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São Paulo state has been the epicenter of the Coronavirus Disease 2019 (COVID-19) in Brazil, ranking first by state with over six million reported cases. In February 2021, the P.4 lineage was reported in 21 cities across the state by public health authorities due to the L452R mutation. Here, by analyzing 17,304 genome sequences of SARS-CoV-2 sampled between February and August of 2021 in 476 distinct cities in São Paulo, we assess the transmission dynamics of the P.4 lineage and other SARS-CoV-2 variants that were, at the time of the study, co-circulating in the state. Additionally, clinical parameters from the city of Araras, São Paulo (N = 251) were considered to estimate the potential risk and mortality rate associated with the P.4 lineage since its higher prevalence was observed in that city. Our data suggest a low frequency (0.55%) of the P.4 lineage across the state, with the gamma variant being the dominant form in all regions (90%) at that time. Furthermore, no evidence of increased transmissibility and disease severity related to the P.4 lineage was observed. The displacement through the time of different lineages in São Paulo highlights how challenging genomic surveillance appears to track the emergence of new SARS-CoV-2 lineages, which could better guide the implementation of control measures.

Crotoxin (CTX), the major toxin of Crotalus durissus terrificus snake venom, induces an inhibitory effect on tumor development and modulates the functions of macrophages (MØs), which play a key role as a defense mechanism against tumor growth. In early tumor progression stage, MØs are avidly phagocytic (inflammatory cell), releasing reactive nitrogen intermediates-RNI/ROI and cytokines TNF-α, IL-1β, and IL-6. However, when the tumor has been developed, tumor-associated MØ (angiogenic cell) presents a decrease in the mentioned activities. We reported that CTX stimulates H

Sepsis is a syndrome of physiological and biochemical abnormalities induced by an infection that represents a major public health concern. It involves the early activation of inflammatory responses. Crotoxin (CTX), the major toxin of the South American rattlesnake Crotalus durissus terrificus venom, presents longstanding anti-inflammatory properties. Since immune system modulation may be a strategic target in sepsis management, and macrophages' functional and secretory activities are related to the disease's progression, we evaluated the effects of CTX on macrophages from septic animals. Balb/c male mice submitted to cecal ligation and puncture (CLP) were treated with CTX (0.9 μg/animal, subcutaneously) 1 h after the procedure and euthanized after 6 h. We used plasma samples to quantify circulating cytokines and eicosanoids. Bone marrow differentiated macrophages (BMDM) were used to evaluate the CTX effect on macrophages' functions. Our data show that CTX administration increased the survival rate of the animals from 40% to 80%. Septic mice presented lower plasma concentrations of IL-6 and TNF-α after CTX treatment, and higher concentrations of LXA

Snake venoms are a natural biological source of bioactive compounds, mainly composed of proteins and peptides with specific pathophysiological functions. The diversity of protein families found in snake venoms is reflected by the range of targets and toxicological effects observed, and consequently, a wide variety of potential pharmacological activities. In this context, in vitro biomimetic models such as spheroid and organoid systems, which are three-dimensional (3D) cell culture models, enable extensive screening and identification of substances with pharmacological potential and the determination of the mechanisms underlying their activities. In this review we summarize the main findings of 3D microenvironment cell culture as a promising model for snake venom research, from producing snake toxins on venom gland organoids to screening pharmacological active compounds on spheroids for drug development.

Sao Paulo State, currently experiences a second COVID‐19 wave overwhelming the healthcare system. Due to the paucity of SARS‐CoV‐2 complete genome sequencing, we established a Network for Pandemic Alert of Emerging SARS‐CoV‐2 Variants to rapidly understand and monitor the spread of SARS‐CoV‐2 variants into the state. Through analysis of 210 SARS‐CoV‐2 complete genomes obtained from the largest regional health departments we identified cocirculation of multiple SARS‐CoV‐2 lineages such as B.1.1 (0.5%), B.1.1.28 (23.2%), B.1.1.7 (alpha variant, 6.2%), B.1.566 (1.4%), B.1.544 (0.5%), C.37 (0.5%) P.1 (gamma variant, 66.2%), and P.2 (zeta variant, 1.0%). Our analysis allowed also the detection, for the first time in Brazil, the South African B.1.351 (beta) variant of concern, B.1.351 (501Y.V2) (0.5%), characterized by the following mutations: ORF1ab: T265I, R724K, S1612L, K1655N, K3353R, SGF 3675_F3677del, P4715L, E5585D; spike: D80A, D215G, L242_L244del, A262D, K417N, E484K, N501Y, D614G, A701V, C1247F; ORF3a: Q57H, S171L, E: P71L; ORF7b: Y10F, N: T205I; ORF14: L52F. The most recent common ancestor of the identified strain was inferred to be mid‐October to late December 2020. Our analysis demonstrated the P.1 lineage predominance and allowed the early detection of the South African strain for the first time in Brazil. We highlight the importance of SARS‐CoV‐2 active monitoring to ensure the rapid detection of potential variants for pandemic control and vaccination strategies. Highlights Identification of B.1.351 (beta) variant of concern in the Sao Paulo State. Dissemination of SARS‐CoV‐2 variants of concern and interest in the Sao Paulo State. Mutational Profile of the circulating variants of concern and interest.

São Paulo is the financial center of Brazil, with a population of over 12 million, that receives travelers from all over the world for business and tourism. It was the first city in Brazil to report a case of COVID-19 that rapidly spread across the city despite the implementation of the restriction measures. Despite many reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the city of São Paulo. Thus, in this study, we provide a retrospective overview of the COVID-19 epidemic in São Paulo City, Southeastern, Brazil, by generating a total of 9995 near-complete genome sequences from all the city’s different macro-regions (North, West, Central, East, South, and Southeast). Our analysis revealed that multiple independent introduction events of different variants (mainly Gamma, Delta, and Omicron) occurred throughout time. Additionally, our estimates of viral movement within the different macro-regions further suggested that the East and the Southeast regions were the largest contributors to the Gamma and Delta viral exchanges to other regions. Meanwhile, the North region had a higher contribution to the dispersion of the Omicron variant. Together, our results reinforce the importance of increasing SARS-CoV-2 genomic monitoring within the city and the country to track the real-time evolution of the virus and to detect earlier any eventual emergency of new variants of concern that could undermine the fight against COVID-19 in Brazil and worldwide.

