treg egress was demonstrated by generating mixed bone marrow chimeras. Chimeric mice had an increased proportion of CCR5(-/-) Tregs in the thymus and lymph nodes and a decreased proportion of Tregs in the lungs prior to and during H. capsulatum infection. Hence, CCR5 signaling regulates pathogen persistence in murine histoplasmosis by regulating Tregs exiting from the thymus and lymph nodes and, consequently, their subsequent homing in the periphery.

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony forming units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Blastomycosis is a serious and potentially fatal infection, and diagnosis can be difficult at times. We evaluated the diagnostic utility of a commercially available assay for detection of Blastomyces dermatitidis antigen, recently modified to permit quantitation, in subjects with newly diagnosed blastomycosis. Twenty-three of 27 (85.1%) subjects had detectable B. dermatitidis antigenuria. In 2 of these 23, positive results were obtained after concentration of the urine specimen. Nine of 11 (81.8%) subjects had detectable B. dermatitidis antigen in serum, including 3 subjects with negative results before treatment of serum with ethylenediaminetetraacetic acid (EDTA) and positive results after EDTA treatment. B. dermatitidis antigen was not detected in specimens from 50 control subjects but was detected in 15 patients with histoplasmosis. B. dermatitidis antigen was detected in most of the patients with blastomycosis and can be a useful tool for timely diagnosis., (Published by Elsevier Inc.)

We describe 16 previously unreported patients with histoplasmosis from Queensland and northern New South Wales, Australia, and review all previous Australian reports, providing 63 cases in total to study (17 cases of acute pulmonary histoplasmosis, 2 cases of chronic pulmonary disease, and 44 cases of systemic disease, including 17 cases of single-organ infection and 27 instances of disseminated disease). All acute pulmonary disease was acquired in Australia, with 52% of systemic disease definitely autochthonous. Most cases of single-organ disease occurred in immunocompetent patients (76%), and were oropharyngeal (53%) in location. Forty-one percent of disseminated disease occurred in patients with human immunodeficiency virus (HIV). Patients with HIV had high rates of systemic symptoms, pancytopenia, fungemia, and hepatosplenomegaly. Oropharyngeal and adrenal involvement as well as systemic symptoms were prominent in immunocompetent patients with disseminated disease, with 6 of 7 cases of adrenal involvement leading to Addison disease. Most systemic disease was diagnosed by culture of Histoplasma capsulatum. Where serology was assessed in cases other than acute pulmonary disease, it was positive in only 32%.Prognosis for patients with single-organ disease was excellent. Disseminated disease was associated with recurrence in 30% and death in 37%. The results of this study confirm several previously known patterns of disease but also provide new insights into this rare but endemic condition in Australia.

Millions of people harbor latent infections of the fungus Histoplasma capsulatum. Such persistent infections represent a stalemate between mechanisms of virulence and the immune response. The differing responses of inbred mouse strains to the same pathogen reflect variation in the genes that control the outcome of infection. Here we show that a 250-fold difference in H. capsulatum susceptibility between inbred mouse strains is attributable to the genotype at the MHC H2 locus. Gene expression analysis of strains varying only at the H2 locus identified genotype-specific and genotype-independent expression signatures, including infection-induced genes such as the fungal pattern recognition receptor Clec7a. Surprisingly, B-cell-specific gene expression was negatively correlated with fungal burden, whereas neutrophil-specific genes were correlated with superior disease outcome. Indeed, disease outcome improved when B cells were eliminated and neutrophils were more active, a previously unknown aspect of the host response. These data refine the understanding of genetic influences on histoplasmosis, reveal how shifts in the composition of immune cell populations compel different disease outcomes, and uncover how innate immunity modulation alters histoplasmosis.

We present a rare case of disseminated cutaneous histoplasmosis in an immunocompetent child who relapsed after treatment with amphotericin B followed by itraconazole and was successfully treated with voriconazole., (© 2010 Wiley Periodicals, Inc.)

Monoclonal antibodies to Histoplasma capsulatum can modify pathogenesis. We now show that monoclonal antibody H1C to a 70-kDa antigen increases intracellular fungal growth and reduces macrophage nitric oxide release but has no effect on fungal burden or survival in murine infection. This further demonstrates the complexities of host-pathogen interactions.

