Sporothrix globosa melanin inhibits antigenpresentation by macrophages and enhances deep organ dissemination.

Melanin is a Sporothrix virulence factor that can inhibit the innate immune functions of macrophages such as phagocytosis and killing. However, no data on melanin's influence on antigen presentation by macrophages are available. In this study, we used conidia, yeasts, and melanin ghosts (MGs) from a black Sporothrix globosa strain (MEL+) and its ultraviolet-induced albino mutant (MEL-), to study the influence of melanin on expression of molecules involved in antigen presentation by mouse macrophages (MHC class II, CD80, CD86), as well as on levels of transcription factors regulating their expression (CIITA and promoters I, III, and IV). A murine infection model was used to assess the virulence of both strains and differences in expression of MHC class II and CD80/86 in vivo. MHC class II, CD86 CIITA, and PIV expressions were lower in macrophages infected with MEL+ than in macrophages infected with MEL- conidia, while CD80 expression was similar. No statistical difference in gene expression was observed between macrophages infected by MEL+ and MEL- yeasts. Infection by MGs alone had no clear effect on expression of antigen presentation-associated molecules. Mice infected with MEL+ S. globosa had significantly higher fungal burdens in the lung, liver, spleen, kidney, and testicle compared with mice infected with MEL- S. globosa 21 days post-infection. MHC class II expression changes in the animal study were similar to those observed in the in vitro experiment. Our results indicate that S. globosa melanin can inhibit expression of antigen presentation-associated molecules during both the early and late stages of infection, representing a new mechanism to evade host immunity and to enhance dissemination. Further investigations of melanin's impact on adaptive immunity will be helpful in understanding this fungal virulence factor.

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host.

We have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of ß-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

Effect of intrahippocampal administration of anti-melanin-concentrating hormone on spatial food-seeking behavior in rats.

Melanin-concentrating hormone (MCH) is a hypothalamic peptide that plays a critical role in the regulation of food intake and energy metabolism. In this study, we investigated the potential role of dense hippocampal MCH innervation in the spatially oriented food-seeking component of feeding behavior. Rats were trained for eight sessions to seek food buried in an arena using the working memory version of the food-seeking behavior (FSB) task. The testing day involved a bilateral anti-MCH injection into the hippocampal formation followed by two trials. The anti-MCH injection did not interfere with the performance during the first trial on the testing day, which was similar to the training trials. However, during the second testing trial, when no food was presented in the arena, the control subjects exhibited a dramatic increase in the latency to initiate digging. Treatment with an anti-MCH antibody did not interfere with either the food-seeking behavior or the spatial orientation of the subjects, but the increase in the latency to start digging observed in the control subjects was prevented. These results are discussed in terms of a potential MCH-mediated hippocampal role in the integration of the sensory information necessary for decision-making in the pre-ingestive component of feeding behavior.

Melanocytes and melanin represent a first line of innate immunity against Candida albicans.

Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicans is an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicans supernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection.

Detection of antibodies against Paracoccidioides brasiliensis melanin in in vitro and in vivo studies during infection.

Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.

Autoantibodies to melanocytes and characterization of melanophages in patients affected by a new variant of endemic pemphigus foliaceus.

BACKGROUND: Melanin and melanophages are commonly seen under the basement membrane zone of the skin in patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF). OBJECTIVE: Our study was conducted to determine the nature of these pigmentary alterations. METHODS: We utilized clinical, histopathologic and immunologic techniques including direct and indirect immunofluorescence, immunohistochemistry, Bielschowsky staining and immunoelectron microscopy studies. RESULTS: In the El Bagre-EPF patients, we detected dermal melanin in melanophages and antigen-presenting cells, in close proximity to neural and vascular markers. The melanophages consisted of a mixed population expressing CD68, myeloid/histoid antigen and S-100 protein. By immunoelectron microscopy, the presence of autoantibodies in proximity to melanin granules was confirmed within the melanocytes utilizing 10-nm gold particles. CONCLUSION: Dermal antigen-presenting cells, including melanophages, seem to contain a diverse combination of molecules, representative of an immunologic process where these cells are engulfing both autoantigens and/or cellular debris in El Bagre-EPF. Autoantibodies to discrete components of melanocytes were also identified; the clinical and immunologic significance of these findings remains unknown. Our work may provide a possible explanation of a darkened complexion in patients affected by endemic pemphigus foliaceus.

Behaviour of albino and melanic variants of Biomphalaria glabrata Say, 1818 (Mollusca: Planorbidae) following infection by Schistosoma mansoni Sambon, 1907.

