BALB/c and C57BL/6 Mice Cytokine Responses to Trypanosoma cruzi Infection Are Independent of Parasite Strain Infectivity

Trypanosoma cruzi is the etiologic agent of Chagas' disease, which affects 6-7 million people worldwide. Different strains of T. cruzi present specific genotypic and phenotypic characteristics that affect the host-pathogen interactions, and thus, the parasite has been classified into six groups (TcI to TcVI). T. cruzi infection presents two clinical phases, acute and chronic, both with distinct characteristics and important participation by the immune system. However, the specific contributions of parasite and host factors in the disease phases are not yet fully understood. The murine model for Chagas' disease is well-established and reproduces important features of the human infection, providing an experimental basis for the study of host lineages and parasite strains. Thus, we evaluated acute and chronic infection by the G (TcI) and CL (TcVI) strains of T. cruzi, which have distinct tropisms and infectivity, in two inbred mice lineages (C57BL/6 and BALB/c) that display variable degrees of susceptibility to different T. cruzi strains. Analysis of the parasite loads in host tissues by qPCR showed that CL strain established an infection faster than the G strainat the same time, the response in BALB/c mice, although diverse in terms of cytokine secretion, was initiated earlier than that in C57BL/6 mice. At the parasitemia peak in the acute phase, we observed, either by confocal microscopy or by qPCR, that the infection was disseminated in all groups analyzed, with some differences concerning parasite tropismat this point, all animals responded to infection by increasing the serum concentrations of cytokines. However, BALB/c mice seemed to better regulate the immune response than C57BL/6 mice. Indeed, in the chronic phase, C57BL/6 mice still presented exacerbated cytokine and chemokine responses. In summary, our results indicate that in these experimental models, the deregulation of immune response that is typical of chronic Chagas' disease may be due to control loss over pro-and anti-inflammatory cytokines early in the acute phase of the disease, depending primarily on the host background rather than the parasite strain.FAPESPCAPESCNPqUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Sao Paulo, BrazilFAPESP: 2011/51475-3FAPESP: 2014/21338-2CNPq: 302068/2016-3Web of Scienc

Monocyte subsets and monocyte-related chemokines in Takayasu arteritis

The pathogenesis of Takayasu arteritis (TAK) is poorly understood and no previous studies have analyzed monocytes in TAK. This study evaluated monocyte subsets and monocyte-related chemokines in the peripheral blood of TAK patients and healthy controls (HC). Monocyte subsets were identified as classical (CD14+CD16−), intermediate (CD14+CD16dim), and non-classical (CD14dimCD16high) in the peripheral blood. The chemokines CCL (C–C chemokine ligand)2, CCL3, CCL4, CCL5, CCL7, CXCL (C-X-C motif ligand)10, and CX3CL (C-X3-C motif ligand)1 were measured in the sera. Thirty-two TAK patients and 30 HC were evaluated. Intermediate monocytes were higher in TAK than HC [25.0 cells ×106/L (16.7–52.0) vs. 17.2 cells ×106/L (9.2–25.3); p = 0.014]. Active disease was associated with monocytosis (p = 0.004), increased classical (p = 0.003), and intermediate (p &lt; 0.001) subsets than HC. Prednisone reduced the percentage of non-classical monocytes (p = 0.011). TAK patients had lower CCL3 (p = 0.033) and CCL4 (p = 0.023) levels than HC, whereas CCL22 levels were higher in active TAK compared to the remission state (p = 0.008). Glucocorticoids were associated with lower CXCL10 levels (p = 0.012). In TAK, CCL4 correlated with total (Rho = 0.489; p = 0.005), classical and intermediate monocytes (Rho = 0.448; p = 0.010 and Rho = 0.412; p = 0.019). In conclusion, TAK is associated with altered counts of monocyte subsets in the peripheral blood compared to HC and CCL22 is the chemokine with the strongest association with active disease in TAK.</p

