Accuracy and Clinical Utility of Reports from Outside Hospitals for CT of the Cervical Spine in Blunt Trauma.

BACKGROUND AND PURPOSE: Multidetector CT is the workhorse for detecting blunt cervical spine injury. There is no standard of care for re-interpretation of radiology images for patients with blunt trauma transferred to a higher level of care. The clinical impact of discrepancies of cervical spine CT reads remains unclear. We evaluated the discordance between primary (from referring hospitals) and secondary radiology interpretations (from a receiving level I tertiary trauma center) of cervical spine CT scans in patients with blunt trauma and assessed the clinical implications of missed cervical spine fractures. MATERIALS AND METHODS: Medical records of patients with blunt trauma transferred to our institution between 2008 and 2015 were reviewed. Primary and secondary interpretations were compared and categorized as concordant and discordant. Two senior neuroradiologists adjudicated discordant reports. The benefit of re-interpretation was determined. For discordant cases, outcomes at discharge, injury severity pattern, treatment, and arrival in a cervical collar were assessed. RESULTS: Six hundred fifty patients were included; 608 (94%) presented with concordant reports: 401 (61.7%) with fractures and 207 (31.8%) with no fractures. There were 42 (6.5%) discordant reports; 18 (2.8%) were cervical spine injuries undetected on the primary interpretation. Following adjudication, the secondary interpretation improved the sensitivity (99.3% versus 95.7%) and specificity (99.1% versus 91.7%) in detecting cervical spine fractures compared with the primary interpretation alone (P < .001). CONCLUSIONS: There was an overall 6.5% discordance rate between primary and secondary interpretations of cervical spine CT scans. The secondary interpretation of the cervical spine CT increased the sensitivity and specificity of detecting cervical spine fractures in patients with blunt trauma transferred to higher-level care.

Risk factors associated with clinical signs of vesicular stomatitis and seroconversion without clinical disease in Colorado horses during the 2014 outbreak.

Vesicular stomatitis (VS) is caused by a contagious rhabdovirus that affects horses, cattle, and swine. Clinical signs of vesicular stomatitis virus (VSV) infection in pigs and cattle are indistinguishable from foot-and-mouth disease (FMD), a foreign animal disease and reportable disease in the United States (Rodriguez et al., 2000). A VS epidemic occurred in the Rocky Mountain region in 2014-15. A study was conducted in Colorado to evaluate horse- and management-level factors associated with VS. For a horse to be considered a clinical VS horse, there were two requirements. First, clinical VS horses had to have clinical signs consistent with VS, including one or more of the following: vesicles, ulcers, erosions or crusting on the muzzle, nares, lips, oral or nasal mucosa, ears, ventrum, udder or penile sheath, or coronary band lesions. Second, clinical VS horses had to have laboratory confirmation of VSV exposure via virus isolation from lesions or a positive complement fixation test performed on sera. All non-clinical horses residing on VSV-affected premises enrolled in the study were evaluated for exposure (i.e., seroconversion) to VSV. Overall, management and housing data were collected from 334 horses on 48 premises in Colorado. Approximately one-third (31.4%) of enrolled horses were clinical cases and two-thirds (68.6%) were controls. Three premises-matched logistic regression models were constructed in SAS using backward elimination (P-value < 0.05) after univariate screening of a priori-selected variables (P-value < 0.20). Model outcomes included differences in characteristics and management of 1) clinical and nonclinical horses, 2) exposed and unexposed horses, and 3) exposed nonclinical and unexposed nonclinical horses. Overall, factors most strongly associated with risk of being a VS clinical horse were access to pasture (P-value = 0.002), and pregnancy status (P-value = 0.001). Factors most strongly associated with VSV exposure among horses were access to pasture (P-value = 0.003) and lack of any insect control (P-value = 0.001). The only factor associated with VSV-exposed nonclinical horses compared with unexposed VSV horses was contact with clinical horses (P-value = 0.013). There were no associations identified regarding clinical horses compared with exposed nonclinical horses. With regard to severity of lesions (severe vs. moderate or mild), no variables met the criteria for inclusion in the multivariable model. Results of this study provide evidence that pasture access and fly control are important factors associated with VSV exposure.

Review of influenza A virus in swine worldwide: a call for increased surveillance and research.

Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high-priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.

Active and latent ovine herpesvirus-2 (OvHV-2) infection in a herd of captive white-tailed deer (Odocoileus virginianus).

