Synthesis, biological evaluation and mechanism of action of benzothiazole derivatives with aromatic hydrazone moiety, a new class of antileishmanial compounds.

Leishmaniasis is a disease caused by protozoa Leishmania spp., considered as a significant and urgent public health problem mainly in developing countries. In the absence of an effective vaccine, the treatment of infected people is one of the most commonly prophylactic measures used to control this disease. However, the therapeutic arsenal is reduced to a few drugs, with serious side effects and variability in efficacy. Attempting to this problem, in this work, a series of benzothiazole derivatives was synthetized and assayed against promastigotes and intracellular amastigotes of L. amazonensis, as well as the toxicity on macrophages. In addition, studies about the mechanism of action were also performed. Among the synthesized molecules, the substitution at position 4 of the aromatic ring appears to be critical for activity. The best compound exhibited IC50 values of 28.86 and 7.70 µM, against promastigotes and amastigotes of L. amazonensis, respectively, being more active than miltefosine, used as reference drug. The in silico analysis of physicochemical and pharmacokinetic (ADMET) properties of this compound suggested a good profile of oral bioavailability and safety. In conclusion, the strategy of using benzothiazole nucleous in the search for new antileishmanial agents was advantageous and preliminar data provide information about the mechanism of action as well as in silico parameters suggest a good profile for preclinical studies.

4-Quinolinylhydrazone analogues kill Leishmania (Leishmania) amazonensis by inducing apoptosis and mitochondria-dependent pathway cell death.

Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 µM to promastigotes, and 14.31-61.98 µM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The in vivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. In vivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent.

Enzootic instability for bovine anaplasmosis on family farms located in southwestern Paraná, Brazil / Instabilidade enzoótica para anaplasmose bovina em propriedades de agricultura familiar do sudoeste paranaense, Brasil

Abstract The objective of this study was to assess the occurence of animals seropositive for Anaplasma marginale in the municipality of Realeza, Paraná State, Brazil. Blood samples were collected from 344 cows on 18 small farms in the municipality of Realeza-PR. The animals’serum samples were forwarded to the Federal University of Fronteira do Sul, in order to investigate the occurrence of anti-A. marginale IgG antibodies by an enzyme-linked immunosorbent assay commercial kit. IgG antibodies to A. marginale were detected in cattle from 77.7% of the farms. To the best author's knowledge, this is the first report of occurrence of A. marginale in cattle in southwestern Paraná. The serological assay showed that 24.4% of the animals were seropositive, thus characterizing the location investigated as an area of enzootic instability for the disease. The family farms located in the municipality of Realeza-PR showed enzootic instability for bovine anaplasmosis. It is necessary to conduct disease monitoring programs in association with preventive measures in order to ensure the sanitary quality of the herds and to reduce economic losses for the farmers. In addition, it is essential to implement educational extension actions that allow farmers to acquire knowledge, attitudes and perceptions regarding the risk factors that contribute towards herd A. marginale-infection.

Resumo O objetivo deste estudo foi avaliar a ocorrência de animais soropositivos para Anaplasma marginale, no município de Realeza, Estado do Paraná, Brasil. Foram colhidas amostras de sangue de 344 fêmeas bovinas provenientes de 18 propriedades rurais do município de Realeza - PR. Amostras de soro dos animais foram encaminhadas à Universidade Federal da Fronteira Sul, para realização do Ensaio Imunoenzimático Indireto para pesquisa de anticorpos IgG anti- Anaplasma marginale por meio de kit comercial. Anticorpos IG anti-A. marginale foram detectados em 77,7% das propriedades. Trata-se do primeiro registro da ocorrência de A. marginale no Sudoeste paranaense. A sorologia evidenciou 24,4% de animais soropositivos, caracterizando o local pesquisado como área de instabilidade enzoótica para a doença. As propriedades de agricultura familiar, localizadas no município de Realeza-PR, apresentaram instabilidade enzoótica para anaplasmose bovina. É necessário que programas de monitoramento da enfermidade sejam realizados em conjunto com medidas de prevenção, visando garantir qualidade sanitária do rebanho e reduzir perdas econômicas dos produtores rurais. Além disso, é fundamental a realização de ações de extensão que viabilizem a aquisição de conhecimento, atitude e percepção dos criadores diante dos fatores de risco contribuintes para a infecção por A. marginale do rebanho.

