The hydrogen sulfide donor 4-carboxyphenyl-isothiocyanate decreases blood pressure and promotes cardioprotective effect through reduction of oxidative stress and nuclear factor kappa B/matrix metalloproteinase (MMP)-2 axis in hypertension.

AIMS: Our aim was to evaluate whether the hydrogen sulfide (H2S) donor, 4-carboxyphenyl-isothiocyanate (4-CPI), exerts cardioprotective effect in the two kidney- one clip (2K-1C) rats through oxidative stress and MMP-2 activity attenuation and compare it with the classical H2S donor, Sodium Hydrosulfide (NaHS). MATERIALS AND METHODS: Renovascular hypertension (two kidneys-one clip; 2K-1C) was surgically induced in male Wistar rats. After two weeks, normotensive (2K) and hypertensive rats were intraperitoneally treated with vehicle (0.6 % dimethyl sulfoxide), NaHS (0.24 mg/Kg/day) or with 4-CPI (0.24 mg/Kg/day), for more 4 weeks. Systolic blood pressure (SBP) was evaluated weekly by tail-cuff plethysmography. Heart function was assessed by using the Millar catheter. Cardiac hypertrophy and fibrosis were evaluated by hematoxylin and eosin, and Picrosirius Red staining, respectively. The H2S was analyzed using WSP-1 fluorimetry and the cardiac oxidative stress was measured by lucigenin chemiluminescence and Amplex Red. MMP-2 activity was measured by in-gel gelatin or in situ zymography assays. Nox1, gp91phox, MMP-2 and the phospho-p65 subunit (Serine 279) nuclear factor kappa B (NF-κB) levels were evaluated by Western blotting. KEY FINDINGS: 4-CPI reduced blood pressure in hypertensive rats, decreased cardiac remodeling and promoted cardioprotection through the enhancement of cardiac H2S levels. An attenuation of oxidative stress, with inactivation of the p65-NF-κB/MMP-2 axis was similarly observed after NaHS or 4-CPI treatment in 2K-1C hypertension. SIGNIFICANCE: H2S is a mediator that promotes cardioprotective effects and decreases blood pressure, and 4-CPI seems to be a good candidate to reverse the maladaptive remodeling and cardiac dysfunction in renovascular hypertension.

Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

Matrix metalloproteinases are associated with severity of disease among COVID-19 patients: A possible pharmacological target.

COVID-19 is a devastating disease and imbalanced matrix metalloproteinase (MMP) activity may contribute to its pathophysiology. This exploratory study examined whether increased circulating concentrations of MMP-2 and MMP-9, and their endogenous inhibitors, the tissue inhibitors of MMP (TIMP)-1, TIMP-2, TIMP-3 and TIMP-4 are persistently found in patients 2 weeks after their recovery from severe or critical COVID-19 as compared with those in healthy controls. Subjects who had severe (n = 26) or critical (n = 25) PCR-confirmed COVID-19 and healthy controls (n = 21) had blood samples drawn 2 weeks after recovery and serum MMP-2, MMP-9, TIMP-1, TIMP-2, TIMP-3 and TIMP-4 were determined using two Human Luminex® Discovery Assays. Circulating MMP activity was also determined by gel zymography. Patients who had severe or critical COVID-19 had increased circulating MMP-9 and MMP-2 concentrations, with increased MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios indicating increased MMP activity, confirmed by gel zymography (all p < 0.05). Higher circulating MMP-9 (but not MMP-2) concentrations were found in critical versus severe COVID-19 (p < 0.05). We found increased circulating MMP-9 and MMP-2 concentrations and activity many days after recovery from the acute disease, with MMP-9 levels associated with disease severity. These biochemical alterations suggest that MMP-2 and MMP-9 may be important pharmacological targets in COVID-19.

Inhibitors of gastric acid secretion increase oxidative stress and matrix metalloproteinase-2 activity leading to vascular remodeling.

