Infection of Mouse Neural Progenitor Cells by Toxoplasma gondii Reduces Proliferation, Migration, and Neuronal Differentiation in Vitro.

Congenital toxoplasmosis constitutes a major cause of pre- and postnatal complications. Fetal infection with Toxoplasma gondii influences development and can lead to microcephaly, encephalitis, and neurologic abnormalities. Systematic studies concerning the effects of neural progenitor cell infection with T. gondii are unavailable. Cortical intermediate progenitor cells cultivated as neurospheres obtained from E16.5 Swiss Webster mice were infected with T. gondii (ME49 strain) tachyzoites to mimic the developing mouse cerebral cortex in vitro. Infection was associated with decreased cell proliferation, detected by Ki-67 staining at 48 and 72 hours after infection in floating neurospheres, and reduced cellularity at 96 hours. Transient decreases in the expression of the neurogenesis-related transcription factors T-box brain protein 1, mouse atonal homolog protein 1, and hairy and enhancer of split protein 1 were found in infected cultures, while the level of transcription factor SOX-2 remained unaltered. Neurogenic potential, assessed in plated neurospheres, was impaired in infected cultures, as indicated by decreased late neuronal marker neurofilament heavy chain immunoreactivity. Infected cultures exhibited decreased overall migration rates at 48 and 120 hours. These findings indicate that T. gondii infection of neural progenitor cells may lead to reduced neurogenesis due to an imbalance in cell proliferation alongside an altered migratory profile. If translated to the in vivo situation, these data could explain, in part, cortical malformations in congenitally infected individuals.

Impact of collaborative actions of Leishmania (Viannia) braziliensis subpopulations on the infection profile.

This study focuses on the role of the population structure of Leishmania spp. on the adaptive capacity of the parasite. Herein, we investigate the contribution of subpopulations of the L. (V.) braziliensis Thor strain (Thor03, Thor10 and Thor22) in the profile of murine macrophages infection. Infection assays were performed with binary combinations of these subpopulations at stationary phases. The initial interaction time showed major effects on the combination assays, as demonstrated by the significant increase in the infection rate at 5 h. Based on the endocytic index (EI), Thor10 (EI = 563.6) and Thor03 (EI = 497) showed a higher infection load compared to Thor22 (EI = 227.3). However, the EI decreased in Thor03 after 48 h (EI = 447) and 72 h (EI = 388.3) of infection, and showed changes in the infection level in all Thor10/Thor22 combinations. Assays with CellTrace CFSE-labelled Thor22 promastigotes indicated an increase (~1.5 fold) in infection by this subpopulation in the presence of Thor10 when compared to the infection profile of Thor03/Thor22 combinations in the same proportions. In addition, the potential of these subpopulations, alone or in binary combinations, to modulate the expression of cytokines and nitric oxide (NO) in vitro was investigated. Lower NO and tumour necrosis factor-α production levels were observed for all Thor10/Thor22 combinations at 24 h compared to these subpopulations alone. In contrast, Thor03/Thor22 combination assays increased IL-10 production at this time. Collectively, these results provide in vitro evidence on the potential of L. (V.) braziliensis population structure to play a relevant role in a host infection by this parasite.

Benznidazole modulates release of inflammatory mediators by cardiac spheroids infected with Trypanosoma cruzi.

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 (LC50 > 200 µM) and high potency in vitro (EC50 = 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.

Toxoplasma gondii infection impairs radial glia differentiation and its potential to modulate brain microvascular endothelial cell function in the cerebral cortex.

Congenital toxoplasmosis is a parasitic disease that occurs due vertical transmission of the protozoan Toxoplasma gondii (T. gondii) during pregnancy. The parasite crosses the placental barrier and reaches the developing brain, infecting progenitor, glial, neuronal and vascular cell types. Although the role of Radial glia (RG) neural stem cells in the development of the brain vasculature has been recently investigated, the impact of T. gondii infection in these events is not yet understood. Herein, we studied the role of T. gondii infection on RG cell function and its interaction with endothelial cells. By infecting isolated RG cultures with T. gondii tachyzoites, we observed a cytotoxic effect with reduced numbers of RG populations together with decrease neuronal and oligodendrocyte progenitor populations. Conditioned medium (CM) from RG control cultures increased ZO-1 protein levels and organization on endothelial bEnd.3 cells membranes, which was impaired by CM from infected RG, accompanied by decreased trans-endothelial electrical resistance (TEER). ELISA assays revealed reduced levels of anti-inflammatory cytokine TGF-ß1 in CM from T. gondii-infected RG cells. Treatment with recombinant TGF-ß1 concomitantly with CM from infected RG cultures led to restoration of ZO-1 staining in bEnd.3 cells. Congenital infection in Swiss Webster mice led to abnormalities in the cortical microvasculature in comparison to uninfected embryos. Our results suggest that infection of RG cells by T. gondii negatively modulates cytokine secretion, which might contribute to endothelial loss of barrier properties, thus leading to impairment of neurovascular interaction establishment.

