Characterizing dopaminergic neuron vulnerability using genome-wide analysis.

Parkinson's disease (PD) is primarily characterized by the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. To identify genes that are associated with DA neuron loss, we screened through 201 wild-caught populations of Drosophila melanogaster as part of the Drosophila Genetic Reference Panel. Here, we identify the top-associated genes containing single-nucleotide polymorphisms that render DA neurons vulnerable. These genes were further analyzed by using mutant analysis and tissue-specific knockdown for functional validation. We found that this loss of DA neurons caused progressive locomotor dysfunction in mutants and gene knockdown analysis. The identification of genes associated with the progressive loss of DA neurons should help to uncover factors that render these neurons vulnerable in PD, and possibly develop strategies to make these neurons more resilient.

New Role of P. brasiliensis α-Glucan: Differentiation of Non-conventional Dendritic Cells.

The cell wall has a critical role in the host immune response to fungal pathogens. In this study, we investigated the influence of two cell wall fractions of the dimorphic fungi Paracoccidioides brasiliensis (Pb) in the in vitro generation of monocyte-derived dendritic cells (MoDCs). Monocytes were purified from the peripheral blood of healthy donors and cultivated for 7 days in medium supplemented with IL-4 and GM-CSF in the presence of Pb cell wall fractions: the alkali-insoluble F1, constituted by ß-1,3-glucans, chitin and proteins, and the alkali-soluble F2, mainly constituted by α-glucan. MoDCs phenotypes were evaluated regarding cell surface expression of CD1a, DC-SIGN, HLA-DR, CD80, and CD83 and production of cytokines. The α-glucan-rich cell wall fraction downregulated the differentiation of CD1a+ MoDCs, a dendritic cell subset that stimulate Th1 responses. The presence of both cell fractions inhibited DC-SIGN and HLA-DR expression, while the expression of maturation markers was differentially induced in CD1a- MoDCs. Differentiation upon F1 and F2 stimulation induced mixed profile of inflammatory cytokines. Altogether, these data demonstrate that Pb cell wall fractions differentially induce a dysregulation in DCs differentiation. Moreover, our results suggest that cell wall α-glucan promote the differentiation of CD1a- DCs, potentially favoring Th2 polarization and contributing to pathogen persistence.

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus.

The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.

Lutzomyia longipalpis Saliva Drives Interleukin-17-Induced Neutrophil Recruitment Favoring Leishmania infantum Infection.

During bloodfeeding, the presence of sand fly saliva in the hemorrhagic pool where Leishmania is also inoculated modulates the development of host immune mechanisms creating a favorable environment for disease progression. To date, information obtained through experimental models suggests that sand fly saliva induces cellular recruitment and modulates production of eicosanoids. However, the effect of sand fly saliva in the different steps of the inflammatory response triggered by Leishmania remains undefined. Here we further investigate if interaction of Lutzomyia longipalpis salivary gland sonicate (SGS) with different host cells present during the initial inflammatory events regulate Leishmania infantum infectivity. Initially, we observed that incubation of human peripheral blood mononuclear cells (PBMC) with Lu. longipalpis SGS in the presence of L. infantum significantly increased IL-10 but did not alter expression of IFN-γ and TNF-α by CD4+ T cells induced by the parasite alone. Interestingly, incubation of PBMC with Lu. longipalpis SGS alone or in the presence of L. infantum resulted in increased IL-17 production. The presence of IL-17 is related to neutrophil recruitment and plays an important role at the site of infection. Here, we also observed increased migration of neutrophil using an in vitro chemotactic assay following incubation with supernatants from PBMC stimulated with L. infantum and Lu. longipalpis SGS. Neutrophil migration was abrogated following neutralization of IL-17 with specific antibodies. Moreover, culture of human neutrophils with L. infantum in the presence of Lu. longipalpis SGS promoted neutrophil apoptosis resulting in increased parasite viability. Neutrophils operate as the first line of defense in the early stages of infection and later interact with different cells, such as macrophages. The crosstalk between neutrophils and macrophages is critical to determine the type of specific immune response that will develop. Here, we observed that co-culture of human macrophages with autologous neutrophils previously infected in the presence of Lu. longipalpis SGS resulted in a higher infection rate, accompanied by increased production of TGF-ß and PGE2. Our results provide new insight into the contribution of Lu. longipalpis SGS to L. infantum-induced regulation of important inflammatory events, creating a favorable environment for parasite survival inside different host cells.

