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Histoplasmosis is a systemic mycosis caused by the thermally dimorphic fungusiHistoplasma capsulatum/i. Although healthy individuals can develop histoplasmosis, the disease is particularly life-threatening in immunocompromised patients, with a wide range of clinical manifestations depending on the inoculum and virulence of the infecting strain. In this review, we discuss the established virulence factors and pathogenesis traits that makeiH. capsulatum/ihighly adapted to a wide variety of hosts, including mammals. Understanding and integrating these mechanisms is a key step toward devising new preventative and therapeutic interventions.

With rising skin cancer rates and interest in preventing photoaging, adjuvants for sunscreens are in high demand. The potential of curcumin has been posited due to its anti-inflammatory, antioxidant and wound healing properties. In prior studies, curcumin decreased UV-induced inflammation, apoptotic changes in human keratinocytes and dermal fibroblasts, and the expression of matrix metalloproteinases. However, curcumin's utility has been hindered by poor aqueous solubility and rapid degradation in vivo. To overcome these limitations, we synthesized curcumin nanoparticles (curc-np), which offer sustained topical delivery and enhanced bioavailability. Curc-np and controls were applied to the skin of BALB/c mice prior to UVB irradiation. Twenty-four hours later, mice pre-treated with curc-np showed less erythema, induration and scale compared to controls. Histopathology showed fewer sunburn cells, and TUNEL assay indicated decreased apoptosis in curc-np treated mice. Immunohistochemistry illustrated less p53 expression in skin pre-treated with curc-np. Furthermore, cytokine analysis revealed significantly less IL-6 and significantly greater anti-inflammatory IL-10 in skin of curc-np-treated mice as compared to controls. Taken together, our results reinforce curcumin's established anti-inflammatory effects in the skin and highlight its potential as a photoprotective adjuvant when delivered through nanoparticles. Further investigation alongside sunscreens against UV-induced damage is warranted.

Methamphetamine (METH) is a major public health and safety problem in the United States. Chronic METH abuse is associated with a 2-fold-higher risk of HIV infection and, possibly, additional infections, particularly those that enter through the respiratory tract or skin. Cryptococcus neoformans is an encapsulated opportunistic yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients, especially in individuals with AIDS. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. l-3,4-Dihydroxyphenylalanine (l-Dopa) is a natural catecholamine that is frequently used to induce melanization in C. neoformans. l-Dopa-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents, and heavy metals. Using a systemic mouse model of infection and in vitro assays to critically assess the impact of METH on C. neoformans melanization and pathogenesis, we demonstrated that METH-treated mice infected with melanized yeast cells showed increased fungal burdens in the blood and brain, exacerbating mortality. Interestingly, analyses of cultures of METH-exposed cryptococci supplemented with l-Dopa revealed that METH accelerates fungal melanization, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.

There is an urgent need to develop novel antifungals. In this study, we screened 1600 compounds for antifungal activity against Cryptococcus neoformans and Candida auris. We evaluated 4 promising compounds against 24 additional isolates of Cr. neoformans, Ca. auris, Cr. deuterogattii, and Cr. gattii. The four compounds, dequalinium chloride (DQC), bleomycin sulfate (BMS), pentamidine isethionate salt (PIS), and clioquinol (CLQ), varied in their efficacy against these pathogens but were generally more effective against cryptococci. The compounds exerted their antifungal effect via multiple mechanisms, including interference with the capsule of cryptococci and induction of hyphal-like morphology in Ca. auris. Our results indicate that DQC, BMS, PIS, and CLQ represent potential prototypes for the future development of antifungals. Lay Summary Fungal infections can be lethal and the options to fight them are scarce. We tested 1600 molecules for their ability to control the growth of two important fungal pathogens, namely Candida auris and species of Cryptococcus. Four of these compounds showed promising antifungal activities.

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

Lipid microdomains or lipid rafts are dynamic and tightly ordered regions of the plasma membrane. In mammalian cells, they are enriched in cholesterol, glycosphingolipids, Glycosylphosphatidylinositol-anchored and signalling-related proteins. Several studies have suggested that mammalian pattern recognition receptors are concentrated or recruited to lipid domains during host-pathogen association to enhance the effectiveness of host effector processes. However, pathogens have also evolved strategies to exploit these domains to invade cells and survive. In fungal organisms, a complex cell wall network usually mediates the first contact with the host cells. This cell wall may contain virulence factors that interfere with the host membrane microdomains dynamics, potentially impacting the infection outcome. Indeed, the microdomain disruption can dampen fungus-host cell adhesion, phagocytosis and cellular immune responses. Here, we provide an overview of regulatory strategies employed by pathogenic fungi to engage with and potentially subvert the lipid microdomains of host cells. TAKE AWAY: Lipid microdomains are ordered regions of the plasma membrane enriched in cholesterol, glycosphingolipids (GSL), GPI-anchored and signalling-related proteins. Pathogen recognition by host immune cells can involve lipid microdomain participation. During this process, these domains can coalesce in larger complexes recruiting receptors and signalling proteins, significantly increasing their signalling abilities. The antifungal innate immune response is mediated by the engagement of pathogen-associated molecular patterns to pattern recognition receptors (PRRs) at the plasma membrane of innate immune cells. Lipid microdomains can concentrate or recruit PRRs during host cell-fungi association through a multi-interactive mechanism. This association can enhance the effectiveness of host effector processes. However, virulence factors at the fungal cell surface and extracellular vesicles can re-assembly these domains, compromising the downstream signalling and favouring the disease development. Lipid microdomains are therefore very attractive targets for novel drugs to combat fungal infections.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterized. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from C. albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here we investigated whether immunization with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunization, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunized with EVs when compared with EVs+Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs+ADJ, while IL-12p70, TGFβ, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80 0 C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations. This article is protected by copyright. All rights reserved.