Background Brazil has been dramatically hit by the SARS-CoV-2 pandemic and is a world leader in COVID-19 morbidity and mortality. Additionally, the largest country of Latin America has been a continuous source of SARS-CoV-2 variants and shows extraordinary variability of the pandemic strains probably related to the country´s outstanding position as a Latin American economical and transportation hub. Not all regions of the country show sufficient infrastructure for SARS-CoV-2 diagnosis and genotyping which can negatively impact the pandemic response. Methods Due to this reason and to disburden the diagnostic system of the inner São Paulo State, the Butantan Institute established the Mobile Laboratory (in Portuguese: LabMovel) for SARS-CoV-2 testing which started a trip of the most important “hotspots” of the most populous Brazilian region. The LabMovel initiated in two important cities of the State: Aparecida do Norte (an important religious center) and the Baixada Santista region which incorporates the port of Santos, the busiest in Latin America. The LabMovel was fully equipped with an automatized system for SARS-CoV-2 diagnosis and sequencing/genotyping. It also integrated the laboratory systems for patient records and results divulgation including in the Federal Brazilian Healthcare System. Results Currently,16,678 samples were tested, among them 1,217 from Aparecida and 4,564 from Baixada Santista. We tracked the delta introductio in the tested regions with its high diversification. The established mobile SARS-CoV-2 laboratory had a major impact on the Public Health System of the included cities including timely delivery of the results to the healthcare agents and the Federal Healthcare system, evaluation of the vaccination status of the positive individuals in the background of exponential vaccination process in Brazil and scientific and technological divulgation of the fieldwork to the most vulnerable populations. Conclusions The SARS-CoV-2 pandemic has demonstrated worldwide the importance of science to fight against this viral agent and the LabMovel shows that it is possible to integrate researchers, clinicians, healthcare workers and patients to take rapid actions that can in fact mitigate this and other epidemiological situations.

Brazil ranks as third in terms of total number of reported SARS-CoV-2 cases globally. The COVID-19 epidemic in Brazil was characterised by the co-circulation of multiple variants as a consequence of multiple independent introduction events occurring through time. Here, we describe the SARS-CoV-2 variants that are currently circulating and co-circulating in the country, with the aim to highlight which variants have driven the different epidemic waves. For this purpose, we retrieved metadata information of Coronavirus sequences collected in Brazil and available at the GISAID database. SARS-CoV-2 lineages have been identified along with eleven variants, labelled as VOCs (Alpha, Gamma, Beta, Delta and Omicron) VOIs (Lambda and Mu) VUMs (B.1.1.318) and FMVs (Zeta, Eta and B.1.1.519). Here we show that, in the Brazilian context, after 24 months of sustained transmission and evolution of SARS-CoV-2, local variants (among them the B.1.1.28 and B.1.1.33) were displaced by recently introduced VOCs firstly with the Gamma, followed by Delta and more recently Omicron. The rapid spread of some of those VOCs (such as Gamma and Omicron) was also mirror by a large increase in the number of cases and deaths in the country. This in turn reinforces that, due to the emergence of variants that appear to induce a substantial evasion against neutralizing antibody response, it is important to strengthen genomic effort within the country and how vaccination still remains a critical process to protect the vulnerable population, still at risk of infection and death.

Made available in DSpace on 2022-04-28T19:51:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Delta VOC is highly diverse with more than 120 sublineages already described as of November 30, 2021. In this study, through active monitoring of circulating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants in the state of São Paulo, southeast Brazil, we identified two emerging sublineages from the ancestral AY.43 strain which were classified as AY.43.1 and AY.43.2. These sublineages were defined by the following characteristic nonsynonymous mutations ORF1ab:A4133V and ORF3a:T14I for the AY.43.1 and ORF1ab:G1155C for the AY.43.2 and our analysis reveals that they might have a likely-Brazilian origin. Much is still unknown regarding their dissemination in the state of São Paulo and Brazil as well as their potential impact on the ongoing vaccination process. However, the results obtained in this study reinforce the importance of genomic surveillance activity for timely identification of emerging SARS-CoV-2 variants which can impact the ongoing SARS-CoV-2 vaccination and public health policies. Center of Scientific Development Butantan Institute, São Paulo Blood Center of Ribeirão Preto Ribeirão Preto Medical School University of São Paulo, São Paulo Centro de Genômica Funcional da ESALQ University of São Paulo, São Paulo NGS Soluções Genômicas, São Paulo Department of Bioprocesses and Biotechnology School of Agricultural Sciences São Paulo State University (UNESP), São Paulo Department of Veterinary Medicine School of Animal Science and Food Engineering University of São Paulo, São Paulo Medicine School of São José do Rio Preto (FAMERP) São José do Rio Preto, São Paulo Coordenação de Vigilância em Saúde—Secretaria Municipal da Saúde, São Paulo Secretaria Municipal da Saúde, São Paulo Molecular Biology Laboratory Applied Biotechnology Laboratory Clinical Hospital of the Botucatu Medical School, São Paulo Laboratório de Genética Celular e Molecular Instituto de Ciências Biológicas Universidade Federal de Minas Gerais, Minas Gerais Laboratório de Flavivírus Instituto Oswaldo Cruz FIOCRUZ, Rio de Janeiro Department of Bioprocesses and Biotechnology School of Agricultural Sciences São Paulo State University (UNESP), São Paulo Molecular Biology Laboratory Applied Biotechnology Laboratory Clinical Hospital of the Botucatu Medical School, São Paulo

