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Abstract Since 2021, the emergence of variants of concern (VOC) has led Brazil to experience record numbers of in COVID-19 cases and deaths. The expanded spread of the SARS-CoV-2 combined with a low vaccination rate has contributed to the emergence of new mutations that may enhance viral fitness, leading to the persistence of the disease. Due to limitations in the real-time genomic monitoring of new variants in some Brazilian states, we aimed to investigate whether genomic surveillance, coupled with epidemiological data and SARS-CoV-2 variants spatiotemporal spread in a smaller region, can reflect the pandemic progression at a national level. Our findings revealed three SARS-CoV-2 variant replacements from 2021 to early 2022, corresponding to the introduction and increase in the frequency of Gamma, Delta, and Omicron variants, as indicated by peaks of the Effective Reproductive Number (Reff). These distinct clade replacements triggered two waves of COVID-19 cases, influenced by the increasing vaccine uptake over time. Our results indicated that the effectiveness of vaccination in preventing new cases during the Delta and Omicron circulations was six and eleven times higher, respectively, than during the period when Gamma was predominant, and it was highly efficient in reducing the number of deaths. Furthermore, we demonstrated that genomic monitoring at a local level can reflect the national trends in the spread and evolution of SARS-CoV-2.

We aimed to describe the landscape, including molecular, epidemiological, and clinical aspects of CHIKV infections in the Ribeirao Preto region, an area endemic to dengue. We randomly screened 3744 plasma samples that had undergone DENV diagnosis to evaluate CHIKV-RNA using an in-house RT-PCR assay. Positive samples were followed clinically, and RNA samples were submitted to whole genome sequencing. Seventeen cases (0.5 %) were positive for CHIKV-RNA despite being negative for DENV-RNA. Notably, half of the patients experienced prolonged arthralgia lasting more than 90 days. Compared with the healthy control group, leukopenia and thrombocytopenia were observed in all CHIKV-positive individuals with statistically significant P values (P

In the original publication [...]

Viral hemorrhagic fever poses a significant public health challenge due to its severe clinical presentation and high mortality rate. The diagnostic process is hindered by similarity of symptoms across different diseases and the broad spectrum of pathogens that can cause hemorrhagic fever. In this study, we applied viral metagenomic analysis to 43 serum samples collected by the Public Health Laboratory (Fundação Ezequiel Dias, FUNED) in Minas Gerais State, Brazil, from patients diagnosed with hemorrhagic fever who had tested negative for the standard local hemorrhagic disease testing panel. This panel includes tests for Dengue virus (DENV) IgM, Zika virus IgM, Chikungunya virus IgM, yellow fever IgM, Hantavirus IgM, Rickettsia rickettsii IgM/IgG, and Leptospira interrogans IgM, in addition to respective molecular tests for these infectious agents. The samples were grouped into 18 pools according to geographic origin and analyzed through next-generation sequencing on the NextSeq 2000 platform. Bioinformatic analysis revealed a prevalent occurrence of commensal viruses across all pools, but, notably, a significant number of reads corresponding to the DENV serotype 2 were identified in one specific pool. Further verification via real-time PCR confirmed the presence of DENV-2 RNA in an index case involving an oncology patient with hemorrhagic fever who had initially tested negative for anti-DENV IgM antibodies, thereby excluding this sample from initial molecular testing. The complete DENV-2 genome isolated from this patient was taxonomically classified within the cosmopolitan genotype that was recently introduced into Brazil. These findings highlight the critical role of considering the patient’s clinical condition when deciding upon the most appropriate testing procedures. Additionally, this study showcases the potential of viral metagenomics in pinpointing the viral agents behind hemorrhagic diseases. Future research is needed to assess the practicality of incorporating metagenomics into standard viral diagnostic protocols.

Growing evidence suggests that metavirome changes could be associated increased risk for malignant cell transformation. Considering Viruses have been proposed as factors for prostate cancer induction. The objective of this study was to examine the composition of the plasma metavirome of patients with prostate cancer. Blood samples were obtained from 49 male patients with primary prostate adenocarcinoma. Thirty blood donors were included as a control group. The obtained next-generation sequencing data were analyzed using a bioinformatic pipeline for virus metagenomics. Viral reads with higher abundance were assembled in contigs and analyzed taxonomically. Viral agents of interest were also confirmed by qPCR. Anelloviruses and the Human Pegivirus-1 (HPgV-1) were the most abundant component of plasma metavirome. Clinically important viruses like hepatitis C virus (HCV), cytomegalovirus and human adenovirus type C were also identified. In comparison, the blood donor virome was exclusively composed of torque teno virus types (TTV) types. The performed HPgV-1 and HCV phylogeny revealed that these viruses belong to commonly detected in Brazil genotypes. Our study sheds light on the plasma viral abundance in patients with prostatic cancer. The obtained viral diversity allowed us to separate the patients and controls, probably suggesting that malignant processes may influence virome composition. More complex and multiple approach investigations are necessary to examine the likely causal relationship between metavirome and its nvolvement in prostate cancer.