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.

Melanization of Histoplasma capsulatum remains poorly described, particularly in regards to the forms of melanin produced. In the present study, 30 clinical and environmental H. capsulatum strains were grown in culture media with or without L-tyrosine under conditions that produced either mycelial or yeast forms. Mycelial cultures were not melanized under the studied conditions. However, all strains cultivated under yeast conditions produced a brownish to black soluble pigment compatible with pyomelanin when grew in presence of L-tyrosine. Sulcotrione inhibited pigment production in yeast cultures, strengthening the hyphothesis that H. capsulatum yeast forms produce pyomelanin. Since pyomelanin is produced by the fungal parasitic form, this pigment may be involved in H. capsulatum virulence.

BACKGROUND Sporotrichosis is a subcutaneous mycosis caused by dimorphic pathogenic fungi belonging to the Sporothrix genus. Pathogenic Sporothrix species typically produce melanin, which is known to be a virulence factor. OBJECTIVES The aim of this study was to perform phenotypic, genotypic, and virulence analyses of two distinct Sporothrix brasiliensis strains isolated from the same lesion on a patient from Rio de Janeiro. METHODS AND FINDINGS Genotypic analyses by partial sequencing of the calmodulin, β-tubulin, and chitin synthase genes, as well as polymerase chain reaction (PCR)-fingerprinting by T3B, M13, and GACA, showed that the isolates were very similar but not identical. Both isolates had similar phenotypic characteristics and effectively produced melanin in their yeast forms, accounting for their ability of causing disease in a murine sporotrichosis model. Remarkably, isolate B was albino in its environmental form but caused more severe disease than the pigmented A isolate. CONCLUSIONS These findings indicate that the patient was infected by two genetically and biologically distinct S. brasiliensis that vary in their production of melanin in their environmental forms. The results underscore the importance of characterizing phenotypically different isolates found in the same clinical specimen or patient.

Staphylococcus aureus is a leading infectious cause of life-threatening disease in humans, yet there is currently no vaccine to combat this bacterium. The pathogenesis of S. aureus is mediated by a diverse array of protein toxins including a large family of secreted pyrogenic superantigens. Neutralization of superantigens, including SEB and TSST-1, has proven to be protective in several animal models of toxic shock and sepsis. We demonstrate, for the first time, that a far more prevalent staphylococcal superantigen, SEK, can also induce lethal shock in mice. Additionally, we describe monoclonal antibodies (mAbs) that inhibit SEK-induced mitogenicity as well as protect against SEK-induced lethality, and enhance survival from S. aureus septicemia in murine models. MAb-4G3 (IgG2b), mAb-5G2 (IgG1), and mAb-9H2 (IgG1), all inhibit SEK-induced proliferation and cytokine production of human immune cells. We then demonstrate that passive immunization with a combination of mAb-4G3 and mAb-5G4, 2 mAbs that do not compete for epitope(s) on SEK, significantly enhance survival in a murine model of SEK-induced toxic shock (p = 0.006). In the setting of sepsis, passive immunization with this combination of mAbs also significantly enhances survival in mice after challenge with CA-MRSA strain USA300 (p = 0.03). Furthermore, septic mice that received mAb treatment in conjunction with vancomycin exhibit less morbidity than mice treated with vancomycin alone. Taken together, these findings suggest that the contribution of SEK to S. aureus pathogenesis may be greater than previously appreciated, and that adjunctive therapy with passive immunotherapy against SEs may be beneficial.

The pathogenic fungus Talaromyces (formerly Penicillium) marneffei is a thermally dimorphic fungus that can cause disseminated infection in patients with secondary immunodeficiency syndrome, in particular in the setting of advanced HIV infection. The areas of highest incidence are in Southeast Asia, Southern China, and Indian subcontinents. Talaromycosis (formerly penicilliosis) is identified as an AIDS-defining illness, and it has recently been recognized in non–HIV-associated patients with impaired cellular-mediated immunity. Microbiological culture is the gold standard method for the diagnosis of T. marneffei infection and usually requires up to 2–4 weeks for detectable growth to occur, which may result in a delay of appropriate treatment. Immunodiagnosis has become an alternative method for confirming talaromycosis. This article reviews various immunological tests for the diagnosis of talaromycosis, including a proposed novel rapid point-of-care assay using a new T. marneffei yeast phase-specific monoclonal antibody.