A Novel Entry/Uncoating Assay Reveals the Presence of at Least Two Species of Viral Capsids During Synchronized HIV-1 Infection.

To better characterize the behavior of HIV-1 capsids we developed EURT, for Entry/Uncoating assay based on core-packaged RNA availability and Translation. EURT is an alternative to Blam-Vpr, but as reporter RNA translation relies on core opening, it can be used to study viral capsids behavior. Our study reveals the existence of two major capsid species, a dead end one in which the viral genome is readily exposed to the cytoplasm and a functional one in which such exposure requires artificial core destabilization. Although reverse transcription drives a faster loss of susceptibility of viral cores to high doses of PF74, it does not lead to higher exposure of the viral genome, implying that viral cores protect the genome irrespectively of reverse transcription. Lastly, IFNα drifts cores from functional to non-functional species, revealing a novel core-destabilizing activity. This assay sheds new light on the behavior of viral cores inside target cells.

Differential Expression of the Eicosanoid Pathway in Patients With Localized or Mucosal Cutaneous Leishmaniasis.

Unfettered inflammation is thought to play critical role in the development of different clinical forms of tegumentary leishmaniasis. Eicosanoids are potent mediators of inflammation and tightly associated with modulation of immune responses. In this cross-sectional exploratory study, we addressed whether targets from the eicosanoid biosynthetic pathway, assessed by multiplexed expression assays in lesion biopsy and plasma specimens, could highlight a distinct biosignature in patients with mucocutaneous leishmaniasis (MCL) or localized cutaneous leishmaniasis (LCL). Differences in immunopathogenesis between MCL and LCL may result from an imbalance between prostaglandins and leukotrienes, which may serve as targets for future host-directed therapies.

Proteome profiling of human cutaneous leishmaniasis lesion.

In this study, we used proteomics and biological network analysis to evaluate the potential biological processes and components present in the identified proteins of biopsies from cutaneous leishmaniasis (CL) patients infected by Leishmania braziliensis in comparison with normal skin. We identified 59 proteins differently expressed in samples from infected and normal skin. Biological network analysis employing identified proteins showed the presence of networks that may be involved in the cell death mediated by cytotoxic T lymphocytes. After immunohistochemical analyses, the expression of caspase-9, caspase-3, and granzyme B was validated in the tissue and positively correlated with the lesion size in CL patients. In conclusion, this work identified differentially expressed proteins in the inflammatory site of CL, revealed enhanced expression of caspase-9, and highlighted mechanisms associated with the progression of tissue damage observed in lesions.

Proteomics of dedifferentiation of SK-N-BE2 neuroblastoma cells.

Neuroblastoma develops through processes which include cellular dedifferentiation. Ability of tumors to form spheroids is one of the manifestations of dedifferentiation and carcinogenic transformation. To study mechanisms of dedifferentiation of neuroblastoma cells, we generated spheroids and performed a proteomics study to compare the spheroids with parental SK-N-BE2 cells. We observed that dedifferentiation induced extensive changes in the proteome profiles of the cells, which affected more than 30% of detected cellular proteins. Using mass spectrometry, we identified 239 proteins affected by dedifferentiation into spheroids as compared to the parental cells. These proteins represented such regulatory processes as transcription, cell cycle regulation, apoptosis, cell adhesion, metabolism, intracellular transport, stress response, and angiogenesis. A number of potent regulators of stemness, differentiation and cancer were detected as subnetworks formed by the identified proteins. Our validation tissue microarray study of 30 neuroblastoma cases confirmed that two of the identified proteins, DISC1 and DNA-PKcs, had their expression increased in advanced malignancies. Thus, our report unveiled extensive changes of the cellular proteome upon dedifferentiation of neuroblastoma cells, indicated top subnetworks and clusters of molecular mechanisms involved in dedifferentiation, and provided candidate biomarkers for clinical studies.

