Isolation and Characterization of IgM and IgY Antibodies from Plasma of Magellanic Penguins (Spheniscus magellanicus).

Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens.

Systemic toxicity of snake venom metalloproteinases: Multi-omics analyses of kidney and blood plasma disturbances in a mouse model.

Snakebite envenomation is classified as a Neglected Tropical Disease. Bothrops jararaca venom induces kidney injury and coagulopathy. HF3, a hemorrhagic metalloproteinase of B. jararaca venom, participates in the envenomation pathogenesis. We evaluated the effects of HF3 in mouse kidney and blood plasma after injection in the thigh muscle, mimicking a snakebite. Transcriptomic analysis showed differential expression of 31 and 137 genes related to kidney pathology after 2 h and 6 h, respectively. However, only subtle changes were observed in kidney proteome, with differential abundance of 15 proteins after 6 h, including kidney injury markers. N-terminomic analysis of kidney proteins showed 420 proteinase-generated peptides compatible with meprin specificity, indicating activation of host proteinases. Plasma analysis revealed differential abundance of 90 and 219 proteins, respectively, after 2 h and 6 h, including coagulation-cascade and complement-system components, and creatine-kinase, whereas a semi-specific search of N-terminal peptides indicated activation of endogenous proteinases. HF3 promoted host reactions, altering the gene expression and the proteolytic profile of kidney tissue, and inducing plasma proteome imbalance driven by changes in abundance and proteolysis. The overall response of the mouse underscores the systemic action of a hemorrhagic toxin that transcends local tissue damage and is related to known venom-induced systemic effects.

Impact of antivenom administration on the evolution of cutaneous lesions in loxoscelism: A prospective observational study.

BACKGROUND: Spiders of the genus Loxosceles are distributed throughout tropical and temperate regions worldwide. Loxosceles spp. bites may evolve to necrosis, with or without intravascular hemolysis. There is no consensus regarding the best treatment to prevent necrosis. The objective of this study was to evaluate the factors associated with the development of necrosis and the impact that antivenom administration has on the evolution of cutaneous loxoscelism. METHODOLOGY/PRINCIPAL FINDINGS: This was a prospective observational study carried out at a referral center for envenoming. Over a 6-year period, we included 146 patients with a presumptive or definitive diagnosis of loxoscelism. Depending on the symptom severity, a polyvalent anti-arachnid antivenom was administered or not-in 74 cases (50.7%) and 72 cases (49.3%), respectively. Cutaneous and systemic manifestations were assessed at admission and weekly thereafter. Adverse reactions to the antivenom were also evaluated. Cutaneous loxoscelism was observed in 141 cases (96.6%), and the spider was identified in 29 (19.9%). The mean time from bite to antivenom administration was 41.6 ± 27.4 h. After discharge, 130 patients (90.9%) were treated with corticosteroids, antihistamines and analgesics being prescribed as needed. The probability of developing necrosis was significantly lower among the patients who were admitted earlier, as well as among those who received antivenom (p = 0.0245). Among the 74 patients receiving antivenom, early and delayed adverse reactions occurred in seven (9.5%) and four (5.4%), respectively. Local infection was observed only in three (2.3%) of the 128 patients for whom that information was available. CONCLUSIONS/SIGNIFICANCE: Necrosis after a Loxosceles sp. bite appears to more common when hospital admission is delayed or when antivenom is not administered. In addition, the administration of a polyvalent anti-arachnid antivenom appears to be safe, with a relatively low rate of adverse reactions.

Autolysis at the disintegrin domain of patagonfibrase, a metalloproteinase from Philodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom.

Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37 degrees C, primarily producing a approximately 32.6 kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca(2+), patagonfibrase was only partially autolysed, giving rise mainly to one fragment of approximately 52.2 kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.

The absence of thrombin-like activity in Bothrops erythromelas venom is due to the deletion of the snake venom thrombin-like enzyme gene.

Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.

Haematological evaluation of patients bitten by the jararaca, Bothrops jararaca, in Brazil.

