PD-L1 pode mediar a exaustão de células T em um caso de leishmaniose cutâneo difusa causada pela Leishmania (L.) Amazonensis

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2019.Introdução: A leishmaniose cutâneo difusa é uma forma rara da leishmaniose tegumentar americana associada à infecção pela Leishmania (L.) amazonensis no Brasil. Representando o polo anérgico da Leishmaniose tegumentar americana, mas explicações para a sua resistência ao tratamento ainda são obscuras. Objetivos: Investigar o papel da via PD1/PD-L1 na exaustão celular da leishmaniose cutâneo difusa causada por Leishmania (L.) amazonensis, através de experimento ex vivo e in vitro após 48h de estímulo com o antígeno de Leishmania (L.) amazonensis. Métodos: A definição do caso de leishmaniose cutânea difusa foi realizado de acordo com os critérios clínicos, laboratoriais e histopatológicos presentes na literatura e através da identificação da espécie de Leishmania responsável. Clinicamente o sujeito apresentava placa eritematosa elevada em dorso nasal de crescimento lento e que surgiu durante viagem à região Amazônica, a imunofluorescência indireta e a intradermo reação de Montenegro foram negativas, a cultura e o exame direto da lesão foram positivos para Leishmania spp. A reação em cadeia da polimerase ITS1PCR identificou Leishmania (L.) amazonensis. O sangue periférico do sujeito da pesquisa foi colhido para o experimento 11 meses após o início do tratamento, quando o mesmo já não apresentava lesão clinicamente evidente. A células mononucleares de sangue periférico foram analisadas ex vivo e in vitro após 48 horas de estimulação com antígeno solúvel de Leishmania (L.) amazonensis. As células foram marcadas para proteínas de superfície celular possivelmente envolvidas na patogênese da leishmaniose cutânea difusa, segundo estudos anteriores [CD14, CD40, CD44, CD80, ligante da proteína de morte celular programada 1 (PD-L1), HLA-DR, CD86, CD3, CD4, CD8, CD45RO, CD45RA, e CD25], bem como para as moléculas intracelulares interferon gama e granzima B e para marcadores de morte celular. Foi realizada revisão narrativa da literatura de forma a correlacionar os achados do estudo aos já descritos por outros trabalhos. Resultados: A análise das moléculas de superfície mostrou expressão de PD-L1, um ligante inibitório, nos monócitos estimulados com o antígeno solúvel de Leishmania (65% das células), enquanto em monócitos não estimulados (controle negativo), esta molécula foi expressa em apenas 35% das células. Por outro lado, observamos uma diminuição de linfócitos CD4+IFN-γ+ (8.32% versus 1.7%) e de linfócitos CD8+ IFN-γ+ (14% versus 1%), quando comparamos as células estimuladas com o controle negativo. Um decréscimo relevante na expressão de granzima B em células T CD8+ após o estímulo com o antígeno solúvel de Leishmania também foi observado, estando presente em 31% no controle negativo, mas somente em 5% no grupo que recebeu o estímulo. A expressão de moléculas coestimulatórias CD40 e CD86 foi semelhante entre as células que receberam o estímulo do SLA e o controle negativo. Conclusão: A ativação da via inibitória PD-L1 após a reestimulação com o antígeno de Leishmania pode estar relacionada a expressão diminuída de IFN-γ em linfócitos T CD8+ e CD4+, e de granzima B em linfócitos T CD8+. A ativação desta via inibitória é uma das hipóteses para explicar a falta de resposta