Alterações hematológicas e bioquímicas causadas por Anaplasma marginale em bovinos com aptidão leiteira da região Sudoeste do Paraná / Changes hematological and biochemical caused by Anaplasma marginale in dairy cattle suitability of southwest region Paraná

O objetivo deste trabalho foi descrever as complicações hematológicas e bioquímicas em bovinos da região Sudoeste do Paraná infectados por Anaplasma marginale, comparando estas alterações com os parâmetros apresentados por bovinos sadios da mesma região. Foram avaliadas 40 vacas com aptidão leiteira, sendo 20 clinicamente suspeitas de estarem infectadas pelo parasito e outras 20 sadias, que serviram como grupo controle. O hemograma foi realizado em contador hematológico automático. Para a contagem diferencial de células brancas e pesquisa do agente realizou-se esfregaço sanguíneo. As análises de albumina, proteínas totais (PT), uréria, creatinina, aspartato-aminotrasferase, e gama-glutamiltransferase séricas, foram realizadas em analisador bioquímico semi-automático. Os resultados obtidos foram avaliados através do programa SPSS, versão 20.0, sendo submetidos ao Teste t de Student. O diagnóstico foi realizado pela identificação do hemoparasito em esfregaço sanguíneo. Os animais infectados apresentaram uma parasitemia que variou de 11 a 20%. Constatou-se diferença estatística significante (p<0,01), entre os seguintes parâmetros: hematócrito das infectadas (17,99±1,49%) e sadias (29,38±0,96); número de hemácias das infectadas (3,71±0,39x106 /µL) e sadias (7,23±0,37x106 /µL); hemoglobina das infectadas (6,08±0,65g/dL) e sadias (9,96±0,33g/dL); VCM das infectadas (52,60±3,07fL) e sadias (41,34±1,31fL); monócitos das infectadas (1353,27±373,61/µL) e sadias (517,84±113,22/µL); PT (proteínas totais) das infectadas (7,60±0,18g/dL) e sadias (5,90±0,28g/dL). Os animais infectados apresentaram acentuada anemia e aumento sérico das PT. Conclui-se que estes hemoparasitos provocam monocitose e sinais clínicos graves decorrentes da anemia acentuada, porém não alteram os marcadores da função hepática e renal de bovinos na forma clínica de Anaplasmose.

The objective of this study was to describe the hematological and biochemical complications in cattle of southwestern Paraná region infected with Anaplasma, comparing these changes with the parameters presented by healthy cattle from the same region. We evaluated 40 cows with milk aptitude, 20 clinically suspected of being infected by the parasite and other 20 healthy, which served as a control group. Blood counts were performed in automated hematology counter. For the differential white cell count and agent search was held smears. Albumin analysis, total protein (TP), urea, creatinine, aspartate-aminotransferase and serum gamma-glutamyl transferase were carried out in semi-automatic biochemical analyzer. The results were evaluated using SPSS, version 20.0, and submitted to the Student t test. The diagnosis was made by identifying the hemoparasite in blood smears. Infected mice had a parasitemia ranging from 11 to 20%. We found statistically significant differences (p<0.01) among the following: hematocrit of infected (17,99±1,49%) and healthy controls (29,38±0,96%); number of red blood cells infected (3,71±0,39x106 / µL) and healthy controls (7,23±0,37x106 /µL); of infected hemoglobin (6,08±0,65g/dL) and healthy controls (9,96±0,33g/dL); VCM infected (52,60±3,07fL) and healthy controls (41,34±1,31fL); of infected monocytes (1353,27±373,61/µL) and healthy controls (517,84±113,22/µL); PT (total protein) of infected (7,60±0,18g/dL) and healthy controls (5,90±0,28g/dL). Infected animals showed marked anemia and serum increase in PT. We conclude that these blood parasites cause monocytosis and severe clinical signs resulting from severe anemia, but do not alter markers of liver and renal function of cattle in the clinical form of Anaplasmosis.

Increased polyclonal immunoglobulin reactivity toward human and bacterial proteins is associated with clinical protection in human Plasmodium infection.

BACKGROUND: Polyclonal B-cell activation is well known to occur in Plasmodium infections, but its role in pathogenesis or protection remains unclear. However, protective properties of natural antibodies have previously been demonstrated in other contexts. METHODS: Sera from asymptomatic and symptomatic Plasmodium-infected subjects locally detected in a survey study in the Brazilian Amazon, and from unexposed and exposed but presently uninfected control subjects, were assayed by a standardized quantitative immunoblot method allowing simultaneous detection of IgG or IgM reactivity to a large number of parasite-unrelated proteins. RESULTS: In subjects free of coinfection with hepatitis B virus, IgG reactivity to human brain antigens and Escherichia coli proteins was strikingly enhanced in asymptomatic Plasmodium-infected individuals when compared to such with clinical malaria symptoms, or to uninfected control subjects. This difference was most characteristic for limited exposure times (less than ten years locally, or 20 years in endemic areas). It was more significant than a similar trend found for IgG to Plasmodium falciparum antigens, and unrelated to parasitaemia levels. Asymptomatic subjects with comparatively short exposure characteristically showed relatively elevated IgG versus IgM reactivity. Polyclonal IgG reactivity appears triggered by previous P. falciparum but not Plasmodium vivax malaria. CONCLUSION: The observed difference in polyclonal antibody production seems related to intrinsic activation states of infected individuals, rather than to parasite-antigen specific immune responses. However, it appears influenced by preceding stimuli. This supports the idea that acquired clinical immunity may not exclusively depend on antigen-specific responses, but also on the individual polyclonal reaction.