Malignant catarrhal fever (MCF) is the clinical manifestation of infection of certain ruminant species with one of a group of pathogenic gammaherpesviruses known as MCF viruses. Cattle and numerous exotic ruminant species are susceptible to clinical disease that may be sporadic or occasionally epidemic in nature. The most common MCF virus worldwide is ovine herpesvirus (OvHV)-2. Reservoir hosts such as sheep, carry and excrete OvHV-2, but do not develop clinical signs, while clinically susceptible species develop severe and often fatal disease. The existence of latent infection in clinically susceptible hosts is poorly understood, but is documented in some ruminant species. Twenty-six animals from a captive herd of white-tailed deer (Odocoileus virginianus) died and were examined from October 2006 to December 2010. Fifteen of these animals (58%) showed clinical signs and gross and microscopical lesions consistent with MCF, while 11 (42%) did not. Polymerase chain reaction (PCR) amplification yielded product consistent with OvHV-2 DNA in samples of spleen from all 26 deer. To examine the possibility of latent infection in this herd, peripheral blood mononuclear cells were examined by PCR for OvHV-2 DNA, and the test was positive in 23/32 (72%) clinically normal deer. Archived serum samples were used to examine the history of MCF exposure in the herd using a competitive enzyme-linked immunosorbent assay, which demonstrated that 10/40 (25%) deer tested had MCF viral antibodies, with nine deer being seropositive over multiple years. Combined with previous observations in deer and other species, these results suggest the existence of latent infection of white-tailed deer with OvHV-2.

Simultaneous infection of pigs and people with triple-reassortant swine influenza virus H1N1 at a U.S. county fair.

Influenza-like illness was noted in people and pigs in attendance at an Ohio county fair in August 2007. The morbidity rate in swine approached 100% within 1-2 days of initial clinical signs being recognized, and approximately two dozen people developed influenza-like illness. Triple-reassortant swine H1N1 influenza viruses were identified in both pigs and people at the fair. The identified viruses (A/Sw/OH/511445/2007, A/Ohio/01/2007, and A/Ohio/02/2007) were similar to H1N1 swine influenza viruses currently found in the U.S. swine population. This case illustrates the possibility of transmission of swine influenza in settings where there is close human/swine interaction.

Identification and analysis of the first 2009 pandemic H1N1 influenza virus from U.S. feral swine.

The first case of pandemic H1N1 influenza (pH1N1) virus in feral swine in the United States was identified in Texas through the United States Department of Agriculture (USDA) Wildlife Services' surveillance program. Two samples were identified as pandemic influenza by reverse transcriptase quantitative PCR (RT-qPCR). Full-genome Sanger sequencing of all eight influenza segments was performed. In addition, Illumina deep sequencing of the original diagnostic samples and their respective virus isolation cultures were performed to assess the feasibility of using an unbiased whole-genome linear target amplification method and multiple sample sequencing in a single Illumina GAIIx lane. Identical sequences were obtained using both techniques. Phylogenetic analysis indicated that all gene segments belonged to the pH1N1 (2009) lineage. In conclusion, we have identified the first pH1N1 isolate in feral swine in the United States and have demonstrated the use of an easy unbiased linear amplification method for deep sequencing of multiple samples.

Neoliberalism and human services: threat and innovation.

BACKGROUND: The turn to neoliberalism in welfare policy suggests that human services need to be based on a market approach. The problem with this suggestion is that it presupposes marketing information such that service providers can market their services for identified client needs. In the field of intellectual disability (ID) services this type of information is not available. METHOD: The method is a reflective analysis of the key presupposition of a market-orientated approach to disability services, namely that service providers know who needs what. Using insights from marketing theory the paper engages in a reflective thought experiment to lay out the intricacies of this presupposition. RESULTS: The analysis results in an argument regarding the validation of a market-based approach to disability services. First, this approach has its limits in view of the question of whether the specific and atypical needs of people with ID, as well as their financial position as potential consumers constitute a market. Second, the approach has limited validity both in view of the ability of people with ID to act as consumers, and of the restrictions imposed upon them by the eligibility criteria for welfare and support programmes. CONCLUSIONS: A market-based approach to disability services and supports can be helpful to spur innovation and further political and philosophical inquiry in human services, but the neoliberal optimism about the market as the only successful mechanism for service distribution is misplaced.

Dietary B vitamin needs of strains of pigs with high and moderate lean growth.