Complete blood counts are used frequently by physicians to assess and manage the development of complications of diseases. We studied 100 patients bitten by Bothrops jararaca snakes, and correlated their haematological values with the severity of envenoming and the development of complications. Patients who developed both local and systemic bleeding showed a greater drop in packed cell volume, red blood cell (RBC) count and haemoglobin concentration than those with who did not bleed. No morphological changes in RBCs were seen in blood films. Total white blood cell (WBC) counts were significantly higher in the clinically "more severe" group than in the "less severe" group on admission. Neutrophilic leucocytosis with left shift was present on admission, concurrently with a decrease in eosinophil and lymphocyte counts. These changes tend to become more marked 6h after antivenom therapy, and are greatest in "more severe" envenoming. Thrombocytopenia on admission is positively associated with the development of systemic bleeding and the severity of envenoming. Thrombocytopenia may also be a useful prognostic indicator for the development of local complications, such as necrosis. The intensity of neutrophilia and eosinopenia might be used to follow the progression of necrosis in victims of snake bite.

Toxic activities of Brazilian centipede venoms.

Centipedes have a venom gland connected to a pair of forceps, which are used to arrest preys. Human victims bitten by centipedes usually manifest burning pain, paresthesia and edema, which may develop into superficial necrosis. The aim of this work was to characterize and compare toxic activities found in venoms of three species of Brazilian centipedes-Otostigmus pradoi, Cryptops iheringi and Scolopendra viridicornis. By SDS-PAGE (4-20%), important differences were noticed among venoms (between 7 and 205kDa). Few bands showed feeble caseinolytic, fibrinogenolytic and gelatinolytic activities by zymography, but strong hyaluronidase activity was observed in S. viridicornis and O. pradoi venoms. In addition, such activities could be inhibited by o-phenanthroline, indicating that these enzymes are metalloproteinases. All venoms induced nociception, edema and myotoxicity in mice, but only S. viridicornis induced mild hemorrhagic activity. No coagulant activity was detected in centipede venoms. Low phospholipase A(2) activity was observed exclusively in S. viridicornis and O. pradoi venoms, but these venoms had intense direct hemolytic activity on human erythrocytes. Cross-reactivity among venoms was observed using species-specific sera raised in rabbits. Differences were noticed among centipede venoms, but S. viridicornis is indeed the most toxic venom and thereby it could induce a more severe envenomation.

Long-lasting anti-inflammatory properties of Crotalus durissus terrificus snake venom in mice.

In the present study, we investigated the effects of Crotalus durissus terrificus venom (CdtV) on vascular and cellular events of inflammation induced by carrageenan (cg) in mice. To evaluate edema, CdtV (75 microg kg(-1)) was administered subcutaneously before (1h, 7 or 14 days) or after (1, 4 or 48 h) subplantar injection of cg (15 mg kg(-1)) into the mouse right hind paw; to analyze leukocyte influx, cg (200 microL) was injected i.p. in mice. The inhibitory action of CdtV on edema, either before or after cg injection, was prolonged, lasting even 72 h after administration. Besides, CdtV significantly inhibited migration of polymorphonuclear cells to peritoneal cavity when administered before or after cg. Such inhibitory effects of CdtV on edema and cell migration were also compared with well-known anti-inflammatory drugs. The results demonstrated that CdtV, when injected either 7 or 14 days or 1h before cg, induced a more effective and long-lasting anti-inflammatory effect than that observed with classical anti-inflammatory drugs. The association of CdtV with different drugs did not potentialize their actions on cell migration. These results demonstrate that CdtV exhibits long-lasting anti-inflammatory effects.

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays.

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80 kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A2 and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents.

Biochemical and biological characterization of Bothriechis schlegelii snake venoms from Colombia and Costa Rica.

Snakebites inflicted by the arboreal viperid snake Bothriechis schlegelii in humans are characterized by pain, edema, and ecchymosis at the site of the bite, rarely with blisters, local necrosis, or defibrination. Herein, a comparative study of Bothriechis schlegelii snake venoms from Colombia (BsCo) and Costa Rica (BsCR) was carried out in order to compare their main activities and to verify the efficacy of Bothrops antivenom produced in Brazil to neutralize them. Biochemical (SDS-PAGE and zymography) and biological parameters (edematogenic, lethal, hemorrhagic, nociceptive, and phospholipase A2 activities) induced by BsCo and BsCR snake venoms were evaluated. The presence of antibodies in Bothrops antivenom that recognize BsCo and BsCR snake venoms by enzyme-linked immunosorbent assay and Western blotting, as well as the ability of this antivenom to neutralize the toxic activities were also verified. SDS-PAGE showed differences between venoms. Distinctive caseinolytic and hyaluronidase patterns were detected by zymography. BsCo and BsCR showed similar phospholipase A2 activity. Strong cross-reactivity between BsCo and BsCR was detected using Bothrops antivenom with many components located between 150 and 35 kDa. BsCR was more edematogenic and almost fourfold more hemorrhagic than BsCo, and both venoms induced nociception. BsCR (LD50 5.60 mg/kg) was more lethal to mice than BsCo (LD50 9.24 mg/kg). Bothrops antivenom was effective in the neutralization of lethal and hemorrhagic activities of BsCo and BsCR and was partially effective in the neutralization of edematogenic and nociceptive activities. In conclusion, geographic distribution influences the composition and activities of Bothriechis schlegelii venoms. Bothrops antivenom cross-reacted with these venoms and was partially effective in neutralizing some toxic activities of BsCo and BsCR.

Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis.

To attain fully functional biological activity, vitamin-K dependent coagulation factors (VKDCF) are γ-carboxylated prior to secretion from liver. Warfarin impairs the γ-carboxylation, and consequently their physiological function. Bothrops jararaca snake venom (BjV) contains several activators of blood coagulation, especially procoagulant enzymes (prothrombin and factor X activators) and thrombin-like enzymes. In order to clarify the relative contribution of prothrombin and factor X activators to the hemostatic disturbances occurring during experimental B. jararaca envenomation, warfarin was used to deplete VKDCF, prior to BjV administration. Male Wistar rats were pretreated with saline (Sal) or warfarin (War) and inoculated subsequently with BjV or saline, thus forming four groups: Sal + Sal (negative control), Sal + BjV (positive control), War + Sal (warfarinization control), and War + BjV. Three hours after inoculation, prothrombin and factor X levels fell 40% and 50%, respectively; levels of both factors decreased more than 97% in the War + Sal and War + BjV groups. Platelet counts dropped 93% and 76% in Sal + BjV and War + BjV, respectively, and plasma fibrinogen levels decreased 86% exclusively in Sal + BjV. After 6 and 24 h, platelet counts and fibrinogen levels increased progressively. A dramatic augmentation in plasma hemoglobin levels and the presence of schizocytes and microcytes in the Sal + BjV group indicated the development of intravascular hemolysis, which was prevented by warfarin pretreatment. Our findings show that intravascular thrombin generation has the foremost role in the pathogenesis of coagulopathy and intravascular hemolysis, but not in the development of thrombocytopenia, in B. jararaca envenomation in rats; in addition, fibrinogenases (metalloproteinases) may contribute to coagulopathy more than thrombin-like enzymes.

Mast cells and histamine play an important role in edema and leukocyte recruitment induced by Potamotrygon motoro stingray venom in mice.

This work aimed to investigate mechanisms underlying the inflammatory response caused by Potamotrygon motoro stingray venom (PmV) in mouse paws. Pre-treatment of animals with a mast cell degranulation inhibitor (cromolyn) diminished edema (62% of inhibition) and leukocyte influx into the site of PmV injection. Promethazine (histamine type 1 receptor antagonist) or thioperamide (histamine type 3 and 4 receptor antagonist) also decreased edema (up to 30%) and leukocyte numbers, mainly neutrophils (40-50 %). Cimetidine (histamine type 2 receptor antagonist) had no effect on PmV-induced inflammation. In the RBL-2H3 lineage of mast cells, PmV caused proper cell activation, in a dose-dependent manner, with release of PGD2 and PGE2. In addition, the role of COXs products on PmV inflammatory response was evaluated. Indomethacin (COX-1/COX-2 inhibitor) or etoricoxib (COX-2 inhibitor) partially diminished edema (around 20%) in PmV-injected mice. Indomethacin, but not etoricoxib, modulated neutrophil influx into the site of venom injection. In conclusion, mast cell degranulation and histamine, besides COXs products, play an important role in PmV-induced reaction. Since PmV mechanism of action remains unknown, hindering accurate treatment, clinical studies can be performed to validate the prescription of antihistaminic drugs, besides NSAIDs, to patients injured by freshwater stingrays.

Bothrops jararaca venom metalloproteinases are essential for coagulopathy and increase plasma tissue factor levels during envenomation.

