Experimental study and computational modelling of cruzain cysteine protease inhibition by dipeptidyl nitriles

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases

Células C em bócio colóide

PURPOSE: The aim of this investigation was to quantitatively evaluate C-cells in colloid goiters, analyzing 36 thyroids that were obtained through thyroidectomy from 24 patients with goiter and 12 normal glands from adult patients without thyroid disease, which were used as the control group. MATERIAL AND METHODS: On average, 6 different thyroid areas were sampled and labeled by immunohistochemistry with a monoclonal anticalcitonin antibody, utilizing the avidin-biotin-peroxidase complex. C-cells were counted in fields measuring 1 square centimeter, and the mean number of cells per field was then calculated. Data were statistically analyzed using the Mann-Whitney test. RESULTS: In the colloid goiter group, the number of C-cells ranged from 0 to 23 per field, while in normal controls they ranged from 20 to 148 per field. CONCLUSIONS: These results demonstrate a significant decrease of C-cell number in the colloid goiter group compared with control group, indicating that the hyperplastic process is restricted to follicular cells, to the detriment of C-cells, which probably cease to receive trophic stimuli.OBJETIVO: Pesquisar, quantitativamente, as células C em bócio colóide com o propósito de investigar a relação destas células na patogênese do bócio. MÉTODO: Foram analisadas 35 tiróides obtidas de tiroidectomia, sendo 24 de pacientes com bócio colóide e 11 tiróides normais de adulto usadas como controle. Seis diferentes áreas foram amostradas em média e coradas com o anticorpo monoclonal anticalcitonina. As células C foram contadas em campos de 1 cm² e o número médio de células/campo foi calculado. Os dados foram estudados estatisticamente pelo teste de Kruskal-Wallis. RESULTADOS: O número de células C variou de 0 a 23/cm² em bócio colóide e em tiróides normais de 20 a 148/cm². CONCLUSÕES: Os resultados demonstraram redução significativa no número de células C em bócio colóide comparando com tiróides normais, indicando que o processo hiperplásico é restrito às células foliculares em detrimento das células C, as quais, provavelmente, deixam de receber estímulos tróficos e se degeneram

MAIT cells are activated in acute Dengue virus infection and after in vitro Zika virus infection.

Dengue virus (DENV) and Zika virus (ZIKV) are members of the Flaviviridae and are predominantly transmitted via mosquito bites. Both viruses are responsible for a growing number of infections in tropical and subtropical regions. DENV infection can cause lethargy with severe morbidity and dengue shock syndrome leading to death in some cases. ZIKV is now linked with Guillain-Barré syndrome and fetal malformations including microcephaly and developmental disorders (congenital Zika syndrome). The protective and pathogenic roles played by the immune response in these infections is unknown. Mucosal-associated invariant T (MAIT) cells are a population of innate T cells with potent anti-bacterial activity. MAIT cells have also been postulated to play a role in the immune response to viral infections. In this study, we evaluated MAIT cell frequency, phenotype, and function in samples from subjects with acute and convalescent DENV infection. We found that in acute DENV infection, MAIT cells had elevated co-expression of the activation markers CD38 and HLA-DR and had a poor IFNγ response following bacterial stimulation. Furthermore, we found that MAIT cells can produce IFNγ in response to in vitro infection with ZIKV. This MAIT cell response was independent of MR1, but dependent on IL-12 and IL-18. Our results suggest that MAIT cells may play an important role in the immune response to Flavivirus infections

An orally active formulation of angiotensin-(1-7) produces an antithrombotic effect

INTRODUCTION AND OBJECTIVE: The heptapeptide angiotensin-(1-7) is a component of the renin-angiotensin system, which promotes many beneficial cardiovascular effects, including antithrombotic activity. We have recently shown that the antithrombotic effect of angiotensin-(1-7) involves receptor Mas-mediated NO-release from platelets. Here, we describe an orally active formulation based on angiotensin-(1-7) inclusion in cyclodextrin [Ang-(1-7)- CyD] as an antithrombotic agent. Cyclodextrins are pharmaceutical tools that are used to enhance drug stability, absorption across biological barriers and gastric protection. METHOD: To test the antithrombotic effect of Ang-(1-7)-CyD, thrombus formation was induced in the abdominal vena cava of spontaneously hypertensive rats that were pretreated either acutely or chronically with Ang-(1-7)-CyD. Male Mas-knockout and wild-type mice were used to verify the role of the Mas receptor on the effect of Ang-(1-7)-CyD. RESULTS: Acute or chronic oral treatment with Ang-(1-7)-CyD promoted an antithrombotic effect (measured by thrombus weight; all values are, respectively, untreated vs. treated animals) in spontaneously hypertensive rats (acute: 2.86 + 0.43 mg vs. 1.14 + 0.40 mg; chronic: 4.27 + 1.03 mg vs. 1.39 + 0.68 mg). This effect was abolished in Mas-knockout mice (thrombus weight in Mas wild-type: 0.76 + 0.10 mg vs. 0.37 + 0.02 mg; thrombus weight in Mas-knockout: 0.96 + 0.11 mg vs. 0.87 + 0.14 mg). Furthermore, the antithrombotic effect of Ang-(1-7)-CyD was associated with an increase in the plasma level of Angiotensin-(1-7). CONCLUSION: These results show for the first time that the oral formulation Ang-(1-7)-CyD has biological activity and produces a Mas-dependent antithrombotic effect