Abstract 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.

The aim of this study was to describe epidemiological characteristics and perform SARS-CoV-2 genomic surveillance in the southeastern region of São Paulo State. During the first months of 2022, we compared weekly SARS-CoV-2 infection prevalence considering age, Ct value, and variants’ lineages. An increase in the number of SARS-CoV-2-positive cases until the fourth epidemiological week of 2022 was observed. From the fourth epidemiological week onwards, the number of tests for SARS-CoV-2 diagnosis began to decrease, but the number of positive samples for SARS-CoV-2 remained high, reaching its most expressive level with a rate of 60% of infected individual cases. In this period, we observed a progressive increase in SARS-CoV-2 infection within the 0–10 age group throughout the epidemiological weeks, from 2.8% in the first epidemiological week to 9.2% in the eighth epidemiological week of 2022. We further observed significantly higher Ct values within younger patient samples compared to other older age groups. According to lineage assignment, SARS-CoV-2 (BA.1) was the most prevalent (74.5%) in the younger group, followed by BA.1.1 (23%), BA.2 (1.7%), and Delta (1%). Phylogenetic analysis showed that BA.2 sequences clustered together, indicating sustained transmission of this Omicron VOC sub-lineage by that time. Our results suggest the initial dissemination steps of the Omicron’s sub-linage BA.2 into the younger group, due to specific genomic features of the detected sequences. These data provide interesting results related to the spread, emergence, and evolution of the Omicron variant in the southeast Brazilian population.

Nipah virus (NiV), a biosafety level 4 agent, was first identified in human clinical cases during an outbreak in 1998 in Malaysia and Singapore. While flying foxes are the primary host and viral vector, the infection is associated with a severe clinical presentation in humans, resulting in a high mortality rate. Therefore, NiV is considered a virus with an elevated epidemic potential which is further underscored by its recent emergence (September 2023) as an outbreak in India. Given the situation, it is paramount to understand the molecular dynamics of the virus to shed more light on its evolution and prevent potential future outbreaks. In this study, we conducted Bayesian phylogenetic analysis on all available NiV complete genomes, including partial N-gene NiV sequences (≥1000 bp) in public databases since the first human case, registered in 1998. We observed the distribution of genomes into three main clades corresponding to the genotypes Malaysia, Bangladesh and India, with the Malaysian clade being the oldest in evolutionary terms. The Bayesian skyline plot showed a recent increase in the viral population size since 2019. Protein analysis showed the presence of specific protein families (Hendra_C) in bats that might keep the infection in an asymptomatic state in bats, which also serve as viral vectors. Our results further indicate a shortage of complete NiV genomes, which would be instrumental in gaining a better understanding of NiV’s molecular evolution and preventing future outbreaks. Our investigation also underscores the critical need to strengthen genomic surveillance based on complete NiV genomes that will aid thorough genetic characterization of the circulating NiV strains and the phylogenetic relationships between the henipaviruses. This approach will better prepare us to tackle the challenges posed by the NiV virus and other emerging viruses.

The emergence of SARS-CoV-2 and the subsequent pandemic have prompted extensive diagnostic and clinical efforts to mitigate viral spread. However, these strategies have largely overlooked the presence of other respiratory viruses. Acute respiratory diseases in pediatric patients can be caused by a diverse range of viral agents, and metagenomics represents a powerful tool for their characterization. This study aimed to investigate the viral abundance in pediatric patients with acute respiratory symptoms who tested negative for SARS-CoV-2 during the Omicron pandemic wave. To achieve this, viral metagenomics and next-generation sequencing were employed on 96 nasopharyngeal swab samples, which were organized into 12 pools, with each pool consisting of eight individual samples. Metagenomic analysis revealed that the most prevalent viruses associated with acute disease in pediatric patients were respiratory syncytial virus (detected in all pools) and enteroviruses, which are known to cause significant morbidity and mortality in children. Additionally, clinically significant viruses such as mumps orthorubulavirus, human metapneumovirus, influenza A, and a wide array of human herpesviruses (1, 3–7) were identified. These findings highlight the extensive potential of viral metagenomics in identifying viruses other than SARS-CoV-2 that contribute to acute infections in children. Consequently, this methodology should garner clinical attention in terms of differential diagnosis and the development of public policies to address such conditions in the global pediatric population.