ao tratamento de paciente com leishmaniose difusa ou LCD.Introduction: Diffuse Cutaneous Leishmaniasis (DCL) is a rare disease form associated with L. (L.) amazonensis in South America. It represents the “anergic” pole of American Tegumentary leishmaniasis and the explanation for its resistance to treatment remains elusive. Objectives: Investigate role of PD-1/PD-L1 pathway in the cellular exhaustion of diffuse cutaneous leishmaniasis caused by Leishmania (L.) amazonensis thought an ex-vivo and in vivo experiment after 48hs stimulation with Leishmania (L.) amazonensis antigen. Objetivos: Investigar o papel da via PD1/PD-L1 na exaustão celular da leishmaniose cutâneo difusa causada por Leishmania (L.) amazonensis através de experimento ex vivo e in vitro após 48h de estímulo com o antígeno de Leishmania (L.) amazonensis. Methods: The definition of diffuse leishmaniasis in the subject was made thought clinical, laboratorial, histopathological criteria reported in the literature and thought the identification of the responsible Leishmania species. Clinically the subject presented with a slow growing elevated erythematous plaque in the dorsum nasi that appeared during a trip to the amazonian region, indirect immunofluorescence was negative, Montinegro’s skin reaction was negative, culture and direct examination of the lesion were positive for Leishmania spp. Polimerase chain reaction ITS1PCR identified Leishmania (L.) Amazonensis. Subjects periferal blood was colected for the experiment 11 months after treatment begining, and he no longer presented with clinical evident lesion at this time. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48h of stimulation with soluble L. (L.) amazonensis antigen. The cells were labeled for cells surface proteins possibly involved in the pathogenesis of diffuse leishmaniasis according to previous studies [CD14, CD40, CD44, CD80, Programed death ligand 1 (PD-L1), HLA-DR, CD86, CD3, CD4, CD8, CD45RO, CD45RA, e CD25] as well as for intracellular molecules IFN-γ and granzyme B and for cell death markers. Their positivity the markers was evaluated with the use of flow cytometry. The immunological findings were compared to the existent in the literature though a narrative review. Results: We observed increased expression of PD-L1, a inhibitory ligand, in monocytes stimulated with soluble leishmania antigen(in 65% of cells), when in the unstimulated monocytes(negative controls), it was expressed in only 35% of cells. Conversely, the expression of CD4+IFN-γ+ in T lymphocytes (8.32% versus 1.7%) and CD8+ IFN-γ+ in T lymphocytes (14% versus 1%) decreaced when compared with negative controls. A relevant decrease in the granzyme B expression in the CD8+T cells after the stimulation with soluble leishmania antigen was also observed, being present in 31% in the negative controls but in only 5% in the group that received the stimulus. There was a similar expression of costimulatory molecules CD40 and CD86 between the cells that received the SLA stimulus and the negative control. Conclusion: The activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation could be related to the reduced levels of IFN-γ in CD8+ and CD4+ T cells and granzyme B in CD8+ T cells. The activation of this inhibitory pathway is one of the hypotheses to explain the lack of response to conventional treatment in a patient with diffuse leishmaniasis