The use of inhibitors of gastric acid secretion (IGAS), especially proton pump inhibitors (PPI), has been associated with increased cardiovascular risk. While the mechanisms involved are not known, there is evidence supporting increased oxidative stress, a major activator of matrix metalloproteinases (MMP), as an important player in such effect. However, there is no study showing whether other IGAS such as histamine H2-receptor blockers (H2RB) cause similar effects. This study aimed at examining whether treatment with the H2RB ranitidine promotes oxidative stress resulting in vascular MMP activation and corresponding functional and structural alterations in the vasculature, as compared with those found with the PPI omeprazole. Male Wistar rats were treated (4 weeks) with vehicle (2% tween 20), omeprazole (10 mg/Kg/day; i.p.) or ranitidine (100 mg/Kg/day; gavage). Then the aorta was collected to perform functional, biochemical, and morphometric analysis. Both ranitidine and omeprazole increased gastric pH and oxidative stress assessed in situ with the fluorescent dye dihydroethidium (DHE) and with lucigenin chemiluminescence assay. Both IGAS augmented vascular activated MMP-2. These findings were associated with aortic remodeling (increased media/lumen ratio and number of cells/µm2). Both IGAS also impaired the endothelium-dependent relaxation induced by acetylcholine (isolated aortic ring preparation). This study provides evidence that the H2RB ranitidine induces vascular dysfunction, redox alterations, and remodeling similar to those found with the PPI omeprazole. These findings strongly suggest that IGAS increase oxidative stress and matrix metalloproteinase-2 activity leading to vascular remodeling, which helps to explain the increased cardiovascular risk associated with the use of those drugs.

Kinetics of IL-6, C-reactive Protein and Fibrinogen Levels in COVID-19 Outpatients Who Evolved to Hypoxemia.

Introduction: Despite the efficacy of the COVID-19, the search for improvements in the management of severe/critical cases continues to be important. The aim is to demonstrate the kinetics of 4 serological markers in patients with COVID-19 who evolved in hypoxemia. Methods: From June to December 2020, the Health Secretariat of Rondônia State, Brazil, established a home medical care service team (HMCS) that provided clinical follow-up for health professionals and military personnel with COVID-19. The clinical and laboratory monitoring was individualized at home by a nursing and medical team. In addition to laboratory parameters, C-reactive protein (CRP), interleukin-6 (IL-6), fibrinogen, and D-dimer levels were periodically taken to monitor the evolution of treatment. Results: Of 218 patients telemonitored, 48 patients needed special care by the HMCS team due to shortness of breath. Chest tomography showed multiple ground-glass shadows and lung parenchymal condensations that was compatible with secondary bacterial infection associated with leukocytosis, for which antibiotics were prescribed. The symptoms were accompanied by increases of CRP and IL-6 levels followed by fibrinogen after a few days, for which an anticoagulant therapy was included. Thirty-three patients evolved to improvements in clinical signs and laboratory results. Between the sixth and eighth day of illness, 15 patients presented signs of hypoxemia with low O2 saturation accompanied with an increase in the respiratory rate, with some of them requiring oxygen therapy. As they did not present signs of clinical severity, but their laboratory markers showed an abrupt IL-6 peak that was higher than the increase in CRP and a new alteration in fibrinogen levels, they received a supplemental dose of anticoagulant and a high dose of corticosteroids, which resulted in clinical improvement. Conclusion: Our study demonstrates that monitoring of IL-6 and CRP may identify precocious hypoxemia in COVID-19 patients and prevented the progressive deterioration of the lung injury.

Gonioscopia diagnóstica e cirúrgica. Atualidades e perspectivas / Diagnostic and surgical gonioscopy. Recent advances and perspective

RESUMO O presente artigo é uma atualização sobre os principais conceitos, as técnicas, os equipamentos, as lentes e as utilidades do exame de gonioscopia, com foco principal na sua importância para as novas terapias antiglaucomatosas: trabeculoplastia seletiva a laser e cirurgias angulares. Se faz necessária esta revisão e atualização por se tratar de um exame imprescindível para a prática diária do oftalmologista, consolidando o conhecimento necessário para realizá-lo e pelo crescente uso da gonioscopia nas novas terapias antiaglaucomatosas.

ABSTRACT This article is an update on the main concepts, techniques, equipment, lenses, and uses of the gonioscopy exam, with a main focus on its importance for new antiglaucoma therapies: selective laser trabeculoplasty and angular surgeries. This review and update is necessary because it is an essential exam for the daily practice of ophthalmologists, consolidating the knowledge necessary to perform it and because of the increasing use of gonioscopy in new anti-aglaucomatous therapies.

Exosome Liberation by Human Neutrophils under L-Amino Acid Oxidase of Calloselasma rhodostoma Venom Action.