BACKGROUND/AIMS: Bleeding tendency, coagulopathy and platelet disorders are recurrent manifestations in snakebites occurring worldwide. We reasoned that by damaging tissues and/or activating cells at the site of the bite and systemically, snake venom toxins might release or decrypt tissue factor (TF), resulting in activation of blood coagulation and aggravation of the bleeding tendency. Thus, we addressed (a) whether TF and protein disulfide isomerase (PDI), an oxireductase involved in TF encryption/decryption, were altered in experimental snake envenomation; (b) the involvement and significance of snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) to hemostatic disturbances. METHODS/PRINCIPAL FINDINGS: Crude Bothrops jararaca venom (BjV) was preincubated with Na2-EDTA or AEBSF, which are inhibitors of SVMP and SVSP, respectively, and injected subcutaneously or intravenously into rats to analyze the contribution of local lesion to the development of hemostatic disturbances. Samples of blood, lung and skin were collected and analyzed at 3 and 6 h. Platelet counts were markedly diminished in rats, and neither Na2-EDTA nor AEBSF could effectively abrogate this fall. However, Na2-EDTA markedly reduced plasma fibrinogen consumption and hemorrhage at the site of BjV inoculation. Na2-EDTA also abolished the marked elevation in TF levels in plasma at 3 and 6 h, by both administration routes. Moreover, increased TF activity was also noticed in lung and skin tissue samples at 6 h. However, factor VII levels did not decrease over time. PDI expression in skin was normal at 3 h, and downregulated at 6 h in all groups treated with BjV. CONCLUSIONS: SVMP induce coagulopathy, hemorrhage and increased TF levels in plasma, but neither SVMP nor SVSP are directly involved in thrombocytopenia. High levels of TF in plasma and TF decryption occur during snake envenomation, like true disseminated intravascular coagulation syndrome, and might be implicated in engendering bleeding manifestations in severely-envenomed patients.

Characterization of inflammatory response induced by Potamotrygon motoro stingray venom in mice.

Freshwater stingray accidents cause intense pain followed by edema, erythema, and necrosis formation. Treatment for stingray envenomation is based on administration of analgesic, antipyretic, and anti-inflammatory drugs. This report evaluated the local inflammatory reaction-including edema formation, leukocyte recruitment, release of inflammatory mediators, and histopathological changes-after the intraplantar injection of Potamotrygon motoro stingray venom in mice. Edema was observed as soon as 15 min after venom injection, peaking at 30 min, and lasted up to 48 h. In addition, P. motoro venom increased neutrophil counts in the site of injection, at all time periods and venom doses analyzed. Increased eosinophil and lymphocyte counts were detected mainly at 24 h. Moreover, monocytes/macrophages were observed in large amounts at 24 and 48 h. Microscopically, the venom induced leukocyte migration to the injured tissue, edema, mast cell degranulation, angiogenesis, and epidermal damage. Inflammatory mediator release (IL-6, MCP-1 and KC) was detected as soon as 1 h after venom injection, and it increased significantly at 4 h. At 24 h, the venom induced only the production of MCP-1. These results show that this stingray venom evokes a complex inflammatory reaction, with rapid and persistent edema formation, leukocyte recruitment, and release of cytokines and chemokines.

Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland.

Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (~65%) and dermonecrosis (~100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.

Venom proteomes of South and North American opisthoglyphous (Colubridae and Dipsadidae) snake species: a preliminary approach to understanding their biological roles.

Opisthoglyphous snake venoms remain under-explored despite being promising sources for ecological, evolutionary and biomedical/biotechnological research. Herein, we compared the protein composition and enzymatic properties of the venoms of Philodryas baroni (PbV), Philodryas olfersii olfersii (PooV) and Philodryas patagoniensis (PpV) from South America, and Hypsiglena torquata texana (HttV) and Trimorphodon biscutatus lambda (TblV) from North America. All venoms degraded azocasein, and this metalloproteinase activity was significantly inhibited by EDTA. PooV exhibited the highest level of catalytic activity towards synthetic substrates for serine proteinases. All venoms hydrolyzed acetylthiocholine at low levels, and only TblV showed phospholipase A(2) activity. 1D and 2D SDS-PAGE profile comparisons demonstrated species-specific components as well as several shared components. Size exclusion chromatograms from the three Philodryas venoms and HttV were similar, but TblV showed a notably different pattern. MALDI-TOF MS of crude venoms revealed as many as 49 distinct protein masses, assigned to six protein families. MALDI-TOF/TOF MS analysis of tryptic peptides confirmed the presence of cysteine-rich secretory proteins in all venoms, as well as a phospholipase A(2) and a three-finger toxin in TblV. Broad patterns of protein composition appear to follow phylogenetic lines, with finer scale variation likely influenced by ecological factors such as diet and habitat.

Loxosceles gaucho spider venom and its sphingomyelinase fraction trigger the main functions of human and rabbit platelets.

