Preclinical evaluation of single domain antibody efficacy in mitigating local tissue damage induced by Bothrops snake envenomation.

Camelid single-domain antibodies (VHH) represent a promising class of immunobiologicals for therapeutic applications due to their remarkable stability, specificity, and therapeutic potential. To enhance the effectiveness of antivenoms for snakebites, various methods have been explored to address limitations associated with serum therapy, particularly focusing on mitigating local damage and ensuring sustainable production. Our study aimed to characterize the pharmacological profile and neutralization capacity of anti-Phospholipase A2 (PLA2) monomeric VHH (Genbank accessions: KC329718). Using a post-envenoming mouse model, we used intravital microscopy to assess leukocyte influx, measured CK and LDH levels, and conducted a histopathology analysis to evaluate VHH KC329718's ability to neutralize myotoxic activity. Our findings demonstrated that VHH KC329718 exhibited heterogeneous distribution in muscle tissue. Treatment with VHH KC329718 reduced leukocyte influx caused by BthTX-I (a Lys-49 PLA2) by 28 %, as observed through intravital microscopy. When administered at a 1:10 ratio [venom or toxin:VHH (w/w)], VHH KC329718 significantly decreased myotoxicity, resulting in a 35-40 % reduction in CK levels from BthTX-I and BthTX-II (an Asp-49 PLA2) and a 60 % decrease in CK levels from B. jararacussu venom. LDH levels also showed reductions of 60%, 80%, and 60% induced by BthTX-I, BthTX-II, and B. jararacussu venom, respectively. Histological analysis confirmed the neutralization potential, displaying a significant reduction in tissue damage and inflammatory cell count in mice treated with VHH KC329718 post B. jararacussu venom inoculation. This study underscores the potential of monomeric anti-PLA2 VHH in mitigating myotoxic effects, suggesting a promising avenue for the development of new generation antivenoms to address current therapeutic limitations.

Development of quantitative multiplex RT-qPCR one step assay for detection of hepatitis delta virus.

Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruses, usually with a more severe clinical outcome when compared to an HBV monoinfection. To date, the real prevalence of HDV infection is underestimated and detection methods are poorly available, especially in more endemic regions. Therefore, a one-step RT-qPCR method for quantification of HDV-RNA was developed. Biological samples were selected between 2017 and 2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada and underwent the test developed by this study and a second quantitative RT-qPCR assay. The slope of the initial quantitative assay was - 3.321 with an efficiency of 100.04% and amplification factor equal to 2. Analysis of the repeatability data revealed a Limit of Quantification of 5 copies/reaction and Limit of Detection (95%) of 2.83 copies per reaction. In the diagnostic sensitivity tests, there was an accuracy of 97.37% when compared to the reference test. This assay proved to be highly efficient and reproducible, making it a valuable tool to monitor hepatitis Delta patients and assess the risk of disease progression, as well as the effectiveness of treatment.

Innovations in Plasmodium spp. diagnosis on diverse detection platforms.

In 2019, 229 million cases of malaria were recorded worldwide. For epidemiologic surveillance and proper treatment of persons infected with Plasmodium spp., rapid detection of infections by Plasmodium spp. is critical. Thus, Plasmodium spp. diagnosis is one of the indispensable measures for malaria control. Although microscopy is the gold standard for diagnosis, it has restrictions related mainly to the lack of qualified human resources, which is a problem in many regions. Thus, this review presents major innovations in diagnostic methods as alternatives to or complementary to microscopy. Detection platforms in lateral flow systems, electrochemical immunosensors, molecular biology and, more recently, those integrated with smartphones, are highlighted, among others. The advanced improvement of these tests aims to provide techniques that are sensitive and specific, but also quick, easy to handle and free from the laboratory environment. In this way, the tracking of malaria cases can become increasingly effective and contribute to controlling the disease.

Molecular quantitative assay for esterase-mediated organophosphate resistance in Rhipicephalus microplus.

The use of pesticides is the main tool to control infestations of the cattle tick Rhipicephalus microplus, and organophosphate (OP) is one of the most used compounds for this purpose. Carboxylesterases (ChEs) are targets for OP pesticides in arthropods, and acetylcholinesterase 2 (AChE2) and esterase 1 (EST1) are metabolic enzymes involved in the xenobiotic detoxification process. The increase in the synthesis of these enzymes can be detected by the quantitative polymerase chain reaction (qPCR) assay, which was used to identify cattle tick populations resistant to OP pesticides. For that, two field populations of R. microplus were used, one previously identified by the larval packet test (LPT) as OP -sensitive (LC50=0.13µg/cm2) and the other OP-resistant (LC50=8.14µg/cm2). To promote the OP enzyme detoxification, groups of 10 females of the resistant strain were immersed in solutions of diazinon in technical grade at concentrations of 1.0mg/ml, 2.5mg/ml, and 5.0mg/ml. The ticks that survived diazinon exposure were submitted to qPCR assay, which enabled observing an increase in AChE2 and EST1 synthesis in the OP-resistant strain when compared to the susceptible strain. The initial results of expression analysis suggest that the qPCR assay can discriminate OP-resistant and susceptible populations. The development and improvement of molecular diagnostic tests to identify pesticide resistant R. microplus populations are priorities and in the near future it will be important to expand the molecular targets involved in OP resistance, which could be used for better selection of effective strategies to control cattle tick populations.

Effect of BjcuL, a lectin isolated from Bothrops jararacussu, on human peripheral blood mononuclear cells.

BjcuL is a C-type lectin with specificity for the binding of ß-d-galactose units isolated from Bothrops jararacussu venom. It triggers cellular infiltration in post capillary venules, increases edema and vascular permeability in murine models, contributes to in vitro neutrophil activation and modulates macrophage functional activation towards an M1 state. The purpose of this study was to investigate the effect of BjcuL on human peripheral blood mononuclear cells (PBMCs) activation with a focus on PBMCs proliferation and inflammatory mediators release. Results showed that BjcuL is not toxic to PBMCs, that BjcuL inhibits PBMCs proliferation and that it stimulates PBMCs to produce superoxide anion and hydrogen peroxide, primarily via lymphocyte stimulation, but does not stimulate the production of nitric oxide and PGE2. These results demonstrate that BjcuL has an immunomodulatory effect on PBMCs. Further studies are needed to confirm the immunomodulatory effect of BjcuL, to elucidate the molecular mechanisms of action responsible for its effects and to determine its potential application as an immunopharmacological and biotechnological tool.