Expression of VEGF and Flk-1 and Flt-1 Receptors during Blood-Brain Barrier (BBB) Impairment Following Phoneutria nigriventer Spider Venom Exposure

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria nigriventer spider venom (PNV) disrupts the blood-brain barrier (BBB) and causes neuroinflammation in central neurons along with excitotoxic signals in rats and humans. All these changes are transient. Herein, we examined the expression of VEGF and its receptors, Flt-1 and Flk-1 in the hippocampal neurons following envenomation by PNV. Adult and neonatal rats were evaluated at time limits of 2, 5 and 24 h. Additionally, BBB integrity was assessed by measuring the expression of occludin, β-catenin and laminin and neuron viability was evaluated by NeuN expression. VEGF, Flt-1 and Flk-1 levels increased in PNV-administered rats, concurrently with respective mRNAs. Flt-1 and Flk-1 immunolabeling was nuclear in neurons of hippocampal regions, instead of the VEGF membrane-bound typical location. These changes occurred simultaneously with the transient decreases in BBB-associated proteins and NeuN positivity. Adult rats showed more prominent expressional increases of the VEGF/Flt-1/Flk-1 system and earlier recovery of BBB-related proteins than neonates. We conclude that the reactive expressional changes seen here suggest that VEGF and receptors could have a role in the excitotoxic mechanism of PNV and that such role would be less efficient in neonate rats.     

Spider neurotoxins block the beta scorpion toxin-induced calcium uptake in rat brain cortical synaptosomes

In this paper we describe the effects of the beta scorpion toxin Tityus gamma (TiTX gamma) and spider neurotoxins Tx3-3 and Tx3-4 in the (45)Ca(2+) uptake in synaptosomes. The TiTX gamma-stimulatory effect on (45)Ca(2+) uptake in synaptosomes was inhibited omega-Conotoxin MVIIC (omega-CgTX MVIIC) (0.1 microM) and omega-Agatoxin IVA (0.1 microM) by 70% and 41%, respectively. omega-CgTX MVIIC (1.0 microM) almost completely blocked the TiTX gamma-induced (45)Ca(2+) uptake in synaptosomes. Verapamil (1.0 microM) and omega-Conotoxin GVIA (0.1 microM) had no effect in the scorpion toxin-induced (45)Ca(2+) influx. The spider neurotoxins Tx3-3 and Tx3-4 inhibited the TiTX gamma-induced calcium uptake with an IC(50) of 10.0 and 30.0 nM, respectively. It is suggested that spider neurotoxins Tx3-3 and Tx3-4 blocking effect in the TiTX gamma-induced calcium uptake involves P/Q-type calcium channels.     

A protective immune response against lethal,dermonecrotic and hemorrhagic effects of Loxosceles intermedia venom elicited by a 27-residue peptide

Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope (²⁵NLGANSIETDVSFDDNANPEYTYHGIP⁵¹) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings.     

Characterization of the venom from the Brazilian Brown Spider Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae)

Accidents caused by brown spiders (Loxosceles genus) are frequent in Brazil and are associated with dermonecrotic lesions and, eventually, systemic reactions that may be lethal. The major species implicated with human envenoming have been: L. intermedia, L. gaucho and L. laeta. In this study we characterized the venom from Loxosceles similis, a species of spider normally found inside caves. L. similis venom was characterized by two-dimensional gel electrophoresis and enzymatic activity (dermonecrosis and haemolysis). The lethal dose to mice and the capacity of commercial anti-serum to neutralize this venom were also analysed. The cross-reactivity with anti-venoms against L. intermedia, L. laeta and L. gaucho were studied. Our results showed that this venom was able to induce severe dermonecrotic lesions and showed the presence of the bacteria Clostridium septicum in association with the fangs. In addition, we have cloned the DNA coding for a dermonecrotic protein (LsD1), using the genomic DNA of L. similis. The deduced amino acid sequence showed a toxin of approximately 31.2 kDa with an estimated pI of 7.37 and sequence similar to LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom, a synanthropic species of medical importance.     