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 μg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 μg/animal: M1 and at 5.0 μg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 μg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

Fulminant hepatitis is a severe clinical disease characterized by a marked decline in liver function and encephalopathy. In a previous survey, using metagenomics in a group of 27 patients with this clinical condition, we observed an expressive quantity of reads of the Human pegivirus-1 (HPgV-1). Therefore, the objective of this study was to evaluate the frequency, molecular features, and HPgV-1 circulating genotypes in patients with fulminant hepatitis. After testing the collected plasma samples, we discovered twelve samples (44.4%) that were positive for HPgV-1 RNA (using both real-time and nested PCR). The positive samples presented a mean cycle threshold (Ct) of 28.5 (±7.3). Genotyping assignments revealed that all HPgV-1 positive samples belonged to the HPgV-1 genotype 2 (both subgenotypes 2A and 2B were identified). Although HPgV-1 is considered a commensal virus, little is known regarding its prevalence and genotypes in cases of fulminant hepatitis. More research is needed to understand whether HPgV-1 can be implicated in clinical disorders and infectious diseases.

Abstract The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.

Dengue virus (DENV) has been a major public health concern in Paraguay, with frequent outbreaks occurring since early 1988. Although control measures have been implemented, dengue remains a significant health threat in the country, and continued efforts are required for prevention and control. In response to that, in collaboration with the Central Public Health Laboratory in Asunción, we conducted a portable whole-genome sequencing and phylodynamic analysis to investigate DENV viral strains circulating in Paraguay over the past epidemics. Our genomic surveillance activities revealed the co-circulation of multiple DENV serotypes: DENV-1 genotype V, the emerging DENV-2 genotype III, BR4-L2 clade, and DENV-4 genotype II. Results additionally highlight the possible role of Brazil as a source for the international dispersion of different viral strains to other countries in the Americas emphasizing the need for increased surveillance across the borders, for the early detection and response to outbreaks. This, in turn, emphasizes the critical role of genomic surveillance in monitoring and understanding arbovirus transmission and persistence locally and over long distances.

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.

Antitumor property of Crotoxin (CTX), the major toxin from Crotalus durissus terrificus snake venom, has been demonstrated in experimental animal models and clinical trials. However, the direct action of this toxin on the significant events involved in neovascularization, which are essential for tumor growth and survival, has not been confirmed. This study investigated the effects of CTX on the key parameters of neovascularization in two- and three-dimensional culture models. Murine endothelial cell lines derived from thymus hemangioma (t.End.1) were treated at different concentrations of CTX (6.25–200 nM). Endothelial cell proliferation, cell adhesion, and actin cytoskeletal dynamics on laminin (10 µg/ml), type I collagen (10 µg/ml), and fibronectin (3 µg/ml) were evaluated along with the endothelial cell migration and formation of capillary-like tubes in 3D Matrigel. CTX concentration of 50 nM inhibited tube formation on 3D Matrigel and impaired cell adhesion, proliferation, and migration under both culture medium and tumor-conditioned medium. These actions were not accountable for the loss of cell viability. Inhibition of cell adhesion to different extracellular matrix components was related to the reduction of αv and α2 integrin distribution and cytoskeletal actin polymerization (F-actin), accompanied by inhibition of focal adhesion kinase (FAK), Rac1 (GTPase) signaling proteins, and actin-related protein 2/3 (Arp 2/3) complex. This study proved that CTX inhibits the major events involved in angiogenesis, particularly against tumor stimuli, highlighting the importance of the anti-angiogenic action of CTX in inhibition of tumor progression.

