Abstract in English:Several health organizations have classified diabetes mellitus, a metabolic syndrome, as the epidemic of the century, since it affects millions of people worldwide and is one of the top ten causes of death. Type 1 diabetes is considered to be an autoimmune disease, in which autoaggressive T cells infiltrate the islets of Langerhans in the pancreas, leading to the destruction of insulin producing beta cells. The risk of the disease is modulated by genetic factors, mainly genes coding for human leukocyte antigens (HLA). However, the incidence of this disease has increased significantly during the recent decades, which cannot be explained only by genetic factors. Environmental perturbations have also been associated to the development of diabetes. Among these factors, viral triggers have been implicated; particularly enteroviruses, which have been associated to the induction of the disease. Supporting the hypothesis, numerous lines of evidence coming from mouse models and patients with this type of diabetes have shown the association. The present review aims to provide some understanding of how type 1 diabetes occurs and the possible role of enterovirus in this pathology.
Abstract in English:Lipocalins are involved in a variety of functions including retinol transport, cryptic coloration, olfaction, pheromone transport, prostaglandin synthesis, regulation of the immune response and cell homeostatic mediation. A full-length cDNA clone (named d-lipo), isolated from the venom gland cDNA library of Deinagkistrodon acutus, contained an insert of 664 bp including an open reading frame that encodes a lipocalin homologue of 177 amino acids. Comparison of d-lipo and other related proteins revealed an overall amino acid identity of less than 21.5%. Primary structures of d-lipo carried three structurally conserved regions (SCR) showing homologies to those of lipocalins. The first conserved Cys residue - the essential amino acid residue for the catalytic activity and unique to lipocalin-type prostaglandin D synthase (L-PGDS) in the lipocalin protein family - was identified in d-lipo at amino acid position 58. Phylogenetic tree analysis showed that d-lipo was in-between the large L-PGDS cluster and the small von Ebner's-gland proteins (VEGP) cluster. Moreover, d-lipo gene presented a high-level expression in the venom gland and a low-level expression in the brain and its expression was significantly increased under pathological conditions, suggesting a possible relationship between d-lipo mRNA expression and the venom gland inflammatory disease. This is also the first report of a lipocalin homologous gene identified in the venom gland of a snake.
Abstract in English:Although it has been demonstrated that venoms and toxins from some snakes are able to influence the growth of tumor cells, few antitumoral compounds from Bothrops leucurus venom have been characterized. Leucurolysin-B (leuc-B) is a metalloproteinase class P-III isolated from B. leucurus which possesses an ECD-disintegrin domain. Both ECD-disentegrin and RGD-disintegrin are able to bind to cell surface integrins and inhibit their adherence to their natural ligands. In the present study, the potential efficacy and the cytotoxic effects of leuc-B on glioblastoma, breast cancer and melanoma cell lines were analyzed. The effect of leuc-B on cancer cell survival was evaluated and its 50% inhibitory concentration (IC50) was determined. Morphological alterations were monitored by contrast phase and fluorescent microscopy. The results demonstrated that leuc-B has potent cytotoxic effect in a micromolar range against all evaluated cancer cell lines. Morphologically, dying cells showed fragmentation, condensation of their contents concomitant with shrinkage and appearance of vacuoles. This study reports for the first time the cytotoxic effect of leuc-B from B. leucurus snake venom on tumor cells.
Abstract in English:The aim of the present study was to investigate the presence of Trypanosoma cruzi in the heart, liver, lung, and kidneys, using hemoculture and PCR analysis, of mice infected with different parasite strains during the acute and chronic phases of infection. Parasitemia curves revealed strain-specific biological behaviors. For the Y and JLP strains, the acute phase of infection started at days six and ten post-infection, parasitemia peaked at days seven and 15 post-infection, the chronic phase started at days nine and 28 post-infection, and animals started dying at days 19 and 120 post-infection, respectively. When the two strains were compared, the JLP strain exhibited reduced and slower replication rates associated with a delayed peak of parasitism and reduced parasite burdens. However, parasites were detected in all studied organs using PCR analysis. The capacity of both strains to infect different organs likely influences disease pathogenesis.
