In silico and in vitro tests can reveal promising anti-leishmania activity for natural products and their derivatives. The aim of this study was to investigate in silico the pharmacological activities of potential new chalcones and their leishmanicidal potential in vitro. The in silico study was carried out using the PASS, MolPredictX and Molegro Virtual Docker 6.0 programs. Antiparasitic activity was assessed in axenic promastigote and amastigote forms of Leishmania braziliensis. The cytotoxicity tests used the J77G8 cell line. The chalcones exhibited 50% cytotoxic concentration values (CC50) values > 50 μM. Chalcone 4 (named FERAI) presented the best activity with concentration for 50% of promastigotes and intracellular parasites forms (EC50) of 9.75 ± 1.7 and 10.13 ± 1.7 μM for promastigote and amastigote, respectively. Reactive oxygen species (ROS) testing presented increased ROS levels in the parasite at the FERAI concentrations of 10 μM (56.33%), 20 μM (61.76%) and 30 μM (67.13%). Molecular docking revealed interactions (binding energy) between FERAI and the enzymes UDP-glycosyl pyrophosphorylase (-56.8384), dihydroorotate-dehydrogenase (-132.276) and trypanothione-reductase (-151.281). Our results demonstrated the anti-leishmanial activity of chalcones, especially FERAI, with a noted raising of ROS levels in the parasite. Molecular docking revealed dihydroorotate dehydrogenase and trypanothione reductase as potential pharmacological targets for FERAI. antileishmania anti leishmania derivatives PASS 60 6 0 6. programs braziliensis JG J G J77G line CC50 CC (CC50 5 named EC50 EC (EC50 975 9 75 9.7 17 1 7 1. 1013 13 10.1 respectively (ROS 56.33%, 5633 56.33% , 56 33 (56.33%) 2 61.76% 6176 61 76 (61.76% 3 67.13%. 6713 67.13% . 67 (67.13%) binding energy UDPglycosyl UDP glycosyl 56.8384, 568384 56.8384 8384 (-56.8384) dihydroorotatedehydrogenase 132.276 132276 132 276 (-132.276 trypanothionereductase 151.281. 151281 151.281 151 281 (-151.281) antileishmanial leishmanial CC5 (CC5 EC5 (EC5 97 9. 101 10. 563 56.33 (56.33% 61.76 617 (61.76 671 67.13 (67.13% 56838 56.838 838 (-56.8384 132.27 13227 27 (-132.27 15128 151.28 15 28 (-151.281 (CC (EC 56.3 (56.33 61.7 (61.7 67.1 (67.13 5683 56.83 83 (-56.838 132.2 1322 (-132.2 1512 151.2 (-151.28 56. (56.3 61. (61. 67. (67.1 568 56.8 8 (-56.83 132. (-132. 151. (-151.2 (56. (61 (67. (-56.8 (-132 (-151. (56 (6 (67 (-56. (-13 (-151 (5 ( (-56 (-1 (-15 (-5 (-

Lantana caatingensis Moldenke (Verbenaceae) is an endemic plant from northeastern Brazil. However, its chemical composition is almost unknown. This study aimed to characterize the chemical compounds found in L. caatingensis and investigate the antileishmanial potential of its hydroalcoholic extract and polar fractions. Compounds present in the hydroalcoholic extract obtained from L. caatingensis leaves and its polar fractions (ethyl acetate and methanolic) were analyzed by liquid chromatography with a photodiode-array detector coupled to electrospray ionization quadrupole time-of-flight mass spectrometry. An antileishmanial assay was performed in a 96-well plate against Leishmania major promastigotes using resazurin on a plate spectrophotometer and the results were expressed as percentage of growth inhibition. Nine compounds were determined in the investigated extract and fractions, classified as flavonoids, flavonoid C-glycosides and phenylethanoid glycosides. The antileishmanial activity revealed that the hydroalcoholic extract and the ethyl acetate fraction were active against L. major promastigotes with inhibitory concentration of 71.78 and 26.86 µg mL-1, respectively, while the methanolic fraction was inactive (478.40 µg mL-1). This is the first report on the chemical composition and antileishmanial activity of L. caatingensis extracts. These results contribute to further knowledge of species belonging to the Lantana genus. Verbenaceae (Verbenaceae Brazil However unknown L photodiodearray photodiode array timeofflight time flight spectrometry 96well well 96 inhibition flavonoids Cglycosides C glycosides 7178 71 78 71.7 2686 26 86 26.8 mL1, mL1 mL 1, 1 mL-1 respectively 478.40 47840 478 40 (478.4 mL1. . mL-1) extracts genus 9 717 7 71. 268 2 8 26. mL- 478.4 4784 47 4 (478. 478. (478 (47 (4 (