Ten sets of 5 littermate pigs from each of 2 genetic strains were utilized to determine the impact of the dietary concentration of 5 B vitamins (riboflavin, niacin, pantothenic acid, cobalamin, and folacin) on growth from 9 to 28 kg of BW in pigs with high or moderate capacity for lean growth. All pigs (penned individually) were reared via a segregated, early weaning scheme, so that the lean growth potential of each strain could be expressed. The basal diet provided the 5 test vitamins at concentrations of total and estimated bioavailability equivalent to a minimum of 100 and 70%, respectively, of their estimated requirements (NRC, 1998) for 5- to 10-kg pigs. At a BW of 9 +/- 0.9 kg, pigs within each litter were allotted to the basal diet supplemented with sources of the 5 test vitamins equivalent to an additional 0, 100, 200, 300, or 400% (bioavailable) of the NRC requirements. Pigs from the high lean strain consumed less feed (P < 0.05) and gained BW faster (P < 0.02) and more efficiently (P < 0.01) than pigs of the moderate lean strain. In both lean strains, the rate and efficiency of growth were improved (P < 0.01) as dietary B vitamin concentrations were increased. However, the dietary B vitamin concentrations needed to optimize G:F were greater (P < 0.03) in the high (>470% of NRC, 1998) vs. moderate (270%) lean strain. Based on these data, the dietary needs for 1 or more of the 5 B vitamins are greater than current NRC (1998) estimates, particularly in pigs expressing a high rate of lean tissue growth. The greater need for these vitamins is not associated with greater dietary energy intake or body energy accretion rate but is potentially due to shifts in the predominant metabolic pathways.

Snake venom fibrin(ogen)olytic enzymes.

Snake venoms contain a number of serine and metalloproteinases and included among these are the fibrin(ogen)olytic proteinases. Some years ago it was postulated that the fibrin(ogen)olytic enzymes may be clinically useful. Over the past 150 years a substantial body of literature has been generated on the identification and characterization of fibrin(ogen)olytic enzymes from a broad spectrum of snake species. In this review we describe the two different classes of fibrin(ogen)olytic enzymes isolated from snake venom and we summarize a number of studies aimed at characterizing the purified enzymes and/or their derivatives. Two distinct classes of venom fibrin(ogen)olytic enzymes have been previously identified, the metalloproteinases and serine proteinases. These two classes of proteinases differ in their mechanism of action and they target different amino acid sequences in fibrin(ogen), but each perform the same role in nature. When a snake envenomates its prey it needs a mechanism to facilitate the spread of the toxic components throughout the circulation. Fibrin(ogen)olytic enzymes break down fibrin rich clots and help to prevent further clot formation by their action on fibrinogen. This characteristic feature has led to development of fibrin(ogen)olytic snake venom enzymes as potential clinical agents to treat occlusive thrombi. Fibrolase, a fibrinolytic metalloproteinase isolated from Agkistrodon contortrix contortrix venom and the serine beta-fibrinogenolytic proteinase from Vipera lebetina have been chosen as representative enzymes from the two classes, and their biochemical and physiochemical properties will be described in detail. Finally, the characterization and development of alfimeprase, a recombinant fibrinolytic enzyme derived from fibrolase, as a clinical agent is described citing the progression from the laboratory bench to its current status as having successfully completed Phase II clinical trials.

Alpha-fibrinogenases.

Snake venoms contain a number of serine and metalloproteinases, included among these are the fibrinolytic metalloproteinases. When the fibrinolytic enzymes were first isolated from viper venoms it was postulated that there may be a clinical application for these enzymes in the treatment of occlusive thrombi, such as those occurring in the great arteries and veins of cardiac and cerebral circulation as well as peripheral arteries and veins. In the ensuing years a substantial body of literature has been generated on the identification and characterization of the fibrinolytic enzymes from a broad spectrum of snake species. In this report we describe the biological properties and positive clinical features of the class of enzymes known as alpha-fibrinogenases. Fibrolase, a fibrinolytic metalloproteinase originally isolated from Agkistrodon contortrix contortrix venom, is the representative fibrinolytic enzyme used for the description and characterization of the alpha-fibrinogenases in this chapter. The biochemical and physiochemical properties and in vivo activity of the enzyme are described as well as in vitro studies using a platelet avid chimera of fibrolase. The chimera was formed by coupling fibrolase to an Arg-Gly-Asp (RGD) like peptide imparting inhibitory activity on platelet aggregation and thrombus formation, while maintaining full fibrinolytic activity. Fibrolase has also been modified through the adduction of polyethylene glycol to reduce the rate of clearance from the circulation. In this review we also include a description of alfimeprase, a recombinant fibrinolytic enzyme derived from fibrolase, and follow the development of the enzyme as a potential clinical agent in the clearance of occlusive thrombi. Alfimeprase is presently in clinical trials for two indications: the treatment of peripheral arterial occlusions (in which phase II is nearing successful completion), and for use in the clearance of occluded vascular access catheters in direct competition with plasminogen activators.

Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the X-ray structure of adamalysin II and atrolysin C.

The fibrinolytic enzyme from southern copperhead snake venom, fibrolase, contains 1 mole of zinc per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), fibrolase has approximately 60% overall sequence identity and nearly 100% sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118-198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158-182 and 160-165). We used Sculpt 2.5 and HyperChem 5.0 to "dock" a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3-dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site.

Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates.

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88-95% nucleotide and 88-96% amino acid sequence identities to each other, 87-97% nucleotide and 84-96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.

A comparison of diagnostic assays for the detection of type A swine influenza virus from nasal swabs and lungs.

Nasal swabs and lung samples from pigs experimentally infected with H1N1 swine influenza virus (SIV) were examined for the presence of SIV by the indirect fluorescent antibody assay, immunohistochemistry, cell culture virus isolation, egg inoculation, and 2 human enzyme immunoassays (membrane enzyme immunoassay, microwell enzyme immunoassay). Egg inoculation was considered to be the gold standard for assay evaluation. The 2 human enzyme immunoassays (EIA) and egg inoculation agreed 100% for the prechallenge nasal swabs. Agreement on SIV identification in nasal swabs with egg inoculation following challenge was considered to be good to excellent for membrane EIA (kappa = 0.85) and microwell EIA (kappa = 0.86). Agreement on SIV identification in lung tissue with egg inoculation following challenge was good to excellent for membrane EIA (kappa = 0.75), fair for microwell EIA, fluorescent antibody, and cell culture virus isolation (kappa = 0.48, 0.64, 0.62, respectively), and poor for immunohistochemistry (kappa = 0.36). No assay was 100% accurate, including the "gold standard," egg inoculation. In light of this information, it is important to consider clinical signs of disease and a thorough herd history in conjunction with diagnostic results to make a diagnosis of SIV infection.

Evolution of swine H3N2 influenza viruses in the United States.

During 1998, severe outbreaks of influenza were observed in four swine herds in the United States. This event was unique because the causative agents, H3N2 influenza viruses, are infrequently isolated from swine in North America. Two antigenically distinct reassortant viruses (H3N2) were isolated from infected animals: a double-reassortant virus containing genes similar to those of human and swine viruses, and a triple-reassortant virus containing genes similar to those of human, swine, and avian influenza viruses (N. N. Zhou, D. A. Senne, J. S. Landgraf, S. L. Swenson, G. Erickson, K. Rossow, L. Liu, K.-J. Yoon, S. Krauss, and R. G. Webster, J. Virol. 73:8851-8856, 1999). Because the U.S. pig population was essentially naive in regard to H3N2 viruses, it was important to determine the extent of viral spread. Hemagglutination inhibition (HI) assays of 4, 382 serum samples from swine in 23 states indicated that 28.3% of these animals had been exposed to classical swine-like H1N1 viruses and 20.5% had been exposed to the triple-reassortant-like H3N2 viruses. The HI data suggested that viruses antigenically related to the double-reassortant H3N2 virus have not become widespread in the U.S. swine population. The seroreactivity levels in swine serum samples and the nucleotide sequences of six additional 1999 isolates, all of which were of the triple-reassortant genotype, suggested that H3N2 viruses containing avian PA and PB2 genes had spread throughout much of the country. These avian-like genes cluster with genes from North American avian viruses. The worldwide predominance of swine viruses containing an avian-like internal gene component suggests that these genes may confer a selective advantage in pigs. Analysis of the 1999 swine H3N2 isolates showed that the internal gene complex of the triple-reassortant viruses was associated with three recent phylogenetically distinct human-like hemagglutinin (HA) molecules. Acquisition of HA genes from the human virus reservoir will significantly affect the efficacy of the current swine H3N2 vaccines. This finding supports continued surveillance of U.S. swine populations for influenza virus activity.

Characterization of immune response of young pigs to porcine circovirus type 2 infection.

