Polyvalent horse F(Ab`)2 snake antivenom: Development of process to produce polyvalent horse F(Ab`)2 antibodies anti-african snake venom

A method to obtain polyvalent anti-Bitis and polyvalent-anti-Naja antibodies was developed by immunizing horses with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambicacrude venoms. Antibody production was followed by the ELISA method during the immunization procedure. Once the desired anti-venom antibody titers were attained, horses were bled and theimmunoglobulins were separated from the sera by (NH4)2SO4 precipitation, cleaved with pepsin and filtered through a 30 kDa ultrafiltration membrane. F(ab´)2 fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 m membranes. The resulting F(ab´)2 preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited high antibody titers, as assayed bythe ELISA method. In addition, the preparations possess a significant capacity to neutralize the lethalityof venoms, as estimated by ED50 determination in mouse assay and are free of toxic substances, pyrogen and bacterial or fungal contaminations.

Expression of the virulence factor, BfpA, by enteropathogenic Escherichia coli is essential for apoptosis signalling but not for NF-kappaB activation in host cells.

Localized adherence (LA) of enteropathogenic Escherichia coli (EPEC) to epithelial cells results in attaching and effacing of the surface of these cells. LA depends on the gene bfpA, which codes for the BfpA protein. We found that EPEC-E. coli adherence factor (EAF)((+)), expressing BfpA, significantly reduced HeLa cell viability in comparison with EPEC-EAF((-)), as evaluated by the mitochondrial-dependent succinate dehydrogenase conversion of 3'-[4,5,-dimethylthiazol-2yl]2,5-diphenyltetrazolium bromide (MTT) to its formazan. Apoptosis accounts for a substantial loss of the cell viability, because the cells incubated with EPEC-EAF((+)) or with cloned BfpA (data not shown), but not with EPEC-EAF((-)), were positive for annexin-V binding, demonstrated chromatin condensation and nuclei fragmentation and exhibited a high level of caspase-3 activity. Because the blockade of bacterial cell-surface-associated BfpA by anti-BfpA immunoglobulin (Ig)Y antibody suppressed apoptotic death induced by EPEC-EAF((+)), BfpA may be the trigger for apoptosis. Both EPEC-EAF((+)) and EPEC-EAF((-)), as well as recombinant BfpA (data not shown), activated nuclear factor (NF)-kappaB in a similar manner as analysed by the electrophoretic mobility shift assay (EMSA). EMSA supershift analysis demonstrated the presence of p65/RelA in a DNA-binding complex. In contrast to DNA binding, NF-kappaB-dependent reporter gene transactivation was stimulated more strongly by EPEC B171/EAF((+)), suggesting a role for this virulence factor in the regulation of transcriptional activity of NF-kappaB. Because suppression of NF-kappaB activation by BAY11-7085, a NF-kappaB inhibitor, neither induced apoptosis by itself nor blocked apoptosis induction by EPEC-EAF((+)), it may be suggested that apoptosis is not regulated by the NF-kappaB pathway in HeLa cells.

Skin morphology of the mutant hairless USP mouse.

The morphology of the skin of the mutant hairless USP mouse was studied by histological, histochemical and immunohistochemical methods and compared to the skin of BALB/c mice. Representative sections of the dorsal skin from mice of both strains aged 18 days, and 1, 3, 6, and 8 months were studied. Sections stained with hematoxylin and eosin showed cystic formations called utricles and dermal cysts in the dermis that increased in size and number during growth. Skin thickness increased significantly at 8 months. Sections stained with picrosirius and examined with polarized light, displayed different colors, suggesting different thicknesses of dermal collagen fibers (probably types I and III). Weigert, Verhoeff and resorcin-fuchsin stains revealed fibers of the elastic system. The PAS and Alcian blue methods revealed neutral and acid glycosaminoglycans in the skin ground substance of both mouse strains. Immunohistochemical staining for fibronectin and laminin did not show differences between the mutant and BALB/c mice. Mast cells stained by the Gomori method and macrophages positive for HAM 56 antibodies were observed in both mouse strains. Except for the presence of enlarged cysts in the hairless strain, no qualitative differences were found during development of the skin of BALB/c and the mutant hairless mice.

