Sponge implant in Swiss mice as a model for studying loxoscelism.

Envenomation by Loxosceles spider bite leads to a set of signs and symptoms, called loxoscelism, which in most cases manifests through the dermonecrotic frame. The development of a smaller size animal model, of easy handling and maintenance, and lower cost is needed to study the loxoscelism pathogenesis. The inflammatory effects of the Loxosceles similis crude venom was evaluated considering neutrophil and macrophage activation, vasodilatation, hyperhaemia, edema and hemorrhage and TNF-α and VEGF production using the murine sponge implant model. Thirty two male Swiss mice (6-8 weeks old) were implanted subcutaneously with polyether-polyurethane sponge discs. Fourteen days post implantation, animals were separated into two groups: (1) control group--16 mice received 30 µL of saline intra-implant; (2) treated group-sixteen mice injected with 0.5 µg/30 µL of L. similis crude venom intra-implant. The animals were euthanized with xylazine/ketamine after 1 and 4 h post- injection. Microscopically, implants of the treated groups presented an acute inflammation characterized by: neutrophilic infiltrate, edema, vasodilatation hyperhaemia, and severe hemorrhage. Some vessels presented ruptured walls. Under morphometric analysis, vessel area was bigger in the treated groups compared with the control ones. The biochemical parameters, hemoglobin content, inflammatory enzyme activities (myeloperoxidase and n-acethyl-ß-D glucosaminidase) and levels of the cytokines, TNF-α and VEGF, were also significantly higher in the venom-treated groups. The effects of Loxosceles venom in the granulation tissue of the implant in mice were similar to those observed in cutaneous loxoscelism in other species (human and rabbits). Consequently, the murine sponge implant model provides a new method to investigate cellular/molecular mechanisms associated with cutaneous loxoscelism.

Biochemical profile of dogs experimentally envenomed with Tityus serrulatus scorpion venom.

The aim of this study was to evaluate the canine blood and urinary profiles after envenomation by Tityus serrulatus venom. Twelve dogs were randomly distributed into two equal groups. Control group animals received 0.5 mL phosphate buffered saline (PBS) injected subcutaneously into the internal portion of the left thigh, whilst dogs in the envenomed group were injected with scorpion venom (250 microg/kg in 0.5 mL PBS). No significant alterations were detected in the urine of envenomed dogs. Levels of plasma glucose and serum urea, creatinine, total protein, potassium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), and amylase were determined. Semi-quantitative analysis of serum cardiac troponin I (cTnI) was performed using an immunochromatographic test. The concentrations of cortisol and insulin were determined using commercial radioimmunoassay kits. Increases in serum cortisol levels in experimental group animals coincided with hyperglycaemia and was probably a response to pain. Increased insulin levels were observed during the hyperglycaemic peaks. Envenomed dogs presented discreet increases in ALT, AST and CK, but no alterations in LDH, amylase, cTnI, urea, creatinine and potassium levels were observed. It was concluded that the venom of T. serrulatus induces blood and urinary biochemical changes in dogs.

Activities against hemostatic proteins and adrenal gland ultrastructural changes caused by the brown widow spider Latrodectus geometricus (Araneae: Theridiidae) venom.

Brown widow spider (BrWS) (Latrodectus geometricus) venom produces intense systemic reactions such as cramps, harsh muscle nociceptive, nauseas, vomiting and hypertension. The proposed pathogenic mechanisms resulting in these accidents have principally been damages occurring at the nervous system. However, it is suspected that there is also damage of the adrenal glands, as a result of the experimental animal's clinical manifestations, which developed symptoms compatible with acute adrenal insufficiency. We have currently found that the adrenal gland is damaged by this venom gland homogenates (VGH) producing severe alterations on cortex cells resulting in death by acute adrenal insufficiency. In general, the ultrastructural study on the glands of mice under transmission electronic microscopy observations showed alterations in the majority of the intracellular membranes within 3 to 24h. BrWSVGH also showed specific actions on extracellular matrix proteins such as fibronectin, laminin and fibrinogen. In addition, zymogram experiments using gelatin as substrates detected gelatinolytic activity. The molecular exclusion fractionation of crude BrWSVGH resulted in 15 fractions, of which F1 and F2 presented alpha/beta-fibrinogenase and fibronectinolytic activities. Fractions F6, F14 and F15 showed only alpha-fibrinogenase activity; in contrast, the gelatinolytic action was only observed in fraction F11. Only metalloproteinase inhibitors abolished all these proteolytic activities. Our results suggest that adrenal cortex lesions may be relevant in the etiopathogenesis of severe brown widow spider envenoming. To our knowledge, this is the first report on adrenal gland damages, fibrinogenolytic activity and interrelations with cell-matrix adhesion proteins caused by L.geometricus VGH. The venom of this spider could be inducing hemostatic system damages on envenomed patients.