Estudo dos fatores clínicos e imunológicos associados à disseminação de Leishmania sp para a mucosa nasal e avaliação da segurança de antígeno de montenegro em camundongos

Tese (doutorado) — Universidade de Brasília, Faculdade de Ciências Médicas, Programa de Pós-Graduação em Ciências Médicas, 2022.Introdução: Uma parte dos pacientes com Leishmaniose tegumentar americana desenvolvem lesões mucosas com consequentes deformidades. A compreensão da fisiopatogênese da forma mucosa é limitada pelo escasso conhecimento dos fatores associados à disseminação do parasito e pelo grande intervalo de tempo que pode existir entre o desenvolvimento das lesões cutânea e mucosa da doença. Objetivo: Avaliar fatores clínicos e imunológicos fatores associados à disseminação de Leishmania spp para a mucosa nasal através de avaliação clínica e laboratorial sistematizada. Além disso procuramos validar novo antígeno de leishmania produzido na UnB. Métodos: Foi realizado estudo transversal onde foram incluídos consecutivamente pacientes atendidos no ambulatório de Leishmaniose do Hospital Universitário de Brasília. Os pacientes foram submetidos a fotografias sistematizadas das lesões e os exames dermatológico/otorrinolaringológico foram registrados em fichas específicas. Citocinas inflamatórias foram medidas no soro. O desfecho principal foi a presença de DNA de leishmania em swab nasal realizada por PCR. Também foi testado antígeno derivado da cepa MHOM/BR/PH8 o qual foi inoculado em camundongos B57BL/6 com avaliação de segurança por parâmetros clínicos, laboratoriais e histopatológicos. Resultados: Para avaliar a associação entre os níveis de citocinas IL-12, IL-6, TNFα, IL-10, IL-1β e IL-8 e a presença de leishmania em mucosa nasal foi construído um modelo de regressão de Poisson considerando interações multivariadas de características clínicas e laboratoriais. Dos 79 pacientes com Leishmaniose cutânea, 24 (30%) apresentavam DNA de L. (V.) braziliensis na mucosa nasal. No modelo multivariado, a presença de DNA do parasito na mucosa foi associada à redução dos níveis de IL-12 (RP = 0,440; p = 0,034), aumento dos níveis de IL-6 (RP = 1,001; p = 0,002) e maior número de afetados segmentos corporais (RP = 1,65; p <0,001). Em um segundo modelo as áreas de infiltração, mácula e eritema foram maiores em pacientes com a disseminação de leishmania para a mucosa nasal, mas a associação não foi significativa. Após a aplicação do antígeno derivado da cepa MHOM/BR/PH8 foi verificada infecção por leishmania em um dos camundongos. Não foram verificados outros sinais clínicos, laboratoriais e histopatológicos de toxicidade em nenhum dos outros camundongos. Conclusões: O modelo multivariado sugeriu que a indução de um perfil Th1 com a produção de IL-12 é importante para prevenir a disseminação de L. (V.) braziliensis para a mucosa. Observamos uma taxa de disseminação precoce de Leishmania spp para a mucosa nasal maior do que a descrita anteriormente. O antígeno testado no estudo produziu possível infecção em um dos camundongos, não sendo seguro para testes em humanos.Introduction: A portion of patients with American Tegumentary Leishmaniasis develop mucosal lesions with consequent deformities. The understanding of the pathophysiogenesis of the mucosal form is limited by the limited knowledge of the factors associated with the dissemination of the parasite and by the large time interval that may exist between the development of the disease's cutaneous and mucosal lesions. Objective: To evaluate clinical and immunological factors related to the spread of Leishmania spp to the nasal mucosa through systematic clinical and laboratory evaluation. In addition, we seek to validate a new leishmania antigen produced at UnB. Methods: A cross-sectional study was carried out in which patients treated at the leishmaniasis outpatient clinic of Hospital Universitário de Brasília were consecutively included. The patients were submitted to systematic photographs of the lesions and dermatological/otorhinolaryngological exams were registered in specific forms. Inflammatory cytokines were measured in serum. The main outcome was the presence of leishmania DNA in a nasal swab performed by PCR. An antigen derived from the MHOM/BR/PH8 strain was also tested and inoculated in B57BL/6 mice with safety assessment by clinical, laboratory and histopathological parameters. Results: To assess the association between the levels of cytokines IL-12, IL-6, TNFα, IL-10, IL-1β and IL-8 and the presence of leishmania in the nasal mucosa, a Poisson regression model was built. considering multivariate interactions of clinical and laboratory characteristics. Of the 79 patients with cutaneous leishmaniasis, 24 (30%) had L. (V.) braziliensis DNA in the nasal mucosa. In the multivariate model, the presence of parasite DNA in the mucosa was associated with reduced IL-12 levels (RP = 0.440; p = 0.034), increased IL-6 levels (PR = 1.001; p = 0.002) and greater number of affected body segments (PR = 1.65; p < 0.001). In a second model, the areas of infiltration, macula and erythema were greater in patients with spread of leishmania to the nasal mucosa, but the association was not significant. After application of the antigen derived from the MHOM/BR/PH8 strain, infection by leishmania was verified in one of the mice. There were no other clinical, laboratory and histopathological signs of toxicity in any of the other mice. Conclusions: The multivariate model suggested that the induction of a Th1 profile with the production of IL-12 is important to prevent the spread of L. (V.) braziliensis to the mucosa. We observed a higher rate of early spread of Leishmania spp to the nasal mucosa than previously described. The antigen tested in the study produced a possible infection in one of the mice, and was not safe for testing in humans