This study details the preparation, antileishmanial assay, and in silico analysis of twenty 2-hydroxy-3-(1,2,3-triazolyl)propyl vanillin derivatives. These compounds were synthesized in three steps and evaluated against Leishmania infantum, Leishmania amazonensis, and Leishmania braziliensis promastigotes. Compounds 3s and 3t were the most effective, showing good activity against all Leishmania species tested. Molecular docking indicated that all compounds bind favorably to the sterol 14α-demethylase (CYP51) enzyme from L. infantum. ADMET (absorption, distribution, metabolism, excretion and toxicity) analysis indicated good oral bioavailability, non blood-brain barrier penetration, and high gastrointestinal absorption. Posaconazole and compounds 3e, 3s, and 3t remained stable in the CYP51 binding region during 100 ns molecular dynamics (MD) simulations. Root mean square deviation (RMSD) and root mean square fluctuations (RMSF) analyses from the MD trajectory revealed significant conformational fluctuations of the CYP51 N-terminal, suggesting occasional expulsion of 3e, potentially explaining its higher IC50 (half-maximal inhibitory concentration) values. Pairwise decomposition analyses from molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations highlighted the importance of hydrophobic residues in interacting with the synthesized derivatives. preparation assay 2hydroxy31,2,3triazolylpropyl 2hydroxy3123triazolylpropyl hydroxytriazolylpropyl 2 hydroxy 3 1,2,3 triazolyl propyl 1 derivatives infantum amazonensis promastigotes s t effective tested 14αdemethylase αdemethylase 14α demethylase α CYP (CYP51 L absorption, absorption (absorption distribution metabolism toxicity bioavailability bloodbrain blood brain penetration 3e e CYP5 10 (MD simulations RMSD (RMSD RMSF (RMSF Nterminal, Nterminal N terminal, terminal N-terminal IC IC5 halfmaximal half maximal concentration values PoissonBoltzmann Poisson Boltzmann MM/PBSA MMPBSA MM PBSA (MM/PBSA 2hydroxy31 3triazolylpropyl triazolylpropyl 123 1,2, (CYP5 2hydroxy3 12 1,2 (CYP 2hydroxy 1,

ABSTRACT This study aimed to determine the performance of Leishmania braziliensis and Leishmania peruviana antigens in the detection of ATL by using serum samples obtained between 2013 - 2016. The obtained soluble and excretion/secretion antigens were transferred to membrane nitrocellulose by immunoblot assay. The evaluation was carried out against sera confirmed for ATL, at a confidence level of 95%, determining that the soluble antigen of Leishmania braziliensis had a sensitivity of 87.7%, specificity of 100% and area under the curve of 0.95; on the other hand, Leishmania peruviana showed values of 92.3%, 95.7% and 0.94, respectively. According to the results, we recommend that the reported immunogenic regions should be characterized and analyzed in order to continue with the development of recombinant and synthetic proteins, aimed at improving the efficiency of the serological diagnosis of the disease.

RESUMEN El presente estudio se planteó determinar el rendimiento de antígenos de Leishmania braziliensis y Leishmania peruviana en la detección de LTA, fue desarrollado a partir de muestras de suero obtenidas entre 2013 - 2016. Los antígenos solubles y de excreción/secreción obtenidos fueron transferidos a membrana de nitrocelulosa mediante un ensayo de inmunotransferencia. Se realizó la evaluación frente a sueros confirmados para LTA, a un nivel de confianza al 95%, logrando determinar que, el antígeno soluble de Leishmania braziliensis presenta una sensibilidad del 87,7%, especificidad del 100% y área bajo la curva de 0,95; mientras que, Leishmania peruviana se encontró valores de 92,3%, 95,7% y 0,94 respectivamente. De acuerdo a los resultados, recomendamos realizar la caracterización y análisis de las regiones inmunogénicas reportadas a fin de continuar con el desarrollo de proteínas recombinantes y sintéticas, orientadas a mejorar la eficiencia del diagnóstico serológico de la enfermedad.

Resumen Introducción: Las úlceras genitales constituyen un reto diagnóstico por sus múltiples etiologías. Uno de los casos a tener en cuenta en la población femenina son las úlceras de Lipschütz. El objetivo es presentar dos casos que se diagnosticaron en el año 2022 en una institución de tercer nivel en Boyacá, Colombia. Caso clínico 1: Paciente de 16 años. Ingresa por cuadro de 5 días con fiebre y aparición de lesiones ulceradas en el área genital. Se tomaron laboratorios para infecciones de transmisión sexual, negativos, con virus Epstein Barr IgG positiva. Se hospitalizó para tratamiento analgésico y antibiótico, y ante la sospecha de infecciones asociadas se tomó la decisión de llevar a desbridamiento y toma de biopsia. Ante la notable mejoría, se dio egreso días después. Caso clínico 2: Paciente de 19 años con discapacidad auditiva y visual, perteneciente a comunidad indígena. Acude por presencia de lesión genital de evolución desconocida. Se solicitaron laboratorios para descartar infecciones de transmisión sexual, Epstein Barr y Leishmania. Se tomó biopsia para descartar malignidad, con resultados negativos. Dermatología considera que se trata de úlcera de Lipschütz, se ajusta manejo ambulatorio y se le da egreso. Conclusiones: La identificación temprana de las lesiones en la región genital en las pacientes menores de edad mejora la sintomatología, tranquiliza a las jóvenes y sus familiares, descarta la asociación con enfermedades de transmisión sexual y disminuye procedimientos e ingresos hospitalarios en ocasiones innecesarios.

