"Bad things come in small packages": predicting venom-induced coagulopathy in Bothrops atrox bites using snake ontogenetic parameters.

Introduction: Snake venom composition shows significant inter- and intra-species variation. In the case of the viperid species Bothrops atrox, responsible for the majority of snakebites in the Amazon region, geographical and ontogenetic variables affect venom composition, with ecological and medical implications. Previous studies had shown that venom from neonate and juvenile Bothrops specimens have a higher in vitro coagulant activity. The aim of this investigation was to assess the association of clinical outcomes, such as venom-induced coagulopathy and local complications, with B. atrox ontogenetic variables.Methods: This study explored the relationship between some clinical parameters in patients suffering envenomations by B. atrox in the Amazon and several morphometric parameters of the snake specimens causing the bites.Results: There were 248 specimens confirmed as agents of envenomation, mostly female snakes (70.5%) and classified as juveniles (62.7%). Patients bitten by neonates compared to adult snakes [OR = 2.70 (95%CI 1.15-6.37); p = .021] and by snakes with white tail tip [OR = 1.98 (95%CI 1.15-3.41); p = .013] were more likely to develop coagulopathy. Time from patient admission to the unclottable blood reversion was not affected by the snake gender (p = .214) or age (p = .254). Patients bitten by neonate (p = .024) or juvenile snakes (p < .0001) presented a lower frequency of moderate to severe edema, as compared to those bitten by adult snakes. In agreement with experimental observations, patients bitten by neonates and by snakes with a white tail tip were more likely to develop coagulopathy than those bitten by adult snakes. In contrast, envenomations by adult snakes were associated with a higher incidence of severe local edema.Conclusion: Despite these variations, no difference was observed in the time needed to recover blood clotting in these patients after Bothrops antivenom administration.

Predicting acute renal failure in Bothrops snakebite patients in a tertiary reference center, Western Brazilian Amazon.

Acute Kidney Injury (AKI) is the main systemic complication and cause of death in viperid envenomation. Although there are hypotheses for the development of AKI, the mechanisms involved are still not established. The aim of this study was to evaluate the clinical-laboratorial-epidemiological factors associated with AKI in victims of Bothrops sp envenomation. This is an observational study carried out at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado. AKI was defined according to the guidelines of the Acute Kidney Injury Network (AKIN). Among the 186 patients evaluated, AKI was observed in 24 (12.9%) after 48 hours of admission. Stage I was present in 17 (70.8%) patients, II in 3 (12.5%) and III in 4 (16.7%). Epidemiological characterization showed predominance of men, occurrence in rural areas, aged between 16-60 years, feet as the most affected anatomical region, and time to medical assistance less than 3 hours. Hypertension and diabetes were the comorbidities identified. Most of the accidents were classified as moderate, and clinical manifestations included severe pain, mild edema, local bleeding and headache. Laboratory results showed blood uncoagulability, hypofibrinogenemia, leukocytosis, increase of creatine kinase, and high lactate dehydrogenase levels. Multivariate analysis showed an association with high LDH levels [AOR = 1.01 (95% CI = 1.01-1.01, p<0.002)], local bleeding [AOR = 0.13 (95%CI = 0.027-0.59, p<0.009)], and the presence of comorbidities [AOR = 60.96 (95%CI = 9.69-383.30; p<0.000)]. Herein, laboratory markers such as high LDH levels along with local bleeding and comorbidities may aid in the diagnosis of AKI.

Poor efficacy of preemptive amoxicillin clavulanate for preventing secondary infection from Bothrops snakebites in the Brazilian Amazon: A randomized controlled clinical trial.

BACKGROUND: Secondary bacterial infections from snakebites contribute to the high complication rates that can lead to permanent function loss and disabilities. Although common in endemic areas, routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy. The aim of this work was to estimate the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, secondarily, identify risk factors for secondary infections from snakebites in the Western Brazilian Amazon. METHODS AND FINDINGS: This was an open-label, two-arm individually randomized superiority trial to prevent secondary infection from Bothrops snakebites. The antibiotic chosen for this clinical trial was oral amoxicillin clavulanate per seven days compared to no intervention. A total of 345 patients were assessed for eligibility in the study period. From this total, 187 accomplished the inclusion criteria and were randomized, 93 in the interventional group and 94 in the untreated control group. All randomized participants completed the 7 days follow-up period. Enzyme immunoassay confirmed Bothrops envenoming diagnosis in all participants. Primary outcome was defined as secondary infection (abscess and/or cellulitis) until day 7 after admission. Secondary infection incidence until 7 days after admission was 35.5% in the intervention group and 44.1% in the control group [RR = 0.80 (95%CI = 0.56 to 1.15; p = 0.235)]. Survival analysis demonstrated that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789).Secondary infections incidence in 7 days of follow-up was independently associated to fibrinogen >400 mg/dL [AOR = 4.78 (95%CI = 2.17 to 10.55; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.52 (95%CI = 1.06 to 5.98; p = 0.037)], C-reactive protein >6.5 mg/L [AOR = 2.98 (95%CI = 1.40 to 6.35; p = 0.005)], moderate pain [AOR = 24.30 (95%CI = 4.69 to 125.84; p<0.001)] and moderate snakebites [AOR = 2.43 (95%CI = 1.07 to 5.50; p = 0.034)]. CONCLUSIONS/SIGNIFICANCE: Preemptive amoxicillin clavulanate was not effective for preventing secondary infections from Bothrops snakebites. Laboratorial markers, such as high fibrinogen, alanine transaminase and C-reactive protein levels, and severity clinical grading of snakebites, may help to accurately diagnose secondary infections. TRIAL REGISTRATION: Brazilian Clinical Trials Registry (ReBec): RBR-3h33wy; UTN Number: U1111-1169-1005.