Excessive production of interleukin-4 impairs clearance of the fungal pathogen Histoplasma capsulatum in mice lacking the chemokine receptor CCR2. An increase in the interleukin-4 level is associated with decreased recruitment of dendritic cells to lungs; therefore, we investigated the possibility that these cells influence interleukin-4 production. Adoptive transfer of wild-type or CCR2(-/-) bone marrow-derived dendritic cells loaded with heat-killed yeast cells to infected CCR2(-/-) mice suppressed interleukin-4 transcription. Surprisingly, transfer of cells did not reduce the fungal burden despite the fact that it limited interleukin-4 transcription. Yeast cell-loaded bone marrow-derived dendritic cell-mediated regulation of interleukin-4 transcription was dependent on major histocompatibility complex II antigen presentation to CD4(+) T cells. We previously showed that CD4(+) T cells were a source of interleukin-4 in infected CCR2(-/-) mice, but their contribution to the TH2 phenotype was unclear. Here we demonstrated that these cells were functionally important since elimination of them prior to infection, but not elimination of them at the time of infection, reduced the interleukin-4 level in infected CCR2(-/-) mice. However, the fungal burden was reduced only in CD4-depleted CCR2(-/-) mice that received yeast cell-loaded bone marrow-derived dendritic cells. Taken together, the data indicate that generation of excess interleukin-4 in lungs of H. capsulatum-infected CCR2(-/-) mice is at least partially a consequence of decreased recruitment of dendritic cells capable of antigen presentation. Furthermore, CD4(+) T cells had a deleterious impact on immunity in infected CCR2(-/-) mice.

CCR5 is a chemotactic mediator for inflammatory cells as well as regulatory T cells (Tregs). In this study, we investigated the role of CCR5 during infection with the fungal pathogen Histoplasma capsulatum. Mice lacking CCR5 or treated with an mAb to CCL4 had impaired infiltration of inflammatory cells to the lungs. Despite displaying an elevated fungal burden prior to activation of an adaptive immune response, CCL4-neutralized and CCR5(-/-) mice resolved infection more efficiently than controls. Accelerated fungal clearance was associated with a reduced number of Tregs in the lungs. Impaired trafficking was not solely responsible for the paucity of Tregs in the lungs, as proliferation of both CD4(+) T cells and Tregs was diminished in CCR5(-/-) lungs during infection. A reduced number of Tregs in CCR5(-/-) lungs was associated with a selective increase of Th17 cytokines, and neutralization of IL-17 increased Treg proliferation and consequently fungal burden in CCR5(-/-) mice. Thus, CCR5 dictates pathogen persistence by tightly regulating the balance between Treg and Th17 cells in H. capsulatum infection.

A clinical review of three potentially severe fungal diseases, which are characterized in many cases by mucosal involvement, is presented. They are paracoccidioidomycosis, histoplasmosis, and mucormycosis. Mucosal involvement for paracoccidioidomycosis and rhinocerebral mucormycosis is frequent. Thus, oral involvement may provide early clue for diagnosis. In paracoccidioidomycosis, the mucosal lesion classically shows superficial ulcers with granular appearance and hemorrhagic points, usually on lips, palate, and jugal mucosa. In mucormycosis, necrosis of the palate followed for purulent discharge is a hallmark of rhinocerebral disease. Treatment with amphotericin B desoxycholate or the new second-generation triazoles is highly efficacious.

A 69-year-old man with chronic lymphocytic leukemia presented with fever and a productive cough. He was diagnosed with a histoplasmosis infection, caused by the dimorphic fungus Histoplasma capsulatum, which is rare in the Netherlands but endemic in parts of the United States and South America. The patient was treated with high doses of itraconazole and gamma globulin infusions. This initially led to a clinical improvement, but eventually he developed a probable progressive histoplasmosis. The patient refused additional treatment and died. In immunocompromised patients, infections of the respiratory tract can be caused by a broad variety of agents. Knowledge of the patient's travel history is crucial to determine or exclude certain causal agents.

