Rhomb-I, a P-I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation.

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

Rhomb-I, a P–I metalloproteinase from Lachesis muta rhombeata venom degrades vessel extra cellular matrix components and impairs platelet aggregation

Rhomb-I, a 23-kDa metalloproteinase was isolated from L. m. rhombeata venom. Its dimethylcasein proteolysis was abolished by metal chelators, and slightly enhanced by Ca2+ and Mg2+ ions, but inhibited by Co2+, Zn2+ and α2-macroglobulin. In aqueous solution, rhomb-I autoproteolyzed to a 20- and 11-kDa fragments at 37 °C. The amino acid sequence showed high homology with other snake venom metalloproteinases. Rhomb-I causes hemorrhage that may be ascribed to hydrolysis of essential basement membrane, extracellular matrix and plasma proteins. It preferentially cleaves the α-chains of fibrin (ogen). Rhomb-I inhibited convulxin- and von Willebrand factor (vWF)-induced aggregation on human platelets without significant effect on collagen-stimulated aggregation or other effectors. It digests vWF into a low-molecular-mass multimers of vWF and a rvWF-A1 domain to a 27-kDa fragment as revealed by western blotting with mouse anti-rvWF A1-domain IgG. Incubation of platelets with rhomb-I resulted in adhesion to and cleavage of platelet receptors glycoprotein (GP)Ibα and GPVI to release a 55-kDa soluble form. Both membrane glycoproteins GPIbα that binds vWF, together with GPVI which binds collagen, play a key role in mediating platelet adhesion/activation and can initiate (patho)physiological thrombus formation. Conclusions: rhomb-I is implicated in the pathophysiology of Lachesis envenoming by disrupting vasculature, hemostasis and platelet aggregation through impairing vWF-GPIb axis and blocking GPVI-collagen binding.

A fibrinolytic snake venom metalloproteinase, mutalysin-II, with antiplatelet activity and targeting capability toward glycoprotein GPIbα and glycoprotein GPVI.

Glycoprotein (GP)Ib that binds von Willebrand factor (vWF) and glycoprotein (GP)VI, that binds collagen play a significant role in platelet activation and aggregation, and are potential targets for antithrombotic treatment. They are targeted by snake venom proteinases. The effect of a such proteinase, mutalysin-II, on platelet aggregation was examined using washed human platelets and platelet-rich plasma. Its proteolytic activity on vWF, on its binding partner GPIbα, and on GPVI was analyzed by SDS-PAGE, and immunodetection with the corresponding antibodies after blotting. Dose- and time-dependently, mutalysin-II inhibits aggregation of washed platelets induced by vWF plus ristocetin and by convulxin, but with no significant effect on platelet-rich-plasma. Furthermore, mutalysin-II cleaves vWF into low molecular mass multimers of vWF and a rvWF-A1 domain to realease a ∼27-kDa fragment detectable by SDS-PAGE and blotting with mouse anti-rvWF-A1-domain IgG. Moreover, GPVI was cut by mutalysin-II into a soluble ∼55-kDa ectodomain and a fragment of ∼35-kDa. Thus, mutalysin-II inhibits vWF-induced platelet aggregation via cleavage of bound vWF-A1, and its receptor GPIbα. The additional cleavage of, GPVI, blocks collagen-induced platelets. Our data highlight mutalysin-II as an interesting platelet-directed tool targeting vWF-GPIbα binding and particularly GPVI. Thus, it might be suited for antithrombotic therapy as its combined inactivation of two receptors does not significantly compromise hemostasis, but shows high efficacy and safety. Studies are needed to further develop and demonstrate its potential benefits.

Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization.

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold ß/ß hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

The Protagonism of the Brazilian Health Regulatory System in the Evolution of Patient Safety in the Country: History, Dilemmas, and Current Challenges.