Our effort in SARS-CoV-2 genomic surveillance in Brazil has detected the Alpha Variant of Concern with a predominance higher than 75% in the population of Ilhabela island (São Paulo State) at a time when the Gamma VOC was already predominating the mainland raised concerns for closer surveillance on this island. Therefore, we intensified the surveillance for 24 weeks by generating data from 34% of local positive cases. Our data show that the patterns of VOC predominance dynamics and infection rates were in general distinct from the mainland. We report here the first known case of Alpha predominance in a Brazilian population, a delay greater than 3 months for the Gamma to dominate the previous variants compared to the mainland, and a faster dispersion rate of Gamma and Delta VOCs compared to the mainland. Phylogenetic analysis revealed the SARS-CoV-2 transmission dynamics in Ilhabela were characterized by multiple independent introduction events of Gamma and Delta, with a few events of Alpha introduction, two of them followed by community transmission. This study evidenced the peculiar behavior of SARS-CoV-2 variants in an isolated population and brought to light the importance of specific programs for SARS-CoV-2 genomic surveillance in isolated populations.

The high numbers of COVID-19 cases and deaths in Brazil have made Latin America an epicentre of the pandemic. SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, but important gaps remain in our understanding of virus transmission dynamics at a national scale. We use 17,135 near-complete genomes sampled from 27 Brazilian states and bordering country Paraguay. From March to November 2020, we detected co-circulation of multiple viral lineages that were linked to multiple importations (predominantly from Europe). After November 2020, we detected large, local transmission clusters within the country. In the absence of effective restriction measures, the epidemic progressed, and in January 2021 there was emergence and onward spread, both within and abroad, of variants of concern and variants under monitoring, including Gamma (P.1) and Zeta (P.2). We also characterized a genomic overview of the epidemic in Paraguay and detected evidence of importation of SARS-CoV-2 ancestor lineages and variants of concern from Brazil. Our findings show that genomic surveillance in Brazil enabled assessment of the real-time spread of emerging SARS-CoV-2 variants.

Background: Melanoma is the most aggressive skin cancer, and BRAF (V600E) is the most frequent mutation that led to the development of BRAF inhibitors (BRAFi). However, patients treated with BRAFi usually present recidivism after 6-9 months. Curcumin is a turmeric substance, and it has been deeply investigated due to its anti-inflammatory and antitumoral effects. Still, the low bioavailability and biodisponibility encouraged the investigation of different analogs. DM-1 is a curcumin analog and has shown an antitumoral impact in previous studies. Methods: Evaluated DM-1 stability and cytotoxic effects for BRAFi-sensitive and resistant melanomas, as well as the role in the metalloproteinases modulation. Results: DM-1 showed growth inhibitory potential for melanoma cells, demonstrated by reduction of colony formation, migration and endothelial tube formation, and cell cycle arrest. Subtoxic doses were able to downregulate important Metalloproteinases (MMPs) related to invasiveness, such as MMP-1, -2 and -9. Negative modulations of TIMP-2 and MMP-14 reduced MMP-2 and -9 activity; however, the reverse effect is seen when increased TIMP-2 and MMP-14 resulted in raised MMP-2. Conclusion: These findings provide essential details into the functional role of DM-1 in melanomas, encouraging further studies in the development of combinatorial treatments for melanomas.

Stingrays skin secretions are largely studied due to the human envenoming medical relevance of the sting puncture that evolves to inflammatory events, including necrosis. Such toxic effects can be correlated to the biochemical composition of the sting mucus, according to the literature. Fish skin plays important biological roles, such as the control of the osmotic pressure gradient, protection against mechanical forces and microorganism infections. The mucus, on the other hand, is a rich and complex fluid, acting on swimming, nutrition and the innate immune system. The elasmobranch's epidermis is a tissue composed mainly by mucus secretory cells, and marine stingrays have already been described to present secretory glands spread throughout the body. Little is known about the biochemical composition of the stingray mucus, but recent studies have corroborated the importance of mucus in the envenomation process. Aiming to assess the mucus composition, a new non-invasive mucus collection method was developed that focused on peptides and proteins, and biological assays were performed to analyze the toxic and immune activities of the Hypanus americanus mucus. Pathophysiological characterization showed the presence of peptidases on the mucus, as well as the induction of edema and leukocyte recruitment in mice. The fractionated mucus improved phagocytosis on macrophages and showed antimicrobial activity against T. rubrumç. neoformans and C. albicans in vitro. The proteomic analyses showed the presence of immune-related proteins like actin, histones, hemoglobin, and ribosomal proteins. This protein pattern is similar to those reported for other fish mucus and stingray venoms. This is the first report depicting the Hypanus stingray mucus composition, highlighting its biochemical composition and importance for the stingray immune system and the possible role on the envenomation process.

Brazil has experienced some of the highest numbers of COVID-19 cases and deaths globally and from May 2021 made Latin America a pandemic epicenter. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of virus transmission dynamics at the national scale. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and a bordering country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed under an absence of effective restriction measures, there was a local emergence and onward international spread of Variants of Concern (VOC) and Variants Under Monitoring (VUM), including Gamma (P.1) and Zeta (P.2). In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the usefulness and need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring that provides a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies.

The authors hereby request the inclusion of two authors (Olivia Teixeira and Maria Cristina Nonato) in the recently published article in Viruses entitled “Nucleocapsid (N) gene mutations of SARS-CoV-2 can affect real-time RT-PCR diagnostic and impact false-negative results” [...]