Abstract in English:Hydrophilic nanoparticles have been widely investigated in recent years as delivery systems for therapeutic macromolecules such as antigens. In the present study Mesobuthus eupeus venom-loaded chitosan nanoparticles were prepared via ionic gelation of tripolyphosphate (TPP) and chitosan. The optimum encapsulation efficiency (91.1%) and loading capacity (76.3%) were obtained by a chitosan concentration of 2 mg/mL, chitosan-to-TPP mass ratio of 2 and M. eupeus venom concentration of 500 µg/mL. The average nanoparticle size at optimum conditions was determined by Zetasizer (Malvern Instruments, UK). The nanoparticle size was about 370 nm (polydispersity index: 0.429) while the zeta potential was positive. Transmission electron microscope (TEM) imaging showed a spherical, smooth and almost homogenous structure for nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles. The in vitro release of nanoparticles showed an initial burst release of approximately 60% in the first ten hours, followed by a slow and much reduced additional release for about 60 hours. It is suggested that the chitosan nanoparticles fabricated in our study may provide a suitable alternative to traditional adjuvant systems.
Abstract in English:Cnidarians comprise an old and diverse animal phylum, and possess a wide variety of biologically active substances. Sea anemones contain a diversity of interesting biologically active compounds including some potent toxins. In the present work, the sea anemones Stichodactyla mertensii and Stichodactyla gigantea, collected from the Mandapam coast, are characterized biomedically and pharmacologically. The crude protein was obtained by using methanol and aqueous extracts. The respective protein contents of S. mertensii and S. gigantea were found to be 2.10 µg/mL and 1.87 µg/mL. The methanol and aqueous extracts of S. mertensii and S. gigantea yielded six and nine bands by SDS-PAGE on 12% gel. In the hemolytic assay, both extracts exhibited hemolytic effect on chicken, goat, cow and human erythrocytes ('A', 'B' and 'O'). The neurotoxic effects of these crude extracts were determined in vivo using the sea shore crab Ocypode macrocera and mortality was observed. The mouse bioassay for lethality was performed on male albino mice. The crude extract of S. mertensii showed higher lethality (58 seconds at 1 mL-dose) than that of S. gigantea (2 minutes and 10 seconds at 0.75 mL-dose). The analgesic activity test was also carried out on albino mice by Eddy's hot plate and tail-flick methods. The extracts showed moderate analgesic effect by both hot-plate and tail-flick methods. These characteristics emphasize the need for the isolation and molecular characterization of new active toxins in S. mertensii and S. gigantea.
Abstract in English:Bmaj-9, a basic PLA2 (13679.33 Da), was isolated from Bothrops marajoensis snake venom through only one chromatographic step in reversed phase HPLC on ¼-Bondapak C-18 column. The amino acid composition showed that Bmaj-9 had a high content of Lys, His, and Arg, typical of a basic PLA2. The sequence of Bmaj-9 contains 124 amino acid residues with a pI value of 8.55, such as DLWQWGQMIL KETGKLPFSY YTAYGCYCGW GGRGGKPKAD TDRCCFVHDC, revealing a high homology with Asp49 PLA2 from other snake venoms. It also exhibited a pronounced phospholipase A2 activity when compared with crude venom. In chick biventer cervicis preparations, the time for 50% and 100% neuromuscular paralysis was respectively (in minutes): 110 ± 10 (1 µg/mL); 40 ± 6 and 90 ± 2 (5 µg/mL); 30 ± 3 and 70 ± 5 (10 µg/mL); 42 ± 1 and 60 ± 2 (20 µg/mL), with no effect on the contractures elicited by either exogenous ACh (110 µM) or KCl (20 mM). Bmaj-9 (10 µg/mL) neither interfered with the muscular response to direct electrical stimulation in curarized preparations nor significantly altered the release of CK at 0, 15, 30 and 60 minutes incubations (27.4 ± 5, 74.2 ± 8, 161.0 ± 21 and 353.0 ± 47, respectively). The histological analysis showed that, even causing blockade at the maximum dosage (5 µg/mL), the toxin does not induce significant morphological alterations such as necrosis or infiltration of inflammatory cells. These results identified Bmaj-9 as a new member of the basic Asp49 PLA2 family able to interact with the motor nerve terminal membrane, thereby inducing a presynaptic neuromuscular blockade.