OBJECTIVE: This article aims to identify and analyze the legal and regulatory frameworks with an interface with patient safety, considering the historical path of the patient safety policy in Brazil. METHODS: This is a historical review based on the relevant literature to the topic such as papers, legislation, and official documents with an interface with public health policies from 1988 to 2019. We also performed a documentary search to include data from the Brazilian Health Regulatory Agency (ANVISA) such as normative and nonnormative regulatory instruments. After organizing the data, the process of content analysis was performed. RESULTS: We debated initially the historical aspects of sanitary surveillance of health services in addition to main actions taken by the Brazilian Health Regulatory System, which includes sanitary regulation and patient safety challenges. We identified a diversity of regulations published by ANVISA in the past decade related to patient safety, in addiction to sanitary actions. These initiatives culminated in the establishment of the National Patient Safety Program in 2013, followed by other health improvements, such as surveillance, incidents monitoring, and safe practices self-assessment. CONCLUSIONS: The regulation and sanitary actions directed to patient safety in Brazil have increased after the creation of ANVISA. In the face of this activities, the social role played by the Brazilian Health Regulatory System toward the advancement in the field of risk minimization in health services can be highlighted as a protagonist in the process of promoting patient safety.

Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function.

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.

NLRP12 negatively regulates proinflammatory cytokine production and host defense against Brucella abortus.

Brucella abortus is the causative agent of brucellosis, which causes abortion in domestic animals and undulant fever in humans. This bacterium infects and proliferates mainly in macrophages and dendritic cells, where it is recognized by pattern recognition receptors (PRRs) including Nod-like receptors (NLRs). Our group recently demonstrated the role of AIM2 and NLRP3 in Brucella recognition. Here, we investigated the participation of NLRP12 in innate immune response to B. abortus. We show that NLRP12 inhibits the early production of IL-12 by bone marrow-derived macrophages upon B. abortus infection. We also observed that NLRP12 suppresses in vitro NF-κB and MAPK signaling in response to Brucella. Moreover, we show that NLRP12 modulates caspase-1 activation and IL-1ß secretion in B. abortus infected-macrophages. Furthermore, we show that mice lacking NLRP12 are more resistant in the early stages of B. abortus infection: NLRP12-/- infected-mice have reduced bacterial burdens in the spleens and increased production of IFN-γ and IL-1ß compared with wild-type controls. In addition, NLRP12 deficiency leads to reduction in granuloma number and size in mouse livers. Altogether, our findings suggest that NLRP12 plays an important role in negatively regulating the early inflammatory responses against B. abortus.

Neutralization of toxicological activities of medically-relevant Bothrops snake venoms and relevant toxins by two polyvalent bothropic antivenoms produced in Peru and Brazil.

Snakebite envenoming is a neglected public pathology, affecting especially rural communities or isolated areas of tropical and subtropical Latin American countries. The parenteral administration of antivenom is the mainstay and the only validated treatment of snake bite envenoming. Here, we assess the efficacy of polyspecific anti-Bothrops serum (α-BS) produced in the Instituto Nacional de Salud (INS, Peru) and at the Fundação Ezequiel Dias (FUNED, Brazil), to neutralize the main toxic activities induced by five medically-relevant venoms of: Bothrops atrox, B. barnetti, and B. pictus from Peru, and the Brazilian B. jararaca and B. leucurus, all of them inhabiting different geographical locations. Protein electrophoretic patterns of these venoms showed significant differences in composition, number and intensity of bands. Another goal was to evaluate the efficacy and safety of lyophilized α-BS developed at INS to neutralize the detrimental effects of these venoms using in vivo and in vitro assays. The availability of lyophilized α-BS has relevant significance in its distribution to distant rural communities where the access to antivenom in health facilities is more difficult. Despite the fact that different antigen mixtures were used for immunization during antivenom production, our data showed high toxin-neutralizing activity of α-BS raised against Bothrops venoms. Moreover, the antivenom cross-reacted even against venoms not included in the immunization mixture. Furthermore, we have evaluated the efficacy of both α-BS to neutralize key toxic compounds belonging to the predominant protein families of Bothrops snakes. Most significantly, both α-BS cross-specifically neutralized the main toxicological activities e.g. lethality and hemorrhage induced by these venoms. Thus, our data indicate that both α-BS are equally effective to treat snake bite victims inflicted by Bothrops snakes particularly B. atrox, responsible for the largest numbers of human envenomations in the Amazon regions of some South American countries including Peru and Brazil.

