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.

BjussuMP-II, a venom metalloproteinase, induces the release and cleavage of pro-inflammatory cytokines and disrupts human umbilical vein endothelial cells.

Snake venom metalloproteases (SVMPs) are hydrolytic enzymes dependent on metal binding, primarily zinc (Zn2+), at their catalytic site. They are classified into three classes (P-I to P-III). BjussuMP-II, a P-I SVMP isolated from Bothrops jararacussu snake venom, has a molecular mass of 24 kDa. It exhibits inhibitory activity on platelet aggregation and hydrolyzes fibrinogen. TNF-α upregulates the expression of adhesion molecules on endothelial cell surfaces, promoting leukocyte adhesion and migration during inflammation. Literature indicates that SVMPs may cleave the TNF-α precursor, possibly due to significant homology between metalloproteases from mammalian extracellular matrix and SVMPs. This study aimed to investigate BjussuMP-II's effects on human umbilical vein endothelial cells (HUVEC), focusing on viability, detachment, adhesion, release, and cleavage of TNF-α, IL-1ß, IL-6, IL-8, and IL-10. HUVEC were incubated with BjussuMP-II (1.5-50 µg/mL) for 3-24 h. Viability was determined using LDH release, MTT metabolization, and 7AAD for membrane integrity. Adhesion and detachment were assessed by incubating cells with BjussuMP-II and staining with Giemsa. Cytokines were quantified in HUVEC supernatants using EIA. TNF-α cleavage was evaluated using supernatants from PMA-stimulated cells or recombinant TNF-α. Results demonstrated BjussuMP-II's proteolytic activity on casein. It was not toxic to HUVEC at any concentration or duration studied but interfered with adhesion and promoted detachment. PMA induced TNF-α release by HUVEC, but this effect was not observed with BjussuMP-II, which cleaved TNF-α. Additionally, BjussuMP-II cleaved IL-1ß, IL-6, and IL-10. These findings suggest that the zinc metalloprotease BjussuMP-II could be a valuable biotechnological tool for treating inflammatory disorders involving cytokine deregulation.

Light Emitting Diode Photobiomodulation Enhances Oxidative Redox Capacity in Murine Macrophages Stimulated with Bothrops jararacussu Venom and Isolated PLA2s.

Photobiomodulation therapy associated with conventional antivenom treatment has been shown to be effective in reducing the local effects caused by bothropic venoms in preclinical studies. In this study, we analyzed the influence of photobiomodulation using light emitting diode (LED) on the oxidative stress produced by murine macrophages stimulated with Bothrops jararacussu venom and it isolated toxins BthTX-I and BthTX-II. Under LED treatment, we evaluated the activity of the antioxidant enzymes catalase, superoxide dismutase, and peroxidase as well as the release of hydrogen peroxide and the enzyme lactate dehydrogenase. To investigate whether NADPH oxidase complex activation and mitochondrial pathways could contribute to hydrogen peroxide production by macrophages, we tested the effect of two selective inhibitors, apocynin and CCCP3, respectively. Our results showed that LED therapy was able to decrease the production of hydrogen peroxide and the liberation of lactate dehydrogenase, indicating less cell damage. In addition, the antioxidant enzymes catalase, superoxide dismutase, and peroxidase increased in response to LED treatment. The effect of LED treatment on macrophages was inhibited by CCCP3, but not by apocynin. These findings show that LED photobiomodulation treatment protects macrophages, at least in part, by reducing oxidative stress caused B. jararacussu venom and toxins.

Increase in the risk of snakebites incidence due to changes in humidity levels: A time series study in four municipalities of the state of Rondônia

Abstract INTRODUCTION: Snakebites represent a serious global public health problem, especially in tropical countries. In Brazil, the incidence of snakebites ranges from 19 to 22 thousand cases per 100000 persons annually. The state of Rondônia, in particular, has had an increasing incidence of snakebites. METHODS: A retrospective cross-sectional study on snakebites was conducted from January 2007 to December 2018. Brazil's Information System for Notifiable Diseases was queried for all snakebites reported in Porto Velho, Ariquemes, Cacoal, and Vilhena. Data on land surface temperatures during the day and night, precipitation, and humidity were obtained using the Google Earth Engine. A Bayesian time series model was constructed to describe the pattern of snakebites and their relationship with climate data. RESULTS: In total, 6326 snakebites were reported in Rondônia. Accidents were commonly caused by Bothrops sp. (n=2171, 81.80%). Snakebites most frequently occurred in rural areas (n=2271, 85.5%). Men, with a median age of 34 years (n=2101, 79.1%), were the most frequent bitten. Moderate clinical manifestation was the most common outcome of an accident (n=1101, 41.50%). There were clear seasonal patterns with respect to rainfall, humidity, and temperature. Rainfall and land surface temperature during the day or night did not increase the risk of snakebites in any city; however, changes in humidity increased the risk of snakebites in all cities. CONCLUSION: This study identified the population exposed to snakes and the influence of anthropic and climatic factors on the incidence of snakebites. According to climate data, changes in humidity increased the risk of snakebites.