The kinin system in the envenomation caused by the Tityus serrulatus scorpion sting.

Several lines of evidence in experimental animals indicate that the kinin system may participate in the pathogenesis of envenomation by the Tityus serrulatus (Ts) scorpion sting, but there are no studies in humans with regard to this system. In this study, we evaluated the plasma levels of high-molecular (HKg) and low-molecular (LKg) weight kininogens (detected by ELISA), the activities of plasma or tissue kallikreins and kininase II (enzymatic action upon selective substrates), and the Ts plasma venom levels (ELISA). A total of 27 patients (12 males) aged 12-72 were evaluated immediately at hospital admittance. According to the severity of envenomation, patients were classified as mild (n = 15), moderate (n = 8), and severe cases (n = 4). Controls were paired for age and sex. Plasma venom levels were associated with the severity of envenomation. Severe cases presented lower levels of LKg in relation to mild and controls. Inverse correlations were seen between LKg levels and the venom concentration. The results of this study suggested that the kinin system may participate in the pathogenesis of human Ts envenomation and knowledge about this system may be useful to develop new strategies to reduce the damage caused by scorpion envenomation.

Production of monoclonal antibodies capable of neutralizing dermonecrotic activity of Loxosceles intermedia spider venom and their use in a specific immunometric assay.

We have produced 13 mAbs for Loxosceles intermedia crude venom. Twelve were reactive against proteins of 32-35 kDa and one of these Li mAb(7) showed high neutralizing potency for the dermonecrotic activity of L. intermedia venom. This Li mAb(7) showed no cross-reactivity, with Loxosceles laeta (Brazil), L. laeta (Perú) and Loxosceles gaucho venoms. The mAbs were produced by immunization with the crude venom and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia whole venom or dermonecrotic fraction (DNF) as antigens coated onto microtitre plates. A sensitive two-site immunometric assay was designed and shown to be useful for identifying and quantifying DNF from L. intermedia in biological samples. The Li mAb(7) coated onto microtitre plates and hyperimmune horse anti-L. intermedia IgGs prepared by immunoaffinity chromatography and conjugated to horseradish peroxidase were used to set up a sandwich-type ELISA. Measurable absorbance signals were obtained with 0.2 ng of L. intermedia crude venom per assay.

Acid-base balance following Tityus serrulatus scorpion envenoming in anaesthetized rats.

In the present work the pH and arterial blood gases were measured in fasted and fed male albino rats, weighing 297 +/- 13 g, anaesthetized with urethane (1.4 g/kg, i.p.) before and after injection of T1 fraction from Titys serrulatus scorpion venom, during 60 min. Arterial blood samples were collected at 0, 5, 15, 30 and 60 min for pH, pCO2, pO2, bicarbonate and base-excess analysis. The data showed that the scorpion toxin induced a continuous drop in the blood pH along the time. Hypercapnia and hypoxemia peaking at 30 min and followed by a recovery towards normal values at 60 min were also observed. A pronounced decrease in the blood bicarbonate levels at 60 min and negative base-excess values along with time were evident at 60 min. The comparisons between fasted and fed animals have shown that in the last group the effects of scorpion toxin on the arterial blood gases were less pronounced. We conclude that T1 fraction of Tityus serrulatus scorpion venom induces in anaesthetized rats an acute respiratory acidosis followed by metabolic acidosis.

ELISA for the detection of venom antigens in experimental and clinical envenoming by Loxosceles intermedia spiders.

Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Loxosceles intermedia spider venom. Hyperimmune horse anti-Loxosceles intermedia IgGs were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate the circulating antigens in mice that were experimentally inoculated with L. intermedia venom from those inoculated with L. gaucho, L. laeta, and Phoneutria nigriventer spider venoms, Tityus serrulatus scorpion venom and Bothrops jararaca, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.8 ng of venom per assay. The ELISA also detected antigens in the sera of patients envenomed by L. intermedia. Therefore, after standardization for clinical use this ELISA may be a valuable tool for clinicians and epidemiologists.

Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus.

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD) corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients.

Search of intravascular hemolysis in patients with the cutaneous form of loxoscelism.

Haptoglobin assay, a highly sensitive method to detect intravascular hemolysis was carried out in the sera of 19 patients referred to Hospital Vital Brazil with the cutaneous form of loxoscelism in order to investigate the occurrence of mild intravascular hemolysis. Data from this series did not show decreased levels haptoglobin, ruling out intravascular hemolysis in these patients with cutaneous form of loxoscelism.

[Hemolysis induced by Loxosceles laeta venom. In vitro experience]. / Estudio de la hemolisis inducida por veneno de Loxosceles laeta. Experiencia in vitro.

To study the effects of loxosceles laeta venom on red blood cells and the possible factors involved in hemolysis during arachnidism, in vitro models were designed to measure the role of loxosceles venom, calcium, complement and antibodies in the mechanism of red blood cell destruction. The degree of basal hemolysis was measured in a 5% suspension of group O, Rh (+) red blood cells in pH 7.4 buffer. In a similar suspension spider venom was added in amounts equivalent to one venom gland. After 72 hours of incubation, basal hemolysis was 5.59 +/- 2.04% which increased to 26.01 +/- 7.9% adding venom (p < 0.001). Adding calcium to the incubation medium, hemolysis increased to 88.5 +/- 7.16% (p < 0.001). Incubating red blood cells with control human serum and venom, hemolysis was 14.58 +/- 2.42%, which decreased significantly to 6.85 +/- 3.35% when serum was heat inactivated; this demonstrates an effect of the presence of complement. We did not find antivenom antibody production in patients with arachnidism 10, 15 or 30 days after the spider bite. It is thus demonstrated that loxosceles laeta venom has a direct lytic action on red blood cells that is calcium and complement dependent and is not mediated by antibodies or other substance.

Methemoglobinemia associated with locoxoscelism

Vinte e cinco pacientes que apresentaram a forma cutânea do loxoscclismo foram estudados após a picada, determinando-se a glicose-6-fosfato desidrogenase, glutationa redutase e fato desidrogenase, glutationa redutase e glutationa peroxidase critrocitárias, haptoglobina e latico desidrogenase séricas, bilirrubina, reticulócitos e meta-hemoglobina. Näo foi observada hemólise aumentada, mas foi detectado aumento da meta-hemoglobina em 54% dos casos: em 7% entre 1,1% e 2%, em 27% variou de 2,1% a 4%, e em 20% de 4,1 a 8%. Amostras de sangue de um indivíduo normal do grupo 0 de uma paciente que exibiu metahemoglobina após picada por Loxosceles foram incubadas separadamente com anti-soros contra Loxosceles gaucho, Crotalus terridicus e Bothrops jararaca, com veneno de Loxosceles gaucho e fenol a 30%, e näo se detectou aumento de meta-hemoglobina depois de 1, 4, 8 e 15 dias em todas as amostras. Por ocasiäo do 25§ dia, todas as amostras, inclusive os controles, exibiram discreta diminuiçäo da atividade da meta-hemoglobina redutase. Os dados sugerem que a meta-hemoglobina que ocorreu em 54% dos pacientes provavelmente decorreu do metabolismo do veneno, uma vez que o veneno in natura näo induziu meta-hemoglobinemia

Methemoglobinemia associated with loxoscelism.

In twenty five patients who presented the cutaneous form of loxoscelism, serum haptoglobin and lactic dehydrogenase, erythrocyte glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, methemoglobin, bilirubin and reticulocytes were investigated after bite. No hemolysis was detected but an increase in methemoglobin was found in 54% of the cases; in 7% it was between 1.1% and 2%, in 27% it ranged from 2.1% to 4%, and in 20% from 4.1% to 8%. Blood samples of a normal, blood group 0 individual and of a patient who exhibited methemoglobinemia after Loxosceles bite were incubated separately with antisera against Loxosceles gaucho, Crotalus terrificus, Bothrops jararaca, with Loxosceles gaucho venom and 0.3% phenol. No methemoglobin was found after 1, 4, 8 and 15 days in both sets of samples. At the 25th day all the samples, including the controls, exhibited similar methemoglobin reductase decrease. The data suggest that the methemoglobinemia which occurs in 50% of the patients probably arises from in vivo venom metabolism, inasmuch as the crude venom does not induce methemoglobinemia.