Abstract in English:Bothropic envenomation induces hemorrhage, coagulant disturbances and necrosis. Regarding therapies against the local damage caused by the venom, there is little information on tissue changes until the complete healing. In the current study, local damage was evaluated by examination of morphological inflammatory alterations, mast cell count, and analysis of collagen deposition. Bleeding was evident four hours after inoculation. After 24 hours, a large area of injury appeared presenting disorganized tissue, significant hemorrhage and acute inflammation. After three days, the damaged area was extensive, with a large amount of inflammatory cells and the presence of scab. In seven days, healing and reepithelization process started. And, 21 days later, the epithelium showed less infiltration and no skin appendages. The number of mast cells was similar to control after four hours, with a drop of 50% at 24 hours, followed by an increase until the 21st day. No differences of collagen deposition were observed among experimental groups. Taken together, wound healing after intradermal injection of Bothrops cotiara venom in mice follows similar parameters to wounds caused by other bothropic venoms. The present work reveals the importance of experimental wound models to the study of neutralizing agents against venom toxins.
Abstract in English:Of the 102 samples collected from mammals and birds, both domestic and captive wild, 48 were found to be positive for Clostridium perfringens. Most of the mammal isolates (84.38%) appeared to have been collected from clinically affected animals, while 33.33% of the bird samples were from clinically affected and 21.43% from apparently healthy birds infected with C. perfringens. Isolates revealed high sensitivity to ciprofloxacin, enrofloxacin and norfloxacin. Among the isolated C. perfringens, 30 (62.50%) showed DNase production. Hemolytic activity was recorded in 14 (24.16%) of the isolates and 28 (58.33%) showed phospholipase C production. All the phospholipase C positive isolates revealed the presence of cpa gene encoding alpha (α) toxin. Of the 102 samples collected from mammals and birds, both domestic and captive wild, 48 were found to be positive for Clostridium perfringens. Most of the mammal isolates (84.38%) appeared to have been collected from clinically affected animals, while 33.33% of the bird samples were from clinically affected and 21.43% from apparently healthy birds infected with C. perfringens. Isolates revealed high sensitivity to ciprofloxacin, enrofloxacin and norfloxacin. Among the isolated C. perfringens, 30 (62.50%) showed DNase production. Hemolytic activity was recorded in 14 (24.16%) of the isolates and 28 (58.33%) showed phospholipase C production. All the phospholipase C positive isolates revealed the presence of cpa gene encoding α toxin.
Abstract in English:Cantharidin provides chemical protection for the coleopteran families Meloidae and Oedemeridae. In the present study, it was observed that cantharidin concentration in Hycleus scabiosae was slightly decreased from mated females (mean = 0.011 mg/mg of dry weight) to males (mean = 0.010 mg/mg) and considerably diminished in relation to virgin females (mean = 0.005 mg/mg). Significant concentrations of palasonin (21.69 ng/mg among virgins and 17.49 ng/mg in mated females) and palasoninimide (14.62 ng/mg in virgins and 9.17 ng/mg in mated females) were found in H. scabiosae. Palasonin, palasoninimide and cantharidinimide content of eggs were measured as 5.61, 7.69 and 7.80 ng/mg respectively. Surprisingly, males showed no trace of cantharidin-related compounds (CRCs); therefore CRCs in H. scabiosae could not be transferred from males to females and based on experiments employing its deuterated form, cantharidin is probably independently synthesized in females from the male nuptial transfer. An inseminated female incorporates about 38.5 ng of cantharidin (0.34% of the maternal content), 196.35 ng of palasonin (91.82% of maternal content) and 269.15 ng of palasoninimide (96.70% maternal content) into each egg mass during oviposition. It seems that eggs of this meloid species exploit a different array of protective chemicals by increasing the ratio of CRCs versus cantharidin. CRCs are less toxic than cantharidin; therefore, such compounds might have been deposited in eggs as a safer substitute for cantharidin to provide effective protection, but does not simultaneously harm the susceptible embryo.
