Influence of the A and B subunits of cholera toxin (CT) and Escherichia coli toxin (LT) on TNF-alpha release from macrophages.

In this study an in vitro model was developed with the aim of investigating the modulatory effect of cholera toxin (CT) and its counterpart the heat labile toxin of Escherichia coli (LT) on TNF-alpha release induced by murine macrophages and primary human monocytes. Previous studies have demonstrated that the enzymatic activity of CT and LT molecules can inhibit TNF-alpha release by macrophages. The results obtained in this study showed that CT and LT are both, in a dose dependent manner, able either to induce or inhibit TNF-alpha release by murine macrophages and primary human monocytes. The results also showed that recombinant B subunits of CT and LT in the absence of their A subunit induce high levels of TNF-alpha release by macrophages and, in addition, increase the level of TNF-alpha release induced by LPS. The ability of both B subunits (CTB and LTB) in inducing TNF-alpha release by macrophages is not related to the level of LPS contamination, since direct measurements of LPS made in the samples employed in this study showed only traces of LPS (3.4 x 10(-8) EU/ml) which is in our system does not induce TNF-alpha release by macrophages. In contrast to the results obtained with the B subunits, incubation of cells with the A subunit of CT (CTA) inhibit TNF-alpha release induced by native CT, native LT, recombinant LTB and LPS. This inhibitory effect must be related to the activity of the A subunit since viability tests performed in terms of metabolic rate demonstrated that high concentrations of CTA are not toxic to the cells. The data presented herein demonstrate that the A subunits of CT and LT have an inhibitory effect on TNF-alpha release in macrophages, whereas their B subunits have a stimulatory effect on TNF-alpha. The results also suggest that the dose dependent bi-modal effect of native CT and native LT on TNF-alpha release by macrophages is a result of the combined effect of their individual A and B subunits.

Epidemiologic and clinical survey of victims of centipede stings admitted to Hospital Vital Brazil (São Paulo, Brazil).

We retrospectively analyzed 98 proven cases of centipede stings admitted to Hospital Vital Brazil, Butantan Institute, São Paulo, Brazil, between 1990 and 2007. Most stings occurred at the metropolitan area of São Paulo city (n=94, 95.9%), in the domiciles of patients (n=67, 68.4%), and during the warm-rainy season (n=60, 61.2%). The mean age of the victims was 32.0+/-18.8-years-old. Cryptops and Otostigmus genera were responsible for most cases. Around 86% of the patients sought medical care within 6h after the sting. Both lower (56.1 %) and upper limbs (41.8 %) were most frequently bitten, especially the feet and hands (89.8%). The most frequent local clinical manifestations were pain (94.9%), erythema (44.9%) and edema (21.4%), and the latter was mainly observed in patients bitten by Otostigmus spp. Supportive treatment was used in only 28.6% of the patients, namely administration of local anesthesia (9.2%) and systemic analgesia (13.3%). No sequels or complications were observed in patients, and the prognostic was benign.

Potamotrygon motoro stingray venom induces both neurogenic and inflammatory pain behavior in rodents.

Freshwater stingray accidents cause an immediate, intense, and unrelieved pain which is followed by edema, erythema and necrosis formation. Treatment for stingray envenomation is based on administration of analgesic, antipyretic and anti-inflammatory drugs. Concerning pain control, it is prescribed to immerse punctured limb on hot water to alleviate pain. There are no studies demonstrating specific targets on which stingray venom acts to promote pain. Therefore, the aim of this work was to investigate some mechanisms of Potamotrygon motoro venom (PmV) that contribute to nociception induction. Evaluating spontaneous pain behavior in mice injected i.pl. with PmV, it was seen that PmV induced both neurogenic and inflammatory pain. PmV also induced hyperalgesia in both mice and rats, evaluated through electronic von Frey and rat paw pressure test, respectively. Partial inhibition of hyperalgesia was observed in mice treated with cromolyn or promethazine, which indicated that mast cell and histamine via H1 receptor participate in the inflammatory pain. To search for some targets involved in PmVinduced hyperalgesia, the participation of TRPV1, calcium channels, neurokinins, CGRP, and norepinephrine, was evaluated in rats. It was seen that PmV-induced hyperalgesia occurs with the participation of neurokinins, mainly via NK1 receptor, CGRP, and calcium influx, through both P/Q and L-type voltage-dependent calcium channels, besides TRPV1 activation. The data presented herein indicate that PmV causes hyperalgesia in rodents which is dependent on the participation of several neuroinflammatory mediators.