A defective TLR4 signaling for IFN-ß expression is responsible for the innately lower ability of BALB/c macrophages to produce NO in response to LPS as compared to C57BL/6.

C57BL/6 mice macrophages innately produce higher levels of NO than BALB/c cells when stimulated with LPS. Here, we investigated the molecular events that account for this intrinsic differential production of NO. We found that the lower production of NO in BALB/c is not due to a subtraction of L-arginine by arginase, and correlates with a lower iNOS accumulation, which is independent of its degradation rate. Instead, the lower accumulation of iNOS is due to the lower levels of iNOS mRNA, previously shown to be also independent of its stability, suggesting that iNOS transcription is less efficient in BALB/c than in C57BL/6 macrophages. Activation of NFκB is more efficient in BALB/c, thus not correlating with iNOS expression. Conversely, activation of STAT-1 does correlate with iNOS expression, being more prominent in C57BL/6 than in BALB/c macrophages. IFN-ß and IL-10 are more highly expressed in C57BL/6 than in BALB/c macrophages, and the opposite is true for TNF-α. Whereas IL-10 and TNF-α do not seem to participate in their differential production of NO, IFN-ß has a determinant role since 1) anti-IFN-ß neutralizing antibodies abolish STAT-1 activation reducing NO production in C57BL/6 macrophages to levels as low as in BALB/c cells and 2) exogenous rIFN-ß confers to LPS-stimulated BALB/c macrophages the ability to phosphorylate STAT-1 and to produce NO as efficiently as C57BL/6 cells. We demonstrate, for the first time, that BALB/c macrophages are innately lower NO producers than C57BL/6 cells because they are defective in the TLR-4-induced IFN-ß-mediated STAT-1 activation pathway.

Serotype and genotype distributions of pneumococcal carriage isolates recovered from Brazilian children attending day-care centres.

Pneumococcal nasopharyngeal carriage isolates recovered from Brazilian children attending day-care centres in 2005 were assessed for serotype, genotype and penicillin susceptibility phenotype. As 124 of the 253 isolates (49 %) were characterized previously with respect to serotype and penicillin susceptibility, the primary objectives were to examine clonal associations and penicillin susceptibility within major serotypes and to assess the suitability of conventional multiplex PCR for deducing carriage serotypes within this population. Using a combination of PCR-based serotyping and the Quellung reaction, serotypes were identified for 81 % (205/253) of the isolates, with serogroups or types 14, 6, 23F, 19F and 18 being predominant. Included within the 205 isolates successfully serotyped by PCR were 28 isolates that had become non-viable. Forty-eight isolates were non-typable using both the PCR method and the Quellung reaction. Penicillin non-susceptibility was observed within 16 of the 18 multilocus sequence types detected. Thus, this study provides further evidence from a diverse collection of pneumococcal clones that PCR-based serotype deduction is useful for providing supportive evidence for pneumococcal conjugate vaccine implementation.

Taxa de mortalidade por asma de acordo com os atestados de óbito nos moradores em Cascavel/PR no período de jan/05 a dez/09 / Asthma mortality rates according to the death certifications among inhabitants of Cascavel, PR during 2005-2009