Distribution of vitamin D3 receptor in the epididymal region of roosters (Gallus domesticus) is cell and segment specific

Vitamin D3 is a steroid hormone well known by its role in maintaining calcium homeostasis, however this hormone may also participate in other biological functions, including control of reproductive processes. The vitamin D3 action is mediated by the vitamin D3 receptor (VDR). VDR is widely distributed in the rodent reproductive tract, however the occurrence of VDR and the role of the vitamin D3 in the avian reproductive tract remain unknown. The aim of the present study was to investigate the expression and cellular distribution of VDR in the epididymal region of roosters. VDR expression was investigated by Western blotting analysis and the tissue distribution of the receptor was determined by immunohistochemistry. The Western blotting assay revealed a major VDR protein band of 61kDa in the epididymal region of rooster. Nuclear VDR expression was found in all segments of the epididymal region, namely rete testis, efferent ductules, connecting ducts and epididymal ducts. Nonciliated cells of the distal efferent ductules showed the highest levels of VDR expression, followed by the proximal efferent ductules and rete testis. The connecting and epididymal ducts showed less intense VDR immunostaining. The differential VDR expression in the epididymal region segments reveals that several extratesticular ducts may be target for vitamin D3 action and suggests that vitamin D3 may have a regional-specific function, such as calcium transport, that is modulated through VDR activity.     

Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom

The present report describes the identification and molecular characterization of LiD1, a protein expressed in the venom gland of the brown spider Loxosceles intermedia. LiD1 belongs to a family of proteins with dermonecrotic activity and members of this family have been found in spiders from the genus Loxosceles. The necrotic lesions caused by this group of proteins may lead to serious socio-economic problems such as surgical tissue reconstitution and even patient death. LiD1 was cloned using a cDNA library constructed from the venom gland of L. intermedia and antibodies against proteins with dermonecrotic activity isolated from the crude venom of this spider. The amino acid sequence deduced from the cDNA revealed a mature protein of approximately 31 kDa, with a pI of 7.37. The cDNA also revealed the existence of a signal peptide, a propeptide and also an untranslated 3′ region with 218 nucleotides. LiD1 was expressed as a protein fused with β-galactoside protein using the vector pBK-CMV, resulting in the recombinant protein recLiD1 with important immunological properties. recLiD1 was strongly recognised by anti-dermonecrotic antibodies and was also able to generate reactive antibodies against native dermonecrotic proteins isolated from the venom of L. intermedia.     

Helicobacter pylori DNA in recurrent aphthous stomatitis

BACKGROUND: Considering not only the fact that recurrent aphthous stomatitis (RAS) and stomach ulcers are immunologically mediated ulcers associated with Helicobacter pylori, but also the recent evidence that anaemia can be associated with both diseases, and the discovery of H. pylori in the oral mucosa led us to hypothesize that this bacteria may be related to RAS pathogenesis. METHODS: Thirty-six consecutive subjects affected by minor and major forms of RAS and 48 healthy volunteers were included in the present study. The nested polymerase chain reaction (PCR) technique was used to detect the presence of H. pylori in the oral lesion, the normal contralateral mucosa of patients affected by RAS and the oral mucosa of control subjects. The chi2- and Fisher's tests were used for statistical analysis. RESULTS: No association between RAS lesions and H. pylori was observed. However, 14 out of 36 (38.9%) of the patients with RAS were found to show the presence of H. pylori DNA in the lesion and/or contralateral mucosa. Sixteen out of 48 (33.3%) of the patients without RAS (control subjects) were positive (P > 0.05). CONCLUSION: The present study does not give support to the assumption that H. pylori could be involved in RAS development.     

Association of CD14, IL1B, IL6, IL10 and TNFA functional gene polymorphisms with symptomatic dental abscesses

AIM: To investigate in individuals with symptomatic dental abscesses the occurrence of functional polymorphisms within five genes involved with the immune response. The functional gene polymorphisms analysed were CD14 (-260 C/T), IL1B (+3954 C/T), IL6 (-174 G/C,), IL10 (-1082 G/A) and TNFA (-308 G/A). METHODOLOGY: Genomic DNA obtained from oral swabs from individuals with symptomatic dental abscesses and asymptomatic inflammatory periapical lesions, without previous exacerbation, was submitted to restriction fragment length polymorphism (RFLP) analyses to determine each individual genotype. The chi-square and principal components analysis tests were used for statistical analysis. RESULTS: A significant association was observed between the occurrence of the GG genotype or the G allele expression of the polymorphic locus-174 (G/C) of the IL6 gene, and the presence of the symptomatic dental abscesses in women and in individuals < or =35 years old. The principal components analysis suggested predominance of the symptomatic dental abscesses in individuals displaying: high-producer IL6 genotype; intermediate and high-producer IL1B genotypes and low-producer TNFA genotype. CONCLUSIONS: The present study suggests that genetic factors are associated with susceptibility to develop symptomatic dental abscesses.