Enzymatic and immunochemical characterization of Bothrops insularis venom and its neutralization by polyspecific Bothrops antivenom.

Herein we compared the biological activities of Bothrops insularis and Bothrops jararaca venoms as well as their neutralization by polyspecific Bothrops antivenom (PBA). On account of that, we investigated their antigenic cross-reactivity and the neutralization of lethal, myotoxic and defibrinating activities by polyspecific and species-specific antivenoms. Silver-stained SDS-PAGE gels evidenced many common bands particularly above 47 kDa between B. jararaca and B. insularis venoms. However, some protein bands between 46 and 28 kDa were observed exclusively in B. jararaca venom. Both venoms presented gelatinolytic, caseinolytic, fibrinogenolytic and phospholipase A(2) activities. No hyaluronidase activity was detected in both venoms by zymography. Polyspecific and species-specific antivenoms showed similar titers to B. jararaca and B. insularis venoms by ELISA, and recognized similar components by immunoblotting. The PBA was effective in neutralizing the lethal, myotoxic and defibrinating activities of both venoms as well as to abrogate microcirculatory disturbances induced by B. insularis venom. No statistically significant difference was observed for minimal hemorrhagic doses between both venoms. Antigenic cross-reactivity was evident between both venoms. Since toxic and enzymatic activities were similar, we speculate that B. insularis venoms can induce a local damage in humans comparable to that observed in other Bothrops venoms. Besides, the PBA was effective in neutralizing the toxic activities of B. insularis venom.

The Loxtox protein family in Loxosceles intermedia (Mello-Leitão) venom.

We isolated cDNA sequences coding for dermonecrotic/sphingomyelinases factor proteins from the brown spider Loxosceles intermedia, here named Loxtox proteins. The amino acid sequences based on cloned cDNA of several Loxtox proteins revealed at least six distinct groups of proteins expressed in the venom gland. The level of similarity among the toxins varied from 99% to 55%. The finding of several isoforms of Loxtox in the venom of this spider may reflect an evolutionary adaptation for different prey types and reinforces the idea of an efficient mutational mechanism in the venom gland of spiders.

Platelet participation in the pathogenesis of dermonecrosis induced by Loxosceles gaucho venom.

Loxosceles gaucho spider venom induces in vitro platelet activation and marked thrombocytopenia in rabbits. Herein, we investigated the involvement of platelets in the development of the dermonecrosis induced by L. gaucho venom, using thrombocytopenic rabbits as a model. L. gaucho venom evoked a drop in platelet and neutrophil counts 4 h after venom injection. Ecchymotic areas at the site of venom inoculation were noticed as soon as 4 h in thrombocytopenic animals but not in animals with initial normal platelet counts. After 5 days, areas of scars in thrombocytopenic animals were also larger, evidencing the marked development of lesions in the condition of thrombocytopenia. Histologically, local hemorrhage, collagen fiber disorganization, and edema were more severe in thrombocytopenic animals. Leukocyte infiltration, predominantly due to polymorphonuclears, was observed in the presence or not of thrombocytopenia. Thrombus formation was demonstrated by immunohistochemistry at the microvasculature, and it occurred even under marked thrombocytopenia. Taken together, platelets have an important role in minimizing not only the hemorrhagic phenomena but also the inflammatory and wound-healing processes, suggesting that cutaneous loxoscelism may be aggravated under thrombocytopenic conditions.