Topical antimicrobials are the ideal mode of onychomycosis treatment for efficient drug delivery and avoidance of sytemic effects associated with oral medications. However, high treatment costs, tissue penetration limitations, and low cure rates have continued to pose major challenges. To capitalize on the progress made by topical efinaconazole solution, efinaconazole was combined with inexpensive, previously-characterized nitric oxide releasing nanoparticles (NO-np), which have been shown to offer sustained nitric oxide release over time and enhanced barrier penetration, while exerting broad spectrum antimicrobial and immunomodulating properties. NO-np were combined with efinaconazole in varying concentrations and applied against reference strains of Trichophyton rubrum using a checkerboard method. Results demonstrated synergism of NO-np+efinaconazole against T. rubrum, which is noteworthy given the barriers present in the topical treatment of onychomycosis, and the multiple potential benefits offered by NO-np. Overall, this study illustrates the untapped potential of nanotechnology in the treatment of disorders of the skin, hair, and nails where drug delivery remains a challenge. J Drugs Dermatol. 2018;17(7):717-720.

Diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus’ EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host’s immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.

Propionibacterium acnes induction of IL-1 cytokines through the NLRP3 (NLR, nucleotide oligomerization domain-like receptor) inflammasome was recently highlighted as a dominant etiological factor for acne vulgaris. Therefore, therapeutics targeting both the stimulus and the cascade would be ideal. Nitric oxide (NO), a potent biological messenger, has documented broad-spectrum antimicrobial and immunomodulatory properties. To harness these characteristics to target acne, we used an established nanotechnology capable of generating/releasing NO over time (NO-np). P. acnes was found to be highly sensitive to all concentrations of NO-np tested, although human keratinocyte, monocyte, and embryonic zebra fish assays revealed no cytotoxicity. NO-np significantly suppressed IL-1β, tumor necrosis factor-α (TNF-α), IL-8, and IL-6 from human monocytes, and IL-8 and IL-6 from human keratinocytes, respectively. Importantly, silencing of NLRP3 expression by small interfering RNA did not limit NO-np inhibition of IL-1 β secretion from monocytes, and neither TNF-α nor IL-6 secretion, nor inhibition by NO-np was found to be dependent on this pathway. The observed mechanism by which NO-np impacts IL-1β secretion was through inhibition of caspase-1 and IL-1β gene expression. Together, these data suggest that NO-np can effectively prevent P. acnes-induced inflammation by both clearing the organism and inhibiting microbial stimulation of the innate immune response.

Wound healing is a complex process driven largely by the migration of a variety of distinct cell types from the wound margin into the wound zone. In this study, we identify the previously uncharacterized microtubule-severing enzyme, Fidgetin-like 2 (FL2), as a fundamental regulator of cell migration that can be targeted in vivo using nanoparticle-encapsulated small interfering RNA (siRNA) to promote wound closure and regeneration. In vitro, depletion of FL2 from mammalian tissue culture cells results in a more than twofold increase in the rate of cell movement, in part due to a significant increase in directional motility. Immunofluorescence analyses indicate that FL2 normally localizes to the cell edge, importantly to the leading edge of polarized cells, where it regulates the organization and dynamics of the microtubule cytoskeleton. To clinically translate these findings, we utilized a nanoparticle-based siRNA delivery platform to locally deplete FL2 in both murine full-thickness excisional and burn wounds. Topical application of FL2 siRNA nanoparticles to either wound type results in a significant enhancement in the rate and quality of wound closure both clinically and histologically relative to controls. Taken together, these results identify FL2 as a promising therapeutic target to promote the regeneration and repair of cutaneous wounds.

Macrophages play a fundamental role in wound healing; therefore, employing a strategy that enhances macrophage recruitment would be ideal. It was previously suggested that the mechanism by which Biafine topical emulsion improves wound healing is via enhanced macrophage infiltration into the wound bed. The purpose of this study was to confirm this observation through gross and histologic assessments of wound healing using murine full-thickness excisional and burn wound models, and compare to common standards, Vaseline and silver sulfadiazine (SSD). Full-thickness excisional and burn wounds were created on two groups of 60 mice. In the excisional arm, mice were divided into untreated control, Biafine, and Vaseline groups. In the burn arm, mice were divided into untreated control, Biafine, and SSD groups. Daily treatments were administered and healing was measured over time. Wound tissue was excised and stained to appropriately visualize morphology, collagen, macrophages, and neutrophils. Collagen deposition was measured and cell counts were performed. Biafine enhanced wound healing in murine full-thickness excisional and burn wounds compared to control, and surpassed Vaseline and SSD in respective wound types. Biafine treatment accelerated wound closure clinically, with greater epidermal/dermal maturity, granulation tissue formation, and collagen quality and arrangement compared to other groups histologically. Biafine application was associated with greater macrophage and lower neutrophil infiltration at earlier stages of healing when compared to other study groups. In conclusion, Biafine can be considered an alternative topical therapy for full-thickness excisional and burn wounds, owing to its advantageous biologically based wound healing properties.

Histoplasmosis, caused by the dimorphic environmental fungus Histoplasma capsulatum, is a major mycosis on the global stage. Acquisition of the fungus by mammalian hosts can be clinically silent or it can lead to life-threatening systemic disease, which can occur in immunologically intact or deficient hosts, albeit severe disease is more likely in the setting of compromised cellular immunity. H. capsulatum yeast cells are highly adapted to the mammalian host as they can effectively survive within intracellular niches in select phagocytic cells. Understanding the biological response by both the host and H. capsulatum will facilitate improved approaches to prevent and/or modify disease. This review presents our current understanding of the major pathogenic mechanisms involved in histoplasmosis.