Viral metagenomics is increasingly being used for the identification of emerging and re-emerging viral pathogens in clinical samples with unknown etiology. The objective of this study was to shield light on the metavirome composition in clinical samples obtained from patients with clinical history compatible with an arboviral infection, but that presented inconclusive results when tested using RT-qPCR. The inconclusive amplification results might be an indication of the presence of an emerging arboviral agent that is inefficiently amplified by conventional PCR techniques. A total of eight serum samples with inconclusive amplification results for the routinely tested arboviruses—dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) obtained during DENV and CHIKV outbreaks registered in the state of Alagoas, Northeast Brazil between July and August 2021—were submitted to metagenomic next-generation sequencing assay using NextSeq 2000 and bioinformatic pipeline for viral discovery. The performed bioinformatic analysis revealed the presence of two arboviruses: DENV type 2 (DENV-2) and CHIKV with a high genome coverage. Further, the metavirome of those samples revealed the presence of multiple commensal viruses apparently without clinical significance. The phylogenetic analysis demonstrated that the DENV-2 genome belonged to the Asian/American genotype and clustered with other Brazilian strains. The identified CHIKV genome was taxonomically assigned as ECSA genotype, which is circulating in Brazil. Together, our results reinforce the utility of metagenomics as a valuable tool for viral identification in samples with inconclusive arboviral amplification. Viral metagenomics is one of the most potent methods for the identification of emerging arboviruses.

Sao Paulo State, the most populous area in Brazil, currently experiences a second wave of the COVID-19 pandemic which overwhelmed the healthcare system. Recently, due to the paucity of SARS-CoV-2 complete genome sequences, we established a Network for Pandemic Alert of Emerging SARS-CoV-2 Variants to rapidly understand the spread of SARS-CoV-2 and monitor in nearly real-time the circulating SARS-CoV-2 variants into the state. Through full genome analysis of 217 SARS-CoV-2 complete genome sequences obtained from the largest regional health departments we were able to identify the co-circulation of multiple SARS-CoV-2 lineages such as i) B.1.1 (0.92%), ii) B.1.1.1 (0.46%), iii) B.1.1.28 (25.34%), iv) B.1.1.7 (5.99%), v) B.1.566 (1.84%), vi) P.1 (64.05%), and P.2 (0.92%). Further our analysis allowed the detection, for the first time in Brazil of the South African variant of concern (VOC), the B.1.351 (501Y.V2) (0.46%). The identified lineage was characterized by the presence of the following mutations: ORF1ab: T265I, R724K, S1612L, K1655N, K3353R, SGF 3675_F3677del, P4715L, E5585D; Spike: D80A, D215G, L242_L244del, A262D, K417N, E484K, N501Y, D614G, A701V, C1247F; ORF3a: Q57H, S171L, E: P71L; ORF7b: Y10F, N: T205I; ORF14: L52F. Origin of the most recent common ancestor of this genomic variant was inferred to be between middle October to late December 2020. Analysis of generated sequences demonstrated the predominance of the P.1 lineage and allowed the early detection of the South African strain for the first time in Brazil. Our findings highlight the importance to increase active monitoring to ensure the rapid detection of new SARS-CoV-2 variants with a potential impact in pandemic control and vaccination strategies.

Background: A stepped-wedge trial is an approach for assessing vaccine effectiveness in the real world. By the end of the study, all participants could receive the intervention, eliminating the ethical dilemma of placebo, especially during a pandemic. Methods: We evaluated the effectiveness of CoronaVac in Serrana, Brazil, amid an uncontrolled community Covid-19 epidemic using a stepped-wedge randomized trial. The city was separated into 25 subareas, divided into four groups, and randomized to receive CoronaVac in a two-dose scheme with a four-week interval. Intervention was initiated in each group with a one-week interval. The primary endpoint was the incidence of symptomatic cases in fully immunized individuals. The secondary endpoints were Covid-19-related hospitalizations and deaths and incidence according to immunization coverage. Findings: The study occurred during epidemiological weeks 6 to 19 in 2021. Up to 27,406 participants received the first dose of the study vaccine, corresponding to 81·3% of the adults and 60·9% of the urban population. Among fully immunized individuals, the vaccine effectiveness was 80·5 (95% CI, 75·1 to 84·7) for preventing symptomatic Covid-19 cases, 95% (95% CI, 86·9 to 98·1) and 94·9% (95% CI, 76·4 to 98·9) for preventing Covid-19-related hospitalizations and deaths, respectively. There was a significant indirect protective effect in unvaccinated people when 52% of the adult population was fully vaccinated. The Gamma variant was dominant during the study. Interpretation: CoronaVac effectively prevented symptomatic Covid-19 cases and protected against severe disease and death during Gamma variant circulation. Unvaccinated individuals benefited from high vaccine coverage levels. Trial Registration: This study was registered with (ClinicalTrials.gov Identifier, NCT04747821) Funding: Fundacao Butantan and Sao Paulo Research Foundation (FAPESP). Declaration of Interest: MCB, BMC, GJV, NNF, PMMG, RH, BALF, and RTC received research funding from Butantan Institute during the conduct of this study. RP, MTRPC, APB, JPS, and ROP were employees of Butantan Institute during the conduct of this study. HAB, EGP, GGP, SCSV and DC are employees of Butantan Institute. All other authors declare no competing interests Ethical Approval: The study was reviewed and approved by the Ethics Committee of the Clinical Hospital, Ribeirao Preto Medical School, University of Sao Paulo (CAAE 42390621.1.0000.5440).