PIGMENTED BOWEN’S DISEASE MIMICKING MELANOMA CLINICALLY AND DERMOSCOPICALLY

Uma variedade de lesões cutâneas pode simular melanoma, tais como nevos melanocíticos, lentigos, ceratose seborreica, nevo azul, carcinoma basocelular pigmentado e dermatofibromas. Relata-se um caso clínico raro de uma paciente do sexo feminino que apresentou lesão em coxa esquerda clínica e dermatoscopicamente compatível com melanoma. A paciente foi submetida à exérese da lesão e o histopatológico diagnosticou doença de Bowen pigmentada. O objetivo é alertar que a doença de Bowen pigmentada, uma forma rara de carcinoma espinocelular in situ, também deve ser lembrada como diagnóstico diferencial de melanoma maligno.A variety of cutaneous lesions can mimic melanoma, such as melanocytic nevi, lentigines, seborrheic keratosis, blue nevi, pigmented basal cell carcinomas and dermatofibromas. This report describes a rare clinical case of a female patient who presented a lesion on the left thigh, which was clinically and dermoscopically compatible with melanoma. The patient underwent excision of the lesion, and histopathology confirmed a diagnosis of pigmented Bowen’s disease. The purpose of this report is to draw attention to the fact that pigmented Bowen’s disease, a rare form of squamous cell carcinoma in situ, should also be considered as a differential diagnosis of malignant melanoma

MELKERSSON-ROSENTHAL SYNDROME

A síndrome de Melkersson-Rosenthal é composta pela tríade: língua plicata, paralisia facial intermitente e edema orofacial. O achado dominante e mais precoce é o edema orofacial. Desta forma, o dermatologista é frequentemente o primeiro profissional a ter contato com o paciente. A condição tem sido pouco descrita em revistas dermatológicas. Apresentamos um caso clássico da tríade completa.The Melkersson-Rosenthal Syndrome is composed of the Triad: linqua plicata, intermittent facial palsy and orofacial swelling. Usually, the dominant and earliest finding of the syndrome is the orofacial swelling. Therefore, it’s frequently the dermatologist the first professional to have contact with the patient. Yet, the condition has been few times described in dermatological literature. We present a classical case of the complete triad

Antibacterial and antioxidant activities of Derriobtusone A isolated from Lonchocarpus obtusus

This study evaluated the effect of derriobtusone A, a flavonoid isolated from Lonchocarpus obtusus, on two important pathogenic bacteria, Staphylococcus aureus and Escherichia coli, as well as its antioxidant activity and toxicity. Planktonic growth assays were performed, and the inhibition of biofilm formation was evaluated. In addition, antioxidant activity was assessed by DPPH radical scavenging assay, ferrous ion chelating assay, ferric-reducing antioxidant power assay, and β-carotene bleaching assay. Toxicity was evaluated by the brine shrimp lethality test. Results showed that derriobtusone A completely inhibited the planktonic growth of S. aureus at 250 and 500 μg/mL; however, it did not have the same activity on E. coli. Derriobtusone A reduced the biomass and colony-forming unit (cfu) of S. aureus biofilm at concentrations of 250 and 500 μg/mL. In various concentrations, it reduced the biofilm biomass of E. coli, and, in all concentrations, it weakly reduced the cfu. Derriobtusone A showed highly efficient antioxidant ability in scavenging DPPH radical and inhibiting β-carotene oxidation. The compound showed no lethality to Artemia sp. nauplii. In conclusion, derriobtusone A may be an effective molecule against S. aureus and its biofilm, as well as a potential antioxidant compound with no toxicity.This study was supported by CAPES (Brazil) through the BEX NT 2052/11NT3 Project and by IBB-CEB and FCT (Portugal) and European Community Fund FEDER, through Program COMPETE, in the ambit of Project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480. Otilia Deusdenia Loiola Pessoa, Benildo Sousa Cavada, and Edson Holanda Teixeira are Senior Fellows of CNPq. Mr. David Martin helped with the English editing of the paper