Histoplasma capsulatum is a primary fungal pathogen of mammals responsible for histoplasmosis. During pathogenesis H. capsulatum yeast proliferate in phagosomes of macrophages. This extensive host/pathogen interaction involves a complex cascade of responses in both organisms. In the mammalian host, infection results in complex branched immunity that is initiated with an innate response and later induces an adaptive response but each response is difficult to resolve during fungal infection. Therefore, in an effort to identify less complex systems and to gain understanding of the host innate response to H. capsulatum, we constructed a mini-host survival assay. With this assay, we found ingestion of virulent Histoplasma capsulatum NAm 1 strain yeasts to be lethal to a Bristol-N2 Caenorhabditis elegans host. The virulent H. capsulatum NAm1 strain shows differential lethality under live/heat-killed infective conditions. Specifically, after ingestion of live yeast lethality is > or = 90% within 48 to 72 h, whereas worms ingesting heat-killed yeast reach equivalent mortality only after 10-14 days. On the other hand, ingestion of live H. capsulatum yeast of the nonvirulent NAm 1 (ura(-)) strain is no more lethal to the nematode than heat-killed yeast. Therefore, C. elegans provides an attractive model for further investigations of the ancient innate immune response during early host/pathogen (H. capsulatum/worm) interaction and pathogenesis.

Histoplasmosis is a pulmonary disease characterised by chronic granulomatous and suppurative inflammatory reactions caused by Histoplasma capsulatum. Regarding new therapies to control fungal infections, the aim of this study was to investigate whether pulmonary administration of leukotriene B(4) (LTB(4))-loaded microspheres (MS) could confer protection to 5-lipoxygenase knockout (5-LO(-/-)) mice infected by H. capsulatum. In this study, MS containing LTB(4) were administered intranasally to mice infected by H. capsulatum. On Day 14 after the infection, fungal recovery from the lungs and histology were evaluated and inflammatory cytokines were measured. Pulmonary administration of LTB(4)-loaded MS was able to reduce fungal recovery from infected lungs. Production of important inflammatory cytokines related to host defence was augmented following MS administration to the lungs. Lung histology also showed that infected mice presented a clear reduction in the fungal burden following the pulmonary release of LTB(4) from MS. Our study provides evidence that the proposed biodegradable microparticulate system, which can release LTB(4) to the lungs, can be employed as therapy, enhancing the antimicrobial activity of host cells during histoplasmosis.

Having reported that pretreatment of serum samples with EDTA at 100 degrees C improved the sensitivity for the detection of Histoplasma antigenemia, we have evaluated this method for the detection of Coccidioides antigenemia. Urine and serum samples from patients with coccidioidomycosis were tested using the MVista Coccidioides enzyme immunoassay, and serum samples with and without EDTA-heat treatment were tested. Antigenemia was detected in 28.6% of patients whose samples were not EDTA-heat treated and in 73.1% of those whose samples were treated. Antigenuria was detected in 50% of patients. Specificity of 100% was obtained in healthy subjects, but cross-reactions were seen in 22.2% of patients with histoplasmosis or blastomycosis. EDTA-heat treatment improves the sensitivity for the detection of Coccidioides antigenemia.

Expression of the chemokine receptor CCR2 can be detrimental or beneficial for infection resolution. Herein, we examined whether CCR2 was requisite for control of infection by the dimorphic fungus Histoplasma capsulatum. H. capsulatum-infected CCR2(-/-) mice manifested defects in inflammatory cell recruitment, increased IL-4, and progressive infection. Increased IL-4 in CCR2(-/-) mice primarily contributed to decreased host resistance as demonstrated by the ability of IL-4-neutralized CCR2(-/-) mice to resolve infection without altering inflammatory cell recruitment. Surprisingly, numerous alveolar macrophages and dendritic cells contributed to IL-4 production in CCR2(-/-) mice. IL-4-mediated impairment of immunity in CCR2(-/-) mice was associated with increased arginase-1 and YM1 transcription and increased transferrin receptor expression by phagocytic cells. Immunity in mice lacking the CCR2 ligand CCL2 was not impaired despite decreased inflammatory cell recruitment. Neutralization of the CCR2 ligand CCL7 in CCL2(-/-) mice, but not wild type, resulted in increased IL-4 and fungal burden. Thus, CCL7 in combination with CCL2 limits IL-4 generation and exerts control of host resistance. Furthermore, increased phagocyte-derived IL-4 in CCR2(-/-) mice is associated with the presence of alternatively activated phagocytic cells.