The Role of CD4 and CD8 T Cells in Human Cutaneous Leishmaniasis.

Leishmaniasis, caused by infection with parasites of the Leishmania genus, affects millions of individuals worldwide. This disease displays distinct clinical manifestations ranging from self-healing skin lesions to severe tissue damage. The control of Leishmania infection is dependent on cellular immune mechanisms, and evidence has shown that CD4 and CD8 T lymphocytes play different roles in the outcome of leishmaniasis. Although the presence of CD4 T cells is important for controlling parasite growth, the results in the literature suggest that the inflammatory response elicited by these cells could contribute to the pathogenesis of lesions. However, recent studies on CD8 T lymphocytes show that these cells are mainly involved in tissue damage through cytotoxic mechanisms. In this review, we focus on the recent advances in the study of the human adaptive immunological response in the pathogenesis of tegumentary leishmaniasis.

CD8(+) granzyme B(+)-mediated tissue injury vs. CD4(+)IFNγ(+)-mediated parasite killing in human cutaneous leishmaniasis.

A protective or deleterious role of CD8(+)T cells in human cutaneous leishmaniasis (CL) has been debated. The present report explores the participation of CD8(+)T cells in disease pathogenesis as well as in parasite killing. CD8(+)T cells accumulated in CL lesions as suggested by a higher frequency of CD8(+)CD45RO(+)T cells and CD8(+)CLA(+)T cells compared with peripheral blood mononuclear cells. Upon Leishmania braziliensis restimulation, most of the CD8(+)T cells from the lesion expressed cytolytic markers, CD107a and granzyme B. Granzyme B expression in CL lesions positively correlated with lesion size and percentage of TUNEL-positive cells. We also observed a significantly higher percentage of TUNEL-positive cells and granzyme B expression in the biopsies of patients showing a more intense necrotic process. Furthermore, coculture of infected macrophages and CD8(+)T lymphocytes resulted in the release of granzyme B, and the use of granzyme B inhibitor, as well as z-VAD, Fas:Fc, or anti-IFN-γ, had no effect upon parasite killing. However, coculture of infected macrophages with CD4(+)T cells strongly increased parasite killing, which was completely reversed by anti-IFN-γ. Our results reveal a dichotomy in human CL: CD8(+) granzyme B(+)T cells mediate tissue injury, whereas CD4(+)IFN-γ(+)T cells mediate parasite killing.

Avaliação do papel das células T CD8+ e análise proteômica nas Biópsias de pacientes com Leishmaniose Cutânea Localizada infectados por L. braziliensis / Assessing the role of CD8 + T cells and proteomic analysis in biopsies from patients with cutaneous leishmaniasis infected by L. braziliensis Located