Ação De Proteínas Isoladas De Plantas Na Hemostasia E Trombose

Protein from plants may act as inhibitors and thus used to assess blood coagulation, thrombosis and inflammation, in order. to determine the role of certain coagulation enzymes in hemostasis. Such inhibitors may be considered for the development of novel antithrombotic drugs. The isolated inhibitors from Enterolobium contortisiliquum (EcTI), Oelonix regia (DrTI), Acacia schweinfurthii (ASTI), and the bifunctional CrataBL protein, a lectin with inhibitory activityextracted from the Crataeva tapia bark, distinctly inhibit proteases, among them, the coagulation enzymes. Our objective was to establish the structure and function relationship of the inhibitors and their effects on thrombus formation. The inhibitors were purified by ion-exchange, molecular exclusion and affinity chromatoqraphies. The homogeneity of the preparations was assessed by SDS-PAGE, reverse phase chromatography and mass spectrometry, depending on the inhibitor. The effect on coagulation was assessed by the partially activated thromboplastin time, TTPa, and thrombin time, TP. The effects on platelet aggregation were investigated in vitro and ex vivo, as well as in the models of arterial thrombosis via injection of rose bengal in mice, and venous thrombosis through vena cava ligation in rats for the estimation of thrombus formation in vivo. DrTI, AsTI and EcTI increased the aPTT in human plasma due to the inhibition of huPK. DrTI differs from AsTI and EcTI by inhibiting FXla, with a Kiapp of 1.3 nM, indicating the dynamic nature of their reactive sites. DrTI, AsTI, EcTI and CrataBL significantly prolonged the time for total carotid artery occiusion in mice compared to the NaCI control. EcTI was also effective in decreasing the venous thrombus (74%, 2 mg/kg). The effects of DrTI, AsTI and EcTI can be attributed to their activities on the contact blood coagulation enzymes and, in the case of DrTI, AsTI and CrataBL, with platelet inhibition, since these proteins inhibited ex-vivo mice platelet aggregation induced by ADP. Although CrataBL has slight inhibitory effect on FXa it has a potent neutralizing effect of heparin activity. In conclusion, the compounds showed antithrombotic action in experimental models of arterial and venous thrombosis, ali interfering in the intrinsic,' but not in the extrinsic coagulation pathway. Only CrataBL was capable of interacting and neutralizing the effect of heparin. As none of the proteins altered the time of bleeding, it is plausible to consider them for therapeutic application, inhibiting thrombus development with a lower hemorrhagic effect. Support by FAPESP, CNPq, CAPES, Ethics Committee N° 179311A utilização de inibidores de origem vegetal em ensaios de coagulação sanguínea, trombose e inflamação contribui para determinar a função específica de enzimas no processo de hemostase e, em consequência, pode levar ao desenvolvimento de drogas antitrombóticas. Os inibidores isolados de Enterolobium contortisiliquum (EcTI), Oelonix regia (DrTI), Acacia schweinfurthii (ASTI), e a proteína bifuncional CrataBL, uma lectina com atividade inibitória extraída da entrecasca Crataeva tapia, inibem distintas proteases como aquelas da coagulação. Nosso objetivo foi estabelecer a relação entre estrutura e atividade dessas proteínas e os seus efeitos na formação de trombos. Os inibidores foram purificados por cromatografias de troca iônica, exclusão molecular e afinidade. A homogeneidade das preparações foi avaliada por SDS-PAGE, cromatografia em fase reversa e espectrometria de massa, dependendo de cada inibidor. A atividade, na coagulação, foi medida pelo tempo de tromboplastina parcialmente ativada (TIPa) e tempo de trombina (TP); na agregação plaquetária, in vitro e ex vivo; nos modelos de trombose arterial, via injeção de rosa de bengala em camundongos e de trombose venosa, via ligadura de veia cava em ratos, para a estimativa da formação de trombos in vivo. DrTI, AsTI e EcTI aumentaram o aPTT no piasma humano devido à inibição de huPK. DrTI difere de AsTI e EcTI pela inibição de FXla, com um Kiapp de 1,3 nM, indicando a natureza dinâmica dos seus sítios reativos. DrTI, AsTI, EcTI ~. CrataBL prolongaram significativamente o tempo de oclusão total da artéria carótida de ratos em comparação com o grupo controle NaCI. EcTI também foi eficaz em diminuir o trombo venoso (74%, 2 mg/kg). Os efeito de DrTI, AsTI e EcTI podem ser atribuídos à suas atividades sobre as enzimas da fase de contato da coagulação sanguínea e, no caso de DrTI, AsTI e CrataBL, associados à inibição plaquetária, uma vez que estas proteínas inibiram a agregação plaquetária induzida por ADP de ratos, ex vivo. Embora CrataBL tenha fraco efeito inibitório sobre o FXa, apresenta potente efeito neutralizador da atividade da heparina. Em conclusão, os compostos mostraram ação antitrombótica em diferentes modelos experimentais de trombose arterial e venosa, todos interferiam na via intrínseca, mas não na via extrínseca da coagulação. Somente CrataBL foi capaz de interagir e neutralizar o efeito da heparina. Nenhuma das proteínas estudadas alterou o tempo de sangramento, sugerindo serem substâncias com potencial valor terapêutico, inibindo o desenvolvimento das tromboses, com menor efeito hemorrágico. Suporte FAPESP, CNPq, CAPES. Comitê ética N° 179311Dados abertos - Sucupira - Teses e dissertações (2017

Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor, Leading to Downregulation of Survival/Death-Related Proteins

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment

Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells