Evaluation of the immunogenicity of peptides derived from Cathepsin L-like from Leishmania infantum in murine model

As espécies do gênero Leishmania parasitam mamíferos em todo o mundo e possuem ciclos de vida com alternância entre hospedeiros vertebrados e invertebrados, sendo que a Leishmania infantum é o agente etiológico da manifestação visceral da leishmaniose. Parte da elevada patogenicidade do protozoário se justifica pela ação catalítica de catepsina L-like, uma cisteíno- protease que participa de uma variedade de processos metabólicos e regulatórios, além de importante mediação nos processos de patogenia e virulência, dentre os quais se destacam a evasão da resposta imune do hospedeiro, diferenciação celular, colonização e invasão de células, transposição de barreiras teciduais, degradação de proteínas hematopoiéticas, ativação de respostas inflamatórias e morte celular programada. Assim, este projeto teve como objetivo metodológico a caracterização in silico de catepsina L-like nos isolados brasileiros de L. infantum, bem como a proposição de fragmentos peptídicos conjugados a carreadores proteicos para imunização de modelos murinos, de modo a avaliar parâmetros de inocuidade e imunogenicidade. As sequências gênicas de catepsina L-like não demonstraram variabilidade intraespecífica, o que se refletiu na proposição de sequências proteicas de caráter consensual. A sequência primária de 354 resíduos demonstrou possuir um pI teórico de 6,69, a presença de três sítios catalíticos, e um caráter hidrofílico e termoestável. A sequência terciária demonstrou a presença de dois domínios proteicos, intercalados por uma região livre com ausência de motivos conhecidos. Com base nestas variáveis bioquímicas e estruturais, se elegeu uma sequência de 12 aminoácidos que correspondeu ao peptídeo 01. Este sofreu uma adição terminal de cisteína acoplada ao carreador Blue carrier protein, de modo a corresponder ao peptídeo 02. Quando estes peptídeos foram utilizados para imunizar camundongos isogênicos BALB/c, não se observou nenhuma resposta colateral, o que atestou a inocuidade dos peptídeos. As amostras séricas dos animais imunizados com estes segmentos peptídicos não demonstraram reatividade nos ensaios de imunofluorescência. Contudo, quando as amostras séricas foram testadas através de ensaios imunoenzimáticos, foi observado um crescimento no valor de absorbância encontrado no grupo imunizado com o peptídeo 01 em relação aos animais imunizados com PBS, o que sugere um eventual potencial imunorreativo deste segmento. Assim, o estudo conclui que estes peptídeos derivados de catepsina L-like de L. infantum demonstram razoável potencial imunogênico, propondo a combinação futura com outras moléculas adjuvantes, de modo a atingir formulações que eventualmente potencializem o caráter imunoestimulante desta molécula. Desse modo, o trabalho contribui com a consolidação de uma metodologia de investigação in silico de segmentos com potencial imunogênico. Além disso, fornece um embasamento para iniciativas posteriores que se disponham a melhorar o perfil de imunorreatividade, voltado tanto para prospecção de substratos antigênicos para padronização de metodologias sorológicas com acurácia diagnóstica, quanto para ensaios de rastreio de antígenos vacinais.Species of genus Leishmania parasitize mammals all over the world and have life cycles with alternation between vertebrate and invertebrate hosts, being that Leishmania infantum is the etiological agent of the visceral manifestation of leishmaniasis. Part of the high pathogenicity of the protozoan is justified by the catalytic action of cathepsin L-like, a cysteine protease that participates in a variety of metabolic and regulatory processes, as well as important mediation in pathogenic and virulence processes, which include: the evasion of the host′s immune response, cell differentiation, cell colonization and invasion, transposition of tissue barriers, degradation of hematopoietic proteins, activation of inflammatory responses and programmed cell death. Thus, the objective of this project is to characterize in silico L-like cathepsin in Brazilian isolates of L. infantum and propose peptide fragments conjugated to protein carriers for immunization of murine models, in order to evaluate parameters of innocuity and immunogenicity. The L-like cathepsin gene sequences did not show intraspecific variability, which was reflected in the proposition of protein sequences of consensual character. The primary sequence of 354 residues have had a theoretical pI of 6.69, the presence of three catalytic sites was found in there, and a hydrophilic and thermostable character was observed. The tertiary sequence demonstrated the presence of two protein domains interspersed by a free region with the absence of known motifs. Based on these biochemical and structural variables, a sequence of 12 amino acids was chosen that corresponded to peptide 01. It underwent a terminal addition of cysteine coupled to the Blue carrier protein carrier, in order to correspond to peptide 02. When these peptides were inoculated to immunize isogenic BALB/c mice, no collateral response was observed, which attested the innocuity of the peptides. Serum samples from mice immunized with these peptide segments did not show reactivity in immunofluorescence assays. However, when the serum samples were tested through enzyme immunoassays, an increase in the absorbance value found in the group immunized with peptide 01 in relation to the animals immunized with PBS was observed, which suggests an eventual immunoreactive potential of this segment. Thus, this study concludes that these peptides derived from L-like cathepsin from L. infantum demonstrate an immunogenic potential, suggesting future combination with other adjuvant molecules to reach formulations that eventually maximize the immunostimulatory character of this molecule. In this way, this work contributes to the consolidation of a methodology for in silico investigation of segments with immunogenic potential. In addition, it provides a basis for further initiatives that aim to improve the immunoreactivity profile to the prospecting antigenic substrates for standardizing serological methodologies with diagnostic accuracy and to the screening assays for vaccine antigens

