Blocking IL-10 signaling with soluble IL-10 receptor restores in vitro specific lymphoproliferative response in dogs with leishmaniasis caused by Leishmania infantum.

rIL-10 plays a major role in restricting exaggerated inflammatory and immune responses, thus preventing tissue damage. However, the restriction of inflammatory and immune responses by IL-10 can also favor the development and/or persistence of chronic infections or neoplasms. Dogs that succumb to canine leishmaniasis (CanL) caused by L. infantum develop exhaustion of T lymphocytes and are unable to mount appropriate cellular immune responses to control the infection. These animals fail to mount specific lymphoproliferative responses and produce interferon gamma and TNF-alpha that would activate macrophages and promote destruction of intracellular parasites. Blocking IL-10 signaling may contribute to the treatment of CanL. In order to obtain a tool for this blockage, the present work endeavored to identify the canine casIL-10R1 amino acid sequence, generate a recombinant baculovirus chromosome encoding this molecule, which was expressed in insect cells and subsequently purified to obtain rcasIL-10R1. In addition, rcasIL-10R1 was able to bind to homologous IL-10 and block IL-10 signaling pathway, as well as to promote lymphoproliferation in dogs with leishmaniasis caused by L. infantum.

Perfil dos manipuladores de alimentos prontos comercializados em feiras livres da cidade de Natal/RN / Profile of food handlers ready marketed in fairs free Christmas / RN city

Atualmente, à comercialização dos alimentos por ambulantes nas ruas é algo cada vez mais frequente, vem se tornando uma alternativa econômica informal e de fácil acesso para a população em geral, sendo uma prática exercida em todo o Brasil. O presente trabalho teve como objetivo avaliar o perfil dos manipuladores de alimentos prontos nas feiras livres da cidade de Natal/RN. Trata-se de uma pesquisa de campo de caráter quantitativa e exploratória, realizada através da aplicação de um questionário sobre o perfil dos feirantes que comercializam alimentos prontos e o nível de conhecimentos sobre as boas práticas de manipulação. Os resultados mostram que a maioria dos feirantes eram do sexo feminino, com idade entre 51 e 70 anos e que compreendem a importância das boas práticas de manipulação, embora não consigam seguir as normas vigentes.

Concomitant treatment with sertraline and social skills training improves social skills acquisition in social anxiety disorder: A double-blind, randomized controlled trial.

OBJECTIVES: To examine whether: (1) sertraline (SER) + psychotherapy is superior to psychotherapy alone; (2) group cognitive-behavioural therapy (GCBT) is superior to group psychodynamic therapy (GPT) and (3) SER+GCBT or SER+GPT is superior to Placebo (PLA)+GCBT or PLA+GPT in social anxiety disorder (SAD). METHODS: A double-blind randomized controlled trial. Participants were assigned either to: SER+GCBT (n = 34); SER+GPT (n = 36); PLA+GCBT (n = 36) or PLA+GPT (n = 41) for 20 weeks. SER (or PLA) was administered at doses from 50 to 200 mg/d. Primary measures were both categorial: remission (CGI score≤2), response of social symptoms (≥50% reduction in Scale of Avoidance and Social Discomfort (SASD)); and continuous: reduction of SASD and Multidimensional Scale of Social Expression(M-MSSE). RESULTS: SER exhibited better improvement of social anxiety symptoms rate than PLA (25.73% vs. 9.46%, P < .05). Neither GCBT differed from GPT (12.33% vs. 22.54%, P = .11) nor SER+GCBT from PLA+GCBT (17.65% vs. 7.69%, P = .20). However, SER+GPT was superior to PLA+GPT (33.33%, vs. 11.43%, P < .05). M-MSSE had superior improvement for SER+GCBT vs PLA+GCBT (P < .01) but not for SER+GPT vs. PLA+GPT (P = .80). SASD scores improvement were greater for SER than PLA (P < .01) and for SER+GCBT vs. PLA+GCBT (P < .05), but neither GCBT differed from GPT(P = .60) nor SER+GPT differed from PLA+GPT (P = .09). CONCLUSIONS: In overall, SER+psychotherapy was superior to psychotherapy alone. SER potentiated GCBT by enhancing social skills acquisition. TRIAL REGISTRATION: ISRCTN 57551461.

Parasitic load and histological aspects in different regions of the spleen of dogs with visceral leishmaniasis.

Leishmania infantum causes from subclinical infection to severe disease in humans and dogs. The spleen is one of the organs most affected by the infection. Although evidence exists that the parasitic load distribution and histological alterations may not be homogeneous in the affected organs of naturally infected individuals, it has not been formally demonstrated using the current techniques used for studying the disease. In six dogs naturally infected with Leishmania, parasitic load and histological changes were compared in samples collected from the lower, middle and upper third of the spleen. Parasitic load in the spleen of the group of dogs was variable, revealing a difference of 61 times between animals with the lowest and the highest parasitism. The set of parasitic load values of each dog showed a cluster trend, when compared to the other animals. Nevertheless, the parasitic load values of each dog showed a variation ranging from 3.2 to 34.7 times between lowest and highest value. Histological changes showed recognizable variation in frequency (granulomas) or intensity (perisplenitis) in the spleen of 2 out of the 6 dogs. The agreement of histological findings between samples collected from the different thirds of the spleen was good (kappa coeficient, 0.61-0.80) very good (0.81-0.99) or perfect (1.00), for most of the parameters analyzed. Variability of parasitic load and, to a lesser extent, histological changes in spleen of dogs with visceral leishmaniasis is observed. Such variability may be taken in account in the design of studies on pathogenesis, vaccine and therapeutic drug development.

Desenvolvimento e caracterização de formulação de hipoclorito de sódio em gel para uso endodôntico / Development and characterization of sodium hypochlorite gel formulation for endodontic use

A presente pesquisa dedicou-se ao desenvolvimento e à caracterização de uma formulação em gel para o NaOCl, que permitisse o maior controle dessa substância durante a irrigação do sistema de canais radiculares sem perder as características que o tornam um agente de limpeza e descontaminação tão eficiente. Foi utilizada uma solução concentrada de NaOCl, a qual foi titulada para determinação do teor de cloro ativo, e um tensoativo, o lauril éter sulfato de sódio, para o qual foi calculada a concentração micelar crítica em água destilada. A partir destes reagentes foram realizadas diluições e manipuladas sete formulações de NaOCl a 2,5% com diferentes concentrações do tensoativo, as quais foram submetidas às análises de pH, tensão superficial, espectroscopia de infravermelho com transformada de Fourier (FTIR), molhabilidade em dentina humana, reologia, potencial antibacteriano contra Enterococcus faecalis e capacidade de dissolução tecidual. Os valores de pH demonstraram que a adição do tensoativo não alterou este parâmetro, mantendo o meio alcalino desejável para a ação do NaOCl. A adição do tensoativo reduziu a tensão superficial em mais de 50% em relação à solução aquosa de NaOCl. A caracterização por FTIR demonstrou que os reagentes das formulações apenas se solubilizam e interagem, não implicando a ocorrência de reação química entre elas. A molhabilidade das formulações sobre a dentina humana foi inferior à medida para a solução de NaOCl. Todas as formulações apresentaram viscosidade e aspecto que permitem a utilização clínica de forma satisfatória. O teste microbiológico de disco-difusão em ágar revelou ação satisfatória e similar à solução aquosa de NaOCl 2,5%. A velocidade de dissolução tecidual foi reduzida pela adição do tensoativo. Concluiu-se que as formulações de NaOCl em gel com o lauril éter sulfato de sódio são promissoras para sua utilização como substâncias químicas auxiliares em Endodontia (AU).