PD-L1 may mediate T-cell exhaustion in a case of early diffuse Leishmaniasis caused by Leishmania (L.) amazonensis

Introduction: Diffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the “anergic” pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry. Case presentation: A 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation. Conclusion: The dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient

The influence of leprosy-related clinical and epidemiological variables in the occurrence and severity of COVID-19 : a prospective realworld cohort study

Background Protective effects of Bacillus Calmette–Gue´rin (BCG) vaccination and clofazimine and dapsone treatment against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been reported. Patients at risk for leprosy represent an interesting model for assessing the effects of these therapies on the occurrence and severity of coronavirus disease 2019 (COVID-19). We assessed the influence of leprosy-related variables in the occurrence and severity of COVID-19. Methodology/Principal findings We performed a 14-month prospective real-world cohort study in which the main risk factor was 2 previous vaccinations with BCG and the main outcome was COVID-19 detection by reverse transcription polymerase chain reaction (RT-PCR). A Cox proportional hazards model was used. Among the 406 included patients, 113 were diagnosed with leprosy. During follow-up, 69 (16.99%) patients contracted COVID-19. Survival analysis showed that leprosy was associated with COVID-19 (p<0.001), but multivariate analysis showed that only COVID-19-positive household contacts (hazard ratio (HR) = 8.04; 95% CI = 4.93– 13.11) and diabetes mellitus (HR = 2.06; 95% CI = 1.04–4.06) were significant risk factors for COVID-19. Conclusions/Significance Leprosy patients are vulnerable to COVID-19 because they have more frequent contact with SARS-CoV-2-infected patients, possibly due to social and economic limitations. Our model showed that the use of corticosteroids, thalidomide, pentoxifylline, clofazimine, or dapsone or BCG vaccination did not affect the occurrence or severity of COVID-19

Leprosy detection rate in patients under immunosuppression for the treatment of dermatological, rheumatological, and gastroenterological diseases : a systematic review of the literature and meta-analysis

Background: Recently developed immunosuppressive drugs, especially TNF antagonists, may enhance the risk of granulomatous infections, including leprosy. We aimed to evaluate the leprosy detection rate in patients under immunosuppression due to rheumatological, dermatological and gastroenterological diseases. Methods: We performed a systematic review of the literature by searching the PubMed, EMBASE, LILACS, Web of Science and Scielo databases through 2018. No date or language restrictions were applied. We included all articles that reported the occurrence of leprosy in patients under medication-induced immunosuppression. Results: The search strategy resulted in 15,103 articles; finally, 20 articles were included, with 4 reporting longitudinal designs. The detection rate of leprosy ranged from 0.13 to 116.18 per 100,000 patients/year in the USA and Brazil, respectively. In the meta-analysis, the detection rate of cases of leprosy per 100,000 immunosuppressed patients with rheumatic diseases was 84 (detection rate = 0.00084; 95% CI = 0.0000–0.00266; I2 = 0%, p = 0.55). Conclusion: Our analysis showed that leprosy was relatively frequently detected in medication-induced immunosuppressed patients suffering from rheumatological diseases, and further studies are needed. The lack of an active search for leprosy in the included articles precluded more precise conclusions. Trial registration: This review is registered in PROSPERO with the registry number CRD42018116275

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio