The histopathological and immunological pattern of CBA mice infected with Leishmania amazonensis after treatment with pyrazole carbohydrazide derivatives

Because there is no vaccine in clinical use, control of Leishmaniasis relies almost exclusively on chemotherapy and the conventional treatments exhibit high toxicity for patients and emerging drug resistance. Recently, we showed that oral treatment with synthetic pyrazole carbohydrazide compounds induced lower parasite load in draining lymph nodes and reduced skin lesion size without causing any toxic effects in an experimental murine infection model with Leishmania amazonensis. In this study, CBA mice were infected in the footpad with L. amazonensis and then orally treated with pyrazole carbohydrazides derivatives, such as BrNO₂, NO₂Cl and NO₂Br and their histopathological and immunological effects were then investigated. Epidermis and dermis had lower levels of inflammatory infiltration compared to the infected untreated control mice. In the dermis of treated animals, the numbers of vacuolated macrophages containing intracellular parasites were far lower than in infected untreated animals. In addition to dermal macrophages, we also observed a mixed inflammatory infiltrate containing lymphocytes and granulocyte cells. Lower numbers of B cells (B220+) and T lymphocytes (CD3+) were identified in the lesions of treated mice compared with the untreated, infected mice. In draining lymph node cells, the number of T lymphocytes (CD3+) was decreased, and the numbers of B cells (CD19+) and CD8+ T cells were increased in infected mice, when compared with the non-infected control group. In additional, we have shown that infected treated and untreated lymph node cells had similar levels of TGF-β and IFN-γ mRNA expression, whereas IL-4 was expressed at a lower level in the treated group. Increased levels of the specific anti-Leishmania IgG2a or IgG3 antibody subclass were observed in NO₂Cl or BrNO₂-treated group, respectively. Overall, our experimental findings suggest that pyrazole carbohydrazides exert modulation of IL-4 expression and B cell levels; however, further evaluation is required to determine the optimal treatment regime.     

Effect of Prediabetes on Membrane Bicarbonate Transporters in Testis and Epididymis

The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. This includes the control of bicarbonate (HCO₃ ^?) concentration, which plays an essential role in the maintenance of extracellular and intracellular pH (pH_i) values. Diabetes mellitus alters pH_i regulation in mammalian cells, mainly by altering the activity of ion transporters, particularly HCO₃ ^?-dependent mechanisms. Yet, little is known about the effects of this pathology and its prodromal stage, prediabetes, on the membrane transport mechanisms of male reproductive tract cells. Herein, we analyzed protein and mRNA levels of the most relevant HCO₃ ^? transporters of the SLC4 family [anion exchanger 2 (AE2), Na⁺-driven Cl^?/HCO₃ ^? exchanger (NDCBE), electrogenic Na⁺/HCO₃ ^? cotransporter 1 (NBCe1), electroneutral Na⁺/HCO₃ ^? cotransporter 1 (NBCn1)] in the testis and epididymis of a prediabetic animal model. Firstly, we identified the HCO₃ ^? transporters of the SLC4 family, in both testicular and epididymal tissue. Secondly, although no alterations were detected in protein expression, mRNA levels of NBCe1, NBCn1 and NDCBE were significantly increased in the testis of prediabetic rats. On the other hand, in the epididymis, prediabetes caused an increase of AE2 and a decrease of NDCBE protein levels. These alterations may be translated into changes of HCO₃ ^? transepithelial epididymal fluxes in vivo, which may represent a threat for sperm survival. Moreover, these results provide evidence of the molecular mechanism that may be responsible for the significant increase in abnormal sperm morphology already reported in prediabetic rats.     

Different effects of catechin on angiogenesis and inflammation depending on VEGF levels

Although physiological and pathological angiogenesis develop through similar processes, during pathological angiogenesis, proangiogenic factors are exacerbated. Polyphenols have been considered therapeutic tools for conditions exhibiting enhanced angiogenesis. However, the possibility that these compounds may also prevent vascularization in physiological situations is a major drawback for their use. The purpose of the current study was to investigate the effects of 0.1–100 μM catechin on endothelial cells (EC) and vascular smooth muscle cells (VSMC) regarding angiogenic and inflammatory processes. Catechin modulation of angiogenesis and inflammation was also evaluated in vivo using different models of angiogenesis: one physiological (skin wound-healing assay) and another one resembling pathological angiogenesis, exhibiting higher vascular endothelial growth factor (VEGF)-A stimulation (Matrigel plug assay). The in vitro results showed that 100 μM catechin increased viability (to 165.58% and to 165.34%) and decreased apoptosis (53.45% and 92.65%) and proliferation (33.19% and 23.36%) of EC and VSMC, respectively. Catechin affected migration and invasion, tending to increase both in EC and decreasing them in VSMC; however, it did not change sprouting angiogenesis. Nevertheless, catechin diminished in vitro inflammatory modulators such as tumor necrosis factor α (58.66% for human umbilical vein endothelial cells and 85.46% for human aortic smooth muscle cells) and nuclear factor kappa-B (38.43% for VSMC). The in vivo results demonstrated that catechin did not change angiogenesis and inflammation in skin wound-healing model and substantially decreased these processes in Matrigel plug assay. Altogether, the current study showed that catechin has different effects in angiogenesis and inflammation depending on VEGF-A levels. The absence of adverse effects in mature vasculature favors catechin potential use against pathological situations where angiogenesis is stimulated.     

Cis-trimethoxystilbene,exhibits higher genotoxic and antiproliferative effects than its isomer trans-trimethoxystilbene in MCF-7 and MCF-10A cell lines

Stilbenes are a class of natural compounds with a wide variety of biological effects, such as antitumor activity. The best-known stilbene is resveratrol, whose clinical application is limited due to its low bioavailability. Methoxylated derivatives of this stilbene, including cis-trimethoxystilbene (cis-TMS) and trans-trimethoxystilbene (trans-TMS) have demonstrated more pronounced cytotoxic and anti-proliferative effects than resveratrol. Thus, the objective of this study is to evaluate and compare the cytotoxicity and antiproliferative effects of cis- and trans-TMS in MCF-7 and its normal counterpart MCF-10A. Both compounds were cytotoxic, genotoxic, and induced G2-M accumulation and cell death in the two cell lines. These results suggested that the genotoxicity of cis- and trans-TMS is involved in the reduction of cellular proliferation of MCF-7 and MCF-10A cells, but notably, such antiproliferative effects are more pronounced for cis- than trans-TMS.     

Evaluation of PCR Based on Gene <Emphasis Type="Italic">apxIVA</Emphasis> Associated with 16S rDNA Sequencing for the Identification of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.     

Glucose-regulated Protein 94 Triage of Mutant Myocilin through Endoplasmic Reticulum-associated Degradation Subverts a More Efficient Autophagic Clearance Mechanism

Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that β-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.