Burn wounds are often complicated by bacterial infection, contributing to morbidity and mortality. Agents commonly used to treat burn wound infection are limited by toxicity, incomplete microbial coverage, inadequate penetration, and rising resistance. Curcumin is a naturally derived substance with innate antimicrobial and wound healing properties. Acting by multiple mechanisms, curcumin is less likely than current antibiotics to select for resistant bacteria. Curcumin's poor aqueous solubility and rapid degradation profile hinder usage; nanoparticle encapsulation overcomes this pitfall and enables extended topical delivery of curcumin. In this study, we synthesized and characterized curcumin nanoparticles (curc-np), which inhibited in vitro growth of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa in dose-dependent fashion, and inhibited MRSA growth and enhanced wound healing in an in vivo murine wound model. Curc-np may represent a novel topical antimicrobial and wound healing adjuvant for infected burn wounds and other cutaneous injuries.

The majority of therapeutic strategies for mycosis require the protracted administration of antifungals, which can result in significant toxicities and have unacceptable failure rates. Hence, there is an urgent need for the development of improved therapeutic approaches, and monoclonal antibody-based drugs are potentially a powerful alternative to standard antifungals. To develop a broad antibody-like reagent against mycosis, wheat germ agglutinin (WGA) was linked to the effector Fc region of murine IgG2a. The resultant WGA-Fc displayed high affinity to purified chitin and bound efficiently to fungal cell walls, co-localizing with chitin, in patterns ranging from circular (Histoplasma capsulatum) to punctate (Cryptococcus neoformans) to labeling at the bud sites (Candida albicans and Saccharomyces cerevisiae). WGA-Fc directly inhibited fungal growth in standard cultures. WGA-Fc opsonization increased fungal phagocytosis, as well augmented the antifungal functions by macrophages. Prophylactic administration of WGA-Fc fully protected mice against H. capsulatum, correlating with a reduction in lung, spleen and liver fungal burdens. Administration of WGA-Fc also dramatically diminished pulmonary inflammation. Hence, the opsonic activity of WGA-Fc effectively modulates fungal cell recognition and promotes the elimination of fungal pathogens. Therefore, we propose WGA-Fc as a potential “pan-fungal” therapeutic that should be further developed for use against invasive mycoses.

Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.

Paracoccidioidomycosis is a systemic granulomatous disease caused by Paracoccidioides spp. A peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4(+) helper-1 immune response in mice and protects against intratracheal challenge with virulent P. brasiliensis. Previously, we evaluated the efficacy of the P10 peptide alone or combined with antifungal drugs in mice immunosuppressed and infected with virulent isolate of P. brasiliensis. In the present work, our data suggest that P10 immunization leads to an effective cellular immune response associated with an enhanced T cell proliferative response. P10-stimulated splenocytes increased nitric oxide (NO) production and induced high levels of IFN-γ, IL-1β and IL-12. Furthermore, significantly increased concentrations of pro-inflammatory cytokines were also observed in lung homogenates of immunized mice. P10 immunization was followed by minimal fibrosis in response to infection. Combined with antifungal drugs, P10 immunization most significantly improved survival of anergic infected mice. Administration of either itraconazole or sulfamethoxazole/trimethoprim together with P10 immunization resulted in 100 % survival up to 200 days post-infection, whereas untreated mice died within 80 days. Hence, our data show that P10 immunization promotes a strong specific immune response even in immunocompromised hosts and thus P10 treatment represents a powerful adjuvant therapy to chemotherapy.

Methamphetamine (METH) is a potent and highly addictive central nervous system (CNS) stimulant. Additionally, METH adversely impacts immunological responses, which might contribute to the higher rate and more rapid progression of certain infections in drug abusers. However no studies have shown the impact of METH on inflammation within specific organs, cellular participation and cytokine production. Using a murine model of METH administration, we demonstrated that METH modifies, with variable degrees, leukocyte recruitment and alters cellular mediators in the lungs, liver, spleen and kidneys of mice. Our findings demonstrate the pleotropic effects of METH on the immune response within diverse tissues. These alterations have profound implications on tissue homeostasis and the capacity of the host to respond to diverse insults, including invading pathogens.

Methamphetamine (METH) is a drug of abuse that is a potent and highly addictive central nervous system (CNS) stimulant. The blood brain barrier (BBB) is a unique interface that in part functions to prevent microbial invasion of the CNS. The effects of METH on brain vasculature have not been studied extensively. We hypothesized that METH alters the BBB integrity, increasing susceptibility to CNS infection. Using a murine model of METH administration, we demonstrated that METH alters BBB integrity and modifies the expression of tight junction and adhesion molecules. Additionally, we showed that BBB disruption accelerates transmigration of the neurotropic fungus Cryptococcus neoformans into the brain parenchyma after systemic infection. Furthermore, METH-treated mice displayed increased mortality as compared to untreated animals. Our findings provide novel evidence of the impact of METH abuse on the integrity of the cells that comprise the BBB and protect the brain from infection.

