Ana Maria Moura da Silva

Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north‐eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

Assessment of efficacy of bothropic antivenom therapy on microcirculatory effects induced by Bothrops jararaca snake venom

Intravenous administration of antibothropic antivenom (BAv) neutralises the systemic effects, but does not efficiently reverse the local symptoms elicited by the Bothrops jararaca venom (BjV). The mechanisms involved in this poor protection have not been clarified. In this work, intravital microscopy studies were carried out to determine the efficacy of different schedules of BAv treatment on local effects evoked by topical application of BjV in the microcirculatory network of the internal spermatic fascia of Wistar rats. Results demonstrated that BAv administration 15 min before, simultaneously with, or 15 min after BjV application did not totally reverse the local symptoms, represented by disturbances of coagulation, development of haemorrhage lesions, vascular permeability increase and increment on leukocyte-endothelium interactions. This lack of effectiveness neither reflects an inadequate amount of specific antibodies in the antivenom against toxins responsible for local effects nor an insufficient dose of circulating BAv during the assays. Administration of fluorescein isothiocyanate (FITC) labelled-BAv showed the dynamics of distribution of the antivenom in the microcirculatory network. Images obtained from prior and simultaneously treated animals showed that the antivenom remains at luminal side of vessels before venom application, and the latency time to antivenom leakage is coincidental to that for local effects evoked by the venom. In addition, images from posterior treatment demonstrated that the intense alterations in the microcirculatory network impair antivenom distribution at the site of injection. Together, our data show that the lack of effectiveness of antivenom therapy is due to impaired and delayed venom and antivenom interaction at the site of injury.

Characterization of antibody isotype responsible for immune clearance in mice infected with Trypanosoma cruzi

Humans and mice chronically infected with Trypanosoma cruzi present a strong humoral immune response mediated by specific antibodies. Passive transfer of homologous immune serum to normal mice containing circulating bloodstream trypomastigotes (Bts) induces a very fast clearance of the parasites. In order to find out the role of the different immunoglobulin classes in the clearance, mice containing a known number of these parasite forms in circulation were injected with total immune serum, IgG-free serum, IgG1, or IgG2 fractions and the speed of removal of the parasites from circulation was determined. The results of these experiments suggest that the immune clearance of T. cruzi is due to antibodies located in the IgG isotype, particularly in the IgG2 subclass.

Snakebites and Scorpion Stings in the Brazilian Amazon: Identifying Research Priorities for a Largely Neglected Problem

Envenomings by snakebites and scorpion stings impose a high burden worldwide and result in considerable social and economic impact [1]. It is estimated that snakebite rates are as high as over 1.8 million cases per year, with associated deaths reaching more than 90,000 cases annually [2]. However, snakebites are a neglected condition with no associated WHO programmes for control and prevention. The countries most affected by snakebites are located in the intertropical zone in areas with high rates of field use for agriculture where the main affected populations are adult men working in agricultural activities [1]. Approximately two billion people are living in areas at risk for scorpion stings, with over one million accidents occurring annually worldwide [3]. However, the true burden of snakebites and scorpion stings is probably higher and difficult to estimate since only a few countries have a reliable system for epidemiological surveillance of these events. In Brazil, the Ministry of Health implemented the National Program for Snakebites Control in 1986, extended to other poisonous animals in 1988. Since then, antivenom (AV) production has been standardized and all the AV production from the three national laboratories (Instituto Butantan, Fundacao Ezequiel Dias, and Instituto Vital Brazil) was acquired by the Ministry of Health for distribution free of charge to patients. Five types of snake AVs are currently available: Bothrops AV (main one), Crotalus AV, Bothrops-Crotalus AV, Bothrops-Lachesis AV, and Micrurus AV. For scorpion stings, there are two types of AVs available in Brazil: Tityus scorpion AV and a polyvalent AV against Loxosceles and Phoneutria spiders and the Tityus scorpion. Table 1 summarizes information on snake and scorpion AVs produced in Brazil. Table 1 Venom pools used for snake and scorpion AVs production in Brazil. In 2013, 27,181 and 78,091 cases of snakebites and scorpion stings were reported by the Brazilian Ministry of Health, respectively [4]. The highest incidence was in the North region (52.6 cases/100,000 inhabitants) followed by the Midwest (16.4/100,000). These values, expected to be higher in remote areas of the Brazilian Amazon [5], may be underestimated due to considerable underreporting. Fig 1 presents the spatial distribution of snakebites and scorpion stings in the Brazilian Amazon. Fig 1 Spatial distribution of snakebites and scorpion stings in the Brazilian Amazon in 2013.

Biological Applications of a Chimeric Probe for the Assessment of Galectin-3 Ligands:

β1–6 branching of N-linked oligosaccharides has been correlated with the progression of different cancers. The leukoagglutinins of Phaseolus vulgaris (L-PHA) have been used to study this pattern of glycosylation whose biological significance is incompletely understood. The animal lectin, galectin-3, also binds to structures recognized by L-PHA. To develop a functional tool for the in situ identification of this pattern of glycosylation, human galectin-3 was fused to bacterial alkaline phosphatase (gal3/AP). Gal3/AP recognized both A and B blood group saccharides (B>A) and lactosamine derivatives. Gal3/AP recognition depended at least in part on the N-linked oligosaccharides of different glycoproteins. The presence and distribution of galectin-3 ligands were analyzed in both murine and human normal and tumor samples. Loss of apical expression of galectin-3 ligands was commonly found in carcinomas. Endothelial and inflammatory cells were enriched in galectin-3 ligands as compared with tumor cells; thus, gal3/AP is a suitable tool for studying tumor micro-environments. Comparative analysis of both gal3/AP and L-PHA binding patterns indicated that although similar, these patterns are not identical. The probe developed was useful for several immunoenzymatic assays and will allow the physiological and clinical significance of the expression pattern of galectin-3 ligands to be established. This manuscript contains online supplemental material at http:/www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 55: 1015–1026, 2007)

Angiostatin-like molecules are generated by snake venom metalloproteinases

Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.

T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4+ and CD8+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, “promiscuous” T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.

The Promigratory Activity of the Matricellular Protein Galectin-3 Depends on the Activation of PI-3 Kinase

Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-3−/− mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-3−/− sarcoma cells were more adherent and less migratory than galectin-3+/+ sarcoma cells on laminin-111. When galectin-3 was transiently expressed in galectin-3−/− sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAK and phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 was inhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111.

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein.

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.

A comparative study of anti-Trypanosoma cruzi serum obtained in acute and chronic phase of infection in mice

The IgG antibody content, specificity, lytic activity, clearance capacity and protective ability of mouse anti-Trypanosoma cruzi serum was determined during the course of infection. The IgG antibody content increased during the course of infection, reaching its highest level in the serum collected in the chronic phase of the infection. The T. cruzi antigens recognized by antibodies using the protein transfer technique also increased with time of infection. Antibodies present in day 22 post-infection (p.i.) serum were already able to recognize all the antigens detected by antibodies present in serum from the chronic phase. The lytic and clearance ability were not detected on day 7 p.i., but appeared on day 14 p.i. and reached their highest level on day 45 p.i. The protective ability was present in serum of the chronic phase, but was absent from the acute serum. The IgG antibody content of the acute serum was four times less than that of the chronic serum. When the IgG antibody concentration of the acute serum was equalized to that of the chronic serum, the acute serum was as able to protect the infected animals as the chronic serum. It is suggested that the disagreement between the protective ability of anti-T. cruzi antisera collected in the acute or in the chronic phase of the infection is due to a quantitative rather than a qualitative difference.