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The P2X7 receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X7 receptor is induced by increasing the intracellular Ca2+ concentration (Faria et al., Am J Physiol Cell Physiol 297:C28–C42, ). Here, we investigated the intracellular signaling pathways associated with P2X7 large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca2+-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X7 receptor large-channel formation in 2BH4 cells and peritoneal macrophages. [ABSTRACT FROM AUTHOR]

During the Zika fever outbreak in Brazil in 2015-2016, only some babies from infected mothers had teratogenic effects, suggesting that cofactors may influence congenital transmission. We investigated the ZIKV infection profile in explants and isolated cells from full-term human placenta to infection with the Brazilian Zika virus strain (ZIKV BR ) and the effect of coinfection with the Brazilian Human alphaherpesvirus 2 strain (HSV-2 BR ) on ZIKV replication. We found that the ZIKV BR infect the explants of amniotic and chorionic membranes, as well as chorionic villi core, but not the trophoblasts layer. It was also observed that ZIKV replication was higher in amniotic cells than chorionic and trophoblasts cells. Upon coinfection, the replication of ZIKV BR was reduced according to exposed HSV-2 BR load in trophoblasts cells and the levels of TNF-α and IL-6 cytokines were also reduced. These findings suggest that the placental cell types and HSV-2 BR coinfection may impact on ZIKV replication., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Chagas disease (CD) caused by Trypanosoma cruzi remains a serious public health problem in Latin America. The available treatment is limited to two old drugs, benznidazole (Bz) and nifurtimox, which exhibit limited efficacy and trigger side effects, justifying the search for new therapies. Also, more accurate and sensitive experimental protocols for drug discovery programs are necessary to shrink the translational gaps found among pre-clinical and clinical trials. Presently, cardiac spheroids were used to evaluate host cell cytotoxicity and anti-T.cruzi activity of benznidazole, exploring its effect on the release of inflammatory mediators. Bz presented low toxic profile on 3D matrices (LC 50  > 200 μM) and high potency in vitro (EC 50  = 0.99 μM) evidenced by qPCR analysis of T.cruzi-infected cardiac spheroids. Flow cytometry appraisal of inflammatory mediators released at the cellular supernatant showed increases in IL - 6 and TNF contents (≈190 and ≈ 25-fold) in parasitized spheroids as compared to uninfected cultures. Bz at 10 μM suppressed parasite load (92%) concomitantly decreasing in IL-6 (36%) and TNF (68%). Our findings corroborate the successful use of 3D cardiac matrices for in vitro identification of novel anti-parasitic agents and potential impact in host cell physiology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Levamisole (Lms) is an anthelminthic drug with immunomodulatory activity. Chagas disease (CD) is caused by Trypanosoma cruzi and there is very low access to the drugs available, benznidazole (Bz) and nifurtimox, both far from ideal. In a drug-repurposing strategy to test potential activity as antiparasitic and immunomodulatory agent for CD, Lms was assayed on acute T. cruzi murine infection, alone and in co-administration with Bz. During protocol standardization, 100 and 10 mpk of Bz given for five consecutive days resulted in parasitaemia suppression and 100% animal survival only with the highest dose. Flow cytometry showed that both optimal (100 mpk) and suboptimal (10 mpk) doses of Bz equally decreased the plasma levels of cytokines commonly elevated in this acute infection model. Lms alone (10-0.5 mpk) did not decrease parasitaemia nor mortality rates. Co-administration was investigated using the suboptimal dose of Bz and different doses of Lms. While Bz 10 mpk did not alter parasitaemia, the combo partially reduced it but only slightly promoted animal survival. This effect could be related to Th1-response modulation since interleukin-6 and interferon-γ were higher after treatment with the combo.

Background: Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection., Methods: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile., Results: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 μL AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases (< 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and IL-6 were detected at 9 dpi in all infected animals as compared to uninfected mice but only Bz displayed a statistically significant diminution ( p =  0.02) in TNF-alpha levels than infected and untreated mice., Conclusions: This study revealed that the use of autologous whole blood (AWB) in the acute model employed was unable to reduce the parasitic load of infected mice, providing only a minor decrease in parasitemia levels (up to 30%) but without protecting against animal mortality. Further in vivo studies will be necessary to elucidate the effective impact of this procedure., Competing Interests: Our present study was submitted to Fiocruz Committee of Ethics for the Use of Animals (CEUA) and approved under the license number CEUA L-032/2016.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Chagas disease is typically associated with cardiac involvement. During the acute phase of murine infection with Trypanosoma cruzi, severe acute myocarditis can develop. Prior to cardiac alteration, however, infected mice present with renal inflammatory infiltration causing acute kidney injury due to an ischemia/reperfusion lesion. Thus, the present study was undertaken in order to evaluate whether the parasites or some of their components would directly affect renal cells. As such, this study employed kidney cell lines (mesangial, epithelial, and proximal tubular) that mimic different regions of the renal system. Mesangial cells are more resistant to infection, showing reduced parasite internalization relative to epithelial and proximal tubular cells. Upon infection, mesangial cells produced more nitric oxide, tumor factor necrosis-α, and interferon-γ and showed decreased viability when compared to the other cell lines. These results indicate that the resistance of mesangial cells to infection may be related to the increased expression of nitric oxide and proinflammatory cytokines. Conversely, the high levels of nitric oxide produced by these cells caused impairment of cell integrity and viability. Higher nitric oxide concentrations promote cellular injury and can be involved in the genesis of ischemia/reperfusion lesions in acute kidney injury.