A longitudinal study was conducted to characterize the immune response of young swine to infection with porcine circovirus type 2 (PCV-2). Five 8-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally and intramuscularly with a field isolate of PCV-2 at a concentration of 10(4) TCID50/mL. Along with monitoring for clinical signs and viremia, serum samples were collected from all pigs at day 0 and thereafter every 7 days postinoculation (PI) until the termination of the study on day 35 PI. No clinical signs were observed in any of the animals during the study period. In all pigs, PCV-2 was detected by polymerase chain reaction (PCR) in serum samples collected on days 7, 14, and 21 PI. Viral DNA and antigens were detected by in situ hybridization and immunohistochemistry in tonsil, spleen, medial iliac lymph nodes, and ileum collected from each pig at the end of the study. Collectively, naïve young swine were shown to be susceptible to PCV-2. Virus-specific antibody was detected by an indirect fluorescent antibody (IFA) assay on day 14 PI, but virus-neutralizing antibody was not detected until day 28 PI. As neutralizing antibodies developed, cross-reactivity with PCV type 1 (PCV-1) also developed on the IFA test. Western immunoblot analysis revealed three PCV-2 proteins with molecular masses of 28 kd, 28.5 kd, and 35 kd. The 35-kd protein was also demonstrated in PCV-1, suggesting that this protein induced the cross-reactivity between PCV types 1 and 2. Antibody to the 28-kd protein was detected on day 14 PI and later, indicating that this protein was the most immunogenic. Because of its immunogenicity and specificity to PCV-2, and 28-kd protein might provide the antigenic basis for the development of diagnostic tests for detection of PCV-2 antibody.

Emergence of H3N2 reassortant influenza A viruses in North American pigs.

In late summer through early winter of 1998, there were several outbreaks of respiratory disease in the swine herds of North Carolina, Texas, Minnesota and Iowa. Four viral isolates from outbreaks in different states were analyzed, both antigenically and genetically. All of the isolates were identified as H3N2 influenza viruses with antigenic profiles similar to those of recent human H3 strains. Genotyping and phylogenetic analysis demonstrated that the four swine viruses had emerged through two different pathways. The North Carolina isolate is the product of genetic reassortment between human and swine influenza viruses, while the others arose from reassortment of human, swine and avian viral genes. The hemagglutinin genes of the four isolates were all derived from the human H3N2 virus circulating in 1995. It remains to be determined if either of these recently emerged viruses will become established in the pigs in North America and whether they will become an economic burden.

Oral vesicular lesions in horses without evidence of vesicular stomatitis virus infection.

OBJECTIVE: To report clinical and serologic findings in horses with oral vesicular lesions that were consistent with vesicular stomatitis (VS) but apparently were not associated with VS virus (VSV) infection. DESIGN: Serial case study. ANIMALS: 8 horses. PROCEDURE: Horses were quarantined after appearance of oral lesions typical of VS. Severity of clinical signs was scored every 2 to 5 days for 3 months. Serum samples were tested for antibodies by use of competitive ELISA (cELISA), capture ELISA for IgM, serum neutralization, and complement fixation (CF). Virus isolation was attempted from swab specimens of active lesions. RESULTS: 2 horses with oral vesicular lesions on day 1 had antibodies (cELISA and CF) against VSV; however, results of CF were negative by day 19. Five of the 6 remaining horses were seronegative but developed oral lesions by day 23. Virus isolation was unsuccessful for all horses. CONCLUSIONS AND CLINICAL RELEVANCE: Horses were quarantined for 75 days in compliance with state and federal regulations. However, evidence suggests that oral lesions were apparently not associated with VSV infection. The occurrence in livestock of a vesicular disease that is not caused by VSV could confound efforts to improve control of VS in the United States and could impact foreign trade. Vesicular stomatitis is of substantial economic and regulatory concern.

Molecular cloning and functional expression of contortrostatin, a homodimeric disintegrin from southern copperhead snake venom.

Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins alphaIIbbeta3, alpha5beta1, alphavbeta3, and alphavbeta5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. The precursor cDNA is 2027 bp with a 1449-bp open reading frame. The disintegrin domain is 195 bp encoding 65 amino acids. Like other members of the disintegrin family, each subunit of contortrostatin has an RGD site, and the cysteine alignment is conserved. The disintegrin domain of the cDNA has been expressed in a eukaryotic expression system as a homodimeric fusion protein with an immunoglobulin. The recombinant protein is recognized by an antiserum against native contortrostatin in Western blot. Both the native and recombinant proteins bind to integrins alphavbeta3 and alphavbeta5. Like native contortrostatin, the recombinant fusion protein inhibits platelet aggregation, blocks cancer cell adhesion to fibronectin and vitronectin, and prevents invasion of cancer cells through a Matrigel barrier. The success of functional expression not only validates the cDNA cloning of this disintegrin, but also provides adequate material for functional studies of contortrostatin.

Contortrostatin, a homodimeric disintegrin, binds to integrin alphavbeta5.

Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.