This research focused on the development and characterization of a gel formulation for NaOCl, which allows greater control during the root canals irrigation without losing the characteristics that make it such na efficient cleaning and decontamination agent. It was used a concentrated solution of NaOCl, which was titrated to determine the active chlorine content, and a surfactant, sodium lauryl ether sulfate, for which the critical micellar concentration in distilled water was calculated. From these reagents were carried out dilutions and manipulated seven formulations of 2.5% NaOCl with different surfactant concentrations, which were submitted to the following analysis: pH, surface tension, Fourier transform infrared spectroscopy (FTIR), wettability on human dentin, rheology and antibacterial potential against Enterococcus faecalis. The pH values showed that the addition of the surfactant did not alter this parameter, keeping the alkaline medium desirable for NaOCl action. The surfactant addition reduced surface tension by more than 50% relative to the NaOCl aqueous solution. The FTIR showed that the reagents only solubilize and interact, not implying the occurrence of chemical reaction between them. The wettability on human dentin was lower than that measured for the NaOCl solution. All formulations showed viscosity and appearance that allow clinical use satisfactorily. The microbiological disk-diffusion test on agar showed a satisfactory action similar to the 2.5% aqueous NaOCl solution. The ability of tissue dissolution was reduced by the addition of the surfactant. It was concluded that NaOCl gel formulations with sodium lauryl ether sulfate are promising for their use as endodontic auxiliary chemicals (AU).

Ultrasound biomicroscopy and claudication test for in vivo follow-up of muscle repair enhancement based on platelet-rich plasma therapy in a rat model of gastrocnemius laceration.

PURPOSE: To track the regeneration process of lateral gastrocnemius due to a muscle laceration in rats, and to treatment with plateletrich plasma (PRP). METHODS: Ultrasound (40 MHz) images were used for measuring pennation angle (PA), muscle thickness (MT) and mean pixel intensity, along with claudication scores, of treated (PRPG) and non-treated (NTG) groups of rats. RESULTS: NTG showed a PA increase for the non-injured leg (p<0.05) and a tendency of MT to increase, whereas for PRPG there were no differences. There was a progressive reduction of the claudication score for the PRPG group throughout the entire period, with an immediate difference after seven days (p<0.05), whereas the NTG had a significant reduction only at day 28 (p<0.05). CONCLUSION: It was observed a compensatory hypertrophic response due to the overload condition imposed to healthy leg for NTG that did not occur in PRPG, suggesting an accelerated repair process of the injured leg due to treatment, anticipating its use.

Ultrasound biomicroscopy and claudication test for in vivo follow-up of muscle repair enhancement based on platelet-rich plasma therapy in a rat model of gastrocnemius laceration

PURPOSE: To track the regeneration process of lateral gastrocnemius due to a muscle laceration in rats, and to treatment with plateletrich plasma (PRP). METHODS: Ultrasound (40 MHz) images were used for measuring pennation angle (PA), muscle thickness (MT) and mean pixel intensity, along with claudication scores, of treated (PRPG) and non-treated (NTG) groups of rats. RESULTS: NTG showed a PA increase for the non-injured leg (p<0.05) and a tendency of MT to increase, whereas for PRPG there were no differences. There was a progressive reduction of the claudication score for the PRPG group throughout the entire period, with an immediate difference after seven days (p<0.05), whereas the NTG had a significant reduction only at day 28 (p<0.05). CONCLUSION: It was observed a compensatory hypertrophic response due to the overload condition imposed to healthy leg for NTG that did not occur in PRPG, suggesting an accelerated repair process of the injured leg due to treatment, anticipating its use.

Optimization of canine interleukin-12 production using a baculovirus insect cell expression system.

BACKGROUND: Interleukin-12 is an important cytokine in mediating cellular immune responses. RESULTS: Recombinant single-chain canine IL-12 was produced in a baculovirus-insect cell system with the aim of conducting further studies on modulation of immune responses in dogs. To optimize the production of recombinant canine IL-12, a classical baculovirus and a modified vector (chitinase A and v-cathepsin knockout) were used containing a native or an optimized insert of canine IL-12. The optimized IL-12 construct contained the GP64 signal peptide and was synthesized with optimized codons for expression in Trichoplusia ni cells. Dot-blot and Western blot analysis showed the highest production levels of recombinant IL-12 protein by the use of the modified baculovirus vector containing the optimized insert, at a multiplicity of infection of five and at 48 h after infection. The recombinant cytokine was successfully purified and showed a good degree of purity, integrity, folding, and yield, with very little endotoxin contamination. Recombinant canine IL-12 induced IFN-γ in canine lymphocytes, indicating that it was biologically active. CONCLUSION: Therefore, this study describes an efficient method to produce adequate amounts of biologically active canine IL-12, useful for immunomodulation studies in dogs.