Paracoccidioidomycosis is a granulomatous systemic mycosis endemic in Brazil and other Latin America countries. A DNA vaccine encoding the immunoprotective peptide 10 (P10) significantly reduced the fungal burden in mice when given prior to or after intratracheal challenge with Paracoccidioides brasiliensis. Presently, the generation/expansion of CD4+ CD44hi memory T cells as well as Foxp3+ Treg cells in mice immunized with the DNA vaccine (pcDNA3-P10) before and after infection with P. brasiliensis was investigated. Memory CD4+ CD44hi T cells simultaneously with Foxp3+ Treg cells increased in the spleens and lungs of pcDNA3-P10 immunized mice on day 0, 30, 60 and 120 postinfection. Histopathology of the lung tissue showed minimal inflammation in immunized mice compared with the unimmunized group, suggesting a role for regulatory T cells in controlling the immunopathology. The DNA vaccine shows that the repeated immunization generates memory cells and regulatory T cells that replace the initially protective pro-inflammatory T cells conferring a long term protection while preserving the integrity of the infected tissue.

Non-mammalian models have been used to investigate fungal virulence. In this work we have explored the use of Galleria mellonella as an infection model for the pathogenic dimorphic fungi Histoplasma capsulatum and Paracoccidioides lutzii. In mammalian models these fungi cause similar infections, and disease outcomes are influenced by the quantity of the infective inocula. We describe a similar aspect in a G. mellonella model and characterize the pathogenesis features in this system. Infection with P. lutzii or H. capsulatum, in all inoculum used, killed larvae at 25 and 37°C. However, there was a lack of correlation between the number of yeast cells used for infection and the time to larvae death, which may indicate that the fungi induce protective responses in a dynamic manner as the lowest concentrations of fungi induced the most rapid death. For both fungi, the degree of larvae melanization was directly proportional to the inocula size, and this effect was visibly more apparent at 37°C. Histological evaluation of the larvae showed a correlation between the inoculum and granuloma-like formation. Our results suggest that G. mellonella is a potentially useful model to study virulence of dimorphic fungi.

Staphylococcus aureus is frequently isolated in the setting of infections of indwelling medical devices, which are mediated by the microbe's ability to form biofilms on a variety of surfaces. Biofilm-embedded bacteria are more resistant to antimicrobial agents than their planktonic counterparts and often cause chronic infections and sepsis, particularly in patients with prolonged hospitalizations. In this study, we demonstrate that sustained nitric oxide-releasing nanoparticles (NO-np) interfere with S. aureus adhesion and prevent biofilm formation on a rat central venous catheter (CVC) model of infection. Confocal and scanning electron microscopy showed that NO-np-treated staphylococcal biofilms displayed considerably reduced thicknesses and bacterial numbers compared to those of control biofilms in vitro and in vivo , respectively. Although both phenotypes, planktonic and biofilm-associated staphylococci, of multiple clinical strains were susceptible to NO-np, bacteria within biofilms were more resistant to killing than their planktonic counterparts. Furthermore, chitosan, a biopolymer found in the exoskeleton of crustaceans and structurally integrated into the nanoparticles, seems to add considerable antimicrobial activity to the technology. Our findings suggest promising development and translational potential of NO-np for use as a prophylactic or therapeutic against bacterial biofilms on CVCs and other medical devices.

Cryptococcus neoformans is an environmental fungus that can cause lethal meningoencephalitis in immunocompromised individuals. The mechanisms by which environmental microbes become pathogenic to mammals are still obscure, but different studies suggest that fungal virulence evolved from selection imposed by environmental predators. The soil-living Acanthamoeba castellanii is a well-known predator of C. neoformans. In this work, we evaluated the participation of C. neoformans virulence-associated structures in the interaction of fungal cells with A. castellanii. Fungal extracellular vesicles (EVs) and the polysaccharide glucuronoxylomannan (GXM) were internalized by A. castellanii with no impact on the viability of amoebal cells. EVs, but not free GXM, modulated antifungal properties of A. castellanii by inducing enhanced yeast survival. Phagocytosis of C. neoformans by amoebal cells and the pathogenic potential in a Galleria mellonella model were not affected by EVs, but previous interactions with A. castellanii rendered fungal cells more efficient in killing this invertebrate host. This observation was apparently associated with marked amoeba-induced changes in surface architecture and increased resistance to both oxygen- and nitrogen-derived molecular species. Our results indicate that multiple components with the potential to impact pathogenesis are involved in C. neoformans environmental interactions.

Made available in DSpace on 2018-12-11T17:13:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Systemic therapies are preferred for treating dermal dermatophytosis due to inadequate penetration of topical agents. However, systemic antifungals are associated with off-target effects and limited tissue penetration, and antimicrobial resistance is a growing concern. To address this, we investigated topical nitric oxide-releasing nanoparticles (NO-np), which have been used against superficial fungal infections and bacterial abscesses. In addition to enhanced penetration and permeation conferred by nanoparticles, nitric oxide, a broad-spectrum multi-mechanistic antimicrobial agent, offers decreased likelihood of resistance development. In the current study, NO-np inhibited Trichophyton rubrum in vitro, as well as in a murine model of dermal dermatophytosis. In mice, NO-np reduced fungal burden after three days, with complete clearance after seven. Furthermore, NO-np decreased tissue IL-2, 6, 10 and TNFα, indicating earlier attenuation of the host inflammatory response and decreased tissue morbidity. Thus, topical NO-np represent an attractive alternative to systemic therapy against dermal T. rubrum infection. Department of Medicine (Division of Dermatology) Albert Einstein College of Medicine Department of Medicine (Division of Infectious Diseases) Albert Einstein College of Medicine Departamento de Análises Clínicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista (UNESP) Department of Microbiology & Immunology Albert Einstein College of Medicine Millennium Institute on Immunology and Immunotherapy Programa de Inmunología Instituto de Ciencias Biomédicas Facultad de Medicina Universidad de Chile Department of Physiology & Biophysics Albert Einstein College of Medicine Department of Dermatology George Washington School of Medicine and Health Sciences Departamento de Análises Clínicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista (UNESP) CNPq: 150261/2016-0 CAPES: 99999.007910/2014-02