Skin morphology of the mutant hairless USP mouse

The morphology of the skin of the mutant hairless USP mouse was studied by histological, histochemical and immunohistochemical methods and compared to the skin of BALB/c mice. Representative sections of the dorsal skin from mice of both strains aged 18 days, and 1, 3, 6, and 8 months were studied. Sections stained with hematoxylin and eosin showed cystic formations called utricles and dermal cysts in the dermis that increased in size and number during growth. Skin thickness increased significantly at 8 months. Sections stained with picrosirius and examined with polarized light, displayed different colors, suggesting different thicknesses of dermal collagen fibers (probably types I and III). Weigert, Verhoeff and resorcin-fuchsin stains revealed fibers of the elastic system. The PAS and Alcian blue methods revealed neutral and acid glycosaminoglycans in the skin ground substance of both mouse strains. Immunohistochemical staining for fibronectin and laminin did not show differences between the mutant and BALB/c mice. Mast cells stained by the Gomori method and macrophages positive for HAM 56 antibodies were observed in both mouse strains. Except for the presence of enlarged cysts in the hairless strain, no qualitative differences were found during development of the skin of BALB/c and the mutant hairless mice.

Two related thrombin-like enzymes present in Bothrops atrox venom

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80 percent homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii

Two related thrombin-like enzymes present in Bothrops atrox venom.

This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical K(m) on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.

Two BCG vaccine formulations prepared from the same strain with different J774 macrophage activation capacities and patterns of NF-kappaB induction.

Two BCG vaccine formulations of the Moreau strain, commercially manufactured for anti-tuberculosis vaccination, ID-BCG, or anti-cancer adjuvant therapy, Onco-BCG, were compared for immunogenic activity in vitro. The growth rates of both vaccines in murine macrophages were the same, however, Onco-BCG induced stronger and longer-lasting secretion of TNF-alpha, IL-6 and nitric oxide. Onco-vaccine was also more potent in inducing NF-kappaB p65/p50 DNA-binding activity whilst in ID-BCG-infected cells the activity was transient and then gradually replaced by the transcriptionally inactive homodimer p50/p50. Comparative analysis of mycobacterial antigens of the two vaccines demonstrated a difference in expression of the 19 kDa and 38 kDa lipoproteins detected only in Onco-BCG extracts. These results suggest that these molecules may be responsible for the vigorous activation of macrophages induced by the Onco-vaccine. The data obtained show that vaccines from the same BCG strain, when manufactured differently, can vary significantly in their antigen expression and, consequently, in their capacity for macrophage activation which could contribute to the difference in their immunopotentiating effects.

Edema induction by the disintegrin-like/cysteine-rich domains from a Bothrops atrox hemorrhagin.

Viperine and crotaline snake venoms contain one or more hemorrhagic metalloproteases called hemorrhagins. The most potent hemorrhagins belong to the P-III class and have, in addition to the protease domain, disintegrin-like and cysteine-rich domains. Although proteolytic degradation of vascular endothelium basement membrane has been established to be the main factor responsible for hemorrhage, several studies reveal other factors that actually do facilitate this process. Recent evidence has shown that the nonprotease domains of the P-III class hemorrhagins are able to inhibit the platelet aggregation by blocking essential procoagulant integrins on platelets. In this study we report the identification of a hemorrhagin from Bothrops atrox venom. This enzyme, a P-III class metalloprotease, undergoes an apparent spontaneous degradation, releasing a proteic fragment containing the disintegrin-like/cysteine-rich domains. This fragment shows the capability to induce an edematogenic process, suggesting the existence of a still unknown nonenzymatic mechanism of vascular permeability increase.

Development of snake antivenom antibodies in chickens and their purification from yolk.

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 microliters/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 microliters/3LD50 and 1.0 ml neutralised 0.075 mg of venom.

Purification and variability in thrombin-like activity of Bothrops atrox venom from different geographic regions.

Bothrops atrox snake venoms from two different Amazon regions, i.e., Manaus, AM (3 degrees 0.6'40"S; 60 degrees 0.1'6.0"W) and Tucurui, PA (3 degrees 0.42'30"S; 49 degrees 0.41'45"W), were analyzed with respect to the thrombin-like activity component by elution profile on gel-filtration and reverse phase HPLC chromatography, electrophoretic mobility on SDS-PAGE, and enzymatic activity on fibrinogen. Despite some individual discrepancies among venom specimens, the thrombin-like activity present in the Manaus pool was eluted earlier compared with the Tucurui pool but its enzymatic specific activity on thrombin was lower (s.a. = 6.0) than that observed in the Tucurui pool (s.a. = 134.0). However, the electrophoretic mobilities of the pools were similar, with most protein bands being concentrated around three main regions, i.e., protein bands with an apparent mr of 100 kDa, of 38-37 kDa and 30 kDa. However, no significant differences were observed in amidolytic activity on the synthetic substrate Tos-Gly-Pro-Arg-pNa, and there was no correlation between thrombin-like and amidolytic activities. A 32 kDa protein endowed with thrombin-like activity and specific activity of 2444 recognized and neutralized by horse anti-B. atrox antivenom, was purified by the successive use of gel filtration, electrofocusing and reverse phase HPLC.

Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom.

We have provided evidence that: (a) lethality of mice to crude Bothrops venom varies according the isogenic strain (A/J > C57Bl/6 > A/Sn > BALB/c > C3H/HePas > DBA/2 > C3H/He); (b)BALB/c mice (LD50=100.0 microg) were injected i.p. with 50 microg of venom produced IL-6, IL-10, INF-gamma, TNF-alpha and NO in the serum. In vitro the cells from the mice injected and challenged with the venom only released IL-10 while peritoneal macrophages released IL-10, INF-gamma and less amounts of IL-6; (c) establishment of local inflammation and necrosis induced by the venom, coincides with the peaks of TNF-alpha, IFN-gamma and NO and the damage was neutralized when the venom was incubated with a monoclonal antibody against a 60 kDa haemorrhagic factor. These results suggest that susceptibility to Bothrops atrox venom is genetically dependent but MHC independent; that IL-6, IL-10, TNF-alpha, IFN-gamma and NO can be involved in the mediation of tissue damage; and that the major venom component inducers of the lesions are haemorrhagins.

Role of membrane-bound IgM in Trypanosoma cruzi evasion from immune clearance.

We have recently described that Trypanosoma cruzi parasites of the reticulotropic Y strain increase their resistance to antibody-induced clearance during their interaction with the vertebrate host immune system. In the present study, we observed that trypomastigotes of the myotropic CL strain isolated from normal host also display an increased resistance to immune clearance when compared to parasites obtained from immunosuppressed donors. Through fluorescence-activated cell sorting analysis, we have observed that the high expression of membrane-bound IgM antibodies on Y and CL trypomastigotes correlates with their enhanced resistance to Ig-induced clearance. Trypomastigotes from normal mice were essentially refractory to the in vitro binding of immunoglobulins, showing that their membrane structures were completely covered by IgM antibodies. These findings suggest that this isotype does not efficiently mediate immune clearance. Moreover, membrane-bound IgM antibodies limited the amount of IgG attached to the parasite and, as a consequence, impaired efficient immune clearance. Through this mechanism, trypomastigotes of T. cruzi could increase their persistence in the bloodstream thus favoring parasite transmission to its hematophagous host vector in the early acute phase of the disease.

Ig-isotype patterns of primary and secondary B cell responses to Plasmodium chabaudi chabaudi correlate with IFN-gamma and IL-4 cytokine production with CD45RB expression by CD4+ spleen cells.

In this work, the authors analysed T and B lymphocyte subsets and cytokine production in the spleen of BALB/c mice during polyclonal lymphocyte activation (primary infection) and parasite-specific response to Plasmodium chabaudi chabaudi (secondary infection). The secondary response was evaluated in fully immunoprotected animals, 60 days after a chloroquine-cured infection. The authors observed that in polyclonal lymphocyte activation antibody-secreting cells of all isotypes increased, with predominance of IgG2a and IgG3 classes. At that time, IFN-gamma was largely produced, but IL-4/IL-5 were just slightly enhanced. In mice re-infected after 60 days, the Ig-isotype pattern was restricted to IgG1 and only IL-4/IL-5 were produced. In both responses, however, the levels of IL-2 were greatly reduced, while those of IL-10 were enhanced to similar levels. The different involvement of Th1 and Th2 cells in both responses was confirmed through analysis of CD45RB expression by CD4+ cells. The authors observed that CD45RBhigh cells were the major CD4+ subpopulation in primary infected mice, while CD45RBlow cells predominated in 60 days re-infected animals. Moreover, the great majority of activated (large) CD4+ cells in the primary infection belonged to the CD45RBhigh subset, while after reinfection most of the CD4+ large had a CD45RBlow phenotype.

Detection of Trypanosoma-decay accelerating factor antibodies in mice and humans infected with Trypanosoma cruzi.