Floristic woody composition of revegetated mining sites in the Brazilian Federal District / Composição florística lenhosa de jazidas revegetadas no Distrito Federal

The extension degraded by mining in the Brazilian Federal District - BFD is proportionately five times larger than country's average. The restoration of plant communities at these sites is ecologically necessary and legally compulsory. Native woody species are commonly used for ecosystem restoration and this study aimed to identify the floristic composition of woody species introduced in BDF mining sites and discuss its outcomes. The floristic survey was conducted by sampling groups of one hundred individuals until sampling sufficiency was achieved. In total 4,500 plants were sampled in ten sites, which housed 92 autochthonous and 21 allochthonous woody species. Plant communities in the sites assembled between 13 and 62 species, planted at low density - 467 ± 222 plants ha-1. The preferential use of autochthonous species in the revegetation projects resembles the standards recommended by successional restoration models, but the floristic diversity and plant density in initial communities fall below the values deemed as ideal. The 92 autochthonous species include representatives of various habitats, ecological groups and dispersal syndromes. These 92 species currently in use may be regrouped in initial plant communities denser and more diverse than those found in the revegetated sites. The surveyed plant communities were massively composed of arboreal species and such pattern may drive succession towards the formation of forest ecosystems where previously inhabited savanna formations.

A extensão minerada no Distrito Federal - DF é proporcionalmente cinco vezes superior à média nacional. A restauração das comunidades vegetais nesses locais é necessária e obrigatória, e o plantio de espécies lenhosas é prática comum de restauração. Dessa forma, este trabalho visou identificar e avaliar a composição florística lenhosa introduzida em jazidas mineradas no DF como meio de restaurar o ecossistema natural. O levantamento florístico nas áreas selecionadas foi realizado por meio da amostragem de grupos de cem indivíduos, até se obter suficiência amostral. Foram amostradas 4.500 plantas em dez jazidas, que abrigavam 92 espécies lenhosas autóctones e 21 alóctones. As comunidades vegetais levantadas eram formadas por 13 a 62 espécies, plantadas a uma baixa densidade - 467 ± 222 plantas ha-1. O uso preferencial de espécies autóctones nos projetos de revegetação os assemelham aos padrões recomendados pelos modelos sucessionais de restauração, mas a diversidade florística e densidade de plantas nas comunidades iniciais estão abaixo dos valores considerados ideais. Entre as 92 espécies identificadas há representantes de variados habitats, grupos ecológicos e síndromes de dispersão. Portanto, essas 92 espécies podem ser reagrupadas em comunidades iniciais mais densas e diversas do que as verificadas nos projetos executados. As comunidades vegetais nos locais investigados eram predominantemente compostas por espécies arbóreas e tal padrão pode levar à formação de ecossistemas florestais onde antes havia formações savanânicas.

Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells.

BACKGROUND: Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs. RESULTS: Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells. CONCLUSIONS: The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs.

Characterization of novel Leishmania infantum recombinant proteins encoded by genes from five families with distinct capacities for serodiagnosis of canine and human visceral leishmaniasis.

To expand the available panel of recombinant proteins that can be useful for identifying Leishmania-infected dogs and for diagnosing human visceral leishmaniasis (VL), we selected recombinant antigens from L. infantum, cDNA, and genomic libraries by using pools of serum samples from infected dogs and humans. The selected DNA fragments encoded homologs of a cytoplasmic heat-shock protein 70, a kinesin, a polyubiquitin, and two novel hypothetical proteins. Histidine-tagged recombinant proteins were produced after subcloning these DNA fragments and evaluated by using an enzyme-linked immunosorbent assays with panels of canine and human serum samples. The enzyme-linked immunosorbent assays with different recombinant proteins had different sensitivities (67.4-93.0% and 36.4-97.2%) and specificities (76.1-100% and 90.4-97.3%) when tested with serum samples from Leishmania-infected dogs and human patients with VL. Overall, no single recombinant antigen was sufficient to serodiagnosis all canine or human VL cases.