L-Amino acid oxidase (LAAO) is an enzyme found in snake venom that has multifaceted effects, including the generation of hydrogen peroxide (H2O2) during oxidative reactions, leading to various biological and pharmacological outcomes such as apoptosis, cytotoxicity, modulation of platelet aggregation, hemorrhage, and neutrophil activation. Human neutrophils respond to LAAO by enhancing chemotaxis, and phagocytosis, and releasing reactive oxygen species (ROS) and pro-inflammatory mediators. Exosomes cellular nanovesicles play vital roles in intercellular communication, including immune responses. This study investigates the impact of Calloselasma rhodostoma snake venom-derived LAAO (Cr-LAAO) on human neutrophil exosome release, including activation patterns, exosome formation, and content. Neutrophils isolated from healthy donors were stimulated with Cr-LAAO (100 µg/mL) for 3 h, followed by exosome isolation and analysis. Results show that Cr-LAAO induces the release of exosomes with distinct protein content compared to the negative control. Proteomic analysis reveals proteins related to the regulation of immune responses and blood coagulation. This study uncovers Cr-LAAO's ability to activate human neutrophils, leading to exosome release and facilitating intercellular communication, offering insights into potential therapeutic approaches for inflammatory and immunological disorders.

Low performance of Policimbac® broth microdilution in determining polymyxin B MIC for Klebsiella pneumoniae.

Klebsiella pneumoniae is a global threat to healthcare, and despite the availability of new drugs, polymyxins are still an important therapeutic option for this and other resistant gram-negative pathogens. Broth microdilution is the only method that is recommended for polymyxins. In this study, we evaluated the accuracy of a commercial Policimbac® plate in determining the polymyxin B MIC for K. pneumoniae clinical isolates. The results were compared with those of the broth microdilution method according to ISO 16782. The Policimbac® plate had an excellent 98.04% categorical agreement, but unacceptable 31.37% essential agreement rates. Almost 2% of major errors as observed. Additionally, 52.94% of the strains overestimated the MIC at 1 µg/mL. Three isolates were excluded from the analysis due to the drying of the Policimbac® plate. To avoid dryness, we included wet gauze for the test, obtaining a 100% of categorical agreement rate; however, a low essential agreement was maintained (25.49%). In conclusion, the Policimbac® plate was unable to correctly determine the polymyxin B MIC for K. pneumoniae isolates. This low performance may interfere with the clinical use of the drug and, thus, with the result of the patient's treatment.

Antibacterial and antifungal action of CTAB-containing silica nanoparticles against human pathogens.

New antibiotic agents are urgently needed worldwide to combat the increasing tolerance and resistance of pathogenic fungi and bacteria to current antimicrobials. Here, we looked at the antibacterial and antifungal effects of minor quantities of cetyltrimethylammonium bromide (CTAB), ca. 93.8 mg g-1, on silica nanoparticles (MPSi-CTAB). Our results show that MPSi-CTAB exhibits antimicrobial activity against Methicillin-resistant Staphylococcus aureus strain (S. aureus ATCC 700698) with MIC and MBC of 0.625 mg mL-1 and 1.25 mg mL-1, respectively. Additionally, for Staphylococcus epidermidis ATCC 35984, MPSi-CTAB reduces MIC and MBC by 99.99% of viable cells on the biofilm. Furthermore, when combined with ampicillin or tetracycline, MPSi-CTAB exhibits reduced MIC values by 32- and 16-folds, respectively. MPSi-CTAB also exhibited in vitro antifungal activity against reference strains of Candida, with MIC values ranging from 0.0625 to 0.5 mg mL-1. This nanomaterial has low cytotoxicity in human fibroblasts, where over 80% of cells remained viable at 0.31 mg mL-1 of MPSi-CTAB. Finally, we developed a gel formulation of MPSi-CTAB, which inhibited in vitro the growth of Staphylococcus and Candida strains. Overall, these results support the efficacy of MPSi-CTAB with potential application in the treatment and/or prevention of infections caused by methicillin-resistant Staphylococcus and/or Candida species.

Substance abuse and susceptibility to false memory formation: a systematic review and meta-analysis.

