Vitamin D-associated genetic variants in the Brazilian population: Investigating potential instruments for Mendelian randomization. / Variantes genéticas asociadas con la vitamina D en la población brasileña: investigación de potenciales instrumentos para aleatorización mendeliana

INTRODUCTION: Vitamin D is required for bone and mineral metabolism and participates in the regulation of the immune response. It is also linked to several chronic diseases and conditions, usually in populations of European descent. Brazil presents a high prevalence of vitamin D deficiency and insufficiency despite the widespread availability of sunlight in the country. Thus, it is important to investigate the role of vitamin D as a risk factor for disease and to establish causal relationships between vitamin D levels and health-related outcomes in the Brazilian population. OBJECTIVE: To examine genetic variants identified as determinants of serum vitamin D in genome-wide association studies of European populations and check whether the same associations are present in Brazil. If so, these single nucleotide polymorphisms (SNPs) could be developed locally as proxies to use in genetically informed causal inference methods, such as Mendelian randomization. MATERIALS AND METHODS: We extracted SNPs associated with vitamin D from the genomewide association studies catalog. We did a literature search to select papers ascertaining these variants and vitamin D concentrations in Brazil. RESULTS: GC was the gene with the strongest association with vitamin D levels, in agreement with existing findings in European populations. However, VDR was the most investigated gene, regardless of its non-existing association with vitamin D in the genomewide association studies. CONCLUSIONS: More research is needed to validate sound proxies for vitamin D levels in Brazil, for example, prioritizing GC rather than VDR.

Introducción. La vitamina D es necesaria para el metabolismo óseo y mineral, y participa en la regulación de la respuesta inmunitaria. También está relacionada con enfermedades crónicas en poblaciones europeas. En Brasil, existe una prevalencia elevada de deficiencia e insuficiencia de vitamina D, a pesar de la amplia disponibilidad de luz solar. Por lo tanto, es importante investigar el papel de la vitamina D como factor de riesgo de diversas enfermedades y establecer relaciones causales entre los niveles de vitamina D y los problemas de salud en la población brasileña. Objetivo. Examinar variantes genéticas relacionadas con la vitamina D sérica en estudios de asociación genómica de poblaciones europeas y comprobar si estas mismas están presentes en Brasil. De ser así, estos SNPs podrían utilizarse como proxies en métodos de inferencia causal, tales como la aleatorización mendeliana. Materiales y métodos. A partir del catálogo de estudios de asociación de genoma completo se extrajeron SNPs relacionados con los niveles de vitamina D. Luego se hizo una búsqueda bibliográfica para identificar los artículos que evaluaran estos SNPs y la concentración de vitamina D en Brasil. Resultados. GC fue el gen más fuertemente asociado con los niveles de vitamina D, en concordancia con los resultados existentes en poblaciones europeas. Sin embargo, el gen VDR fue el más investigado, aunque no esté vinculado con la vitamina D en los estudios de asociación de genoma completo. Conclusiones. Se necesita más investigación para validar proxies genéticos de los niveles de vitamina D en Brasil y se recomienda priorizar el gen GC en lugar de VDR.

In Situ versus Systemic Immune Response in the Pathogenesis of Cutaneous Leishmaniasis.

The role of the immune response in the pathogenesis of cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis is predominantly carried out via blood cells. Here, we evaluate whether cytokine production by peripheral blood mononuclear cells (PBMCs) reflects what has been documented at the lesion site. The participants included 22 CL patients diagnosed with a positive PCR. PBMCs were stimulated for 72 h with a soluble leishmania antigen (SLA). Biopsies obtained from the edge of the ulcers were incubated for the same period. Cytokines in supernatants were assessed via ELISA. TNF, IL-1ß, IL-6, IL-17, and granzyme B (GzmB) were higher in the supernatants of biopsies than in PBMCs, but IFN-γ was higher in the supernatants of PBMCs than in biopsies. There was a positive correlation between IFN-γ and TNF in PBMCs, and an inverse correlation between TNF and IL-10 in the cells from the lesion site. A strong correlation between IL-1ß, IL-17, and GzmB was observed in the biopsies, and a positive correlation was detected between these cytokines and the lesion size. Our results indicate that the immune response in L. braziliensis lesions is different from that observed in peripheral blood, and our data suggest that in addition to IL-1ß and GzmB, IL-17 participates in the pathology of CL.

Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Interaction between diterpene icetexanes and old yellow enzymes of Leishmania braziliensis and Trypanosoma cruzi.

Old Yellow Enzymes (OYEs) are flavin-dependent redox enzymes that promote the asymmetric reduction of activated alkenes. Due to the high importance of flavoenzymes in the metabolism of organisms, the interaction between OYEs from the parasites Trypanosoma cruzi and Leishmania braziliensis and three diterpene icetexanes (brussonol and two analogs), were evaluated in the present study, and differences in the binding mechanism and inhibition capacity of these molecules were examined. Although the aforementioned compounds showed poor and negligible activities against T. cruzi and L. braziliensis cells, respectively, the experiments with the purified enzymes indicated that the interaction occurs by divergent mechanisms. Overall, the ligands' inhibitory effect depends on their accessibility to the N5 position of the flavin's isoalloxazine ring. The results also indicated that the OYEs found in both parasites share structural similarities and showed affinities for the diterpene icetexanes in the same range. Nevertheless, the interaction between OYEs and ligands is directed by enthalpy and/or entropy in distinct ways. In conclusion, the binding site of both OYEs exhibits remarkable plasticity, and a large range of different molecules, including that can be substrates and inhibitors, can bind this site. This plasticity should be considered in drug design using OYE as a target.

Chlorella vulgaris extract and Imiquimod as new therapeutic targets for leishmaniasis: An immunological approach.

The therapeutic regimen for the treatment of American Tegumentary Leishmaniasis (ATL) is targeted at the death of the parasite; therefore, it is essential to develop a treatment that can act on the parasite, combined with the modulation of the inflammatory profile. Thus, the aim of this study was to make an in vitro evaluation of the therapeutic potential of Chlorella vulgaris extract (CV) and Imiquimod for ATL. Selectivity indices (SI) were determined by inhibitory concentration assays (IC50) in L. braziliensis cells and cytotoxic concentrations (CC50) were measured in human cells using the MTT method, based on the CV microalgae extract (IC50 concentrations of 15.63 to 500 µg/mL; CC50 concentrations of 62.5-1000 µg/mL) in comparison with the reference drugs and Imiquimod. The immune response was evaluated in healthy human cells by gene expression (RT-qPCR) and cytokine production (Flow Cytometry). The CV extract (SI = 6.89) indicated promising results by showing higher SI than meglumine antimoniate (SI = 3.44) (reference drug). In all analyses, CV presented a protective profile by stimulating the production of Th1 profile cytokines to a larger extent than the reference drugs. Imiquimod showed a high expression for Tbx21, GATA3, RORc and Foxp3 genes, with increased production only of the TNF cytokine. Therefore, the data highlight the natural extract and Imiquimod as strong therapeutic or adjuvant candidates against ATL, owing to modulation of immune response profiles, low toxicity in human cells and toxic action on the parasite.

Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.

Leishmania braziliensis exosomes activate human macrophages to produce proinflammatory mediators.

Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.

CCR5 promotes the migration of CD8+ T cells to the leishmanial lesions.

Cytolytic CD8+ T cells mediate immunopathology in cutaneous leishmaniasis without controlling parasites. Here, we identify factors involved in CD8+ T cell migration to the lesion that could be targeted to ameliorate disease severity. CCR5 was the most highly expressed chemokine receptor in patient lesions, and the high expression of CCL3 and CCL4, CCR5 ligands, was associated with delayed healing of lesions. To test the requirement for CCR5, Leishmania-infected Rag1-/- mice were reconstituted with CCR5-/- CD8+ T cells. We found that these mice developed smaller lesions accompanied by a reduction in CD8+ T cell numbers compared to controls. We confirmed these findings by showing that the inhibition of CCR5 with maraviroc, a selective inhibitor of CCR5, reduced lesion development without affecting the parasite burden. Together, these results reveal that CD8+ T cells migrate to leishmanial lesions in a CCR5-dependent manner and that blocking CCR5 prevents CD8+ T cell-mediated pathology.

Clinical Profile and Diagnosis of Recurrent Cutaneous Leishmaniasis.

This case-control study compared the clinical profile, parasite load, polymerase chain reaction positivity, and response to therapy in patients with recurrent cutaneous leishmaniasis (RCL) with primary cutaneous leishmaniasis (CL). The RCL patients had milder diseases with lower parasite loads, a lower number of lesions, and more self-healing diseases than primary CL patients.