Biological activity of chronic phase antibodies eluted from sensitized Trypanosoma cruzi trypomastigotes.

Chronic infection with Trypanosoma cruzi induces high levels of antibodies that have lytic and clearance activities on bloodstream trypomastigote forms. These two activities were tested with antibodies eluted from parasites sensitized with serum obtained from mice in the chronic phase of infection. Parasites submitted to treatment for antibody elution were also tested. Our results show that antibodies eluted from the parasites are very efficient to induce lysis but unable to induce clearance. In addition, we observed that after being submitted to treatment for antibody elution the parasites still presented a slower but significant clearance and a high lytic activity. These results allow us to suggest that clearance inducing antibodies are mostly high affinity antibodies that could not be eluted from the parasites in our experimental conditions.

Envenoming by Bothrops jararaca in Brazil: association between venom antigenaemia and severity at admission to hospital.

The association between the clinical severity of Bothrops jararaca envenoming at admission and serum venom and plasma fibrinogen concentrations before antivenom administration is reported in 137 patients admitted to Hospital Vital Brazil, Instituto Butantan, São Paulo, Brazil, between 1989 and 1990. Other variables such as age, gender, site of the bite, use of tourniquet and the time interval between the bite and start of antivenom therapy, spontaneous systemic bleeding, and the 20 minute whole blood clotting test (20WBCT) at admission showed no association with either severity or serum venom antigen concentration (SVAC). Mean SVAC in patients with mild envenoming was significantly lower than in the group with moderate envenoming (P = 0.0007). Patients with plasma fibrinogen concentrations > 1.5 g/L had a lower mean SVAC than patients with plasma fibrinogen concentrations < or = 1.5 g/L (P = 0.02). Those admitted with a tourniquet in place had significantly higher plasma fibrinogen concentrations than those without a tourniquet (P = 0.002). A multiple logistic regression model showed independent risk factors for severity: bites at sites other than legs or forearms, SVACs > or = 400 ng/mL, and the use of a tourniquet. Rapid quantification of SVAC before antivenom therapy might improve initial evaluation of severity in B. jararaca bites.

A clinical and epidemiological study of Loxosceles spider envenoming in Santa Catarina, Brazil.

A clinical and epidemiological study of 267 cases of envenomation by Loxosceles spp. (loxoscelism), notified to Centro de Informações Toxicológicas de Florianópolis (Santa Catarina State, Brazil), was conducted between January 1985 and December 1995. Most of the incidents occurred along the coast of the mid-southern region of the state, during the warmest months. L. laeta and L. intermedia were identified as the causative agents. Cutaneous loxoscelism was clinically diagnosed in 232 (86.9%) patients with local pain (86.5%), oedema (80.5%), hyperaemia (79.8%) and necrosis (56.9%). Cutaneous-visceral loxoscelism was detected in 35 patients (13.1%) with intravascular haemolysis, manifested by jaundice (68.6%), oliguria (45.7%), dark urine (28.6%), haemorrhage (25.7%), anuria (8.6%) and shock (2.9%), besides the cutaneous effects. Specific antivenom was given to 125 patients (46.8%) and only 8 (6.5%) had mild reactions. Acute renal failure was observed in 17 cases (6.4%); 4 patients (1.5%) died, all of whom were children under 14 years old.

Changes of anti-Trypanosoma cruzi antibodies after gamma-irradiation of mice in the chronic phase of the infection.

The effect of sublethal whole body irradiation (800 rads) on the level and biological activities of antibodies in mice chronically infected with the CL strain of Trypanosoma cruzi was studied. Irradiated mice died, although a high parasitemia did not always preceded death. Before and after irradiation, a constant level of antibodies was detected by enzyme-linked immunosorbent assay and complement mediated lysis, but after irradiation the level of clearance antibodies was decreased. These results suggest that clearance antibodies are important in the control of the chronic phase of the infection.

Antigenic cross-reactivity of venoms from medically important Loxosceles (Araneae) species in Brazil.