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (β-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

Made available in DSpace on 2022-04-28T19:47:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Financiadora de Estudos e Projetos The SARS-CoV-2 alpha VOC (also known as lineage B.1.1.7) initially described in the autumn, 2020 in UK, rapidly became the dominant lineage across much of Europe. Despite multiple studies reporting molecular evidence suggestive of its circulation in Brazil, much is still unknown about its genomic diversity in the state of São Paulo, the main Brazilian economic and transportation hub. To get more insight regarding its transmission dynamics into the State we performed phylogenetic analysis on all alpha VOC strains obtained between February and August 2021 from the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants. The performed phylogenetic analysis showed that most of the alpha VOC genomes were interspersed with viral strains sampled from different Brazilian states and other countries suggesting that multiple independent Alpha VOC introductions from Brazil and overseas have occurred in the São Paulo State over time. Nevertheless, large monophyletic clusters were also observed especially from the Central-West part of the São Paulo State (the city of Bauru) and the metropolitan region of the São Paulo city. Our results highlight the Alpha VOC molecular epidemiology in the São Paulo state and reinforce the need for continued genomic surveillance strategies for the real-time monitoring of potential emerging SARS-CoV-2 variants during the ever-growing vaccination process. University of São Paulo Ribeirão Preto Medical School Blood Center of Ribeirão Preto Butantan Institute University of São Paulo Centro de Genômica Funcional da ESALQ NGS Soluções Genômicas Mendelics Análise Genômica SA São Paulo State University (UNESP) School of Agricultural Sciences Department of Bioprocesses and Biotechnology Department of Veterinary Medicine School of Animal Science and Food Engineering University of Sao Paulo Medicine School of São José do Rio Preto (FAMERP) São José do Rio Preto Molecular Biology Laboratory Applied Biotechnology Laboratory Clinical Hospital of the Botucatu Medical School Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Belo Horizonte Instituto Oswaldo Cruz FIOCRUZ Rio de Janeiro University of São Paulo Ribeirão Preto Medical School São Paulo State University (UNESP) School of Agricultural Sciences Department of Bioprocesses and Biotechnology Molecular Biology Laboratory Applied Biotechnology Laboratory Clinical Hospital of the Botucatu Medical School FAPESP: 2017/26950–6 FAPESP: 2018/15826–5 FAPESP: 2019/08528–0 FAPESP: 2020/10127; 2020/05367–3 Financiadora de Estudos e Projetos: 207.234

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin, however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Neutrophils have a critical role in the innate immune response; these cells represent the primary line of defense against invading pathogens or tissue injury. Crotoxin (CTX), the major toxin of the South American rattlesnake (Crotalus durissus terrificus) venom, presents longstanding anti-inflammatory properties, inhibiting neutrophil migration and phagocytosis by peritoneal neutrophils for 14 days. Herein, to elucidate these sustained inhibitory effects induced by CTX, we performed in vitro and in vivo studies evaluating the functionality of bone marrow neutrophils and possible molecular mechanisms associated with these effects. CTX inhibited the processes of chemotaxis, adhesion to fibronectin, and phagocytosis of opsonized particles; however, it did not affect ROS production or degranulation in bone marrow neutrophils. To understand the molecular mechanisms that orchestrate this effect, we investigated the expression of CR3 on the neutrophil surface and the total expression and activity of signaling proteins from the Syk-GTPase pathway, which is involved in actin polymerization. CTX down-regulated both subunits of CR3, as well as, the activity of Syk, Vav1, Cdc42, Rac1 and RhoA, and the expression of the subunit 1B from Arp2/3. Together, our findings demonstrated that CTX inhibits the functionally of bone marrow neutrophils and that this effect may be associated with an impairment of the Syk-GTPase pathway. This study demonstrates, for the first time, that the sustained down-modulatory effect of CTX on circulating and peritoneal neutrophils is associated with functional modifications of neutrophils still in the bone marrow, and it also contributes to a better understanding of the anti-inflammatory effect of CTX.

Federal University of Para. Institute of Biological Sciences. Laboratory of Parasitology and Laboratory of Structural Biology. Bel?m, PA, Brazil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Parasitology and Laboratory of Structural Biology. Bel?m, PA, Brazil. University of S?o Paulo. Department,Institute of Biomedical Sciences. Cell and Developmental Biology. S?o Paulo, SP, Brazil. Butantan Institute. Laboratory of Pathophysiology. S?o Paulo, SP, Brazil / University of S?o Paulo. Institute of Biomedical Sciences. Department of Pharmacology. S?o Paulo, SP, Brazil. Federal University of Para. Institute of Biological Sciences. Laboratory of Parasitology and Laboratory of Structural Biology. Bel?m, PA, Brazil / National Institute of Science and Technology in Structural Biology and Bioimaging. Rio de Janeiro, RJ, Brazil. American tegumentary leishmaniasis is caused by different species of Leishmania. This protozoan employs several mechanisms to subvert the microbicidal activity of macrophages and, given the limited efficacy of current therapies, the development of alternative treatments is essential. Animal venoms are known to exhibit a variety of pharmacological activities, including antiparasitic effects. Crotoxin (CTX) is the main component of Crotalus durissus terrificus venom, and it has several biological effects. Nevertheless, there is no report of CTX activity during macrophage - Leishmania interactions. Thus, the main objective of this study was to evaluate whether CTX has a role in macrophage M1 polarization during Leishmania infection murine macrophages, Leishmania amazonensis promastigotes and L. amazonensis-infected macrophages were challenged with CTX. MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] toxicity assays were performed on murine macrophages, and no damage was observed in these cells. Promastigotes, however, were affected by treatment with CTX (IC50 = 22?86 ?g mL-1) as were intracellular amastigotes. Macrophages treated with CTX also demonstrated increased reactive oxygen species production. After they were infected with Leishmania, macrophages exhibited an increase in nitric oxide production that converged into an M1 activation profile, as suggested by their elevated production of the cytokines interleukin-6 and tumour necrosis factor-? and changes in their morphology. CTX was able to reverse the L. amazonensis-mediated inhibition of macrophage immune responses and is capable of polarizing macrophages to the M1 profile, which is associated with a better prognosis for cutaneous leishmaniasis treatment.