Transcriptome Analysis Identifies the Crosstalk between Dendritic and Natural Killer Cells in Human Cutaneous Leishmaniasis.

Skin ulcers of cutaneous leishmaniasis (CL) are characterized by a localized inflammatory response mediated by innate and adaptive immune cells, including dendritic cells (DC) and natural killer (NK) cells. Bidirectional interactions between DCs and NK cells contribute to tailor leishmaniasis outcome. Despite advances in the Leishmania biology field in recent decades, the mechanisms involved in DC/NK-mediated control of Leishmania sp. pathogenesis as well as the cellular and molecular players involved in such interaction remain unclear. The present study sought to investigate canonical pathways associated with CL arising from Leishmania braziliensis infection. Initially, two publicly available microarray datasets of skin biopsies from active CL lesions were analyzed, and five pathways were identified using differentially expressed genes. The "Crosstalk between DCs and NK cells" pathway was notable due to a high number of modulated genes. The molecules significantly involved in this pathway were identified, and our findings were validated in newly obtained CL biopsies. We found increased expression of TLR4, TNFRSF1B, IL-15, IL-6, CD40, CCR7, TNF and IFNG, confirming the analysis of publicly available datasets. These findings reveal the "crosstalk between DCs and NK cells" as a potential pathway to be further explored in the pathogenesis of CL, especially the expression of CCR7, which is correlated with lesion development.

Synthesis of 1,2,3-Triazole-Containing Methoxylated Cinnamides and Their Antileishmanial Activity against the Leishmania braziliensis Species.

Leishmaniasis is a group of infectious diseases caused by protozoan parasites that belong to the genus Leishmania. Currently, there is no human vaccine, and the available treatments are associated with toxicity, high cost, and the emergence of resistant strains. These factors highlight the need to identify new antileishmanial candidates. In this study, we synthesized twenty-four methoxylated cinnamides containing 1,2,3-triazole fragments and evaluated their antileishmanial activity against the Leishmania braziliensis species, which is the main etiological agent responsible for American Tegumentary Leishmaniasis (ATL). The cinnamides were synthetically prepared using nucleophilic acyl substitution and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. The compounds were characterized using infrared, nuclear magnetic resonance, and high-resolution mass spectrometry techniques. We performed preliminary studies to evaluate the biological activity of these compounds against L. braziliensis promastigotes and axenic amastigotes. Compound 28, N-((1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1H-1,2,3-triazole-4-yl) methyl)-3,4-dimethoxy cinnamide, demonstrated relevant antileishmanial activity with low toxicity in murine cells. The selectivity index values for this compound were superior compared with data obtained using amphotericin B. Furthermore, this cinnamide derivative reduced the infection percentage and number of recovered amastigotes in L. braziliensis-infected macrophages. It also induced an increase in reactive oxygen species production, depolarization of the mitochondrial potential, and disruption of the parasite membrane. Taken together, these findings suggest that this synthetic compound holds potential as an antileishmanial candidate and should be considered for future studies in the treatment of ATL.

Accuracy of serological tests in diagnosing mucosal leishmaniasis.

In this serum panel-based study, we evaluated the accuracy of serological tests originally developed for visceral leishmaniasis (VL), for diagnosis of mucosal leishmaniasis (ML). A total of five tests were evaluated, four of which are registered at the National Agency of Sanitary Surveillance (Agência Nacional de Vigilância Sanitária-ANVISA) (RIDASCREEN® Leishmania Ab from R-Biopharm AG., Leishmania ELISA IgG + IgM from Vircell S.L., IFI Leishmaniose Humana-BioManguinhos, and IT-LEISH® from Bio-Rad Laboratories, Inc.), and the other a direct agglutination test (DAT-LPC) prototype kit developed at Fiocruz. The panel was composed of 40 serum samples from patients with confirmed ML and 20 from patients with mucosal involvement and negative parasitological/molecular tests for leishmaniasis and confirmation of another etiology. All cases were treated from 2009 to 2016 in a referral center for leishmaniasis in Belo Horizonte, Minas Gerais, Brazil (Instituto René Rachou, Fiocruz). Diagnostic accuracy, based on the cut-off point for VL diagnosis, was 86.2% with RIDASCREEN® Leishmania Ab, 73.3% with Leishmania ELISA IgG + IgM, and 66.7% with IFI Leishmaniose Humana, while IT-LEISH® and DAT-LPC had the lowest accuracy (38.3%), despite high specificity (100% and 95%, respectively). New cut-off points defined with sera from ML patients improved accuracy from 86.2 to 89% (p = 0.64) and 73.3 to 88% (p = 0.04) for RIDASCREEN® Leishmania Ab and Leishmania ELISA IgG + IgM, respectively. Moreover, these tests presented greater sensitivity and immunoreactivity in patients with moderate/severe clinical ML forms. The data of this study suggest that ELISA assays can contribute to laboratory diagnosis, especially for patients with moderate or severe mucosal involvement.