Antiparasitic and anti-inflammatory activities of ß-lapachone-derived naphthoimidazoles in experimental acute Trypanosoma cruzi infection.

BACKGROUND: Chagas disease, which is caused by the protozoan Trypanosoma cruzi, is endemic to Latin America and mainly affects low-income populations. Chemotherapy is based on two nitrocompounds, but their reduced efficacy encourages the continuous search for alternative drugs. Our group has characterised the trypanocidal effect of naphthoquinones and their derivatives, with naphthoimidazoles derived from ß-lapachone (N1, N2 and N3) being the most active in vitro. OBJECTIVES: In the present work, the effects of N1, N2 and N3 on acutely infected mice were investigated. METHODS: in vivo activity of the compounds was assessed by parasitological, biochemical, histopathological, immunophenotypical, electrocardiographic (ECG) and behavioral analyses. FINDINGS: Naphthoimidazoles led to a decrease in parasitaemia (8 dpi) by reducing the number of bloodstream trypomastigotes by 25-50% but not by reducing mortality. N1 protected mice from heart injury (15 dpi) by decreasing inflammation. Bradycardia was also partially reversed after treatment with N1 and N2. Furthermore, the three compounds did not reverse hepatic and renal lesions or promote the improvement of other evaluated parameters. MAIN CONCLUSION: N1 showed moderate trypanocidal and promising immunomodulatory activities, and its use in combination with benznidazole and/or anti-arrhythmic drugs as well as the efficacy of its alternative formulations must be investigated in the near future.

Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases, including Muscular Dystrophies.

Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krüppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close.

Impact of levamisole in co-administration with benznidazole on experimental Chagas disease.

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.

Chemotaxis and Immunoregulatory Function of Cardiac γδ T Cells in Dystrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by mutations in the dystrophin gene that lead to degeneration of skeletal and cardiac muscles and to chronic inflammation. Despite the importance of γδ T cells in many diseases, this cellular subpopulation has not been described in DMD patients or in mdx mice, a widely used mouse model for studying DMD. Therefore, in this study, we aimed to evaluate the migration of γδ T cells to the cardiac muscle of mdx mice and to characterize their phenotype and functional activity. We observed no migration of γδ T cells to skeletal muscles, but these cells were found in the hearts of mdx mice during the study period, reaching a peak in 12-wk-old mice. These cells migrate primarily owing to CCL2 and CCL5 chemokines produced by cardiac tissue, and they are Vγ1+/CD27+ and thus produce high levels of IFN-γ. In vivo depletion of the γδ T cells revealed γδ T cell-dependent cardiac inflammatory immunoregulation, with increased numbers of CD3+CD4+, CD3+CD8+, and, in particular, F4/80+ cells in the heart and increased cardiac damage in mdx mice. We also observed in vitro that purified cardiac Γδ T cells are cytotoxic against adherent endomysial cardiac cells, mostly macrophages, but not against peritoneal cells, in a perforin/granzyme-dependent manner. Our present data indicate that γδ T cells exert protective effects on the hearts of mdx mice, possibly by selectively killing pathogenic macrophages, and this function may be important for the late onset of cardiac damage in DMD.

Defective T-lymphocyte migration to muscles in dystrophin-deficient mice.

Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting 1 in 3500 males, is caused by mutations in the dystrophin gene. DMD leads to degeneration of skeletal and cardiac muscles and to chronic inflammation. The mdx/mdx mouse has been widely used to study DMD; this model mimics most characteristics of the disease, including low numbers of T cells in damaged muscles. In this study, we aimed to assess migration of T cells to the heart and to identify any alterations in adhesion molecules that could possibly modulate this process. In 6-week-old mdx/mdx mice, blood leukocytes, including T cells, were CD62L(+), but by 12 weeks of age down-modulation was evident, with only approximately 40% of T cells retaining this molecule. Our in vitro and in vivo results point to a P2X7-dependent shedding of CD62L (with high levels in the serum), which in 12-week-old mdx/mdx mice reduces blood T cell competence to adhere to cardiac vessels in vitro and to reach cardiac tissue in vivo, even after Trypanosoma cruzi infection, a known inducer of lymphoid myocarditis. In mdx/mdx mice treated with Brilliant Blue G, a P2X7 blocker, these blood lymphocytes retained CD62L and were capable of migrating to the heart. These results provide new insights into the mechanisms of inflammatory infiltration and immune regulation in DMD.