From a country with one of the highest SARS-CoV-2 morbidity and mortality rates, Brazil has implemented one of the most successful vaccination programs. Brazil’s first model city vaccination program was performed by the CoronaVac vaccine (Sinovac Biotech) in the town of Serrana, São Paulo State. To evaluate the vaccination effect on the SARS-CoV-2 molecular dynamics and clinical outcomes, we performed SARS-CoV-2 molecular surveillance on 4375 complete genomes obtained between June 2020 and April 2022 in this location. This study included the period between the initial SARS-CoV-2 introduction and during the vaccination process. We observed that the SARS-CoV-2 substitution dynamics in Serrana followed the viral molecular epidemiology in Brazil, including the initial identification of the ancestral lineages (B.1.1.28 and B.1.1.33) and epidemic waves of variants of concern (VOC) including the Gamma, Delta, and, more recently, Omicron. Most probably, as a result of the immunization campaign, the mortality during the Gamma and Delta VOC was significantly reduced compared to the rest of Brazil, which was also related to lower morbidity. Our phylogenetic analysis revealed the evolutionary history of the SARS-CoV-2 in this location and showed that multiple introduction events have occurred over time. The evaluation of the COVID-19 clinical outcome revealed that most cases were mild (88.9%, 98.1%, 99.1% to Gamma, Delta, and Omicron, respectively) regardless of the infecting VOC. In conclusion, we observed that vaccination was responsible for reducing the death toll rate and related COVID-19 morbidity, especially during the gamma and Delta VOC; however, it does not prevent the rapid substitution rate and morbidity of the Omicron VOC.

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.

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.

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.

The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.

Epithelial–mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer.

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.

TSPO (Translocator 18 KDa; tryptophan-rich sensory protein oxygen sensor) is a constitutive outer mitochondrial membrane protein overexpressed in inflammatory cells during local or systemic processes. Despite its expression is characterized, role of TSPO in inflammation remains elusive. For this study, we investigated the role of TSPO ligands on neutrophil functions elicited by two different inflammatory pathways. Peritoneal neutrophils were isolated from male Balb-C mice, treated with TSPO ligand diazepam, Ro5-4864 or PK11195 (1,100 or 1000 nM; 2 h) and further stimulated with lipopolysaccharide from Escherichia coli (LPS), a binding for Toll-Like Receptor-4 (TLR4), or leukotriene B4 (LTB4), a G-protein coupled receptor (GPCR) ligand. LPS treatment did not lead to overexpression of TSPO on neutrophils, and pre-treatment with any TSPO ligand did not alter cytokine expression, adhesion molecule expression, or the production of reactive oxygen and nitrogen species caused by LPS stimulation. Conversely, all TSPO ligands impaired LTB4’s actions, as visualized by reductions in L-selectin shedding, β2 integrin overexpression, neutrophil chemotaxis, and actin filament assembly. TSPO ligands showed distinct intracellular effects on LTB4-induced neutrophil locomotion, with diazepam enhancing cofilin but not modifying Arp2/3 expression, and Ro5-4864 and PK11195 reducing both cofilin and Arp2/3 expression. Taken together, our data exclude a direct role of TSPO ligands in TLR4-elicited pathways, and indicate that TSPO activation inhibits GPCR inflammatory pathways in neutrophils, with a relevant role in neutrophil influx into inflammatory sites.

Abstract 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.

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.

The protein S100A9 plays a key role in the control of inflammatory response. The C-terminus of the murine S100A9 protein (mS100A9p) downregulates the spreading and phagocytic activity of adherent peritoneal cells. Murine peritoneal cells are constituted by macrophages and B-1 cells, and the latter exert an inhibitory effect on macrophage functions by secreting interleukin- (IL-) 10. Here, we investigated the influence of B-1 cells on the inhibitory effect evoked by mS100A9p on macrophages. mS100A9p did not alter spreading and phagocytosis either by peritoneal macrophages obtained from mice deprived of B-1 cells or by bone marrow-derived macrophages (BMDMϕ). Nevertheless, when BMDMϕ were cocultivated by direct or indirect contact with B-1 cells treated with mS100A9p, the phagocytosis by BMDMϕ was decreased, showing that the effect of mS100A9p on macrophages was modulated by B-1 cells and/or their secretory compounds. Furthermore, the inhibitory action of mS100A9p on phagocytosis by adherent peritoneal cells was abolished in cells obtained from IL-10 knockout mice. Taken together, the results show that mS100A9p has no direct inhibitory effect on macrophages; however, mS100A9p modulates B-1 cells, which in turn downregulates macrophages, at least in part, via IL-10. These data contribute to the characterization of S100A9 functions involving B-1 cells in the regulation of the inflammatory process.