Abstract Introduction: Genital ulcers constitute a diagnostic challenge given their multiple etiologies. One of the cases to take into account in the female population is Lipschütz ulcers. The objective is to present two cases that were diagnosed in the year 2022 in a third level institution in Boyacá, Colombia. Case report 1: 16-year-old patient was admitted with fever for 5 days and the appearance of ulcerated lesions in the genital area. Laboratory tests for sexually transmitted infections were negative, with Epstein Barr virus IgG positive. The patient was hospitalized for analgesic and antibiotic treatment, and given the suspicion of associated infections, the decision was made to undergo debridement and take a biopsy. Given the notable improvement, he was discharged. Case report 2: 19-year-old patient with hearing and visual disabilities, belonging to an indigenous community, presented with a genital lesion of unknown evolution. Laboratories were requested to rule out sexually transmitted infections, Epstein Barr and Leishmania. A biopsy was taken to rule out malignancy, with negative results. Dermatology considers it to be a Lipschütz ulcer, therefore outpatient management is adjusted and gives discharge. Conclusions: The early identification of lesions in the genital region in underage patients improves symptoms, reassures young women and their families, ruling out the association with sexually transmitted diseases, and reducing procedures and hospital admissions on occasions unnecessary.

Abstract Leishmaniasis is a disease caused by a flagellated protozoan of the genus Leishmania. Its clinical presentation and global distribution vary depending on the Leishmania species. There are three classic clinical forms: cutaneous, mucocutaneous, and visceral, the most serious form. In Chile, its not an endemic disease, so a high clinical suspicion must be had. The diagnosis can be confirmed by detection of amastigotes in clinical samples, culture and molecular techniques. There are multiple therapeutic options, both systemic and local. The case of a 60-year-old woman who traveled to Spain and developed cutaneous leishmaniasis due to Leishamnia infantum, being the first case reported in Chile, treated successfully with miltefosine,

Resumen La leishmaniasis es una enfermedad causada por un protozoo flagelado del género Leishmania. Su presentación clínica y distribución mundial varían dependiendo de las especies de Leishmania. Existen tres formas clínicas clásicas: cutánea, mucocutánea y visceral, la forma más grave. En Chile no es una enfermedad endémica, por lo que se debe tener una alta sospecha clínica. El diagnóstico se puede confirmar por la detección de amastigotes en muestras clínicas, cultivo o técnicas moleculares. Existen múltiples opciones terapéuticas, tanto sistémicas como locales. Se presenta el caso de una mujer de 60 años con antecedentes de un viaje a España y que desarrolló una leishmaniasis cutánea por Leishmania infantum. Es el primer caso de esta especie reportado en Chile, tratado exitosamente con miltefosina.

RESUMEN En proyectos hidroeléctricos se han realizado pocas investigaciones para determinar la composición taxonómica de dípteros de la familia Psychodidae, subfamilia Phlebotominae. En este trabajo se realizó un inventario taxonómico de flebótomos en el área de influencia de la central hidroeléctrica de Ituango, departamento de Antioquia con el fin de actualizar su distribución y resaltar su importancia médica como vectores del protozoo del género Leishmania. Para esto, se realizaron monitoreos entomológicos con trampas CDC y trampas Shannon, en diez municipios de la subregión occidente (Santa Fe de Antioquia, Peque, Olaya, Liborina y Sabanalarga) y de la subregión norte (municipios de Ituango, Briceño, Valdivia, Toledo y San Andrés de Cuerquia). Para la identificación taxonómica se utilizaron las claves de Young y Duncan y Galati. En total se recolectaron 7993 flebótomos, distribuidos en 39 especies según Galati, destacándose seis especies consideradas vectores de especies de Leishmania, que causan la leishmaniasis cutánea. Estas especies fueron Lutzomyia (Trl.) gomezi, Lu. (Hel.) hartmanni, Psychodopygus panamensis, Pintomyia (Pif.) columbiana, Nyssomyia trapidoi y Ny. yuilli yuilli. Los datos indican que la zona de muestreo es endémica para la leishmaniasis debido a la presencia de vectores transmisores de este parásito. El inventario sobre la distribución de flebótomos servirá de base para estudios posteriores que permitan la implementación de estrategias de vigilancia y control.