P2X7 Receptor Modulation of the Gut Microbiota and the Inflammasome Determines the Severity of Toxoplasma gondii-Induced Ileitis.

In mice, oral Toxoplasma gondii infection induces severe ileitis. The aim of the present study was to investigate the impact of the P2X7 receptor (P2X7) on the inflammatory response to T. gondii-induced ileitis. Cysts of the ME49 strain of T. gondii were used to induce ileitis. The infected mice were euthanized on day 8 and ileal tissue and peripheral blood were collected for histopathological and immunohistochemical analyses. Ileal contractility, inflammatory mediators, inflammasome activation, quantitative PCR analysis of gene expression, and fecal microbiota were assessed using appropriate techniques, respectively. The infected P2X7-/- mice had greater disease severity, parasitic burden, liver damage, and intestinal contractility than the infected wild-type (WT) mice. Infection increased serum IL-6 and IFN-γ and tissue caspase-1 but not NLRP3 in P2X7-/- mice compared to WT mice. Bacteroidaceae, Rikenellaceae, and Rhodospirillales increased while Muribaculaceae and Lactobacillaceae decreased in the infected WT and P2X7-/- mice. Bacteroidia and Tannerellaceae increased in the P2X7-/- mice with ileitis. By contrast, Clostridiales and Mollicutes were absent in the P2X7-/- mice but increased in the WT mice. P2X7 protects mice against T. gondii infection by activating the inflammasome and regulating the local and systemic immune responses. Specific gut bacterial populations modulated by P2X7 determine disease severity.

In the Acute Phase of Trypanosoma cruzi Infection, Liver Lymphoid and Myeloid Cells Display an Ambiguous Phenotype Combining Pro- and Anti-Inflammatory Markers.

Multiple cell populations, cellular biochemical pathways, and the autonomic nervous system contribute to maintaining the immunological tolerance in the liver. This tolerance is coherent because the organ is exposed to high levels of bacterial pathogen-associated molecular pattern (PAMP) molecules from the intestinal microbiota, such as lipopolysaccharide endotoxin (LPS). In the case of Trypanosoma cruzi infection, although there is a dramatic acute immune response in the liver, we observed intrahepatic cell populations combining pro- and anti-inflammatory markers. There was loss of fully mature Kupffer cells and an increase in other myeloid cells, which are likely to include monocytes. Among dendritic cells (DCs), the cDC1 population expanded relative to the others, and these cells lost both some macrophage markers (F4/80) and immunosuppressive cytokines (IL-10, TGF-ß1). In parallel, a massive T cell response occured with loss of naïve cells and increase in several post-activation subsets. However, these activated T cells expressed both markers programmed cell death protein (PD-1) and cytokines consistent with immunosuppressive function (IL-10, TGF-ß1). NK and NK-T cells broadly followed the pattern of T cell activation, while TCR-γδ cells appeared to be bystanders. While no data were obtained concerning IL-2, several cell populations also synthesized IFN-γ and TNF-α, which has been linked to host defense but also to tissue injury. It therefore appears that T. cruzi exerts control over liver immunity, causing T cell activation via cDC1 but subverting multiple populations of T cells into immunosuppressive pathways. In this way, T. cruzi engages a mechanism of hepatic T cell tolerance that is familiar from liver allograft tolerance, in which activation and proliferation are followed by T cell inactivation.

Experimental Combination Therapy with Amiodarone and Low-Dose Benznidazole in a Mouse Model of Trypanosoma cruzi Acute Infection.