Genes of Cysteine proteases (Cathepsin L-like) from Leishmania infantum chagasi: characterization, phylogenetic relations and molecular diagnosis

Os parasitas pertencentes ao gênero Leishmania têm distribuição ubíqua. Este táxon inclui Leishmania infantum chagasi, agente etiológico da leishmaniose visceral nas Américas, uma zoonose negligenciada cujas metodologias diagnósticas acumulam uma série de limitações, requerendo a validação e padronização de metodologias diagnósticas satisfatórias. Vários fatores estão relacionados à patogênese causada por este protozoário, entre eles a catepsina L-like, uma cisteíno protease envolvida em processos regulatórios metabólicos e infecciosos. Portanto, este trabalho teve como objetivo avaliar a eficácia do gene de catepsina L-like isoforma CPA como alvo de diagnóstico molecular e como marcador filogenético que permita a compreensão das variações intraespecíficas e elucidem a história evolutiva de L. infantum chagasi no Brasil. Foram utilizados 44 isolados de L. infantum chagasi de diferentes estados brasileiros. Os fragmentos do gene de catepsina L-like foram amplificados, purificados, sequenciados, alinhados manualmente e analisados por métodos filogenéticos de máxima parcimônia e inferência bayesiana. As sequências geradas foram usadas para pesquisar e sintetizar iniciadores a serem usados em reações específicas para o parasita alvo. O gene de catepsina L-like não mostrou variabilidade intraespecífica entre os isolados analisados, sugerindo um evento recente de introdução do mesmo nas Américas. O par de iniciadores propostos amplificou o DNA alvo de isolados de L. infantum chagasi, sendo efetivo na amplificação de DNA em concentrações de até 10-11g / µl. O marcador proposto não apresentou reações cruzadas com outros hemoparasitas de importância clínica. Quando utilizado para o diagnóstico em um painel de amostras clínicas de cães, obteve-se uma frequência de positividade de 49,03% (102/208), contrastando com o valor de 14,42% (30/208) obtido com o marcador para o gene do espaçador ribossomal interno ITS. Quando testado em amostras de flebotomíneos se obteve um valor de 6,25% e em amostras de pacientes humanos o valor foi de 14,28%. Os marcadores também foram eficazes em amplificar DNA extraído de amostras de urina, de sangue fixado em papel filtro e mesmo em amostras de swab de lesões conjuntivas. Este conjunto de parâmetros permite inferir que o CatLeish- PCR é sensível e específico para o diagnóstico de L. infantum chagasi podendo ser aplicado tanto em pesquisas clínicas quanto em inquéritos epidemiológicos de vigilância.The parasites belonging to the Leishmania genus have a wide distribution. This taxon includes Leishmania infantum chagasi, the etiologic agent of Visceral Leishmaniasis in the Americas, a neglected zoonosis that requires the validation and standardization of satisfactory diagnostic methodologies. Several factors are related to the pathogenesis caused by this protozoan, as Catepsin L-like, a cysteine protease involved in regulatory and infectious processes. Given this information this work aimed to evaluate the effectiveness of Cathepsin L-like isoform CPA as a target for molecular diagnosis and as a phylogenetic marker that allows understanding the intraspecific variations and the evolutionary history of L. infantum chagasi in Brazil. We used 44 isolates of L. infantum chagasi from different Brazilian states. The cathepsin L-like gene fragments were amplified, purified, sequenced, manually aligned and analyzed by maximum parsimony and Bayesian inference methods. The sequences generated were researched to construction of oligonucleotide primers to be used in reactions specific to the target parasite. The Cathepsin L-like gene did not show intraspecific variability among the isolates analyzed, suggesting a recent event of introduction of the same in the Americas. The pair of proposed primers amplified the target DNA of L. infantum chagasi isolates, being effective in DNA amplification at concentrations of up to 10-11g/µl. The proposed marker did not present cross-reactions with other hemoparasites of clinical importance. When used for the diagnosis in a panel of clinical samples of dogs obtained a positive frequency of 49.03% (102/208), against the 14.42% (30/208) to ribosomal ITS marker. Samples of sandflies obtained a value of 6.25% and in humans the value was 14.28%. The markers were also effective in blood samples fixed on filter paper and even in samples from conjunctival lesion swabs. This set of parameters allows to infer that CatLeish-PCR is a sensitive and specific marker for the diagnosis of L. infantum chagasi in clinical and epidemiological surveys