Antigenic cross-reactivity between the components of venoms from three spiders of the genus Loxosceles, L. gaucho, L. laeta and L. intermedia, was studied. Species-specific antisera were prepared by immunization of rabbits with each venom. Anti-L. gaucho horse hyperimmune serum provided by the Butantan Institute for treatment of accidents with these spiders was also used. Separation by SDS-PAGE showed the existence of many common components in the three antigens. No individual antigen was observed. Analysis of the antisera by ELISA and Western blotting showed cross-reactivity as well as several common bands between the three venoms. The horse anti-L. gaucho venom serum recognized many common proteins when antigens of the other two species were used. Antigens in the range of 33,000-35,000 mol. wt showed most cross-reactivity. Both horse and rabbit anti-venom sera contained antibodies able to neutralize the lethal and dermonecrotic activities of the venom of the three species studied.

Neutralization of dermonecrotic and lethal activities and differences among 32-35 kDa toxins of medically important Loxosceles spider venoms in Brazil revealed by monoclonal antibodies.

Neutralization of dermonecrotic and lethal activities and differences among the principal toxic proteins (32-35 kDa) of medically important Loxosceles spider venoms in Brazil (Loxosceles gaucho, Loxosceles laeta and Loxosceles intermedia) were studied using monoclonal antibodies (MAbs) produced against the dermonecrotic component (35 kDa) of L. gaucho venom. MAb titers were 512,000 to homologous venom, between 2000 and 64,000 for L. intermedia venom and between 1000 and 64,000 for L. laeta venom. By Western blotting, MAbs could recognize mainly the 35 kDa protein of L. gaucho venom and with less intensity the 35 kDa protein of L. intermedia venom. These MAbs also recognized weakly or did not recognize the 32 kDa component of L. laeta venom. Only MoALg1 showed high affinity for L. gaucho venom and neutralized in vivo 90-97% of the dermonecrotic activity, besides delaying the lethality induced by homologous venom. MoALg1 maintained its capacity to neutralize the dermonecrotic activity, even when administered (i.v.) 6h after envenoming (i.d.). All MAbs obtained failed to neutralize the toxic activities of the heterologous venoms.These results suggest that different epitopes are present in the protein responsible for the dermonecrotic activity of Loxosceles venoms, and confirm the participation of other venom components during the local reaction process. This study also confirms the importance of antibodies for neutralization of dermonecrotic activity, even when administered some hours after envenoming, and emphasizes the differences of composition and toxicity of medically important Loxosceles venoms. These findings must be considered in order to improve loxoscelism immunotherapy.

Adjuvant effect of Loxosceles gaucho (South American brown spider) venom.

Injection of L. gaucho venom and antigens (ovalbumin, ovomucoid and bovine gamma globulin) into rabbit skin induced an intense local inflammatory lesion and resulted in a significant increase in the level of IgG antibodies to the antigen in both the primary and secondary humoral immune response. The adjuvant activity of the venom was associated with its high mol. wt components, which are responsible for the inflammatory lesion. Rabbits rendered unresponsive to the venom and injected with venom plus antigen presented a very mild local inflammatory reaction and no increase in antibody formation. When venom and antigen were injected simultaneously but at different skin sites no adjuvant effect was induced. However, when antigen was injected 4 hr after venom injection but at the same skin site a significant adjuvant effect was produced. Furthermore, when venom plus antigen was injected intradermally into mice, a species in which the venom does not cause an inflammatory skin lesion, no adjuvant effect was detected. It is suggested that the adjuvant effect of L. gaucho venom in rabbits is probably due to its ability to cause a local severe inflammatory reaction.

Dermonecrotic and lethal components of Loxosceles gaucho spider venom.