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately 6 to 7 million people in Latin America, with cardiomyopathy being the clinical manifestation most commonly associated with patient death during the acute phase. The etiological treatment of CD is restricted to benznidazole (Bz) and nifurtimox (Nif), which involve long periods of administration, frequent side effects, and low efficacy in the chronic phase. Thus, combined therapies emerge as an important tool in the treatment of CD, allowing the reduction of Bz dose and treatment duration. In this sense, amiodarone (AMD), the most efficient antiarrhythmic drug currently available and prescribed to CD patients, is a potential candidate for combined treatment due to its known trypanocidal activity. However, the efficacy of AMD during the acute phase of CD and its interaction with Bz or Nif are still unknown. In the present study, using a well-established murine model of the acute phase of CD, we observed that the Bz/AMD combination was more effective in reducing the peak parasitemia than both monotherapy treatments. Additionally, the Bz/AMD combination reduced (i) interleukin-6 (IL-6) levels in cardiac tissue, (ii) P-wave duration, and (iii) frequency of arrhythmia in infected animals and (iv) restored gap junction integrity in cardiac tissue. Therefore, our study validates AMD as a promising candidate for combined therapy with Bz, reinforcing the strategy of combined therapy for CD. IMPORTANCE Chagas disease affects approximately 6 to 7 million people worldwide, with cardiomyopathy being the clinical manifestation that most commonly leads to patient death. The etiological treatment of Chagas disease is limited to drugs (benznidazole and nifurtimox) with relatively high toxicity and therapeutic failures. In this sense, amiodarone, the most effective currently available antiarrhythmic drug prescribed to patients with Chagas disease, is a potential candidate for combined treatment due to its known trypanocidal effect. In the present study, we show that combined treatment with benznidazole and amiodarone improves the trypanocidal effect and reduces cardiac damage in acutely T. cruzi-infected mice.

ZIKV replication is differential in explants and cells of human placental which is suppressed by HSV-2 coinfection.

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 (ZIKVBR) and the effect of coinfection with the Brazilian Human alphaherpesvirus 2 strain (HSV-2BR) on ZIKV replication. We found that the ZIKVBR 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 ZIKVBR was reduced according to exposed HSV-2BR 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-2BR coinfection may impact on ZIKV replication.

Induction of proinflammatory cytokines and nitric oxide by Trypanosoma cruzi in renal cells.

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.

Serum 1,3-beta-D-glucan as a noninvasive test to predict histologic activity in patients with inflammatory bowel disease.

BACKGROUND: 1,3-beta-D-glucan (BG) is a ubiquitous cell wall component of gut micro-organisms. We hypothesized that the serum levels of BG could reflect active intestinal inflammation in patients with inflammatory bowel disease. AIM: To determine whether the serum BG concentrations correlate with intestinal inflammation. METHODS: A prospective observational study was performed in a tertiary referral center, from 2016 to 2019, in which serum BG was determined in 115 patients with Crohn's disease (CD), 51 with ulcerative colitis (UC), and 82 controls using a photometric detection kit. Inflammatory activity was determined by ileocolonoscopy, histopathology, magnetic resonance enterography, and biomarkers, including fecal calprotectin (FC), C-reactive protein, and a panel of cytokines. The ability of BG to detect active vs inactive disease was assessed using the area under the receiver operating characteristic curve. In subgroup analysis, serial BG was used to assess the response to therapeutic interventions. RESULTS: The serum BG levels were higher in CD patients than in controls (P = 0.0001). The BG levels paralleled the endoscopic activity in CD patients and histologic activity and combined endoscopic and histologic activity in both CD and UC patients. The area under the curve (AUC) in receiver operating characteristic analysis to predict endoscopic activity was 0.694 [95% confidence interval (CI): 0.60-0.79; P = 0.001] in CD, and 0.662 (95%CI: 0.51-0.81; P = 0.066) in UC patients. The AUC in receiver operating characteristic analysis to predict histologic activity was 0.860 (95%CI: 0.77-0.95; P < 0.001) in CD, and 0.786 (95%CI: 0.57-0.99; P = 0.015) in UC patients. The cut-off values of BG for both endoscopic and histologic activity were 60 µg/mL in CD, and 40 µg/mL in UC patients. Performance analysis showed that the results based on BG of 40 and 60 µg/mL were more specific for predicting endoscopic activity (71.8% and 87.2% for CD; and 87.5% and 87.5% for UC, respectively) than FC (53.3% and 66.7% for CD; and 20% and 80% for UC, respectively); and also histologic activity (60.5% and 76.3% for CD; and 90.0% and 95.0% for UC, respectively) than FC (41.7% and 50.0% for CD; and 25% and 50% for UC, respectively). Regarding the clinical, endoscopic, and histologic activities, the BG levels were reduced following therapeutic intervention in patients with CD (P < 0.0001) and UC (P = 0.003). Compared with endoscopic (AUC: 0.693; P = 0.002) and histologic (AUC: 0.868; P < 0.001) activity, no significant correlation was found between serum BG and transmural healing based on magnetic resonance enterography (AUC: 0.576; P = 0.192). Positive correlations were detected between BG and IL-17 in the CD (r: 0.737; P = 0.001) and the UC group (r: 0.574; P = 0.005), and between BG and interferon-gamma in the CD group (r: 0.597; P = 0.015). CONCLUSION: Serum BG may represent an important novel noninvasive approach for detecting mucosal inflammation and therapeutically monitoring inflammatory bowel diseases, particularly in CD.