EVALUATION OF IN VITRO DISSOLUTION OF BENZNIDAZOLE AND BINARY MIXTURES: SOLID DISPERSIONS WITH HYDROXYPROPYLMETHYLCELLULOSE AND β-CYCLODEXTRIN INCLUSION COMPLEXES

Objective: To increase the solubility/dissolution of benznidazole (BNZ) in water using two systems: solid dispersions (SD) with hydroxypropylmethylcellulose (HPMC) and β-cyclodextrin (β-CD) inclusion complexes (IC).Methods: The samples were obtained by physical mixtures (PM), kneading (KN), evaporation (EV) and by spray-dryer (SY) atomization The analysis was based on results of in vitro dissolution and molecular modeling techniques.Results: Molecular modeling showed that BNZ can form β-CD complexes in different ways such as in an aqueous solution or a vacuum. In vitro dissolution showed significant improvement in BNZ solubility in the PM, SD and IC, and also that the β-CD IC promoted better solubility than SD with HPMC.Conclusion: Considering the data obtained, it is possible to consider the technique for the formation of β-CD IC as a more effective technique in promoting the improvement of BNZ solubility compared with getting SD with HPMC which, in turn, may increase the bioavailability of the drug and improve their pharmaceutical potential

Performance and plasma urea nitrogen of immunocastrated males pigs of medium genetic potential

ABSTRACT Objective. A study was carried out to evaluate the performance and the plasma urea nitrogen (PUN) of male pigs of medium genetic potential for lean meat deposition in carcass, which underwent immunocastration. Materials and methods. Forty-five seventy-days old Large White x Landrace crossbred were used. The pigs were distributed in a randomized design in three treatments: castrated males, females and immunocastrated males. Each treatment group was replicated three times with five pigs per replicate. The trial period was of 70 days, divided into phases of growing (70 to 110 days old) and finishing (111 to 140 days old). The pigs were weighed four times: at the beginning of the trial, at the first immunocastration vaccine dose (80 days old), at the second immunocastration vaccine dose (110 days old) and just before slaughter (140 days old). Blood samples were taken on the same day that the animals were weighed. Results. Between 80 and 110 days old, there was an increase in PUN value, only for castrated males and females. No differences were found in weight gain between the studied groups within the periods. Immunocastrated males had lower feed intake than females and these had a lower feed intake than castrated males. To 110 days old, immunocastrated animals showed feed conversion ratio similar to females and better than castrated males. However, after the second dose of the vaccine, feed conversion was similar between groups. Conclusions. The benefits of immunocastration are prominent in animals with low to medium genetic potential. RESUMEN Objetivo. Se realizó un estudio para evaluar el rendimiento y la concentración de urea en plasma (PUN) de los cerdos machos de medio potencial genético de carne magra en la canal sometidos a la inmunocastración. Materiales y métodos. Se utilizaron 45 cerdos de 70 días de edad Landrace x Large White. Los animales se distribuyeron en un diseño completamente al azar con tres tratamientos: machos castrados, hembras y machos inmunocastrados. Cada tratamiento consistió en tres repeticiones, con cinco animales por réplica. El período experimental fue de 70 días, divididos en las etapas de crecimiento (70 a 110 días de edad) y terminación (111 a 140 días de edad). Los cerdos fueron pesados cuatro veces: al inicio del experimento, en la primera dosis de vacuna de inmunocastración (80 días de edad), en la segunda dosis de la vacuna de inmunocastración (110 días de edad) y antes de el sacrificio (140 días de edad). Las muestras de sangre se recogieron en el mismo día en que se pesaron los animales. Resultados. Entre 80 y 110 días de edad, hubo un aumento en la cantidad de PUN, sólo para machos castrados y hembras. No hubo diferencias en la ganancia de peso entre los grupos en ninguno de los períodos estudiados. Machos inmunocastrados tuvieron menor consumo de alimento que las hembras y éstas mostraron un menor consumo que los machos castrados. En 110 días de edad, los animales inmunocastrados mostraron la conversión de alimento similar a las hembras y mejor que los machos castrados. Sin embargo, después de la segunda dosis de la vacuna, la conversión alimenticia fue similar entre los grupos. Conclusiones. Los beneficios de inmunocastración son prominentes en animales con bajo a médio potencial genético.