Antileishmanial Activity of Clinanthus milagroanthus S. Leiva & Meerow (Amaryllidaceae) Collected in Peru.

Leishmaniasis is a worldwide infectious parasitic disease caused by different species of protozoa of the genus Leishmania, which are transmitted to animals and humans through the bite of insects of the Psychodidae family. In the present work, the antileishmanial activity of an alkaloid extract of the bulbs of Clinanthus milagroanthus S. Leiva & Meerow (Amaryllidaceae) was evaluated in vitro, in vivo, and in silico against the parasite Leishmania braziliensis, and the chemical profile of the sample was determined by GC-MS analysis. At concentrations of 1, 10, and 100 µg·mL−1, the alkaloid extract presented inhibition percentages of 8.7%, 23.1%, and 98.8%, respectively, against L. braziliensis with a p < 0.05, and IC50 values of 18.5 ± 0.3 µg·mL−1. Furthermore, at a dose of 1.0 mg·kg−1, a greater decrease in lesion size was observed (90%) for in vivo assays, as well as a decrease in infection (96%), finding no significant differences (p > 0.05) in comparison with amphotericin B (92% and 98%, respectively). Eleven alkaloids were identified in C. milagroanthus bulbs: galanthamine, vittatine/crinine, 8-O-demethylmaritidine, anhydrolycorine, 11,12-dehydroanhydrolycorine, hippamine, lycorine, 2-hydroxyanhydrolycorine, 7-hydroxyclivonine, 2α-hydroxyhomolycorine, and 7-hydroxyclivonine isomer. A molecular model of Leishmania braziliensis trypanothione reductase (TRLb) was built using computational experiments to evaluate in silico the potential of the Amaryllidaceae alkaloid identified in C. milagroanthus toward this enzyme. The structures galanthamine, 7-hydroxyclivonine isomer, and crinine showed better estimated free energy of binding than the reference compound, amphotericin B. In conclusion, this is the first in vitro, in vivo, and in silico report about the antileishmanial potential and alkaloid profiling of the extract of C. milagroanthus bulbs, which could become an interesting source of bioactive molecules.

Uso de anfotericina B liposomal en leishmaniasis cutánea diseminada causada por Leishmania braziliensis en un paciente pediátrico con síndrome de Down / Use of liposomal anfotericin B in diseminated cutaneous leishmaniasis caused by Leishmania braziliensis in a pediatric patient with Down syndrome

Se presenta un caso de leishmaniasis selvática cutánea diseminada con manifestación extensa en una paciente pediátrica con síndrome de Down. El caso se confirmó a través de estudios parasitológicos e inmunológicos, mientras que la identificación se realizó mediante la técnica de reacción en cadena de la polimerasa-polimorfismos de longitud de fragmentos de restricción (PCR-RFLP, por sus siglas en inglés), determinándose la especie como Leishmania (Viannia) braziliensis. La manifestación clínica agresiva y prolongada con poca respuesta a estibogluconato y anfotericina desoxicolato pueden deberse al déficit inmunológico que se presenta como parte del síndrome de Down. La paciente eventualmente recibió tratamiento con anfotericina B liposomal y al término de la terapia, mostró mejoría clínica de las lesiones. El presente reporte ilustra los desafíos tanto de diagnóstico como tratamiento de leishmaniasis cutánea en pacientes pediátricos inmunosuprimidos, especialmente en un entorno de difícil acceso social, económico y geográfico, a los servicios de salud. Se recomienda considerar a la leishmaniasis en el diagnóstico diferencial cuando se atienda ulceras crónicas dermatológicas atípicas; así como tener en cuenta el uso de anfotericina liposomal en pacientes inmunocomprometidos.

We present a case of disseminated cutaneous leishmaniasis with extensive manifestation in a pediatric patient with Down syndrome. The case was confirmed by parasitological and immunological tests. The species was identified as Leishmania (Viannia) braziliensis by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). The immune deficit that occurs as part of Down syndrome may have been the reason for the aggressive and prolonged clinical manifestations as well as the poor response to stibogluconate and deoxycholate amphotericin. The patient was treated with liposomal amphotericin B and at the end of therapy, showed clinical improvement of the lesions. This report highlights the challenges of the diagnosis and treatment of cutaneous leishmaniasis in immunosuppressed pediatric patients, especially under difficult social, economic and geographic conditions. Leishmaniasis should be considered as a differential diagnosis when treating atypical chronic dermatologic ulcers; the use of liposomal amphotericin in immunocompromised patients should also be considered in these cases.

Desenvolvimento e avaliação de uma proteína quimérica recombinante para o diagnóstico e prognóstico das leishmanioses tegumentar e visceral / Development and evaluation of a recombinant chimeric protein for the diagnosis and prognosis of tegumentary and visceral leishmaniasis

As leishmanioses são doenças tropicais negligenciadas com alta endemicidade e que afetam milhares de pessoas no mundo. Sua infecção é causada por parasitos protozoários do gênero Leishmania. A diversidade biológica entre as espécies é quem permite determinar as manifestações clínicas, sendo elas na forma de leishmaniose visceral (LV) ou leishmaniose tegumentar (LT). Dentre estas manifestações, a LV é considerada a mais grave, devido sua alta letalidade e grande emergência em indivíduos com a infecção provocada pelo vírus da imunodeficiência humana (HIV). Atualmente, as medidas de controle e prevenção adotadas pela Organização Mundial da Saúde (OMS), baseiam-se em uma combinação de estratégias de intervenção contra a infecção, uma vez que o diagnóstico eficaz e precoce é indispensável para que se possa intervir com o tratamento adequado, diminuindo índices de mortalidade e a evolução de complicações clínicas. Entretanto, os testes sorológicos utilizados apresentam sensibilidade e especificidade prejudicadas em pacientes com leishmanioses e/ou coinfectados LV/HIV, devido a baixos ní-veis de anticorpos antileishmanial ou pela presença de doenças que causem reação cruzada, levando a resultados falso-positivos. A sensibilidade torna-se também variável em pacientes tratados, uma vez que a sorologia pode manter-se positiva por meses ou anos após o fim do tratamento e cura da doença. Buscando resolver tal problemática, a identificação de novos antígenos, por meio de análises de bioinformática associadas à imunoproteômica, tem permitido a detecção de novas proteínas com potencial aplicação diagnóstica. Em estudos anteriores, as proteínas hipotéticas LiHyT, LiHyD, LiHyV e LiHyP foram encontradas em espécies de Leishmania spp, e avaliadas em suas versões recombinantes por meio de ensaios de ELISA, obtendo resultados satisfatórios para a detecção da LV humana e canina. Com base nessas informações, o presente trabalho teve como objetivo desenvolver uma proteína quimera recombinante base-ada na predição de epítopos lineares específicos de células B das quatro proteínas antigênicas de L. infantum citadas e avaliar o potencial diagnóstico, assim como dos peptídeos individuais que a constituíram, frente à leishmaniose humana, bem como com a coinfecção com HIV, além de testar os antígenos como marcadores prognóstico após o tratamento da LV e LT. As sequências de aminoácidos das proteínas foram avaliadas e oito epítopos de células B foram preditos e utilizados na construção de uma nova proteína quimérica. A proteína foi expressa, purificada e avaliada como antígeno recombinante em ELISA para o diagnóstico de LV, LT, coinfecção LV/HIV e prognóstico em amostras de pacientes tratados de LV e LT. Os epítopos de células B usados na construção da quimera foram sintetizados e também testados em ELISA frente às mesmas amostras, assim como um extrato antigênico solúvel de Leishmania braziliensis (SLA). Os resultados mostraram que a proteína quimera apresentou sensibilidade e especificidade de 100% para diagnosticar a LV, LT e LV/HIV, enquanto os peptídeos sintéticos apresentaram sensibilidade variando entre eles de 9,1% a 90,9% para amostras de LT e 76,8% a 99,2% para amostras de LV e LV/HIV, já os valores de especificidade atingiram 98,3% a 99,1% para LT e 67,1% a 95,7% para LV e LV/HIV. O SLA apresentou sensibilidade e especificidade de 18,2% e 98,3% para LT, e 56,8% a 69,5% para amostras de LV e LV/HIV, respectivamente. Uma avaliação prognóstica preliminar mostrou ainda que os anticorpos anti-quimera diminuíram em níveis significativos, quando comparada a reatividade sorológica antes e seis meses após o tratamento, sugerindo um possível papel prognóstico da quimera para as leishmanioses. O presente estudo, mostrou-se eficaz na construção e avaliação de novos candidatos, que demonstram ter um bom desempenho na detecção diagnóstica e prognóstica para as leishmanioses e dos casos de coinfecção LV/HIV.