Host defenses against Histoplasma capsulatum require the action of several cytokines. Here, we explored the influence of interleukin (IL)-17 and IL-23 on immunity to H. capsulatum infection in mice. In lungs, synthesis of IL-17 was up-regulated during acute infection, and the cells producing it were predominantly CD3(+). Neutralization of IL-17A blunted fungal clearance but did not promote progressive infection. Decreased inflammatory cell recruitment and increased levels of IL-6 and IL-10 were associated with impaired clearance. To determine whether the elevated cytokine levels were important in the action of IL-17A, IL-6(-/-) or IL-10(-/-) mice were treated with anti-IL-17A; neutralization of IL-17A did not alter fungal burden in either group of knockout mice. We explored the relationship between IL-17 and IL-23 because they have been reported to form a regulatory network. IL-23 transcription and protein level were increased in the lungs of infected mice. Mice producing IL-23 in the absence of IL-12 manifested prolonged survival that was IL-17 dependent. Thus, IL-17 is requisite for the generation of optimal inflammatory and protective responses. Generation of functional IL-17(+) cells is dependent on IL-6 and IL-10. Our findings also establish the existence a regulatory IL-17/IL-23 axis in histoplasmosis.

Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The alpha-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the beta-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.

Heat shock proteins with molecular masses of approximately 60 kDa (Hsp60) are widely distributed in nature and are highly conserved immunogenic molecules that can function as molecular chaperones and enhance cellular survival under physiological stress conditions. The fungus Histoplasma capsulatum displays an Hsp60 on its cell surface that is a key target of the cellular immune response during histoplasmosis, and immunization with this protein is protective. However, the role of humoral responses to Hsp60 has not been fully elucidated. We generated immunoglobulin G (IgG) isotype monoclonal antibodies (MAbs) to H. capsulatum Hsp60. IgG1 and IgG2a MAbs significantly prolonged the survival of mice infected with H. capsulatum. An IgG2b MAb was not protective. The protective MAbs reduced intracellular fungal survival and increased phagolysosomal fusion of macrophages in vitro. Histological examination of infected mice showed that protective MAbs reduced the fungal burden and organ damage. Organs of infected animals treated with protective MAbs had significantly increased levels of interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha and decreased levels of IL-4 and IL-10. Hence, IgG1 and IgG2a MAbs to Hsp60 can modify H. capsulatum pathogenesis in part by altering the intracellular fate of the fungus and inducing the production of Th1-associated cytokines.

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of worldwide significance. Infection with Hc produces a broad spectrum of diseases and may progress to a life-threatening systemic disease, particularly in individuals with HIV infection. Resolution of histoplasmosis is associated with the activation of cell-mediated immunity, and leukotriene B(4) plays an important role in this event. Lipid bodies (LBs) are increasingly being recognized as multifunctional organelles with roles in inflammation and infection. In this study, we investigated LB formation in histoplasmosis and its putative function in innate immunity. LB formation in leukocytes harvested from Hc-infected C57BL/6 mice peaks on day 2 postinfection and correlates with enhanced generation of lipid mediators, including leukotriene B(4) and PGE(2). Pretreatment of leukocytes with platelet-activating factor and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signaling for LB formation. Alveolar leukocytes cultured with live or dead Hc also presented an increase in LB numbers. The yeast alkali-insoluble fraction 1, which contains mainly beta-glucan isolated from the Hc cell wall, induced a dose- and time-dependent increase in LB numbers, indicating that beta-glucan plays a signaling role in LB formation. In agreement with this hypothesis, beta-glucan-elicited LB formation was inhibited in leukocytes from 5-LO(-/-), CD18(low) and TLR2(-/-) mice, as well as in leukocytes pretreated with anti-Dectin-1 Ab. Interestingly, human monocytes from HIV-1-infected patients failed to produce LBs after beta-glucan stimulation. These results demonstrate that Hc induces LB formation, an event correlated with eicosanoid production, and suggest a role for these lipid-enriched organelles in host defense during fungal infection.

The sensitivity for detection of Histoplasma antigen is lower in serum than in urine. In other antigen assays, treatment of serum at 104 degrees C in the presence of EDTA was required for detection of antigenemia. Sensitivity and specificity for detection of Histoplasma antigenemia were examined with or without EDTA heat treatment of the serum using the MVista Histoplasma antigen enzyme immunoassay. A total of 94.6% of serum specimens from patients with AIDS and histoplasmosis that were negative untreated were positive after EDTA-heat treatment. Two-thirds of the negative serum specimens from patients with probable histoplasmosis, based upon clinical suspicion and Histoplasma antigenuria, were positive after heat treatment. Specificity was 99.0% in controls, including healthy subjects and patients in whom histoplasmosis or blastomycosis, were excluded. Precision and reproducibility were good and excellent, respectively. These findings demonstrate improvement in sensitivity without reduction in specificity, precision, or reproducibility after heat-EDTA treatment.