In the presence of Trypanosoma cruzi antigens, activated peripheral T lymphocytes retained in the liver induce a proinflammatory phenotypic and functional shift in intrahepatic T lymphocyte.

In secondary lymphoid organs, pathogen-derived and endogenous danger molecules are recognized by pattern recognition receptors, leading to adaptive proinflammatory immune responses. This conceptual rule does not apply directly to the liver, as hepatic immune cells tolerate gut-derived bacterial molecules from the flora. Therefore, the recognition of danger and proinflammatory stimuli differs between the periphery and the liver. However, the tolerant nature of the liver must be overcome in the case of infections or cancer, for example. The central paradigm is the basis for danger recognition and the balance between inflammation and tolerance in the liver. Here, we observed functional integration, with activated peripheral T lymphocytes playing a role in the induction of a proinflammatory environment in the liver in the presence of Trypanosoma cruzi antigens. When only parasite extract was orally administered, it led to the up-regulation of hepatic tolerance markers, but oral treatment plus adoptively transferred activated splenic T lymphocytes led to a proinflammatory response. Moreover, treated/recipient mice showed increased levels of TNF, IFN-γ, IL-6, and CCL2 in the liver and increased numbers of effector and/or effector memory T lymphocytes and F4/80+ cells. There was a reduction in FoxP3+ Treg cells, NKT cells, and γδ T lymphocytes with increased liver damage in the presence of activated peripheral T cells. Our results show that the induction of a proinflammatory liver response against T. cruzi danger molecules is at least partially dependent on cooperation with activated peripheral T cells.

Rapamycin Treatment Reduces Acute Myocarditis Induced by Trypanosoma cruzi Infection.

Chagas disease affects millions of people mainly in Latin America and is a protozoan illness caused by the parasite Trypanosoma cruzi. Chagasic cardiomyopathy is the leading cause of mortality of infected patients, due to compromised electrical and mechanical cardiac function induced by tissue remodeling, especially fibrosis, and lymphocytic infiltration. Some cellular biochemical pathways can be protective to the heart, and we tested if the in vivo activation of the autophagic machinery by rapamycin could reduce parasite-induced myocarditis. Regarding the expression of LC3, an autophagy marker, we observed its upregulation in the cardiac tissue of infected untreated mice. However, after rapamycin treatment, an autophagy inducer, infected mice showed reduced electrical cardiac dysfunctions, myocarditis, cardiac damage, and reduced production of pro-inflammatory cytokines by the heart. On the other hand, the parasite's life cycle was not affected, and we observed no modulations in cardiac tissue or blood parasitemia. Our data indicate that, at least partially, autophagy induction controls inflammation in the heart¸ illustrating the complexity of the pathways that concur to the development of the infection.

After Experimental Trypanosoma cruzi Infection, Dying Hepatic CD3+TCRαß+B220+ T Lymphocytes Are Rescued from Death by Peripheral T Cells and Become Activated.

The unusual phenotype of CD3+ T lymphocyte expressing B220, a marker originally attributed to B lymphocytes, was first observed in the liver of Fas/Fas-L-deficient mice as a marker of apoptotic T lymphocytes. However, other CD3+B220+ T lymphocyte populations were later described in the periphery as functional cytotoxic or regulatory cells, for example. Then, in this work, we studied whether hepatic CD3+B220+ T lymphocytes could play a role in experimental Trypanosoma cruzi infection. In control and infected mice, we observed two subpopulations that could be discerned based on CD117 expression, which were conventional apoptotic CD3+B220+(CD117-) and thymus-independent CD3+B220+CD117+ T lymphocytes. Regardless of CD117 expression, most B220+ T lymphocytes were 7AAD+, confirming this molecule as a marker of dying T cells. However, after infection, we found that around 15% of the CD3+B220+CD117+ hepatic population became B220 and 7AAD negative, turned into CD90.2+, and upregulated the expression of CD44, CD49d, and CD11a, a phenotype consistent with activated T lymphocytes. Moreover, we observed that the hepatic CD3+B220+CD117+ population was rescued from death by previously activated peripheral T lymphocytes. Our results extend the comprehension of the hepatic CD3+B220+ T lymphocyte subpopulations and illustrate the complex interactions that occur in the liver.