A prevalência estimada de asma no Brasil (21%) coloca-o em 8º lugar no ranking mundial. As taxas de mortalidade por asma costumam variar entre os países e são moduladas por fatores externos à doença, tais como o nível de desenvolvimento dos recursos de saúde disponíveis e a qualidade do sistema de registros de óbitos. Este trabalho teve como objetivo quantificar e analisar a taxa de mortalidade por asma nos moradores da cidade de Cascavel/PR. Os dados foram selecionados dos atestados de óbito dos moradores no município de Cascavel, PR, ocorridos de janeiro de 2005 a dezembro de 2009, sem limitação de faixa etária, que contivessem em qualquer das partes a citação de asma (J45), na 10ª revisão da Classificação Internacional de Doenças (CID 10). Os dados foram obtidos no Sistema de Informações sobre Mortalidade (SIM) da Secretaria Municipal de Saúde. A asma foi identificada como causa associada em 17 óbitos, correspondendo ao coeficiente médio de mortalidade de 1,11/100.000 habitantes, diferente do observado em outros países. Observou-se que a mortalidade associada à asma foi maior nas faixas etárias mais altas. O gênero não pareceu ser uma variável importante. A maioria das mortes ocorreu em hospitais e não houve maior ocorrência de óbitos em determinada época do ano. Não se pôde avaliar com acurácia o diagnóstico de asma dos óbitos, nem se a asma contribuiu ou não para o óbito, pois não se teve acesso à identificação e dados de internação dessas pessoas.

Brazil is the 8th country in the world in asthma prevalence (approximately 21%). Mortality rates for asthma vary among countries and are modulated by external factors, such as the level of development of health resources available and the quality of the system of death records. This study aimed to analyze and quantify the asthma mortality rate in people living in thecity of Cascavel, PR, from 2005 to 2009. Data were selected from death certificates of residents of Cascavel, PR, registered from January 2005 to December 2009, which contained in any field asthma (ICD 10, code J45), without limitation of age. Data were collected from Mortality Information System (SIM) in the Health secretary of Cascavel. Asthma was identified as an associated cause in 17 deaths, corresponding to an average mortality rate of 1.11 per 100,000 inhabitants, different from the observed in other countries. It can be observed that the mortality associated with asthma was higher in higher age groups. The genderdoes not seem to be an important variable. Most deaths occurred in hospitals and there was not a higher incidence of deaths related to certain period of the year. We were unable to assess accurately the diagnosis of asthma deaths, nor if asthma contributedto death or not, because there were no identification available and admission data of such people.

Molecular epidemiology and risk factors for nasal carriage of staphylococcus aureus and methicillin-resistant S. aureus in infants attending day care centers in Brazil.

Investigations regarding Staphylococcus aureus carriage among Brazilian children are scarce. We evaluated the determinants of S. aureus and methicillin-resistant S. aureus (MRSA) nasal carriage in infants attending day care centers (DCCs) and the molecular features of the MRSA strains. A total of 1,192 children aged 2 months to 5 years attending 62 DCCs were screened for S. aureus and MRSA nasal carriage. MRSA isolates were characterized by pulsed-field gel electrophoresis, multilocus sequence typing, spa typing, staphylococcal cassette chromosome (SCC) mec typing and the presence of the Panton-Valentine leukocidin gene. Logistic regression was performed to determine risk factors associated with S. aureus and MRSA colonization. S. aureus and MRSA carriage were detected in 371 (31.1%) and 14 (1.2%) children, respectively. Variables found to be independently associated with an increased risk for S. aureus carriage included being older than 24 months (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.3 to 2.6) and previous DCC attendance (OR, 1.5; 95% CI, 1.0 to 2.2). Having a mother with a high level of education was a protective factor for nasal colonization (OR, 0.4; 95% CI, 0.2 to 0.8). Moreover, we observed that more children carrying MRSA had younger siblings than children not colonized by MRSA. Among the 14 MRSA strains, three SCCmec types (IIIA, IV, and V) were detected, together with a multidrug-resistant dominant MRSA lineage sharing 82.7% genetic similarity with the Brazilian clone (ST239-MRSA-IIIA; ST indicates the sequence type determined by multilocus sequence typing). Although SCCmec type V was recovered from one healthy child who had been exposed to known risk factors for hospital-associated MRSA, its genetic background was compatible with community-related MRSA. Our data suggest that DCC attendees could be contributing to MRSA cross-transmission between health care and community settings.