Leishmaniasis are neglected tropical diseases with high endemicity that affect thousands of people in the world. Infection is caused by protozoan parasites of the genus Leishmania. The biological diversity between species is what allows determining the clinical manifestations, either in the form of visceral leishmaniasis (VL) or tegumentary leishmaniasis (TL). Among these clinical manifestations, VL is considered the most serious, due to its high lethality and great emergence in individuals with infection caused by the human immunodeficiency virus (HIV). Currently, the control and prevention measures adopted by the World Health Organiza-tion (WHO) are based on a combination of intervention strategies against the infection, since an effective and early diagnosis is essential to intervene with the appropriate treatment, decrea-sing mortality rates and evolution of clinical complications. However, the serological tests used show impaired sensitivity and specificity in patients with leishmaniasis and/or coinfected with VL/HIV, due to low levels of anti-leishmanial antibodies or the presence of diseases that cause cross-reaction, leading to false-positive results. The sensitivity also becomes variable in treated patients, since the serology can remain positive for months or years after the end of the trea-tment and cure of the disease. Seeking to solve this problem, the identification of new antigens, through bioinformatic analysis associated with immunoproteomic, has allowed the detection of new proteins with potential diagnostic application. In previous studies, the hypothetical proteins LiHyT, LiHyD, LiHyV and LiHyP were found in species of Leishmania spp, and evaluated in their recombinant versions through ELISA assays, and satisfactory results were obtained for the detection of human and canine VL. Based on this information, the present work aimed to develop a recombinant chimera protein through on the prediction of specific linear epitopes of B cells derived from these four antigenic proteins of L. infantum and to evaluate its diagnostic potential, as well as the individual peptides that constitute it, against human leishmaniasis, as well as co-infection with HIV, in addition to testing them as possible prognostic markers of patients after VL and TL treatment. The amino acid sequences of the proteins were evaluated and eight B cell epitopes were predicted and used in the construction of a new chimeric protein. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA for the diagnosis of VL, TL, VL/HIV co-infection and prognosis in samples from patients treated for VL and TL. The B cell epitopes used in the construction of the chimera were synthesized and also tested in ELISA against the same samples, as well as a soluble Leishmania braziliensis antigenic extract (SLA). The results showed that the chimera protein apresented sensitivity and specificity of 100% for diagnosing VL, TL and VL/HIV, while the synthetic peptides showed sensitivity ranging from 9.1% to 90.9% for TL samples and 76.8 % to 99.2% for VL and VL/HIV samples, while the specificity values reached from 98.3% to 99.1% for TL and 67.1% to 95.7% for VL and VL/HIV. The SLA showed sensitivity and specificity of 18.2% and 98.3% for TL, and 56.8% to 69.5% for VL and VL/HIV samples, respectively. A preliminary prog-nostic evaluation also showed that anti-chimera antibodies significantly decreased when com-pared to serological reactivity before and six months after treatment, suggesting a possible prognostic role of the antigen for leishmaniasis. The present study proved to be effective in the construction and evaluation of new candidates, who demonstrate good performance in diagnos-tic and prognostic detection for leishmaniasis and VL/HIV co-infection.

Development of Ag-ZnO/AgO Nanocomposites Effectives for Leishmania braziliensis Treatment.