Engineered mammalian vector to express EGFP-tagged proteins as biomarkers.

Due to its specialized post-translational machinery, mammalian cells represent an interesting and not fully explored system to express snake toxins. Therefore, in this work, we built up a new mammalian expression vector that enhances the feasibility to use mammalian cells to express proteins as biomarkers. Among the modifications, an Igκ signal peptide and a 6xHis tag were inserted into this vector in order to drive the protein to the supernatant and simplify its purification, respectively. In addition, to facilitate selection of high producing clones and also tag proteins which may function as a biomarker, the sequence of enhanced green fluorescent protein (EGFP) was added. The efficiency of the resulting vector (pToxEGFP) was tested by cloning and expressing the viper venom disintegrin echistatin (Ech) that due to its affinity to integrin αvß3 was tested as a molecular marker. Expression of EGFP-Ech was achieved in CHO-DXB11 cells resulting in a yield of 22 mg/L. The binding activity of this chimera protein was successfully achieved on human umbilical vein endothelial cells which highly express αvß3. The results indicate that pToxEGFP may constitute an efficient and versatile expression vector to express tagged proteins with potential biomarker activity.

The promigratory activity of the matricellular protein galectin-3 depends on the activation of PI-3 kinase.

Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-3(-/-) mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-3(-/-) sarcoma cells were more adherent and less migratory than galectin-3(+/+) sarcoma cells on laminin-111. When galectin-3 was transiently expressed in galectin-3(-/-) sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAK and phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 was inhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111.

Molecular engineering of an EGFP/disintegrin-based integrin marker.

Disintegrins are viper venom peptides, which bind integrins with high affinity (10(-8) M) and selectivity. Among them, eristostatin (Er) selectively binds and inhibits alphaIIbbeta3 integrin function. In this work we have engineered an enhanced green fluorescence protein (EGFP)-tagged Er as an alphaIIbbeta3 biomarker to be used in bioassays involving fluorescence detectors. For this, we have first constructed an EGFP bacterial expression vector, which resulted in a 6xHis tag-coding region followed by the EGFP gene and a 3' multiple cloning site (MCS) comprising nine restriction sites. This vector, termed pAZ, was used to clone the Er gene, resulting in a 32 kDa EGFP-Er fusion protein when expressed as characterized by SDS-PAGE and Western blot. Both EGFP-Er and EGFP (expressed from the empty pAZ vector) were purified by immobilized metal affinity chromatography (IMAC) and their fluorescence was measured showing similar values, thus suggesting that the Er portion is not affecting the EGFP activity. EGFP-Er, but not EGFP selectively bound to immobilized platelets as detected by confocal microscopy indicating the preservation of Er disintegrin activity and its potential use as a marker for alphaIIbbeta3 integrin. Our data suggest the use of the pAZ vector for expressing soluble EGFP-labeled proteins and the use of EGFP-fused disintegrins as markers for integrins.

The C-terminus of murine S100A9 inhibits hyperalgesia and edema induced by jararhagin.

The effect of a synthetic peptide (H92-G110) identical to the C-terminus of murine S100A9 (mS100A9p) was investigated on hyperalgesia and edema induced by either jararhagin or papain in the rat paw. mS100A9p not only reverted hyperalgesia and edema induced by jararhagin, but also the highest concentration induced antinociception. Hemorrhage induced by jararhagin and its hydrolytic activity were inhibited by mS100A9p. These data suggest that mS100A9p might block jararhagin-induced hyperalgesia and edema by inhibiting jararhagin catalytic activity, since papain-induced hyperalgesia and edema were not inhibited by mS100A9p.

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein.

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.