The hepatocyte growth factor (HGF)/c-met pathway, which mainly consists of HGF activator (HGFA) and its substrate HGF, protects various types of cells via anti-apoptotic and anti-inflammatory signals. Thrombin is the main physiological activator of such plasmatic pathway, and increased plasma concentrations of HGF have been considered as a molecular marker for some pathological conditions, such as disseminated intravascular coagulation. Since thrombin generation is often linked to tissue injury, and these events are common during snake venom-induced consumption coagulopathies (VICC), our goals were to examine whether Bothrops jararaca venom (Bjv), which induces VICC in vivo: (i) activates the HGF/c-met pathway in vivo and (ii) cleaves zymogen forms of HGFA and HGF (proHGFA and proHGF, respectively) in vitro. Two experimental groups (n = 6, each) of male adult Wistar rats were subcutaneously injected with 500 μL of 0.9% NaCl solution (control) or sub-lethal doses (1.6 mg/kg) of Bjv. Three hours after envenomation, whole blood samples were collected from the carotid arteries to evaluate relevant coagulation parameters using rotational thromboelastometry and fibrinogen level (colorimetric assay). Additionally, the plasma concentration of HGF was assayed (ELISA). Thromboelastometric assays showed that blood clotting and fibrin polymerization were severely impaired 3 h after Bjv injection. Total plasma HGF concentrations were almost 6-fold higher in the Bjv-injected group (410.0 ± 91) compared with control values (68 ± 18 pg/mL, p 0.05). Western blotting assay showed that Bjv processed proHGFA and proHGF, generating bands resembling those generated by thrombin and kallikrein, respectively. In contrast to the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), the metalloprotease inhibitor ethylenediaminetetraacetic acid disodium salt (Na

In 1986 and 1987, Philip Newsholme et al. reported macrophages utilize glutamine, as well as glucose, at high rates. These authors measured key enzyme activities and consumption and production levels of metabolites in incubated or cultured macrophages isolated from the mouse or rat intraperitoneal cavity. Metabolic pathways essential for macrophage function were then determined. Macrophages utilize glucose to generate (i) ATP in the pathways of glycolysis and mitochondrial oxidative phosphorylation, (ii) glycerol 3-phosphate for the synthesis of phospholipids and triacylglycerols, (iii) NADPH for the production of reactive oxygen species (ROS) and (iv) ribose for the synthesis of RNA and subsequently production and secretion of protein mediators (e.g. cytokines). Glutamine plays an essential role in macrophage metabolism and function, as it is required for energy production but also provides nitrogen for synthesis of purines, pyrimidines and thus RNA. Macrophages also utilize fatty acids for both energy production in the mitochondria and lipid synthesis essential to plasma membrane turnover and lipid meditator production. Recent studies utilizing metabolomic approaches, transcriptional and metabolite tracking technologies have detailed mitochondrial release of tricarboxylic acid (TCA) intermediates (e.g. citrate and succinate) to the cytosol, which then regulate pro-inflammatory responses. Macrophages can reprogramme their metabolism and function according to environmental conditions and stimuli in order to polarize phenotype so generating pro- or anti-inflammatory cells. Changes in macrophage metabolism result in modified function/phenotype and vice versa. The plasticity of macrophage metabolism allows the cell to quickly respond to changes in environmental conditions such as those induced by hormones and/or inflammation. A past and present overview of macrophage metabolism and impact of endocrine regulation and the relevance to human disease are described in this review.

Endotoxin tolerance is defined as a reduced capacity of the host to respond to LPS activation following a first exposure to this stimulus. It affects all leukocytes and regarding macrophages, most studies focus on the reduced ability of these cells to secrete pro-inflammatory cytokines. Therefore, we evaluated other macrophages functions (fungicidal capacity, reactive oxygen species production and antigen presentation) in cells from tolerant mice. We have performed a tolerance model in our laboratory that does not stimulate directly the place from where the cells will be removed (peritoneal cavity). Mouse received subcutaneous injections of LPS in the scruff for 5 days and we analyze the capacity of peritoneal macrophages to phagocyte using three different receptors: Fc, C3b and mannose receptors. We found a reduction in the phagocytosis of erythrocytes and Candida albicans related to the Fc and mannose receptors. These differences can be due to a macrophage reprogramming, as demonstrated by altered expression of cytokines and chemokines. Despite this reduction in phagocytosis capacity, macrophages from tolerant animals exhibited enhanced hydrogen peroxide production and expression of antigen presentation molecules, suggesting that their ability to combat an infection is improved. In summary, our data indicates that LPS tolerance drives macrophages from a predominant release of proinflammatory mediators that amplify inflammation and host damage toward a better killing and antigen presentation state.

Advanced glycation end-products (AGEs) are proteins/lipids that are glycated upon sugar exposure and are often increased during inflammatory diseases such as osteoarthritis and neurodegenerative disorders. Here, we developed an extracellular matrix (ECM) using glycated type I collagen (ECM-GC), which produced similar levels of AGEs to those detected in the sera of arthritic mice. In order to determine whether AGEs were sufficient to stimulate sensory neurons, dorsal root ganglia (DRGs) cells were cultured on ECM-GC or ECM-NC-coated plates. ECM-GC or ECM-NC were favorable for DRG cells expansion. However, ECM-GC cultivated neurons displayed thinner F-actin filaments, rounded morphology, and reduced neuron interconnection compared to ECM-NC. In addition, ECM-GC did not affect RAGE expression levels in the neurons, although induced rapid p38, MAPK and ERK activation. Finally, ECM-GC stimulated the secretion of nitrite and TNF-α by DRG cells. Taken together, our in vitro glycated ECM model suitably mimics the in vivo microenvironment of inflammatory disorders and provides new insights into the role of ECM impairment as a nociceptive stimulus.