Loxosceles gaucho spider venom causes a typical dermonecrotic lesion in bitten patients and rarely causes lethal systemic effects. Gel filtration on Sephadex G 100 of L. gaucho spider venom resulted in three fractions: fraction A, containing the higher mol. wt components (approximately 35,000); fraction B, containing lower mol. wt components (approximately 15,000); and fraction C, containing very low mol. wt components (probably small peptides). The dermonecrotic and lethal activities were detected exclusively in fraction A. The venom and fraction A produced large dermonecrotic lesions in rabbits with necrosis spreading by gravity to the skin of the lateral body wall. Analysis by SDS-PAGE showed that the proteins contained in fraction A are approximately 35,000 and 33,000 mol. wt. Immunoblotting analysis showed that the proteins responsible for the dermonecrotic and lethal activity are very immunogenic and the first to be detected by antibodies during the course of immunization.

IgG antibodies to Loxosceles sp. spider venom in human envenoming.

The presence and specificity of IgG antibodies produced by patients with loxoscelism were studied. The loxoscelism diagnosis was supported mainly by clinical parameters. A search for IgG antibodies anti-Loxosceles gaucho venom in patients with loxoscelism submitted to serumtherapy showed antibodies in four out of 20 patients. The IgG antibodies were detected as early as 9 days and as late as 120 days after bite. The highest IgG antibody titer was 1:640 and the lowest was 1:80. Immunoblotting tests showed that human anti-L. gaucho IgG antibodies recognize preferentially the components responsible for the dermonecrotic and lethal activities of the venom. A comparison of the clinical picture, the level of serum IgG antibodies and the dose of antivenom administered suggest that there is no relationship between these parameters.

Thalassophryne nattereri fish venom: biological and biochemical characterization and serum neutralization of its toxic activities.

Envenomation by Thalassophryne nattereri fishes are an important medical problem in northeast of Brazil, causing in human victims considerable pain and edema followed by necrosis. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. Intradermal injection of the venom in the foot pad of mice induced local edema and hemorrhage followed a few hours later by necrosis. Subcutaneous injection of the venom induced systemic effects consisting in jerking motions, paralysis of hind limbs, erection of hair, rotational movements and violent convulsions followed by death. Dead animals showed hyperemia of the small intestine and lungs. The venom showed distinct edematous, necrotizing and hemolytic activities, a low level of hemorrhagic, myotoxic and proteolytic activities and no detectable phospholipase A2 activity. SDS-PAGE analysis of the crude venom showed at least 17 components with the major band located around Mw = 19,000. Almost all proteins stained by amido black were also revealed by Western blotting with antibodies to T. nattereri venom. Fractionation of the venom by either gel filtration or cation exchange chromatography resulted in a few distinct peaks but in both situations the biological activities were located in only one of the peaks which corresponded to basic proteins with approximately Mw = 47,000. Heating of the venom at 56 degrees C for 60 min completely destroyed its biological activities. All venom toxic activities except edema were completely neutralized after in vitro incubation with anti-T. nattereri serum.

Influence of sphingomyelin and TNF-alpha release on lethality and local inflammatory reaction induced by Loxosceles gaucho spider venom in mice.

It is well known that Loxosceles venom induces local dermonecrosis in rabbits, guinea pigs and humans but not in mice, although, depending on the dose, Loxosceles venom can be lethal to mice. In this work we demonstrate that mice injected intradermally in the dorsal area of the back can survive a lethal dose of Loxosceles gaucho venom and also develop an inflammatory reaction (with infiltration of leukocytes shown by histological analysis) at the local injection site when the venom is co-administered with sphingomyelin. It was observed that more venom was retained for a longer period of time at the local injection site when venom was co-administered with sphingomyelin. The presence of exogenous sphingomyelin did not influence significantly the release of TNF-alpha induced by L. gaucho venom. These results suggest that the action of venom on sphingomyelin, producing ceramide phosphate, causes the development of an inflammatory reaction, which in turn traps the venom in the local area for a long period of time and does not allow it to disperse systemically in a dose sufficient to cause death. Our findings also indicate that the size and availability of the local sphingomyelin pool may be important in determining the outcome of Loxosceles envenoming in different mammalian species.