Anthelmintic effect of Cassia fistula and Combretum leprosum protein fractions against goat gastrointestinal nematodes

<div><p>Abstract In this study, we evaluated the ovicidal and larvicidal activity of protein preparations obtained from Cassia fistula L. and Combretum leprosum Mart. leaves on the gastrointestinal parasites of goats. Protein preparations were obtained after the extraction of C. fistula L. and C. leprosum Mart. leaves, followed by protein fractionation (with ammonium sulfate saturation percentages of 30%, 30%-60%, and 60%-90%) and dialysis, which resulted in protein fractions (called F1, F2, and F3, respectively). The fractions were evaluated by egg hatching (the eggs were recovered in stool samples from naturally infected goats) and larval development tests. The results reveled that the inhibition of hatching of eggs caused by the protein fractions of C. fistula (38%) were similar to that of the control drug, thiabendazole. In addition, the fractions of C. fistula caused significant inhibition (61-69%) of larval development also. However, C. leprosum did not reveal significant inhibition of egg hatching and larval development. We conclude that C. fistula L. showed better ovicidal and larvicidal activity against endoparasites.</p></div

First measurement of the CPCP-violating phase ϕsdd‾\phi_s^{d\overline{d}} in Bs0→(K+π−)(K−π+)B_s^0\to(K^+\pi^-)(K^-\pi^+) decays

International audienceA flavour-tagged decay-time-dependent amplitude analysis of B$_{s}^{0}$  → (K$^{+}$π$^{−}$)(K$^{−}$π$^{+}$) decays is presented in the K$^{±}$π$^{∓}$ mass range from 750 to 1600MeV/c$^{2}$. The analysis uses pp collision data collected with the LHCb detector at centre-of-mass energies of 7 and 8 TeV, corresponding to an integrated luminosity of 3.0 fb$^{−1}$. Several quasi-two-body decay modes are considered, corresponding to K$^{±}$π$^{∓}$ combinations with spin 0, 1 and 2, which are dominated by the K$_{0}^{*}$ (800)$^{0}$ and K$_{0}^{*}$ (1430)$^{0}$, the K$^{*}$(892)$^{0}$ and the K$_{2}^{*}$ (1430)$^{0}$ resonances, respectively. The longitudinal polarisation fraction for the ${B}_s^0\to {K}^{\ast }(892){}^{\circ}{\overline{K}}^{\ast }{(892)}^0$ decay is measured as f$_{L}$ = 0.208 ± 0.032 ± 0.046, where the first uncertainty is statistical and the second is systematic. The first measurement of the mixing-induced CP-violating phase, ${\phi}_s^{d\overline{d}}$ , in $b\to d\overline{d}s$ transitions is performed, yielding a value of ${\phi}_s^{d\overline{d}}=-0.10\pm 0.13\left(\mathrm{stat}\right)\pm 0.14$ (syst) rad