ABSTRACT In hydroelectric projects, few investigations have been carried out to determine the taxonomic composition of diptera of the family Psychodidae, subfamily Phlebotominae. In this work, a taxonomic inventory of phlebotomine sandflies was conducted in the area of influence of the Ituango hydroelectric power plant, in the department of Antioquia, Colombia. We highlight their medical importance as vectors of the protozoan of the genus Leishmania and update their distribution. Entomological monitoring was carried out in ten municipalities from the western subregion (Santa Fe de Antioquia, Peque, Olaya, Liborina and Sabanalarga) and the northern subregion (the municipalities of Ituango, Briceño, Valdivia, Toledo and San Andrés de Cuerquia). CDC traps and Shannon traps were used for monitoring. For taxonomic identification, the keys of Young and Duncan and Galati were used. A total of 7993 phlebotomine sandflies were collected, distributed in 39 species according to Galati, highlighting six species considered vectors of Leishmania spp. that causes cutaneous leishmaniasis, which are: Lutzomyia (Trl.) gomezi, Lu. (Hel.) hartmanni, Psychodopygus panamensis, Pintomyia (Pif.) columbiana, Nyssomyia trapidoi and Ny. yuilli yuilli. We conclude that the sampling area is endemic for leishmaniasis due to the presence of vectors transmitting this parasite. The inventory on the distribution of phlebotomine sandflies will serve as a basis for further studies that allow the implementation of surveillance and control strategies.

Abstract The objective of this investigation was to detect positive cases of 3 species of hemopathogens, from the records of 300 dog samples, without distinction of breed, sex and age from the metropolitan area of Asunción, using the conventional PCR (cPCR) technique. for the detection of Ehrlichia spp. and Anaplasma platys from whole blood and Leishmania spp. in whole blood and spinal aspirate; in addition to blood serum for the detection of antibodies to Leishmania spp. and Ehrlichia spp. using immunochromatographic tests, processed by the Biotechnology Laboratory Division of the Faculty of Veterinary Sciences of the National University of Asunción during the first semester of 2021. Out of the 300 records analyzed, 66 were positive for at least one hemoparasite. The detection of positive cases, according to the type of microorganism identified, was for Leishmania spp. 13%, Ehrlichia spp. 9% and Anaplasma platys 0%. Regarding the type of samples analyzed, using cPCR or immunochromatography interchangeably, positive cases were mostly through whole blood 15%, followed by blood serum 6% and finally bone marrow aspirate 3%. Finally, 27 breeds were recorded, of which the mixed breed had the highest number of positive results for hemoparasites (40/66). The findings from this study provide relevant data for understanding the presence and distribution of hemoparasites in canines in the metropolitan area.

Resumen La presente investigación tuvo como objetivo detectar casos positivos de 3 especies de hemopatógenos, a partir de los registros de 300 muestras de perros, sin distinción de raza, sexo y edad procedentes del área metropolitana de Asunción, empleando la técnica PCR convencional (cPCR), para la detección de Ehrlichia spp. y Anaplasma platys a partir de sangre entera y Leishmania spp. en sangre entera y aspirado medular; además de suero sanguíneo para la detección de anticuerpos para Leishmania spp. y Ehrlichia spp. utilizando test inmunocromatográfico, procesadas por la División Laboratorio de Biotecnología de la Facultad de Ciencias Veterinarias de la Universidad Nacional de Asunción durante el primer semestre del 2021. De las 300 muestras analizadas, 66 fueron positivas para al menos un hemopatógeno. La detección de casos positivos, según tipo de microorganismo identificado fue, para Leishmania spp. del 13 %, Ehrlichia spp. del 9 % y Anaplasma platys del 0 %. En cuanto al tipo de muestras analizada, empleando cPCR o inmunocromatografía indistintamente, los casos positivos fueron mayoritariamente a través de sangre entera 15 %, seguida por suero sanguíneo 6 % y finalmente aspirado medular 3 %. Por último, fueron registradas 27 razas, de las cuales la mestiza presentó mayor número de positivos a hemoparásitos (40/66). Los hallazgos encontrados a partir del presente trabajo aportan datos relevantes para el conocimiento de la presencia de hemoparásitos en caninos del área metropolitana y su distribución.

ABSTRACT Introduction. The Toll-like receptor (TLR) interacting with the promastigote of Leishmania spp. determines the cellular activation pathway. Objective. To determine the transcriptional expression of TLR-3, TLR-4, TLR-9, IL-12 and TNF-α in macrophages infected with a native strain of L. braziliensis (Lbn). Materials and Methods. Identification of Lbn was performed by qPCR for kinetoplast DNA sequences. Mouse peritoneal macrophages were infected with promastigotes (MI) and nitric oxide (NO) production was measured; transcript levels for TLRs and cytokines were quantified by qRT-PCR. Results. Lbn showed 96% homology to L. braziliensis. High ON production was observed in IMs at 2 h; significant transcriptional expression especially of TLR-3 and TLR-9, which corresponded with expression for cytokines. Conclusions. Lbn strongly activated macrophages via endosomal TLRs, which can be applied in the design of agonists to treat the disease.