Biological and immunochemical characterization of Micrurus altirostris venom and serum neutralization of its toxic activities.

Micrurus altirostris venom from Rio Grande do Sul State, Brazil, was characterized by its biological activities, immunochemical properties and electrophoretic pattern. The results showed a high edematogenic activity, whose peak was observed after 30min of venom injection, as well as a high indirect hemolytic activity. This venom was myotoxic, as shown by a peak of CK release at 6h after injection, and also by the appearance of muscular lesions characterized by necrosis, loss of striated muscle fibers, and the presence of vacuolization, edema and inflammatory infiltrate. This venom showed minimum proteolytic activity and no hemorrhagic, dermonecrotic or coagulant activities. Nonetheless, M. altirostris venom presented high lethal activity. Electrophoretic patterns of Micrurus frontalis and M. altirostris venoms showed different protein bands. Anti-elapidic serum could recognize M. frontalis (homologous) and M. altirostris (heterologous) venoms by Western blotting, and both venoms presented similar titers when assayed by ELISA. The results observed on neutralization tests showed that the anti-elapidic serum produced by Instituto Butantan neutralized myotoxic and hemolytic activities. However, this antivenom could not neutralize the lethal activity of M. altirostris venom. Thus, these data suggest that M. altirostris venom presents different biological, enzymatic and immunological characteristics from other Micrurus venoms, and some activities are not neutralized by the commercial anti-elapidic serum produced in Brazil.

Effect of Loxosceles gaucho venom on cell morphology and behaviour in vitro in the presence and absence of sphingomyelin.

This study was performed to investigate whether the toxic effects of Loxosceles gaucho venom on cells might be exerted via stimulators of TNF-alpha release generated by sphingomyelinase D--a major component of the venom. It was demonstrated that L. gaucho venom alone is unable to induce TNF-alpha release by J774A.1 cells, while in the presence of exogenous sphingomyelin it induces a high level of TNF-alpha release which is significantly increased by incubation with non-inactivated serum. Ceramide phosphate also induces TNF-alpha release in J774A.1 cells, but (unlike sphingomyelin/sphingomyelinase) the level of release is not influenced by the presence or otherwise of non-inactivated serum. L. gaucho venom does not induce proliferation of J774A.1 cells and even at high concentrations it does not affect their viability. J774A.1 cells, which prior to venom treatment were elongated and clumped, round up after venom treatment, but, revert to their original morphology after incubation with fresh medium. TNF-alpha resistant MRC-5 cells and TNF-alpha sensitive MCF-7 cells are susceptible to the toxic effect of both L. gaucho venom and ceramide phosphate. The results obtained in this study demonstrate that exogenous sphingomyelin can modulate, in vitro, the release of TNF-alpha induced by L. gaucho venom in mouse macrophages. In addition, the results also indicate that ceramide phosphate and L. gaucho venom are toxic to several different cell types, via a variety of mechanisms, some, but not all, of which may involve TNF-alpha as an intermediary.

Specificity and role of anti-Trypanosoma cruzi clearance antibodies.

Two strains of Trypanosoma cruzi (Y and CL) were used to study the specificity and role of anti-T. cruzi clearance antibodies. Clearance antibodies were only induced after immunization with living blood-stream trypomastigotes (Btrys) but not with dead parasites. Btrys of either strain were readily cleared from the circulation after passive immunization with anti-Y or anti-CL serum provided that the homologous strain was used. CL or Y Btrys sensitized in vitro with the homologous or heterologous antiserum and transferred to normal mice were cleared from the circulation only when the homologous antiserum was used. Clearance antibodies were removed from serum by absorption with the homologous but not with the heterologous strain. Clearance antibodies were removed from serum by absorption with living Btrys but not with fixed parasites. These results suggest that: a) the parasite epitopes involved in the clearance are peculiar to each strain, b) the clearance antibodies are specific to these epitopes, and c) a proper conformation of the parasite antigens is required for the induction and effector activity of the clearance antibodies.