Células T CD8+ são essenCIaIS na defesa contra vírus, mas pouco se sabe sobre a sua participação na defesa do hospedeiro contra parasitas, como a Leishmania. Nesse trabalho, investigou-se a participação das células T CD8+ no processo inflamatório e na indução da morte do parasita, bem como os perfis proteômicos das lesões dos pacientes com leishmaniose cutânea localizada (LCL) infectados por L. braziliensis. Observa-se uma alta porcentagem de células T CD8+ nas lesões dos pacientes com LCL, como sugerido pela maior frequência de células T CD8+CD45RO+ e células T CD8+CLA + em comparação com CMSP do próprio indivíduo. Após re-estimulação com L. braziliensis, a maioria das células T CD8+ presentes na lesão expressam marcadores citolíticos, CD 107a e Granzima B. Além disso, a co-cultura de macrófagos infectados com linfócitos T CD8+ resultou na libertação de granzima B. A utilização do inibidor da granzima B, assim como Z-V AO, Fas:Fc ou anti­ IFN-y não teve nenhum efeito sobre a morte do parasita. Por outro lado, a co-cultura de macrófagos infectados com células T CD4+ diminuiu a taxa de infecção, efeito que foi completamente revertido ao se utilizar o anti-IFN-y, enfatizando o papel determinante de células T CD4+IFN-/ na morte da Leishmania. Além disso, observou-se um total de 150 proteínas diferencialmente expressas entre as lesões de pacientes com LCL e as amostras de pele normal. Destas, cinquenta e nove proteínas foram identificadas. Entre elas, 13 apresentaram uma maior ou menor expressão nas amostras dos pacientes em relação a pele normal. Vinte e sete proteínas foram encontradas somente nas lesões dos pacientes e 18 somente nas amostras de pele normal. Essas proteínas foram associadas com processos de regulação biológica; incluindo apoptose, ciclo celular e resposta imune. Para explorar as interações entre as proteínas identificadas e as proteínas e os genes que poderiam ser afetados por elas, redes e sub-redes de interações foram geradas. Após análises de imuno-histoquímica, a presença de caspas e 9, caspase-3 e granzima B foi validada no sítio da lesão. A expressão da granzima B nas lesões dos pacientes correlacionou-se positivamente com a expressão da caspase 9 e a porcentagem de células positivas para TUNEL. Observou-se também uma maior porcentagem de células postivas para TUNEL expressando granzima B nas biópsias dos pacientes que apresentam um processo mais intenso de necrose. Além disso, a presença de granzime B, caspase 9 e caspase-3 foram correlacionados positivamente com o tamanho da, lesão. Neste estudo, podemos concluir que as células T CD8+Granzima B+ estão envolvidas na patogênese da L. braziliensis, através da ativação da apoptose no sítio inflamatório, favorecendo a progressão do dano tecidual observado nos pacientes com LCL

Human mucosal leishmaniasis: neutrophils infiltrate areas of tissue damage that express high levels of Th17-related cytokines.

Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL-17-type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL-17 and IL-17-inducing cytokines (IL-1ß, IL-23, IL-6 and TGF-ß) were detected by immunohistochemistry in ML patients. IL-17(+) cells exhibited CD4(+), CD8(+) or CD14(+) phenotypes, and numerous IL-17(+) cells co-expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17-mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP-9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL-17 is involved in ML pathogenesis.

IFN-gamma expression is up-regulated by peripheral blood mononuclear cells (PBMC) from non-exposed dogs upon Leishmania chagasi promastigote stimulation in vitro.

While the response to Leishmania spp. is well characterized in mice and humans, much less is known concerning the canine immune response, particularly soon after exposure to the parasite. Early events are considered to be a determinant of infection outcome. To investigate the dog's early immune response to L. chagasi, an in vitro priming system (PIV) using dog naïve PBMC was established. Until now, dog PIV immune response to L. chagasi has not been assessed. We co-cultivated PBMC primarily stimulated with L. chagasi in vitro with autologous infected macrophages and found that IFN-gamma mRNA is up-regulated in these cells compared to control unstimulated cells. IL-4 and IL-10 mRNA expression by L. chagasi-stimulated PBMC was similar to control unstimulated PBMC when incubated with infected macrophages. Surprisingly, correlation studies showed that a lower IFN-gamma/IL-4 expression ratio correlated with a lower percentage of infection. We propose that the direct correlation between IFN-gamma/IL-4 ratio and parasite load is dependent on the higher correlation of both IFN-gamma and IL-4 expression with lower parasite infection. This PIV system was shown to be useful in evaluating the dog immune response to L. chagasi, and results indicate that a balance between IFN-gamma and IL-4 is associated with control of parasite infection in vitro.

Saliva from Lutzomyia longipalpis induces CC chemokine ligand 2/monocyte chemoattractant protein-1 expression and macrophage recruitment.

Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.