Background: Substance abuse has an impact on various cognitive domains, including memory. Even though this impact has been extensively examined across different subdomains, false memory has been sparsely studied. This systematic review and meta-analysis seek to synthesize the current scientific data concerning false memory formation in individuals with a history of substance abuse. Methods: PubMed, Scopus, the Cochrane Library, Web of Science, and PsycINFO were searched to identify all experimental and observational studies in English, Portuguese, and Spanish. Studies were then examined by four independent reviewers and, if they met the inclusion criteria, assessed for their quality. The Cochrane Risk of Bias Tool for randomized controlled trials (RCT) and the Joanna Briggs Institute (JBI) critical appraisal checklists for quasi-experimental and analytic cross-sectional studies were used to assess the risk of bias. Results: From the 443 screened studies, 27 (and two more from other sources) were considered eligible for full-text review. A final 18 studies were included in the present review. Of these, 10 were conducted with alcoholics or heavy drinkers, four focused on ecstasy/polydrug users, three were done with cannabis users and one focused on methadone maintenance patients with current cocaine dependence. Regarding false memory type, 15 studies focused on false recognition/recall, and three on provoked confabulation. Conclusions: None but one of the studies considering false recognition/recall of critical lures found any significant differences between individuals with a history of substance abuse and healthy controls. However, most of the studies taking into account false recognition/recall of related and unrelated events found that individuals with a history of substance abuse showed significantly higher rates of false memories than controls. Future research should continue to consider different types of false memories as well as their potential association with relevant clinical variables. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=266503, identifier: CRD42021266503.

Effectiveness of smartphone interventions as continuing care for substance use disorders: A systematic review.

As a chronic disease with consistent relapse rates, substance use disorders (SUD) require a continuity-of-care approach. Unfortunately, many patients do not have access to continuing care. This systematic review analysed the current scientific knowledge to better understand if app-based smartphone interventions can be an effective alternative. The databases Cochrane Library, PubMed, Web of Science, and PsycINFO were used to find experimental and quasi-experimental studies investigating the effectiveness of a smartphone intervention in individuals who had completed treatment for SUD. After removing duplicates, a total of 1488 studies were screened, with 48 being selected for a full-text review. Four studies met all the criteria, with one other being added by identification through other resources, making a total of 5 studies included in the present review. Out of the four studies using a control group, only one found no significant differences in favour of the experimental group. That study used an active control group and compared the smartphone intervention to its therapeutic group equivalent. There were no significant differences between the two experimental groups. Overall, the results indicate that app-based smartphone interventions can be an effective alternative to traditional forms of continuing care. However, literature is still scarce, and more research needs to be made on this subject. This systematic review is registered at PROSPERO with the identifier [CRD42021272070].

Phenotypic, functional and plasticity features of human PBMCs induced by venom secreted PLA2s.

Bothrops venom contains a high amount of secreted phospholipase A2 (sPLA2s) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLA2s are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLA2s (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-ß and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLA2s trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming.

Quercetin decreases cardiac hypertrophic mediators and maladaptive coronary arterial remodeling in renovascular hypertensive rats without improving cardiac function.

Oxidative stress and MMP activity are found in the hearts and arteries in hypertension and contribute to the resulting hypertrophy and dysfunction. Quercetin is a flavonoid that reduces MMP-2 activity and ameliorates hypertrophic vascular remodeling of hypertension. The hypothesis is that treatment of hypertensive rats with quercetin ameliorates coronary maladaptive remodeling and decreases hypertrophic cardiac dysfunction by decreasing oxidative stress and MMP activity. Male Sprague-Dawley two-kidney, one-clip (2K1C) and Sham rats were treated with quercetin (10 mg/kg/day) or its vehicle for 8 weeks by gavage. Rats were analyzed at 10 weeks of hypertension. Systolic blood pressure (SBP) was examined by tail-cuff plethysmography. Cardiac left ventricles were used to determine MMP activity by in situ zymography and oxidative stress by dihydroethidium. Immunofluorescence was performed to detect transforming growth factor (TGF)-ß and nuclear factor kappa B (NFkB). Morphological analyses of heart and coronary arteries were done by H&E and picrosirius red, and cardiac function was measured by Langendorff. SBP was increased in 2K1C rats, and quercetin did not reduce it. However, quercetin decreased both oxidative stress and TGF-ß in the left ventricles of 2K1C rats. Quercetin also decreased the accentuated MMP activity in left ventricles and coronary arteries of 2K1C rats. Quercetin ameliorated hypertension-induced coronary arterial hypertrophic remodeling, although it did not reduce cardiac hypertrophic remodeling and dysfunction. Quercetin decreases cardiac oxidative stress and TGF-ß and MMP activity in addition to improving coronary remodeling, yet does not ameliorate cardiac dysfunction in 2K1C rats.

Enhanced biological nitrogen and phosphorus removal from sewage driven by fermented glycerol: comparative assessment between sequencing batch- and continuously fed-structured fixed bed reactor.

The nutrient biological removal from sewage, especially from anaerobic reactor effluents, still represents a major challenge in conventional sewage treatment plants. In this work, the nitrogen and phosphorus removal from anaerobic pre-treated domestic sewage in an up-flow anaerobic sludge blanket (UASB) reactor was assessed in a structured fixed bed reactor (SFBR) operated in a continuous and in a batch mode using polyurethane foam as material support for biomass and fermented glycerol as the exogenous carbon source. The SFBR was operated as a sequencing batch reactor with cycles of 90, 120, and 150 min under anaerobic, oxic, and anoxic conditions, respectively, reaching average efficiencies for total nitrogen and phosphorus removal of 88% and 56%, respectively. Fermented glycerol was added during the non-aerated periods. Under continuous feeding, the SFBR was operated with aeration/non-aeration periods of 2/1 (h) and 3/1 (h), hydraulic retention time of 12 h, and a recirculation ratio of 3. Without fermented glycerol addition, the maximum removal of total nitrogen (TN) reached 42%, while adding glycerol in the non-aerated period improved TN removal to 64.9% (2/1 h) and 69.5% (3/1 h). During continuous operation, no phosphorus removal was observed, which was released during the non-aerated period, remaining in the effluent. Optical microscopy analyses confirmed the presence of polyphosphate granules and of the phosphorus accumulating organisms in the reactor biofilm. It was concluded that the batch feeding method was determinant for phosphorus removal. The structured fixed bed reactor with polyurethane foam proved to be feasible in the removal of organic matter and nutrients remaining in the UASB reactor effluent.

Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom.

l-Amino acid oxidase isolated from Calloselasma rhodostoma (Cr-LAAO) snake venom is a potent stimulus for neutrophil activation and production of inflammatory mediators, contributing to local inflammatory effects in victims of envenoming. Cr-LAAO triggered the activation of nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase complex and protein kinase C (PKC)-α signaling protein for reactive oxygen species (ROS) production. This study aims to evaluate the ROS participation in the NLRP3 inflammasome complex activation in human neutrophil. Human neutrophils were isolated and stimulated for 1 or 2 h with RPMI (negative control), LPS (1 µg/mL, positive control) or Cr-LAAO (50 µg/mL). The neutrophil transcriptome was examined using the microarray technique, and RT-qPCR for confirmation of gene expression. Immunofluorescence assays for NLRP3, caspase-1, IL-1ß and GSDMD proteins was performed by Western blot in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase. IL-1ß release was also detected in the presence and/or absence of NLRP3, caspase-1 and NADPH oxidase inhibitors. Results showed that Cr-LAAO upregulated the expression of genes that participate in the NADPH oxidase complex formation and inflammasome assembly. NLRP3 was activated and accumulated in the cytosol forming punctas, indicating its activation. Gasdermin D was not cleaved but lactate dehydrogenase was released. Furthermore, ROS inhibition decreased the expression of NLRP3 inflammasome complex proteins, as observed by protein expression in the presence and/or absence of apocynin, an NADPH oxidase inhibitor. IL-1ß was also released, and pharmacological inhibition of NLRP3, caspase-1, and ROS reduced the amount of released cytokine. This is the first report demonstrating the activation of the NLRP3 inflammasome complex via ROS generation by Cr-LAAO, which may lead to the development of local inflammatory effects observed in snakebite victims.

Escherichia coli ST224 and IncF/blaCTX-M-55 plasmids drive resistance to extended-spectrum cephalosporins in poultry flocks in Parana, Brazil.