RESUMEN Introducción. El receptor de tipo Toll (TLR) que interactúe con el promastigote de Leishmania spp. determina la vía de activación celular. Objetivo. Identificar la expresión transcripcional de TLR-3, TLR-4, TLR-9, IL-12 y TNF-α en macrófagos infectados con una cepa nativa de L. braziliensis (Lbn). Métodos. La identificación de Lbn se hizo empleando qPCR para secuencias del DNA del cinetoplasto. Los macrófagos peritoneales de ratones fueron infectados con promastigotes y se midieron la producción de óxido nítrico (ON). Se cuantificaron los niveles transcripcionales para TLRs y citoquinas empleando qRT-PCR. Resultados. Lbn presentó 96% de homología con L. braziliensis. En los infectados con promastigotes se observó elevada producción de ON a las 2 h; significativa expresión transcripcional especialmente de TLR-3 y TLR-9 que se correspondió con la expresión para citoquinas. Conclusión. Lbn activó fuertemente a los macrófagos mediante los TLRs endosomales lo cual puede ser aplicado en el diseño de agonistas para tratar la enfermedad.

Abstract Leishmaniosis is a disease caused by protozoan parasites of the genus Leishmania and, in the New World, is transmitted to mammals through the bite of blood-sucking dipteran insects of the genus Lutzomyia. Canines are susceptible to infection, developing the visceral form (VL) and the cutaneous form (CL) of the disease. Diagnostic methods include direct observation of the parasite by light microscopy, serology, or molecular biology techniques in samples from dogs with clinical signs and/or clinical suspicion. In this word, the usefulness of a nested PCR designed for the diagnosis of this pathology in dogs was evaluated. A total of 18 samples of EDTA pre-treated dog’s whole blood were analyzed. Dogs were from the city of Corrientes and presented clinical suspicion of suffering from leishmaniasis. From the samples, deoxyribonucleic acid (DNA) was extracted and two successive rounds of amplification of a sequence that codes for the small subunit of ribosomal RNA were carried out. The results obtained from the nested PCRs were compared with this obtained from the direct parasitological method (gold standard). The applied technique showed a sensitivity of 100%, specificity of 75%, positive predictive value of 67% and negative predictive value of 100%, turning it into a valuable tool for diagnosing canine leishmaniosis.

Resumen La leishmaniosis es una enfermedad causada por parásitos protozoarios del género Leishmania y transmitida a mamíferos mediante la picadura de insectos dípteros hematófagos del género Lutzomyia en el nuevo mundo. Los caninos son susceptibles de adquirir la forma visceral y la forma cutánea de la enfermedad. Los métodos de diagnóstico incluyen observación directa del parásito por microscopía óptica, serología o técnicas de biología molecular en muestras de perros con signos clínicos evidentes y/o sospecha clínica. En este estudio se evaluó la utilidad de una reacción en cadena de la polimerasa anidada (PCRn) para el diagnóstico de dicha patología en perros. Se analizaron 18 muestras de sangre entera anticoagulada con EDTA de perros de la ciudad de Corrientes con sospecha clínica de padecer leishmaniosis. A partir de las mismas, se extrajo ácido desoxiribonucleico (ADN) y se realizaron dos rondas de amplificación sucesivas de una secuencia que codifica para la subunidad pequeña de ARN ribosomal. Los resultados obtenidos de las PCR anidadas fueron comparados con los resultados obtenidos a partir del método parasitológico directo considerado como gold standard. La técnica aplicada mostró sensibilidad del 100%, especificidad del 75%, valor predictivo positivo del 67% y valor predictivo negativo del 100% lo que la convierte en una herramienta útil para el diagnóstico de leishmaniosis canina.

