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.

Bothrops atrox mice experimental envenoming treatment using light-emitting diode (led) as an adjunct therapy to conventional serum therapy.

The use of anti-venom is one of the main control measures for snakebite envenoming when applied immediately after the snakebite. Systemic effects of the envenoming are usually reversed; however, neutralization of local effects is hardly achieved. The need for adjuvant therapies associated with serum therapy can improve the treatment for local effects of envenoming, with greater effectiveness in preventing or delaying the progression of damage, reducing the clinical signs and symptoms of victims of snakebites. The purpose of the study was to evaluate the photobiomodulation therapy using LED and/or dexamethasone associated with conventional serum therapy for the treatment of local damage caused by Bothrops atrox envenomation in a murine model. For this, experimental envenoming was carried out in the gastrocnemius muscle of male Swiss mice weighing 18 to 22 g divided into 8 groups of animals, distributed in groups non-treat, treated with anti-bothropic serum, dexamethasone, and LED, or the associated treatments, by intramuscular inoculation of 50 µg of venom or sterile PBS (control). After 30 min, the proposed treatments were administered alone or in combination. After 3 h, blood and muscle samples were collected for myotoxicity, cytotoxicity, histological analysis, and IL-1ß assays. The evaluation of the treatment alone showed that serum therapy is not effective for the treatment of local damage and photobiomodulation demonstrated to be an effective therapy to reduce leukocyte infiltration, hemorrhage, and myotoxicity in experimental envenoming; dexamethasone proved to be a good resource for the treatment of the inflammatory process reducing the leukocyte infiltration. The association of serum therapy, LED, and dexamethasone was the best treatment to reduce the local effects caused by Bothrops atrox venom. All in all, the association of photobiomodulation therapy using LED with conventional serum therapy and the anti-inflammatory drug is the best treatment for reducing the undesirable local effects caused by snakebite accidents involving B. atrox species.

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.

NLRP3 inflammasome activation in human peripheral blood mononuclear cells induced by venoms secreted PLA2s.

Bothropic venoms contains high amount of secreted phospholipases A2 (sPLA2s) that play a significant role in leukocyte activation and inflammation. Monocytes and lymphocytes are highly functional immune system cells that mediate and provide efficient responses during the inflammation. NLRP3 inflammasome is a multiprotein complex found in immune system cells that is triggered by pathogen- and damage-associated molecular patterns, PAMPs and DAMPs, respectively. PLA2s' effect on human peripheral blood mononuclear cells (PBMCs) is still incompletely understood. PBMCs were isolated by density gradient and incubated with RPMI (control), LPS, BthTX-I (PLA2-Lys49) or BthTX-II (PLA2-Asp49) isolated from Bothrops jararacussu venom, to evaluate viability, and the results showed that there was no cell death. RT-qPCR and immunoblot were used to assess the gene and protein expression of NLRP3 components. Results indicated that there was substantial amplification of ASC, Caspase-1, IL-6, and IL-1ß in 1 h and NLRP3 in 2 h. Protein expression was measured, and the results revealed substantial expression of the NLRP3 inflammasome complex after 4 h. IL-1ß and LDH was quantified in the supernatant of the cells. Taken together, the findings demonstrate that BthTX-I and BthTX-II activate the NLRP3 inflammasome complex in human PBMCs and contribute to the inflammatory response seen in envenoming.

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.

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.

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.