Chloroquine and Sulfadoxine Derivatives Inhibit ZIKV Replication in Cervical Cells.

Despite the severe morbidity caused by Zika fever, its specific treatment is still a challenge for public health. Several research groups have investigated the drug repurposing of chloroquine. However, the highly toxic side effect induced by chloroquine paves the way for the improvement of this drug for use in Zika fever clinics. Our aim is to evaluate the anti-Zika virus (ZIKV) effect of hybrid compounds derived from chloroquine and sulfadoxine antimalarial drugs. The antiviral activity of hybrid compounds (C-Sd1 to C-Sd7) was assessed in an in-vitro model of human cervical and Vero cell lines infected with a Brazilian (BR) ZIKV strain. First, we evaluated the cytotoxic effect on cultures treated with up to 200 µM of C-Sds and observed CC50 values that ranged from 112.0 ± 1.8 to >200 µM in cervical cells and 43.2 ± 0.4 to 143.0 ± 1.3 µM in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 µM of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 µM induced a reduction of 79.8% ± 4.2% to 90.7% ± 1.5% of ZIKV-envelope glycoprotein expression in infected cells as compared to 36.8% ± 2.9% of infection in vehicle control. The viral load was also investigated and revealed a reduction of 2- to 3-logs of ZIKV genome copies/mL in culture supernatants compared to 6.7 ± 0.7 × 108 copies/mL in vehicle control. The dose-response curve by plaque-forming reduction (PFR) in cervical cells revealed a potent dose-dependent activity of C-Sds in inhibiting ZIKV replication, with PFR above 50% and 90% at 6 and 12 µM, respectively, while 25 µM inhibited 100% of viral progeny. The treatment of Vero cells at 12 µM led to 100% PFR, confirming the C-Sds activity in another cell type. Regarding effective concentration in cervical cells, the EC50 values ranged from 3.2 ± 0.1 to 5.0 ± 0.2 µM, and the EC90 values ranged from 7.2 ± 0.1 to 11.6 ± 0.1 µM, with selectivity index above 40 for most C-Sds, showing a good therapeutic window. Here, our aim is to investigate the anti-ZIKV activity of new hybrid compounds that show highly potent efficacy as inhibitors of ZIKV in-vitro infection. However, further studies will be needed to investigate whether these new chemical structures can lead to the improvement of chloroquine antiviral activity.

Glucagon reduces airway hyperreactivity, inflammation, and remodeling induced by ovalbumin.

Glucagon has been shown to be beneficial as a treatment for bronchospasm in asthmatics. Here, we investigate if glucagon would prevent airway hyperreactivity (AHR), lung inflammation, and remodeling in a murine model of asthma. Glucagon (10 and 100 µg/Kg, i.n.) significantly prevented AHR and eosinophilia in BAL and peribronchiolar region induced by ovalbumin (OVA) challenge, while only the dose of 100 µg/Kg of glucagon inhibited subepithelial fibrosis and T lymphocytes accumulation in BAL and lung. The inhibitory action of glucagon occurred in parallel with reduction of OVA-induced generation of IL-4, IL-5, IL-13, TNF-α, eotaxin-1/CCL11, and eotaxin-2/CCL24 but not MDC/CCL22 and TARC/CCL17. The inhibitory effect of glucagon (100 µg/Kg, i.n.) on OVA-induced AHR and collagen deposition was reversed by pre-treatment with indomethacin (10 mg/Kg, i.p.). Glucagon increased intracellular cAMP levels and inhibits anti-CD3 plus anti-CD28-induced proliferation and production of IL-2, IL-4, IL-10, and TNF- α from TCD4+ cells in vitro. These findings suggest that glucagon reduces crucial features of asthma, including AHR, lung inflammation, and remodeling, in a mechanism probably associated with inhibition of eosinophils accumulation and TCD4+ cell proliferation and function. Glucagon should be further investigated as an option for asthma therapy.