Resumo A leishmaniose cutânea (LC) é considerada um problema de saúde pública. Os tratamentos atuais apresentam desvantagens por serem invasivos e possuem efeitos colaterais graves, levando a pesquisa de novas alternativas farmacológicas mais eficazes. As plantas são fontes promissoras de bioativos, e por meio de reações químicas pode-se obter novos análogos. O presente estudo objetivou avaliar os efeitos antileishmania do análogo éter n-butil do dilapiol (EBD) extraído das folhas de Piper aduncum. Foi avaliado o potencial citotóxico do EBD nas concentrações de 15,62 a 500 µM em macrófagos peritoneais por 48 h, e em macrófagos RAW 264.7 por 72 h utilizando o método dose-resposta. A atividade antileishmania em promastigotas de L. amazonensis usou-se concentrações de 0,2 a 4,5 µM durante 24, 48 e 72 h e a quantificação da taxa de infecção celular usou-se uma concentração de 4,5 µM do EBD contra as formas amastigotas interiorizado em macrófagos por 24 h e 48 h. O óxido nítrico foi quantificado a partir de macrófagos tratados previamente com o EBD durante 24 h e 48 h. A dosagem das espécies reativas de oxigênio utilizou-se a concentração de 4,5 µM do EBD incubado junto ao acetato de diclorofluoresceína por 1, 3, 6, e 24 h. Para a modelagem molecular do EBD, usou-se a proteína de origem de Leishmania, disponível no banco de dados “Protein Data Bank”. A molécula do estudo não foi tóxica para as células, apresentou uma CC50 de 413 µM em macrófagos peritoneais e de 373,5 µM em RAW 264.7. O análogo inibiu as formas promastigotas de L. amazonensis com uma CI50 de 1,6 µM por 72 horas. O EBD apresentou uma taxa de infecção de 17% e 12%, dilapiol de 24% e 14% e Pentacarinat® de 10% e 9% durante 48 h. O EBD demonstrou uma ligação de energia de -7,8 pelo sítio de ação da enzima U53. Conclui-se que o análogo mostrou atividade antileishmania promissora para futuros testes in vivo. LC (LC pública graves eficazes bioativos podese pode se análogos nbutil n butil (EBD aduncum 1562 15 62 15,6 50 4 2647 264 7 264. doseresposta. doseresposta dose resposta. resposta dose-resposta L usouse usou 02 0 2 0, 45 5 4, utilizouse utilizou 1 3 6 Leishmania Protein Bank. Bank . Bank” células CC CC5 41 3735 373 373, CI CI5 16 horas 17 12 12% 14 Pentacarinat 10 9 7,8 78 8 -7, U53 U Concluise Conclui vivo 156 15, 26 37 7, -7 U5 -

Abstract Cutaneous leishmaniasis (CL) is considered a public health problem. Current treatments have disadvantages because they are invasive and have serious side effects, and thus there is a need for research into new, more effective pharmacological alternatives. Plants are promising sources of bioactive substances, and new analogues can be obtained through chemical reactions. The present study aimed to evaluate the antileishmanial effects of the analog dillapiole n-butyl ether (DBE) extracted from Piper aduncum leaves. The cytotoxic potential of DBE was evaluated at concentrations of 15.62 to 500 µM in peritoneal macrophages for 48 h, and in RAW 264.7 macrophages for 72 h using a dose-response method. The antileishmanial activity in L. amazonensis promastigotes used concentrations of 0.2 to 4.5 µM for 24, 48 and 72 h and the quantification of the cellular infection rate used a concentration of 4.5 µM of DBE against the amastigote forms internalized in macrophages for 24 h and 48 h. Nitric oxide was quantified from macrophages previously treated with DBE for 24 h and 48 h. The dosage of reactive oxygen species used a concentration of 4.5 µM of DBE incubated together with dichlorofluorescein acetate for 1, 3, 6, and 24 h. For the molecular modeling of DBE, the Leishmania protein, available in the “Protein Data Bank” database, was used. The studied molecule was not toxic to cells and presented a CC50 of 413 µM in peritoneal macrophages and 373.5 µM in RAW 264.7. The analogue inhibited promastigote forms of L. amazonensis with an IC50 of 1.6 µM for 72 h. DBE presented an infection rate of 17% and 12%, dillapiole of 24% and 14% and Pentacarinat® of 10% and 9% over 48 h. DBE demonstrated a binding energy of -7.8 for the U53 enzyme. It is concluded that the analogue showed promising antileishmanial activity for future in vivo tests. CL (CL problem alternatives substances reactions nbutyl n butyl (DBE leaves 1562 15 62 15.6 50 4 2647 264 7 264. doseresponse dose response method L 02 0 2 0. 45 5 4. 1 3 6 protein Protein Bank database CC CC5 41 3735 373 373. IC IC5 16 1. 17 12 12% 14 Pentacarinat 10 9 7.8 78 8 -7. U U5 enzyme tests 156 15. 26 37 7. -7 -

Resumo O estudo transversal atual foi realizado entre setembro de 2019 e janeiro de 2020 no Departamento de Patologia Mardan Medical Complex (MMC), Mardan, e no District Headquarter Hospital North Waziristan, Khyber Pakhtunkhwa (KP), Paquistão. Os objetivos do presente estudo foram determinar a prevalência da leishmaniose e seus fatores de risco associados em distritos selecionados da província de KP, Paquistão. Ao todo, 374 (n = 374) pacientes com leishmaniose foram incluídos no presente estudo. Amostras de pele da borda da úlcera foram coletadas. As lâminas foram coradas pelo corante Giemsa e examinadas quanto à presença de amastigotas. As prevalências de pacientes infectados por leishmania em diferentes regiões de KP foram as seguintes: região do Waziristão Norte 53,7 (n = 201), distrito Mardan 34,7% (n = 130); distrito Nowshera 6,7% (n = 25), distrito Swabi 1,1% (n = 4) e outros distritos, ou seja, Dir, Malakand, Buner e Bajawarr foram 3,7% (n = 14). A frequência de leishmaniose foi maior no sexo masculino e a maioria dos pacientes infectados estava na faixa etária < 10 anos. Entre n = 374 pacientes, 95,7% (n = 358) tinham leishmaniose cutânea enquanto 3% (n = 11) tinham infecção do tipo mucocutâneo e 1,3% (n = 5) pacientes tinham infecção cutânea e mucocutânea. Após tratamento com estibogluconato de sódio (SSG), 97% (n = 362) apresentaram sinais clínicos de recuperação completa ou parcial de suas lesões cutâneas. Conclusivamente, a maior incidência de infecção por leishmania ocorreu durante o curto período de estudo e a maioria dos casos apresentou resposta positiva ao tratamento.

Abstract Current cross-sectional study was carried out between September 2019 to January 2020 at the Department of Pathology, Mardan Medical Complex (MMC), Mardan, and District Headquarter Hospital North Waziristan, Khyber Pakhtunkhwa (KP), Pakistan. The objectives of the current study were to determine the prevalence of leishmaniasis and its associated risk factors in selected districts of KP province, Pakistan. Altogether, three hundred and seventy-four (n=374) leishmaniosis patients were included in the current study. Skin specimen from the ulcer border were collected. The slides were stained by Giemsa stain and examined for the presence of amastigote. The prevalence of leishmania infected patients in different region of KP were as follows: North Waziristan region 53.7 (n=201) District Mardan 34.7% (n=130); District Nowshera 6.7% (n=25), District Swabi 1.1% (n=4) and other Districts i.e. Dir, Malakand, Buner and Bajawarr were 3.7% (n=14). The frequency of leishmaniasis were more in male and majority of the infected patients were in the age group of <10 years. Among n=374 patients 95.7% (n= 358) had cutaneous leishmaniasis while 3% (n= 11) had mucocutaneous type of infection and 1.3% (n= 5) patients had both cutaneous and mucocutaneous infection. Upon treatment by Sodium stibogluconate (SSG) 97% (n=362) showed clinical signs of complete or partial recovery of their skin lesions. Conclusively, highest incidence of leishmania infection occurred during short study period and majority of the cases showed positive response to treatment.

Abstract The clinical manifestations of cutaneous leishmaniasis (CL) depend not only on the infecting species involved, but also on the immune response of the individual. Although not yet well understood in humans, parasite survival and persistence are related to the cytokine profile and T cell proliferation, with the Th1 profile being related to cure, and the Th2 profile to disease progression. Considering the need for studies focused on the species with the highest circulation in the state of Amazonas, this study aimed to analyze the immunoregulation stimulated by soluble antigens (SLAs) of Leishmania (L.) amazonensis and Leishmania (V.) guyanensis in human lymphocytes in vitro, in order to understand the immune response of patients with CL. Lymphoproliferation was evaluated against stimuli of SLAs from L. amazonensis (100 µg/mL), SLAs from L. guyanensis (100 µg/mL) and phytohemagglutinin (10 µg/mL) using a BrdU Cell Proliferation ELISA kit after 72 h of incubation. Quantification of the cytokines IL-1b, IL-6, IL-8, IL-10, IL-12 and TNF was performed using the BD™ cytometric bead array human Th1/Th2/Th17 cytokine kit. Our results demonstrated that soluble antigens from L. amazonensis and L. guyanensis stimulated the lymphoproliferation of PBMCs from patients primo-infected with CL. Among the cytokines dosed, the highest concentrations were of IL-6 and IL-8, thus demonstrating that the soluble antigens evaluated are capable of inducing regulatory mechanisms. CL (CL involved individual humans proliferation Th cure progression Amazonas (SLAs L (L. V. V (V. vitro 100 µg/mL, µgmL µg/mL , µg mL 10 (1 7 incubation IL1b, IL1b ILb IL 1b, 1b b IL-1b IL6, IL6 6, 6 IL8, IL8 8, 8 IL-8 IL10, IL10 10, IL-10 IL12 12 IL-1 BD Th1Th2Th17 ThThTh Th17 Th1/Th2/Th1 primoinfected primo infected dosed IL- mechanisms (L (V 1 ( IL1 Th1Th2Th1 Th1/Th2/Th Th1Th2Th

Resumo As manifestações clínicas da leishmaniose cutânea (LC) dependem não apenas da espécie infectante envolvida, mas também da resposta imune do indivíduo. Apesar de ainda não bem esclarecido em humanos, a sobrevivência e persistência do parasito estão relacionados ao perfil de citocinas e proliferação de células T, relacionando-se a cura ao perfil Th1, e a progressão da doença ao perfil Th2. Considerando a necessidade de estudos voltados para as espécies de maior circulação no estado do Amazonas, este trabalho teve como objetivo analisar a imunorregulação estimulada por antígenos solúveis (SLAs) de Leishmania (L.) amazonensis e Leishmania (V.) guyanensis em linfócitos humanos in vitro, afim de compreender a resposta imune de pacientes com LC. A linfoproliferação foi avaliada frente a estímulos de SLAs de L. amazonensis (100 μg/mL), SLAs de L. guyanensis (100 μg/mL) e Phytohemagglutinin (10 μg/mL) utilizando BrdU Cell Proliferation ELISA Kit após 72h de incubação. A quantificação das citocinas IL-1b, IL-6, IL-8, IL-10, IL-12 e TNF foi realizada por BD™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit. Nossos resultados demonstraram que os antígenos solúveis de L. amazonensis e L. guyanensis estimularam a linfoproliferação de PBMCs de pacientes primoinfectados com LC. Entre as citocinas dosadas, as de maiores concentrações foram de IL-6 e IL-8, demonstrando que os antígenos solúveis avaliados são capazes de induzir mecanismos regulatórios. LC (LC envolvida indivíduo T relacionandose relacionando se Th1 Th Th2 Amazonas (SLAs L (L. V. V (V. vitro 100 μg/mL, μgmL μg/mL , μg mL 10 (1 h incubação IL1b, IL1b ILb IL 1b, 1b b IL-1b IL6, IL6 6, 6 IL8, IL8 8, 8 IL-8 IL10, IL10 10, IL-10 IL12 12 IL-1 BD CBA (CBA Th1Th2Th17 ThThTh Th17 Th1/Th2/Th1 dosadas IL- regulatórios (L (V 1 ( IL1 Th1Th2Th1 Th1/Th2/Th Th1Th2Th

ABSTRACT This research aimed to carry out an epidemiological investigation of dogs with canine leishmaniasis in an area of Rio Grande do Sul with no history of notification of the disease. Leishmaniasis is considered an endemic zoonosis in different countries. In Brazil, it occurs mainly in the Southeast and Midwest regions, having drastically increased the number of notifications in recent years. In the state of Rio Grande do Sul, there are reports of the disease in the metropolitan region of Porto Alegre and in the border region in the west of the state, as well as in the central region. The investigation was conducted in 100 dogs from the municipalities of Frederico Westphalen and region. Whole blood was collected for immunochromatographic testing (TR DPP® Canine Visceral Leishmaniasis) and lymph node aspiration. Positives were confirmed by ELISA test and indirect immunofluorescence test with total dilution or immunohistochemistry after skin biopsy. Two animals were diagnosed with visceral leishmaniasis, considered the first reported cases of the disease in the canine population in the region. Therefore, it becomes necessary to research and control the potential presence of sandflies in the area. There is a hypothesis that the animals were contaminated in other regions of the country, remaining as a reservoir until the moment of diagnosis. countries Brazil years 10 TR DPP aspiration biopsy Therefore country diagnosis 1

RESUMO Esta pesquisa objetivou realizar uma investigação de cães com leishmaniose canina, em uma área do Rio Grande do Sul, sem histórico de notificação da doença. A leishmaniose é considerada uma zoonose endêmica em diferentes países. No Brasil, ocorre principalmente nas regiões Sudeste e Centro-Oeste, tendo aumentado drasticamente o número de notificações nos últimos anos. No estado do Rio Grande do Sul, existem relatos da doença na região metropolitana de Porto Alegre e na região de fronteira no oeste do estado, bem como na região central. A investigação ocorreu em 100 cães, provenientes de municípios de Frederico Westphalen e região. Foram colhidos sangue total para realização de teste imunocromatográfico (TR DPP® Leishmaniose Visceral Canina) e punção aspirativa de linfonodo. Os positivos foram confirmados por meio do teste ELISA e do teste de imunofluorescência indireta com diluição total ou imuno-histoquímica após biopsia cutânea. Foram diagnosticados dois animais com leishmaniose visceral, sendo considerados os primeiros relatos de caso da doença canina na região, portanto torna-se necessária a pesquisa e o controle de potencial presença de flebotomíneos na região. Há hipótese de os animais terem sido contaminados em outras regiões do país, permanecendo como reservatório até o momento do diagnóstico. Sul países Brasil CentroOeste, CentroOeste Centro Oeste, Oeste Centro-Oeste anos central 10 TR DPP Canina linfonodo imunohistoquímica imuno histoquímica cutânea visceral tornase torna se país diagnóstico 1