Cancer pain is an important clinical problem and may not respond satisfactorily to the current analgesic therapy. We have characterized a novel and potent analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in a rat model of cancer pain induced by intraplantar injection of Walker 256 carcinoma cells. Intraplantar injection of tumor cells caused the development of hyperalgesia and allodynia, detected on day 5 after tumor cell inoculation. Crotalphine (6 μg/kg), administered p.o., blocked both phenomena. The antinociceptive effect was detected 1 h after treatment and lasted for up to 48 h. Intraplantar injection of nor-binaltorphimine (50 g/paw), a selective antagonist of κ-opioid receptors, antagonized the antinociceptive effect of the peptide, whereas N,N-diallyl-Tyr-Aib-Phe-Leu (ICI 174,864, 10 μg/paw), a selective antagonist of δ-opioid receptors, partially reversed this effect. On the other hand, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP, 20 g/paw), an antagonist of μ-opioid receptors, did not modify crotalphine-induced antinociception. These data indicate that crotalphine induces a potent and long lasting opioid-mediated antinociception in cancer pain.

In this study, we evaluated the effect of the Crotalus durissus terrificus (Cdt) venom on the chronic paw edema induced by the injection of bacillus Calmette-Guerin (BCG) into the footpad of mice. The BCG injection evoked chronic edema, which was significantly diminished in animals treated subcutaneously (s.c.) with Cdt venom 1 h before or after the BCG injection. This inhibition persisted throughout the evaluation period (15 days). In mice injected with Cdt venom 6 or 11 days after injection of BCG, we observed a significant reduction in edema only in the period after the venom injection. While studying possible mechanisms involved in this inhibition, we observed that pre-treatment with dexamethasone, zileuton or Boc2 (a selective antagonist of formyl peptide receptors), but not with indomethacin, canceled out the inhibitory effect of Cdt venom on the edema induced by BCG. These results strongly suggest that this rattlesnake venom can stimulate the generation of mediators from the lipoxygenase pathway, which can down-regulate this chronic inflammatory edema. Using fractionated venom, the results indicated that crotoxin was the only component of Cdt venom responsible for this inhibitory effect. These results indicated that crotoxin, the main toxin of the C. durissus terrificus venom, has a significant inhibitory effect on BCG-induced chronic edema, possibly by generating anti-inflammatory mediators from the lipoxygenase pathway.

Background Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. Methods The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Results Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). Conclusion These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

Deficient wound healing is a common multifactorial complication in diabetic patients, but the cellular and molecular mechanisms involved are poorly defined. In the present study, we analyzed the effects of hyperglycemia on integrins expression in rat dermal fibroblasts and addressed its role in cell adhesion and migration. Diabetes Mellitus was induced in rats by streptozotocin injection and maintained for 30 days. Primary cultures of dermal fibroblasts from control and diabetic rats were maintained under low glucose (5 mM D-glucose) or high glucose (30 mM D-glucose) for 7 days. Cell adhesion and migration were studied by kymography, transwell, and time-lapse assays, and the expressions of integrin subunits αv and α5 were studied by immunocytochemistry and western blotting. Fibroblasts derived from diabetic rats confirmed a reduced migration speed and delayed spreading compared to fibroblasts derived from control rats. The membrane fraction of diabetic-derived fibroblasts showed a decrease of integrin subunits α5 and αv, which was confirmed by immunocytochemistry assays. A reduction in the pericellular fibronectin matrix was also observed. The exposure of diabetic-derived cells to a higher concentration of exogenous fibronectin improved migration velocity and the expression of αv but did not completely restore their migration capacity. In conclusion, the mechanisms involved in the deleterious effects of Diabetes Mellitus on wound healing include the ability of fibroblasts to secrete and to adhere to fibronectin.

We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs.

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor α (TNF-α) and cytokine-induced neutrophil chemoattractant-2 (CINC-2αβ)] by rat neutrophils. The involvement of nuclear factor κB (NF-κB) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-α, CINC-2αβ and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-κB activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions.

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric beta-neurotoxin that consists of a weakly toxic basic phospholipase A(2) and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin.

In the present study, it was investigated which components are responsible for the anti-inflammatory properties of Crotalus durissus terrificus venom (CdtV). The effect of crotoxin, as well as of other CdtV components was evaluated on edema, cell migration and alterations in leukocyte-endothelium interactions induced by carrageenan. Crotoxin (40 microg kg(-1)) was injected at different time periods before or after the injection of carrageenan (15 mg kg(-1)) into the mouse hind paw, peritoneum or scrotum. Results showed that crotoxin, but not other CdtV components, significantly inhibited inflammatory edema and cell migration when administered before or after carrageenan injection in mice. This toxin also prevented the occurrence of alterations in leukocyte-endothelium interactions induced by carrageenan injection, such as the increase in adhered cells. In animals pretreated with Boc2 (a selective antagonist of formyl peptide receptors), crotoxin showed neither inhibitory effects on edema and cell migration, nor prevented alterations in leukocyte-endothelium interactions induced by carrageenan. These findings demonstrate that crotoxin is the component responsible for the long-lasting anti-inflammatory activity of crude C. durissus terrificus venom, and activation of formyl peptide receptors seems to play a major role in this effect.

Neuropathic pain is an important clinical problem and it is usually resistant to the current therapy. We have recently characterized a novel analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus . In the present work, the antinociceptive effect of crotalphine was evaluated in an experimental model of neuropathic pain induced in rats by chronic constriction of sciatic nerve. The effect of the peptide was compared to that induced by the crude venom, which confirmed that crotalphine is responsible for the antinociceptive effect of the crotalid venom on neuropathic pain. For characterization of neuropathic pain, the presence of hyperalgesia, allodynia and spontaneous pain was assessed at different times after nerve constriction. These phenomena were detected 24 h after surgery and persisted at least for 14 days. The pharmacological treatments were performed on day 14 after surgery. Crotalphine (0.2–5 µg/kg) and the crude venom (400–1600 µg/kg) administered p.o. inhibited hyperalgesia, allodynia and spontaneous pain induced by nerve constriction. The antinociceptive effect of the peptide and crude venom was long lasting, since it was detected up to 3 days after treatment. Intraplantar injection of naloxone (1 µg/paw) blocked the antinociceptive effect, indicating the involvement of opioid receptors in this phenomenon. Gabapentin (200 mg/kg, p.o.), and morphine (5 mg/kg, s.c.), used as positive controls, blocked hyperalgesia and partially inhibited allodynia induced by nerve constriction. These data indicate that crotalphine induces a potent and long lasting opioid antinociceptive effect in neuropathic pain that surpasses that observed with standard analgesic drugs.

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1-2h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A(2) (PLA(2)). PLA(2), but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte-endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin.

In the present study, we investigated the effects of Crotalus durissus terrificus venom ( Cdt V) on vascular and cellular events of inflammation induced by carrageenan (cg) in mice. To evaluate edema, Cdt V (75 μg kg −1 ) was administered subcutaneously before (1 h, 7 or 14 days) or after (1, 4 or 48 h) subplantar injection of cg (15 mg kg −1 ) into the mouse right hind paw; to analyze leukocyte influx, cg (200 μL) was injected i.p. in mice. The inhibitory action of Cdt V on edema, either before or after cg injection, was prolonged, lasting even 72 h after administration. Besides, Cdt V significantly inhibited migration of polymorphonuclear cells to peritoneal cavity when administered before or after cg. Such inhibitory effects of Cdt V on edema and cell migration were also compared with well-known anti-inflammatory drugs. The results demonstrated that Cdt V, when injected either 7 or 14 days or 1 h before cg, induced a more effective and long-lasting anti-inflammatory effect than that observed with classical anti-inflammatory drugs. The association of Cdt V with different drugs did not potentialize their actions on cell migration. These results demonstrate that Cdt V exhibits long-lasting anti-inflammatory effects.

An animal model of cancer pain induced by injection of Walker 256 carcinoma cells into the plantar surface of rat hind paw is described. Tumor growth and the occurrence of metastasis were investigated by histopathological analysis. Tumor cell growth was also analyzed plethysmographically by the increase in paw volume. For characterization of pain symptoms, hyperalgesia, allodynia, and spontaneous pain were evaluated 5 to 8 days after cell injection. The volume of the inoculated paw started to increase on day 2 after inoculation, being 40% higher on day 5 after injection. At this time, there was a marked proliferation of tumor cells, with the presence of anaplastic and pleomorphic cells, nucleoli, and atypical mitotic features. On days 7 and 8 after injection, histopathological analysis of popliteal lymph nodes showed the presence of tumor cells. The intraplantar injection of Walker 256 cells caused hyperalgesia at day 5 after cell inoculation. Low-threshold mechanical allodynia was significant 2 days after cell injection, being increased on day 5. In addition, inoculation of tumor cells induced gross behavior, characterized by a significant increase in licking and lifting of the injected paw 5 days after injection. The pain-enhancing effect caused by cell inoculation was partially inhibited by indomethacin on day 2 after cell injection, whereas morphine blocked allodynia on days 2 and 5. These results indicate that intraplantar injection of Walker 256 cells cause pain symptoms characteristic of cancer pain. This experimental model can then be used to investigate new analgesic or anti-tumor drugs. Perspective This article presents a new animal model for studying cancer pain and metastasis. This model could help in understanding the mechanisms involved in cancer pain symptoms and may be used for the investigation of new analgesic or anti-tumor drugs.

Crotalus durissus terrificus snake venom and its major toxin, crotoxin or type II PLA2 subunit of this toxin, induce an inhibitory effect on spreading and phagocytosis in 2h incubated macrophages. The involvement of arachidonate-derived mediators on the inhibitory action of the venom or toxins on rat peritoneal macrophage phagocytosis was presently investigated. Peritoneal cells harvested from naive rats and incubated with the venom or toxins or harvested from the peritoneal cavity of rats pre-treated with the toxins were used. Zileuton, a 5-lipoxygenase inhibitor but not indomethacin, a cyclooxygenase inhibitor, given in vivo and in vitro abolished the inhibitory effect of venom or toxins on phagocytosis. Resident peritoneal macrophages incubated with the venom or toxins showed increased levels of prostaglandin E2 and lipoxin A4, with no change in leukotriene B4. These results suggest that lipoxygenase-derived eicosanoids are involved in the inhibitory effect of C.d. terrificus venom, crotoxin or PLA2 on macrophage phagocytosis.

Previous work of our group demonstrated that Crotalus durissus terrificus venom has a dual effect on macrophage function: it inhibits spreading and phagocytosis and stimulates hydrogen peroxide and nitric oxide production, antimicrobial activity and glucose and glutamine metabolism of these cells. Crotalid venom also induces analgesia and this effect is mediated by opioid receptors. The involvement of opioidergic mechanism and the determination of the active component responsible for the inhibitory effect of crotalid venom on macrophage function were investigated. The venom reduced the spreading and phagocytic activities of peritoneal macrophages. This effect was observed in vitro, 2 h after incubation of resident peritoneal macrophages with the venom, and in vivo, 2 h after subcutaneous injection of the venom. The inhibition of phagocytosis was not modified by naloxone, an antagonist of opioid receptors. Venom neutralization with crotalid antivenom abolished the inhibitory effect of the venom, indicating that venom toxins are involved in this effect. Crotoxin, the main toxin of crotalid venom, s.c. injected to rats or added to the medium of peritoneal cell incubation, inhibited macrophage function in a similar manner to that observed for crude venom. The present results suggest that crotoxin causes a direct inhibition of macrophage spreading and phagocytic activities and may contribute to the inhibitory effect of crotalid venom on macrophage function.