The behaviour of the albino and melanic variants of Biomphalaria glabrata of Belo Horizonte (MG. Brazil) was studied comparatively, in terms of their respective susceptibilities to infection by Schistosoma mansoni of the same origin, through observation of the elimination of cercariae for a three-month period and the calculation of mortality and infection rates, in control and in infected snails. The number of amoebocytes, granulocytes and hyalinocytes in the circulating hemolymph during different periods of infection was analyzed. The evolution of the infection in the tissues was observed by means of histological cross-sections. The melanic variant showed greater susceptibility to infection and a higher mortality rate. The albino variant showed a higher number of circulating amoebocytes, both granulocytes and hyalinocytes. A higher number of degenerated sporocysts were seen in the histological cross-sections of the albino variant. The results suggest that the melanic variant of B. glabrata was more susceptible to infection by S. mansoni than was the albino variant.

Behaviour of albino and melanic variants of Biomphalaria glabrata Say, 1818 (Mollusca: Planorbidae) following infection by Schistosoma mansoni Sambon, 1907 / Comportamento de Biomphalaria glabrata variantes albina e melânica frente à infecção pelo Schistosoma mansoni

The behaviour of the albino and melanic variants of Biomphalaria glabrata of Belo Horizonte (MG. Brazil) was studied comparatively, in terms of their respective susceptibilities to infection by Schistosoma mansoni of the same origin, through observation of the elimination of cercariae for a three-month period and the calculation of mortality and infection rates, in control and in infected snails. The number of amoebocytes, granulocytes and hyalinocytes in the circulating hemolymph during different periods of infection was analyzed. The evolution of the infection in the tissues was observed by means of histological cross-sections. The melanic variant showed greater susceptibility to infection and a higher mortality rate. The albino variant showed a higher number of circulating amoebocytes, both granulocytes and hyalinocytes. A higher number of degenerated sporocysts were seen in the histological cross-sections of the albino variant. The results suggest that the melanic variant of B. glabrata was more susceptible to infection by S. mansoni than was the albino variant.

Foi estudado comparativamente o comportamento das variantes albina e melânica de Biomphalaria glabrata de Belo Horizonte (MG. Brasil), em relação à susceptibilidade à infecção pelo Schistosoma mansoni de mesma origem, através da observação da eliminação de cercárias por um período de três meses e a verificação das taxas de mortalidade e infecção nos moluscos controles e infectados. Analisou-se também, o número de amebócitos granulócitos e hialinócitos na hemolinfa circulante em diferentes períodos de infecção. A evolução da infecção nos tecidos foi observada através de cortes histológicos. A variante melânica apresentou maior susceptibilidade à infecção e maior taxa de mortalidade. A variante albina apresentou maior número de amebócitos circulantes, tanto granulócitos quanto hialinócitos. Nos cortes histológicos foi verificado maior número de esporocistos degenerados na variedade albina. Por estes resultados sugere-se que nas populações estudadas a variante melânica de B. glabrata, foi mais susceptível à infecção pelo S. mansoni.

Guanosine promotes B16F10 melanoma cell differentiation through PKC-ERK 1/2 pathway.

Malignant melanoma is one of the most lethal cancers. Nowadays, several anti-melanoma therapies have been employed. However, the poor prognosis and/or the increased toxicity of those treatments clearly demonstrate the requirement of searching for new drugs or novel combined chemotherapeutic protocols, contemplating both effectiveness and low toxicity. Guanosine (Guo) has been used in combination with acriflavina to potentiate the latter's antitumor activity, through still unknown mechanisms. Here, we show that Guo induces B16F10 melanoma cell differentiation, attested by growth arrest, dendrite-like outgrowth and increased melanogenesis, and also reduced motility. A sustained ERK 1/2 phosphorylation was observed after Guo treatment and ERK inhibition led to blockage of dendritogenesis. Intracellular cyclic AMP was not involved in ERK activation, since its levels remained unchanged. Protein kinase C (PKC), in contrast to phospholipase C (PLC), inhibition completely prevented ERK activation. While the classical melanoma differentiation agent forskolin activates cAMP-PKA-Raf-MEK-ERK pathway in B16F10 cells, here we suggest that a cAMP-independent, PKC-ERK axis is involved in Guo-induced B16F10 differentiation. Altogether, our results show that Guo acts as a differentiating agent, with cytostatic rather than cytotoxic properties, leading to a decreased melanoma malignancy. Thus, we propose that Guo may be envisaged in combination with lower doses of conventional anti-melanoma drugs, in an attempt to prevent or diminish their adverse effects.

Melanin from Fonsecaea pedrosoi induces production of human antifungal antibodies and enhances the antimicrobial efficacy of phagocytes.

Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients' sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients' lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.