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungi Paracoccidioides spp. The duration of antifungal treatment ranges from months to years and relapses may nevertheless occur despite protracted therapy. Thus, there remains an urgent need for new therapeutic options. Miltefosine (MLT), an analogue of alkylphospholipids, has antifungal activity against species of yeast and filamentous fungi. The aim of this study was to evaluate the antifungal effects of MLT on the yeast forms of Paracoccidioides brasiliensis and Paracoccidioides lutzii. MLT demonstrated inhibitory activity from 0.12 to 1 µg/mL, which was similar to amphotericin B or the combination trimethoprim/sulfamethoxazole but was not more effective than itraconazole. The fungicidal activity of MLT occurred at concentrations ≥1 µg/mL. Ultrastructural alterations were observed following exposure of the fungus to a subinhibitory concentration of MLT, such as cytoplasmic membrane alteration, mitochondrial swelling, electron-lucent vacuole accumulation and increasing melanosome-like structures. Melanin production by yeasts following MLT exposure was confirmed by labelling with antibodies to melanin. In addition, the combination of a subinhibitory concentration of MLT and tricyclazole, an inhibitor of DHN-melanin biosynthesis, drastically reduced yeast viability. In conclusion, MLT had a fungicidal effect against both Paracoccidioides spp., and a subinhibitory concentration impacted melanogenesis. These findings suggest that additional investigations should be pursued to establish a role for MLT in the treatment of PCM.

Cryptococcus neoformans (Cn) and Histoplasma capsulatum (Hc) co-exist in the environment and occasionally co-infect individuals, which can lead to severe disease/lethal outcomes. We investigated specific interactions between Cn-Hc to determine the impact of synchronous infection in virulence and disease. Co-infected mice had significantly higher mortality than infection with either species or acapsular Cn-Hc. Coating of Hc with cryptococcal glycans (Cn-gly) resulted in higher pulmonary fungal burden in co-infected animals relative to control. Co-cultivation or addition of Cn-gly resulted in enhanced pellicle formation with a hybrid polysaccharide matrix with higher reactivity to GXM mAbs. Transfer and incorporation of Cn polysaccharide onto Hc surface was time and temperature dependent. Cn-gly transfer altered the zeta potential of Hc and was associated with increased resistance to phagocytosis and killing by macrophages. Mice infected with Hc and subsequently injected with purified Cn-gly died significantly more rapidly than Hc alone infected, establishing the precedent that virulence factors from one fungus can enhance the virulence of unrelated species. These findings suggest a new mechanism of microbial interaction involving the transfer of virulence traits that translates into enhanced lethality during mixed fungal infections and highlights the importance of studying heterogeneous microbial populations in the setting of infection.

The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for the sharing of antigenically similar components across phylogenetically distant fungal species. Since GXM has been shown to be important for the pathogenesis of C. neoformans and to elicit protective antibodies, the finding of similar molecules in P. brasiliensis raises the possibility that these glycans play similar functions in paracoccidiomycosis.

Wound healing is a complex process, coordinated by various biological factors. In immunocompromised states wound healing can be interrupted as a result of decreased numbers of immune cells, impairing the production of effector molecules such as nitric oxide (NO). Therefore, topical NO-releasing platforms, such as diethylenetriamine (DETA NONOate), have been investigated to enhance wound healing. Recently, we demonstrated a nanoparticle platform that releases NO (NO-NPs) in a sustained manner, accelerating wound healing in both uninfected and infected murine wound models. Here, NO-NPs were investigated and compared to DETA NONOate in an immunocompromised wound model using non-obese, diabetic, severe combined immunodeficiency mice. NO-NP treatment accelerated wound closure as compared to controls and DETA NONOate treatment. In addition, histological assessment revealed that wounds treated with NO-NPs had less inflammation, more collagen deposition, and more blood vessel formation as compared to other groups, consistent with our previous data in immunocompetent animals. These data suggest that NO-NPs may serve as a novel wound-healing therapy in the setting of immunocompromised states associated with impaired wound healing. From the Clinical Editor Wound healing in an immunocompromised host is often incomplete and is a source of major concern in such conditions. This work demonstrates in a murine model that in these settings NO releasing nanoparticles significantly enhance wound healing.

Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans , two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans . In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans , depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans , increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans , as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.

Nitric oxide (NO) is a critical component of host defense against invading pathogens; however, its therapeutic utility is limited due to a lack of practical delivery systems. Recently, a NO-releasing nanoparticulate platform (NO-np) was shown to have in vitro broad-spectrum antimicrobial activity and in vivo pre-clinical efficacy in a dermal abscess model. To extend these findings, both topical (TP) and intralesional (IL) NO-np administration was evaluated in a MRSA intramuscular murine abscess model and compared with vancomycin. All treatment arms accelerated abscess clearance clinically, histologically, and by microbiological assays on both days 4 and 7 following infection. However, abscesses treated with NO-np via either route demonstrated a more substantial, statistically significant decrease in bacterial survival based on colony forming unit assays and histologically revealed less inflammatory cell infiltration and preserved muscular architecture. These data suggest that the NO-np may be an effective addition to our armament for deep soft tissue infections.