Studies of the resonance structure in D0→K∓π±π±π∓D^{0} \rightarrow K^\mp \pi ^\pm \pi ^\pm \pi ^\mp decays

International audienceAmplitude models are constructed to describe the resonance structure of ${D^{0}\rightarrow K^{-}\pi ^{+}\pi ^{+}\pi ^{-}}$ and ${D^{0} \rightarrow K^{+}\pi ^{-}\pi ^{-}\pi ^{+}}$ decays using pp collision data collected at centre-of-mass energies of 7 and 8 TeV with the LHCb experiment, corresponding to an integrated luminosity of 3.0  $fb^{-1}$ . The largest contributions to both decay amplitudes are found to come from axial resonances, with decay modes $D^{0} \rightarrow a_1(1260)^{+} K^{-}$ and $D^{0} \rightarrow K_1(1270/1400)^{+} \pi ^{-}$ being prominent in ${D^{0}\rightarrow K^{-}\pi ^{+}\pi ^{+}\pi ^{-}}$ and $D^{0}\rightarrow K^{+}\pi ^{-}\pi ^{-}\pi ^{+}$ , respectively. Precise measurements of the lineshape parameters and couplings of the $a_1(1260)^{+}$ , $K_1(1270)^{-}$ and $K(1460)^{-}$ resonances are made, and a quasi model-independent study of the $K(1460)^{-}$ resonance is performed. The coherence factor of the decays is calculated from the amplitude models to be $R_{K3\pi } = 0.459\pm 0.010\,(\mathrm {stat}) \pm 0.012\,(\mathrm {syst}) \pm 0.020\,(\mathrm {model})$ , which is consistent with direct measurements. These models will be useful in future measurements of the unitary-triangle angle $\gamma$ and studies of charm mixing and $C\!P$ violation

Search for excited Bc+B_{c}^{+} states

International audienceA search is performed in the invariant mass spectrum of the B$_{c}^{+}$ π$^{+}$π$^{−}$ system for the excited B$_{c}^{+}$ states B$_{c}$ (2$^{1}$S$_{0}$)$^{+}$ and B$_{c}$ (2$^{3}$S$_{1}$)$^{+}$ using a data sample of pp collisions collected by the LHCb experiment at the centre-of-mass energy of $\sqrt{s}=8$ TeV, corresponding to an integrated luminosity of 2 fb$^{−1}$. No evidence is seen for either state. Upper limits on the ratios of the production cross-sections of the B$_{c}$ (2$^{1}$S$_{0}$)$^{+}$ and B$_{c}$ (2$^{3}$S$_{1}$)$^{+}$ states times the branching fractions of B$_{c}$ (2$^{1}$S$_{0}$)$^{+}$ → B$_{c}^{+}$ π$^{+}$π$^{−}$ and B$_{c}$ (2$^{3}$S$_{1}$)$^{+}$ → B_{c}^{*}^{+}π$^{+}$π$^{−}$ over the production cross-section of the B$_{c}^{+}$ state are given as a function of their masses. They are found to be between 0.02 and 0.14 at 95% confidence level for B$_{c}$ (2$^{1}$S$_{0}$)$^{+}$ and B$_{c}$ (2$^{3}$S$_{1}$)$^{+}$ in the mass ranges [6830, 6890] MeV/c$^{2}$ and [6795, 6890] MeV/c$^{2}$, respectively