Resistant Enterobacterales of avian intestinal origin can contaminate carcasses during broiler processing and thereby spread through the human food chain. This study aimed at assessing the prevalence, diversity and genomic characteristics of ESBL/AmpC Enterobacterales in poultry flocks from different farms and cities in the state of Paraná, Brazil. Enterobacterales isolated from cloacal samples were subjected to antimicrobial susceptibility testing (AST). ESBL/AmpC isolates were whole-genome sequenced and subjected to S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blotting to determine the location of resistant genes on plasmids. A surprisingly high proportion of E. coli (40.6 %) collected on non-selective plates presented an ESBL/AmpC phenotype. Multidrug resistance was statistically not higher in ESBL/AmpC E. coli having the potential to be Avian Pathogenic (APEC-like) compared to non-APEC-like ESBL/AmpC E. coli isolates. Resistance to antibiotics not authorized for use in poultry in the State of Paraná was observed, suggesting that antimicrobial resistance (AMR) is co-selected by the use of veterinary-licensed antibiotics. Phylogenetic analyzes revealed the presence of identical or highly similar ESBL/AmpC E. coli clones on farms distant up to 100 km of each other; this strongly suggests that the centralization and verticalization of the poultry industry can facilitate the spread of resistant bacteria among different farms, companies, and cities. The molecular characterization of clones and plasmids proved the dominance of the ST224 E. coli lineage and the IncF/blaCTX-M-55 plasmid, possibly indicating the emergence of successful clones and plasmids adapted to the chicken host. Our data contribute to the epidemiological tracking of resistance mechanisms in Enterobacterales from poultry and to knowledge for further One Health studies to control the spread of resistant bacteria from food animals to humans.

Dynamics of action of a Lys-49 and an Asp-49 PLA2s on inflammasome NLRP3 activation in murine macrophages.

Phospholipases A2 (PLA2s) are proteins found in snake venoms with hemolytic, anticoagulant, myotoxic, edematogenic, bactericidal and inflammatory actions. In Bothrops jararacussu snake venom were isolated a Lys49-PLA2 (BthTX-I) and an Asp49-PLA2 (BthTX-II) with myotoxic and inflammatory actions. Both PLA2s can activate the NLRP3 inflammasome, an intracytoplasmic platform that recognizes molecules released when tissue is damaged liberating IL-1ß that contributes to the inflammatory response observed in envenoming. The dynamic of action of BthTX-I and BthTX-II in both thioglycollate (TG)-elicited macrophages and C2C12 myoblasts and the involvement of EP1 and EP2 receptors, and PGE2 in NLRP3 inflammasome activation were evaluated. Both toxins induced PGE2 liberation and inflammasome components (NLRP3, Caspase-1, ASC, IL-1ß, and IL18), IL-6, P2X7, COX-1, COX-2, EP2 and EP4 gene expression in TG-elicited macrophages but not in C2C12 myoblasts. EP2 (PF04418948) and EP4 (GW627368X) inhibitors abolished this effect. Both PLA2s also induced NLRP3 inflammasome protein expression that was abolished with the inhibitors used. Immunofluorescence and IL-1ß assays confirmed the NLRP3 activation in TG-elicited macrophages with the participation of both EP2 and EP4 receptors confirming their involvement in this effect. All in all, BthTX-I and BthTX-II activate macrophages and induce the NLRP3 inflammasome complex activation with the participation of the PGE2 via COX pathway and EP2 and EP4, both PGE2 receptors, contributing to the local inflammatory effects observed in envenoming.

Inflammasome NLRP3 activation induced by Convulxin, a C-type lectin-like isolated from Crotalus durissus terrificus snake venom.

Convulxin (CVX), a C-type lectin-like protein isolated from the venom of the snake species, Crotalus durissus terrificus, stimulates platelet aggregation by acting as a collagen receptor agonist for glycoprotein VI found in the platelets. The effect of CVX on platelets has been studied, but its effect on human peripheral blood mononuclear cells (PBMCs) remains unclear. Given the significance of PBMCs in inflammation, this study explored the effect of CVX on PBMCs, specifically regarding NLRP3 inflammasome activation by assessing cell viability, ability to induce cell proliferation, reactive oxygen species (ROS) and nitric oxide production, interleukin (IL)-2 and IL-10 secretion, NLRP3 complex activation, and the role of C-type lectin-like receptors (CTLRs) in these. CVX was not toxic to PBMCs at the investigated concentrations and did not increase PBMC growth or IL-2 release; however, CVX induced IL-10 release and ROS generation via monocyte activation. It also activated the NLRP3 complex, resulting in IL-1ß induction. Furthermore, the interaction between CVX and Dectin-2, a CTLR, induced IL-10 production. CVX interaction with CTLR has been demonstrated by laminarin therapy. Because of the involvement of residues near the Dectin-2 carbohydrate-recognition site, the generation of ROS resulted in inflammasome activation and IL-1ß secretion. Overall, this work helps elucidate the function of CVX in immune system cells.