Highly purified Trypanosoma-decay accelerating factor (T-DAF), a 87-93-kD glycoprotein present on the surface of metacyclic and trypomastigote forms of Trypanosoma cruzi, was used as antigen to evaluate the presence of specific serum antibodies in experimentally infected mice and patients with Chagas' disease by enzyme-linked immunosorbent assay (ELISA). Mouse T-DAF antibodies were first recorded on day 7 postinfection, reached maximal concentration on day 30, and maintained at positive titers thereafter. High immunogenicity was clearly demonstrated by the detection of T-DAF antibodies in 96% of the sera collected from chagasic patients in either the acute or the chronic phase of disease. Control sera from normal individuals and from patients with leishmaniasis or other chronic infections did not give positive results. Serologic evaluation using T-DAF as antigen did not discriminate between patients with the cardiac and the digestive forms of the disease. The performance of the T-DAF ELISA was compared with that of conventional screening tests for Chagas' disease (indirect immunofluorescence and hemagglutination). The T-DAF ELISA test showed a sensitivity of 96%, a specificity of 100%, an efficiency of 99%, a positive predicted value of 100%, a negative predicted value of 98%, and a kappa index of 0.96, thus indicating that it can be successfully used for the serodiagnosis of T. cruzi infection in humans.

A new mutant hairless mouse with lymph node hyperplasia and late onset of autoimmune pathology.

Adult BALB/c male mice were injected with a single dose of ethyl nitroso urea (ENU; 250 mg/kg, i.p.) and mated to C57BL/6, DBA/2 and A/J adult females 13 weeks later. F1 males were mated with BALB/c females and F2 females were than backcrossed to the F1 parents. One BALB/c male mouse thus treated gave origin to a mutant presenting hair and skin alterations similar to those of natural hairless mutants. The new mutation is located on chromosome 14 near the Es10 locus, and probably at the same locus for the hairless mutation. Similar to the hairless mouse, this new mutant has a normal phenotype at birth and after three weeks starts to loose hair which is never replaced. Additionally, the skin becomes thickened and wrinkled. One feature that distinguishes this mutant from other hairless mice is the peculiar enlargement of its axillary and cervical lymph nodes. The new mutant develops membranoproliferative glomerulonephritis similar to the rhino mouse, one of the hairless allele mutants already described in the literature, but with a much later onset.

A new mutant hairless mouse with limph node hyperplasia and late onset of autoimmune pathology

Adult BALB/c male mice were injected with a single dose of ethyl nitroso urea (ENU; 250 mg/Kg, ip) and mated to C57BL/6, DBA/2 AND A/J adult females 13 weeks later. F1 males were mated with BALB/c females and F2 females were then backcrossed to the F1 parents. One BALB/c male mouse thus teated gave origin to a mutant presenting hair and skin alterations similar to those of natural hairless mutants. The new mutation is located on chromosome 14 near the Es10 locus, and probably at the same locus for the hairless mutation. Similar to the hairless mouse, this new mutant has a normal phenotype at birth and after three weeks starts to loose hair which is never replaced. Additionally, the skin becomes thickened and wrinkled. One feature that distinguishes this mutant from other hairless mice is the peculiar enlargement of its axillary and cervical lymph nodes. The new mutant develops membranoproliferative glomerulonephritis similar to the rhino mouse, one of the hariless allele mutants already described in the literature, but with a much later onset

Antigenic cross-reactivity among the venoms from several species of Brazilian scorpions.

The venoms of seven species of scorpions living in different regions of Brazil were analysed with regard to their lethality, antigenic cross-reactivity and ability to induce antibody production. In mice, the tested scorpion venoms can be grouped as: (a) highly toxic: Tityus stigmurus Thorell (LD50 = 0.773 mg/kg), Tityus bahiensis (Perty) (LD50 = 1.062 mg/kg), Tityus serrulatus Lutz and Mello (LD50 = 1.160 mg/kg), and Tityus costatus (Karsch) (LD50 = 1.590 mg/kg); (b) moderately toxic: Tityus cambridgei Pocock (LD50 = 12.136 mg/kg); and (c) practically nontoxic: Rhopalurus agamemnon (Koch) (LD50 = 36.363 mg/kg), and Brotheas amazonicus Lourenço (LD50 = 90.909 mg/kg). On electrophoresis the venoms showed many protein bands displayed along the chromatogram, most of them cross-reacting in immunoelectrophoresis and immunoblotting using horse anti-T. serrulatus, anti-T. bahiensis or anti-T. serrulatus+T. bahiensis sera as probes. The antibodies present in these antivenoms combine with venom components as measured in vitro by the ELISA assay, and neutralize their lethal effects in vivo. These results indicate that horse anti-venoms against a mixture of T. serrulatus and T. bahiensis venoms or only against T. serrulatus venom yield an antibody population able to neutralize the toxic effects found in all venoms studied.