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.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.(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.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.

The host factors that contribute to the increased susceptibility of preterm neonates to invasive candidiasis have not been fully identified. In addition, there has been a lack of suitable models to study this problem. We show that rat pups, similar to premature neonates, display increased susceptibility to experimental Candida albicans infection. Further, we find that both C. albicans and Candida parapsilosis lipase disruptant mutants exhibit decreased virulence in rat pups, demonstrating the utility of the model to evaluate the impact of specific genes in disease pathogenesis. Our findings highlight the contribution of lipases to the virulence of C. albicans and C. parapsilosis and provide a new system to study the increased susceptibility of neonates to Candida infections.

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

Unsaturated fatty acids (UFA) are essential components of cells. In Saccharomyces cerevisiae , stearoyl-coenzyme A (CoA) desaturase 1 ( OLE1 ) affects cell viability through the regulation of oleic (18:1) or palmitoleic (16:1) acid production. In this study, we used a targeted gene deletion approach to determine the impact of OLE1 on the emerging human pathogenic fungus Candida parapsilosis . We found that the deletion of OLE1 resulted in an auxotrophic yeast strain (designated OLE1 KO) that required unsaturated fatty acids for growth but not saturated fatty acids. Additionally, the production of UFA by OLE1 KO yeast cells was markedly reduced, suggesting that Ole1 is essential for UFA production. In contrast to wild-type C. parapsilosis , which produced pseudohyphal growth on UFA-supplemented medium agar, pseudohyphal formation in the OLE1 KO cells was severely impaired, suggesting that Ole1 regulates morphology. Furthermore, the OLE1 KO cells were hypersensitive to various stress-inducing factors, such as salts, SDS, and H 2 O 2 , especially at the physiological temperature. The results indicate that OLE1 is essential for the stress response, perhaps through the production of UFA for cell membrane biosynthesis. The OLE1 KO cells also were hypersensitive to human and fetal bovine serum, suggesting that targeting Ole1 could suppress the dissemination of yeast cells in the bloodstream. Murine-like macrophage J774.16 more efficiently killed the OLE1 KO yeasts, and significantly larger amounts of nitric oxide were detected in cocultures of macrophages and OLE1 KO cells than with wild-type or heterozygous strains. Moreover, the disruption of OLE1 significantly reduced fungal virulence in systemic murine infection. Taken together, these results demonstrate that Ole1 regulates the pathobiology of C. parapsilosis via UFA and that the OLE1 pathway is a promising antifungal target.

Recognition and internalization of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalization of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organization, which is characteristic of lipid microdomains, was observed during the first steps of Hc-macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc-macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc-macrophage interaction. Using optical tweezers (OT) to study cell-to-cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalization was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide-glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1 (−/−) mice) showed a deficient ability to interact with Hc. Co-incubation of oligo-GM1 and treatment with Cholera toxin subunit B, which recognizes the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1 (−/−) macrophages. In addition, macrophages with reduced CD18 expression (CD18(low)) were associated with Hc at levels similar to wild type cells. Finally, CD11b and CD18 co-localized with GM1 during Hc-macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilize Hc-macrophage adhesion and mediate efficient internalization during histoplasmosis.

Purpose: Protection of bone marrowagainst radiotoxicity during radioimmunotherapy and in some cases external beamradiationtherapy suchashemi-body irradiationwould permitadministrationof significantlyhigher dosesto tumors, resulting in increased efficacy and safety of treatment. Melanin, a naturally occurring pigment, possesses radioprotective properties. We hypothesized that melanin, which is insoluble, could be delivered to the bone marrow by intravenously administrated melanin-covered nanoparticles (MNs) because of the human body’s ‘‘selfsieving’’ ability, protecting it against ionizing radiation. Methods and Materials: The synthesis of MNs was performed via enzymatic polymerization of 3,4-dihydroxyphenylalanine and/or 5-S-cysteinyl-3,4-dihydroxyphenylalanine on the surface of 20-nm plain silica nanoparticles. The biodistribution of radiolabeled MNs in mice was done at 3 and 24 h. Healthy CD-1 mice (Charles River Laboratories International, Inc., Wilmington, MA) or melanoma tumor‐bearing nude mice were given MNs intravenously, 50 mg/kg of body weight, 3 h before either whole-body exposure to 125 cGy or treatment with 1 mCi of 188 Re-labeled 6D2 melanin-binding antibody. Results: Polymerization of melanin precursors on the surface of silica nanoparticles resulted in formation of a 15nm-thick melanin layer as confirmed by light scattering, transmission electron microscopy, and immunofluorescence. The biodistribution after intravenous administration showed than MN uptake in bone marrow was 0.3% and 0.2% of injected dose per gram at 3 and 24 h, respectively, whereas pre-injection with pluronic acid increased the uptake to 6% and 3% of injected dose per gram, respectively. Systemic MN administration reduced hematologic toxicity in mice treated with external radiation or radioimmunotherapy, whereas no tumor protection by MNs was observed. Conclusions: MNs or similar structures provide a novel approach to protection of bone marrow from ionizing radiation based on prevention of free radical formation by melanin. 2010 Elsevier Inc. Melanin, Nanoparticles, Bone marrow, Radiation protection, Radioimmunotherapy.

Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.

Purpose: Melanin has emerged as an attractive target for radioimmunotherapy (RIT) of melanoma, and a radiolabeled monoclonal antibody (mAb) 6D2 to melanin is currently in clinical evaluation. We investigated two approaches to improve the targeting of radiation to tumors using melanin-binding mAbs: (a) the use of an additional mAb to melanin could provide information on whether using antibodies to melanin can serve as a general approach to development of therapeutics for melanoma, and (b) as melanin targeting involves the antibody binding to extracellular melanin released from necrotic melanoma cells, we hypothesized that the administration of a chemotherapeutic agent followed by RIT would facilitate the delivery of radiation to the tumors due to the increased presence of free melanin. Experimental Design: We evaluated the therapeutic efficacy of two melanin-binding IgM mAbs labeled with 188Re (6D2 and 11B11). We compared the efficacy of RIT with 188Re-6D2 to chemotherapy with dacarbazine (DTIC) and to combined chemotherapy and RIT in human metastatic melanoma-bearing nude mice. Results: Therapeutic efficacy of 188Re-labeled 6D2 and 11B11 was comparable despite differences in their affinity and binding site numbers. Comparison of chemotherapy with DTIC and RIT revealed that RIT was more effective in slowing tumor growth in mice. Administration of DTIC followed by RIT was more effective than either modality alone. Conclusions: These results provide encouragement for the development of RIT for melanoma with melanin-binding mAbs and suggest that combining chemotherapy and RIT may be a promising approach for the treatment of metastatic melanoma.

Heat shock proteins with molecular masses of ∼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 fungusHistoplasma capsulatumdisplays 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) toH. capsulatumHsp60. IgG1 and IgG2a MAbs significantly prolonged the survival of mice infected withH. 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 modifyH. capsulatumpathogenesis in part by altering the intracellular fate of the fungus and inducing the production of Th1-associated cytokines.

Monoclonal antibodies (MAbs) to a cell surface histone onHistoplasma capsulatummodify murine infection and decrease the growth ofH. capsulatumwithin macrophages. Without the MAbs,H. capsulatumsurvives within macrophages by modifying the intraphagosomal environment. In the present study, we aimed to analyze the affects of a MAb on macrophage phagosomes. Using transmission electron and fluorescence microscopy, we showed that phagosome activation and maturation are significantly greater whenH. capsulatumyeast are opsonized with MAb. The MAb reduced the ability of the organism to regulate the phagosomal pH. Additionally, increased antigen processing and reduced negative costimulation occur in macrophages that phagocytose yeast cells opsonized with MAb, resulting in more-efficient T-cell activation. The MAb alters the intracellular fate ofH. capsulatumby affecting the ability of the fungus to regulate the milieu of the phagosome.

The PD-1 costimulatory receptor inhibits T cell receptor signaling upon interacting with its ligands PD-L1 and PD-L2. The PD-1/PD-L pathway is critical in maintaining self-tolerance. In this study, we examined the role of PD-1 in a mouse model of acute infection withHistoplasma capsulatum, a major human pathogenic fungus. In a lethal model of histoplasmosis, all PD-1-deficient mice survived infection, whereas the wild-type mice died with disseminated disease. PD-L expression on macrophages and splenocytes was up-regulated during infection, and macrophages from infected mice inhibitedin vitroT cell activation. Of interest, antibody blocking of PD-1 significantly increased survival of lethally infected wild-type mice. Thus, our studies extend the role of the PD-1/PD-L pathway in regulating antimicrobial immunity to fungal pathogens. The results show that the PD-1/PD-L pathway has a key role in the regulation of antifungal immunity, and suggest that manipulation of this pathway represents a strategy of immunotherapy for histoplasmosis.

Candida parapsilosis is a major cause of human disease, yet little is known about the pathogen’s virulence. We have developed an efficient gene deletion system for C. parapsilosis based on the repeated use of the dominant nourseothricin resistance marker (caSAT1) and its subsequent deletion by FLP-mediated, site-specific recombination. Using this technique, we deleted the lipase locus in the C. parapsilosis genome consisting of adjacent genes CpLIP1 and CpLIP2. Additionally we reconstructed the CpLIP2 gene, which restored lipase activity. Lipolytic activity was absent in the null mutants, whereas the WT, heterozygous, and reconstructed mutants showed similar lipase production. Biofilm formation was inhibited with lipase-negative mutants and their growth was significantly reduced in lipid-rich media. The knockout mutants were more efficiently ingested and killed by J774.16 and RAW 264.7 macrophage-like cells. Additionally, the lipase-negative mutants were significantly less virulent in infection models that involve inoculation of reconstituted human oral epithelium or murine intraperitoneal challenge. These studies represent what we believe to be the first targeted disruption of a gene in C. parapsilosis and show that C. parapsilosis–secreted lipase is involved in disease pathogenesis. This efficient system for targeted gene deletion holds great promise for rapidly enhancing our knowledge of the biology and virulence of this increasingly common invasive fungal pathogen.