Local tissue damage induced by crotaline snake venoms includes edema, myonecrosis, hemorrhage, and an inflammatory response associated with a prominent cellular infiltrate. The role of neutrophils in the local tissue damage induced by Bothrops asper snake venom and by myotoxin I, a phospholipase A2 isolated from this venom, was investigated. Male Swiss mice were pretreated with either an antimouse granulocyte rat monoclonal immunoglobulin G (IgG) antibody or with isotype-matched control antibody. No significant differences in these local effects were observed between mice pretreated with antigranulocyte antibodies and those receiving control IgG. Moreover, myotoxicity induced by B. asper myotoxin I was similar in neutrophil-depleted and control mice. The role of neutrophils in the process of skeletal muscle regeneration was also assessed. Muscle regeneration was assessed by quantifying the muscle levels of creatine kinase and by morphometric histological analysis of the area comprised by regenerating cells in damaged regions of skeletal muscle. Mice depleted of neutrophils and then injected with B. asper venom showed a more deficient regenerative response than mice pretreated with control IgG. Moreover, a drastic difference in the regenerative response was observed in mice injected with myotoxin I, because animals pretreated with control IgG showed a successful regeneration, whereas those depleted of neutrophils had abundant areas of necrotic tissue that had not been removed 7 days after injection, associated with reduced contents of creatine kinase. It is concluded that (1) neutrophils do not play a significant role in the acute local pathological alterations induced by the venom of B. asper, and (2) neutrophils play a prominent role in the process of skeletal muscle regeneration after injection of B. asper venom and myotoxin I, probably related to the phagocytosis of necrotic material and the recruitment of other inflammatory cells, two events directly associated with a successful muscle regenerative response.

Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. The selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 × 10−10 M, detected as little as 6.2 ng of Stx1 and was stable up to 50 ºC. In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of 6.1 × 10−10 M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 µg 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.

Recombinant rabies virus glycoprotein (rRVGP) was expressed in Drosophila melanogaster Schneider 2 (S2) cells. The cDNA encoding the entire RVGP gene was cloned in an expression plasmid under the control of the constitutive actin promoter (Ac), which was co‐transfected into S2 cells together with a hygromycin selection plasmid. Selected S2 cell populations (S2AcRVGP) had a decreased ability to grow and consume substrates, when compared to the non‐transfected cells (S2). They were shown, by PCR, to express the RVGP gene and mRNA and, by immunoblotting, to synthesize the rRVGP in its expected molecular mass of 65 kDa. ELISA kinetic studies showed the rRVGP expression in cell lysates and supernatants attaining concentrations of 300 μg/L. By flow cytometry analysis, about 30% of the cells in the co‐transfected populations were shown to express the rRVGP. Cell populations selected by limiting dilution expressed higher rRVGP yields. Mice immunized with rRVGP were shown to synthesize antibodies against rabies virus and be protected against experimental infection with rabies virus. The data presented here show that S2 cells can be suitable hosts for the rRVGP expression, allowing its synthesis in a high degree of physical and biological integrity.

No abstract

Monoclonal antibodies (MAb) against Taenia crassiceps and Taenia solium cysticerci were produced and showed cross-reactivity with a 14-kDa protein from T. solium and with 18-and 14-kDa proteins from T. crassiceps. These MAbs and antibodies from cerebrospinal fluid (CSF) as well as serum samples from patients with neurocysticercosis (NC) reacted with 18-and 14-kDa T. crassiceps proteins purified by immunoaffinity chromatography using a Sepharose column coupled with MAbs (anti-excretory/secretory or anti-vesicular fluid antigens). Immunoaffinity-purified 18-and 14-kDa proteins were used in the design of a diagnostic enzymelinked immunosorbent assay (ELISA) to detect antibodies in 23 CSF and 20 serum samples from patients with NC, showing 100% sensitivity. The test specificity was determined using 42 noninflammatory CSF samples and 70 inflammatory CSF samples from patients with other neurological disorders (OND), showing 100% and 99.1% (confidence interval, 97.3% to 100%) specificity, respectively. A false-positive CSF sample result in the OND group was from a human immunodeficiency virus-positive patient with meningoencephalitis. By using serum samples from 194 healthy individuals, the specificity was 100%. Analysis of an additional 16 serum samples from individuals with other parasitic diseases (13 with intestinal parasitosis and 3 with schistosomiasis) showed negative results. Three (10%) serum samples from patients with hydatidosis were positive in our ELISA and in ELISA with T. solium cysticerci antigens. Two of them were also positive by immunoblotting. The use of 18-and 14-kDa T. crassiceps immunoaffinity-purified proteins for detection of anti-cysticercus antibodies in CSF and/or serum samples using an ELISA system showed a good performance and high specificity for serum samples, dispensing with the use of confirmatory tests, such as immunoblotting, for checking specificity.

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to alpha(2)beta(1) integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to alpha(2)beta(1) integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant alpha(2)beta(1) integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant alpha(2)beta(1) integrin nor to cell-surface-exposed alpha(2)beta(1) integrin (alpha(2)-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and alpha(2)beta(1) integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.

No abstract

The antarctic fish Notothenia coriiceps neglecta synthesizes eight antifreeze glycopeptides (AFGP 1-8; Mr 2600-34,000) to avoid freezing in its ice-laden freezing habitat. We report here the sequence of one of its AFGP genes. The structural gene contains 46 tandemly repeated segments, each encoding one AFGP peptide plus a 3-amino acid spacer. Most of the repeats (44/46) code for peptides of AFGP 8; the remaining 2 code for peptides of AFGP 7. At least 2 of the 3 amino acids in the spacers could act as substrate for chymotrypsin-like proteases. The nucleotide sequence between the translation initiation codon (ATG) and the first AFGP-coding segment is G + T-rich and encodes a presumptive 37-residue signal peptide of unusual sequence. Primer extension establishes the transcription start site at nucleotide 43 upstream from ATG. CAAT and TATA boxes begin at nucleotides 53 and 49, respectively, upstream from the transcription start site. The polyadenylylation signal, AATAAA, is located approximately 240 nucleotides downstream from the termination codon. A mRNA (approximately 3 kilobases) was found that matches the size of this AFGP gene. Thus, this AFGP gene encodes a secreted, high-copy-number polyprotein that is processed posttranslationally to produce active AFGPs.