Antigenic cross-reactivity among the venoms from several species of Brazilian scorpions

The venoms of seven species of scorpions living in different regions of Brazil were analysed with regard to their lethality, antigenic cross-reactivity and ability to induce antibody production. In mice, the tested scorpion venoms can be grouped as: (a) highly toxic: Tityus stigmurus Thorell (), Tityus bahiensis (Perty) , Tityus serrulatus Lutz and Mello (, and Tityus costatus (Karsch) ; (b) moderately toxic: Tityus cambridgei Pocock ; and (c) practically nontoxic: Rhopalurus agamemnon (Koch) , and Brotheas amazonicus Lourenço . On electrophoresis the venoms showed many protein bands displayed along the chromatogram, most of them cross-reacting in immunoelectrophoresis and immunoblotting using horse anti-T. serrulatus, anti-T. bahiensis or anti-T. serrulatus + T. bahiensis sera as probes. The antibodies present in these antivenoms combine with venom components as measured in vitro by the ELISA assay, and neutralize their lethal effects in vivo. These results indicate that horse antivenoms against a mixture of T. serrulatus and T. bahiensis venoms or only against T. serrulatus venom yield an antibody population able to neutralize the toxic effects found in all venoms studied.

Pro-inflammatory activities in elapid snake venoms.

1. Snake venoms from the genera Micrurus (M. ibiboboca and M. spixii) and Naja (N. naja, N. melanoleuca and N. nigricollis) were analysed, using biological and immunochemical methods, to detect pro-inflammatory activities, cobra venom factor (COF), proteolytic enzymes, thrombin-like substances, haemorrhagic and oedema-producing substances. 2. The venoms of the five snake species activate the complement system (C) in normal human serum (NHS) in a dose-related fashion, at concentrations ranging from 5 micrograms to 200 micrograms ml-1 serum. Electrophoretic conversion of C3 was observed with all venoms in NHS containing normal concentrations of Ca2+ and Mg2+, but only by venoms from N. naja and N. melanoleuca when Ca2+ was chelated by adding Mg(2+)-EGTA. 3. Purified human C3 was electrophoretically converted, in the absence of other C components, by the venoms from N. naja, N. nigricollis and M. ibiboboca. However, only the venoms from N. naja and N. melanoleuca contained a 144 kDa protein revealed in Western blot with sera against COF or human C3. 4. All venoms, at minimum concentrations of 30 ng ml-1, were capable of lysing sheep red blood cells, also in a dose-related fashion, when incubated with these cells in presence of egg yolk as a source of lecithin. Although the venoms from M. spixii and N. nigricollis showed detectable thrombin-like activity, these and the other venoms were free of proteolytic activity when fibrin, gelatin and casein, were used as substrates. 5. When tested on mice skin, all five venoms were capable of inducing an increase in vascular permeability and oedema, but were devoid of haemorrhagic producing substances (haemorrhagins). 6. These data provide evidence indicating that Elapidae venoms contain various pro-inflammatory factors which may be important in the spreading of neurotoxins throughout the tissues of the prey or human victim.

A critical evaluation of the expression of parasitemia in experimental Chagas' disease.

This paper aims to study the best way to express the parasitemia of Trypanosoma cruzi's experimentally infected animals. Individual scores may have a great variability, not emphasized by the majority of the authors. A group of 50 rats infected with 1 x 10(6) trypomastigotes of T. cruzi Y strain was used and the parasitemia was estimated by BRENER's method. The results showed that the median can avoid false results due to very high or low parasitemias but it does not have the mathematic properties necessary for analysis of variance. The comparison of the means of the original and transformed data, with their respective coefficients of variability (CV), showed that the logarithmic mean (Mlog) have the minor value of CV. Therefore, the Mlog is the best way to express the parasitemia when the data show great variability. The number of the animal for group did not affect the variability of data when the Mlog and CV were used.