Avaliação da resposta imune em camundongos utilizando diferentes protocolos de imunização com antígenos recombinantes de Leishmania chagasi / Evaluation of immune response in mice using different protocols of immunization with recombinant antigens of Leishmania chagasi

A leishmaniose visceral é uma doença parasitária causada por Leishmania chagasi. Os cães são considerados como principais reservatórios do parasito. Uma vacina efetiva para o cão pode contribuir para o controle da LV tanto no homem como no cão. Em um estudo prévio, antígenos recombinantes, obtidos a partir de uma biblioteca de cDNA da forma amastigota de Leishmania chagasi, foram selecionados a fim de serem avaliados como candidatos a componente de uma vacina contra leishmaniose visceral canina. No presente trabalho, a resposta imune humoral e celular de camundongos foi avaliada após injeção de alguns desses antígenos recombinantes, em cinco diferentes protocolos de imunização. Grupos de camundongos BALB/c foram injetados com as proteínas rLci4A-NH6 ou rLci2B-NH6, isoladamente ou associadas aos adjuvantes saponina, ODN 1826 ou diferentes doses de um plasmídeo codificando IL-12 murina. Além disso, animais foram sensibilizados com plasmídeo codificando Lci2B e receberam reforço com a proteína rLci2B-NH6 associada à saponina. Por último, os animais foram injetados com plasmídeos codificando novos antígenos, Lci2-NT-5R-CT ou Lci2-NT-CT, e recebram reforço com as respectivas proteínas recombinantes rLci2-NT-5R-CT-NH6 ou rLci2-NT-CT-NH6 associadas à saponina. Nesse último protocolo, os plasmídeos utilizados continham o gene para uma proteína transmembrana associada a lisossomos, LAMP, visando à produção in vivo de quimeras de dos antígenos de Leishmania com LAMP, a fim de direcionar a apresentação dos peptídeos para linfócitos T CD4+ via moléculas de MHC-II. Em um dos protocolos, os animais foram desafiados com 1 x 107 Leishmanias para avaliação da carga parasitária no baço após imunização. Os resultados encontrados mostraram que animais que receberam rLci4A-NH6, isoladamente ou com diferentes doses de plasmídeo codificando IL-12, apresentaram níveis signficantes de IgG e IgG1, ao passo que o uso do antígeno com os adjuvantes saponina ou ODN 1826 induziu a produção de IgG, IgG2a e IgG1, além de produção de IL-5 pelo grupo que recebeu saponina. Animais que receberam rLci2B-NH6, isoladamente ou associado aos plasmídeos contendo inserto de IL-12, produziram IgG e IgG1 antígeno específicos e falharam também na produção de citocinas e proliferação celular. A utilização de saponina com esse antígeno induziu produção de IgG, IgG2a e IgG1, assim como de IFN-γ. Já o uso de ODN 1826, induziu a produção de IgG e IgG1, favoreceu a produção de IgG2a, mas falhou na indução de resposta imune celular significante. Por outro lado, animais que receberam plasmídeo codificando Lci2B e reforço com a proteína associada à saponina apresentaram uma resposta imune com predominância de anticorpos IgG2a e produção de IFN-γ por alguns animais, a qual não conferiu proteção contra infecção pelo parasito. Por fim, a utilização de quimeras de LAMP/Lci2-NT-5R-CT ou LAMP/Lci2-5R-CT induziu uma fraca resposta imune humoral, com produção de IgG, IgG1 e IgG2a por alguns animais. No entanto, a utilização da quimera LAMP/Lci2-NT-CT induziu proliferação celular e tendência a produção de IFN-γ. Em conclusão, nenhum dos cinco protocolos utilizados foi capaz de induzir uma resposta imune específica predominantemente celular do tipo Th1 ou capaz de conferir proteção contra infecção dos animais.

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

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