Abstract in English:Members of the subfamily Crotalinae are considered to be essentially nocturnal and most of the data about these snakes have been collected from the field. Information on how nutritional status affects the movement rate and activity patterns is a key point to elucidating the ecophysiology of snakes. In this study, we distributed 28 lancehead Bothrops moojeni into three groups under distinct feeding regimens after a month of fasting. Groups were divided as follows: ingestion of meals weighing (A) 40%, (B) 20%, or (C) 10% of the snake body mass. Groups were monitored for five days before and after food intake and the activity periods and movement rates were recorded. Our results show that B. moojeni is prevalently nocturnal, and the activity peak occurs in the first three hours of the scotophase. After feeding, a significant decrease in activity levels in groups A and B was detected. The current results corroborate previous field data that describe B. moojeni as a nocturnal species with low movement rates. The relationship between motion and the amount of food consumed by the snake may be associated with its hunting strategy.
Abstract in English:Cytomegalovirus causes significant morbidity and mortality in AIDS patients and those having undergone bone marrow or another transplant. PP65 antigenemia is based on detecting viral antigen in peripheral blood leukocytes through immunochemistry and by monitoring the infection in immunocompromised individuals. The present study aimed to set up this diagnostic technique in AIDS patients with active cytomegalovirus infection and verify its occurrence in the Botucatu region of São Paulo state, Brazil. Fifty patients, 35 men and 15 women aged from 24 to 69 years, were recruited from those attended at the Department of Tropical Diseases of Botucatu Medical School, UNESP, and divided into three groups according to CD4+ T lymphocyte counts and antiretroviral treatment. The control group comprised bone marrow transplant patients. Fourteen AIDS patients with low CD4+ cell counts tested positive for PP65 antigenemia, which could predict cytomegalovirus infection and indicate prophylactic treatment.
Abstract in English:Structural characteristics of discharged and undischarged nematocysts from the hydrozoans Millepora alcicornis and Millepora complanata, two fire corals collected in the Mexican Caribbean, were examined using transmission electron, scanning and light microscopy. In this study, we report for the first time images of the nematocysts found in these Mexican Caribbean venomous species. Two types of nematocysts were observed in both species, the more abundant identified as macrobasic mastigophore and the other a stenotele type. Macrobasic mastigophores were present in medium and large size classes while stenoteles appeared in only one size.
Abstract in English:Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.
Abstract in English:Leptospirosis is a globally distributed emerging zoonosis. Dogs are commonly affected and although other serovars can cause canine leptospirosis, Leptospira interrogans serovar Canicola is primary found in these animals. A retrospective study was conducted using a database of 1195 dogs tested for Leptospira infection from 2003 to 2010 at the Laboratory of Zoonosis Diagnosis at the Veterinary Hospital of São Paulo State University (UNESP) in Botucatu, São Paulo state, Brazil. The seroprevalence of infected dogs was 20.08% (240/1195), and the most prevalent serovars were Canicola (6.7%), Copenhageni (5.0%), Icterohaemorrhagiae (2.9%), Autumnalis (2.9%), Pyrogenes (2.8%), Pomona (2.0%), Hardjo (2.0%), Australis (1.8%), Bratislava (1.6%), Cynopteri (1.4%), Grippotyphosa (1.3%) and Djasiman (1.0%). By univariate analysis, the variables age and breed were not statistically related to the infection, while gender and season were. The effects of gender were also noticeable related to serovars Australis, Canicola and Hardjo. In multivariate analysis, the level of significance (p-value) of season was suppressed by gender, indicating possible collinearity between those two variables.