Loxosceles venoms can promote severe local and systemic damages. We have previously reported that Loxosceles gaucho spider venom causes a severe early thrombocytopenia in rabbits. Herein, we investigated the in vitro effects of this venom and its sphingomyelinase fraction on the main functions of platelets. Whole venom and its fraction induced aggregation of both human and rabbit platelets. Aggregation was dependent of plasma component(s) but independent of venom-induced lysophosphatidic acid generation. There was no increase in the levels of lactate dehydrogenase during platelet aggregation, ruling out the possibility of platelet lysis. The increased expression of ligand-induced binding site 1 (LIBS1) induced by L. gaucho venom and its sphingomyelinase fraction, as well as of P-selectin by the whole venom, evidenced the activation state of both human and rabbit platelets. Adhesion assays showed an irregular response when platelets were exposed to the whole venom, whereas the sphingomyelinase fraction induced a dose-dependent increase in the platelet adhesion to collagen. These findings evidence that L. gaucho venom and its sphingomyelinase fraction trigger adhesion, activation, and aggregation of both human and rabbit platelets. Thus, this work justifies the use of rabbits to investigate Loxosceles venom-induced platelet disturbances, and it also supports research on the role of platelets in the pathogenesis of loxoscelism.

Clinical picture and laboratorial evaluation in human loxoscelism.

Loxosceles spiders are found globally, especially in South and North America. In Brazil, approximately 10,000 cases of Loxosceles spp. spider bites are reported annually. Herein we analyzed 81 patients diagnosed as either cutaneous or cutaneous-hemolytic loxoscelism, in a geographical area where most accidents are caused by Loxosceles gaucho, and we report their clinical and laboratory data obtained during week 1 and 2 after the bite. Massive hemolysis was noticed in only 2 cases, but high serum bilirubin and LDH levels, suggestive of hemolysis, were noticed in 25 cases on admission. Anemia was not frequent (14.7%), and reticulocytosis was particularly noticed during week 2 (in 56% of patients). High D-dimer levels were suggestive of endothelial cell activation and intravascular thrombin generation, but thrombocytopenia was noticed in only 17.6% of patients in week 1. Acute kidney injury (AKI) only occurred in patients with massive hemolysis. The definitive diagnosis of overt disseminated intravascular coagulation (DIC) could not be established on admission. Fever was associated with the presence of hemolysis (p = 0.03). Altogether, these findings provide evidence that mild hemolysis is frequent in loxoscelism and suggest that AKI is uncommon, exclusively occurring in patients with massive hemolysis.

Comparative analysis of newborn and adult Bothrops jararaca snake venoms.

Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercial antivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 5'-nucleotidase activities. However, newborn snake venom showed higher l-amino oxidase, hyaluronidase, platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities of B. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciously neutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.

Evaluation of albuminuria and its relationship with blood pressure in dogs with chronic kidney disease.

BACKGROUND: Microalbuminuria and hypertension have long been associated with a guarded prognosis in human patients with a variety of diseases. In veterinary medicine, tests for microalbuminuria have been used for detecting early kidney damage, but there is little information regarding its association with high blood pressure in dogs with chronic kidney disease (CKD). OBJECTIVE: The objective of this study was to evaluate albuminuria and its association with arterial hypertension in dogs with CKD. METHODS: Urinary albumin:creatinine (UAC) ratio, urinary protein:creatinine (UPC) ratio, and systolic blood pressure were determined in 39 clinically healthy dogs and 40 dogs with CKD. RESULTS: UAC in dogs with CKD (range, 0.002-7.99; median, 0.38) was statistically different from that of control dogs (range, 0.0005-0.01; median, 0.002). Microalbuminuria (UAC 0.03-0.3) and macroalbuminuria (UAC>0.3) were detected in 32.5% and 50% of dogs with CKD, respectively. Sixty percent (24/40) of dogs with CKD had systolic pressure > or =180 mmHg; in these dogs, UAC ratio (range, 0.006-7.99; median, 1.72) was significantly higher than in dogs with CKD and systolic pressure<180 mmHg (range, 0.002-4.83; median, 0.10). Of hypertensive dogs with CKD, those with UPC>1.0 usually had macroalbuminuria, those with UPC 0.5-1.0 usually had microalbuminuria, and those with UPC<0.5 usually lacked albuminuria. CONCLUSIONS: UAC ratio was higher in hypertensive than in normotensive dogs with CKD. Tests designed to detect microalbuminuria may be useful for hypertensive dogs with CKD and a UPC < or = 1.0 to detect the onset and magnitude of albuminuria. Once macroalbuminuria is overt, the UPC ratio itself can be used for the same purpose.