Tegumentary leishmaniasis (TL) is caused by parasites of the genus Leishmania. Leishmania braziliensis (L.b) is one of the most clinically relevant pathogens that affects the skin and mucosa, causing single or multiple disfiguring and life-threatening injuries. Even so, the few treatment options for patients have significant toxicity, high dropout rates, high cost, and the emergence of resistant strains, which implies the need for studies to promote new and better treatments to combat the disease. Zinc oxide nanocrystals are microbicidal and immunomodulatory agents. Here, we develop new Ag-ZnO/xAgO nanocomposites (NCPs) with three different percentages of silver oxide (AgO) nanocrystals (x = 49%, 65%, and 68%) that could act as an option for tegumentary leishmaniasis treatment. Our findings showed that 65% and 68% of AgO inhibit the extra and intracellular replication of L.b. and present a high selectivity index. Ag-ZnO/65%AgO NCPs modulate activation, expression of surface receptors, and cytokine production by human peripheral blood mononuclear cells toward a proinflammatory phenotype. These results point to new Ag-ZnO/AgO nanocomposites as a promising option for L. braziliensis treatment.

Comparison between Colorimetric In Situ Hybridization, Histopathology, and Immunohistochemistry for the Diagnosis of New World Cutaneous Leishmaniasis in Human Skin Samples.

New world cutaneous leishmaniasis (NWCL) is an anthropozoonosis caused by different species of the protozoan Leishmania. Colorimetric in situ hybridization (CISH) was shown to satisfactorily detect amastigote forms of Leishmania spp. in animal tissues, yet it was not tested for the diagnosis of human NWCL. The aim of this study was to compare CISH, histopathology (HP), and immunohistochemistry (IHC) techniques to diagnose NWCL in human cutaneous lesions. The sample comprised fifty formalin-fixed, paraffin-embedded skin biopsy specimens from patients with NWCL caused by L. (V.) braziliensis. These specimens were analyzed by CISH, using a generic probe for Leishmania, IHC, and HP to assess the sensitivity of these methods by using a parasitological culture as a standard reference. Additional specimens from three patients diagnosed with cutaneous mycoses were also included to evaluate cross-reactions between CISH and IHC. The sensitivities of IHC, CISH, and HP for detecting amastigotes was 66%, 54%, and 50%, respectively. IHC, unlike CISH, cross-reacted with different species of fungi. Together, these results demonstrate that CISH may be a complementary assay for the detection of amastigote in the laboratorial diagnosis routine of human NWCL caused by L. (V.) braziliensis.

Serodiagnosis and therapeutic monitoring of New-World tegumentary leishmaniasis using synthetic type-2 glycoinositolphospholipid-based neoglycoproteins.

American tegumentary leishmaniasis (TL) caused by Leishmania braziliensis is characterized by a spectrum of clinical presentations, ranging from localized cutaneous ulcers (CL), mucosal (ML), or disseminated (DL) disease, to a subclinical (SC) asymptomatic form. Current diagnosis based on parasite culture and/or microscopy lacks sensitivity and specificity. Previous studies showed that patients with CL and ML have very high levels of Leishmania-specific anti-α-Gal antibodies. However, the native parasite α-Gal glycotope(s) is(are) still elusive, thus they have not yet been explored for a more accurate TL diagnosis. Using a chemiluminescent immunoassay, we evaluated the seroreactivity of TL patients across its clinical spectrum, and of endemic (EC) and nonendemic healthy controls (NEC) against three synthetic neoglycoproteins (NGP29b, NGP30b, and NGP28b), respectively comprising the L. major-derived type-2 glycoinositolphospholipid (GIPL)-1 (Galfß1,3Manα), GIPL-2 (Galα1,3Galfß1,3Manα), and GIPL-3 (Galα1,6Galα1,3Galfß) glycotopes. Contrary to NGP29b and NGP30b, NGP28b exhibited high sensitivity and specificity to a CL serum pool. More importantly, NGP28b reacted strongly and specifically with individual sera from distinct clinical forms of TL, especially with SC sera, with 94% sensitivity and 97% specificity, by post-two-graph receiver-operating characteristic curve analysis. Contrary to NGP29b, NGP28b showed low cross-reactivity with Chagas disease and control (NEC/EC) sera. Additionally, seroreactivity of CL patients against NGP28b was significantly decreased after successful chemotherapy, indicating that L. braziliensis-specific anti-α-Gal antibodies may serve as an early biomarker of cure in CL. Our data also points towards the applicability of L. major type-2 GIPL-3-derived Galα1,6Galα1,3Galfß glycotope for the serological diagnosis of American TL, particularly of the subclinical form.