A mouse model to study the alterations in haemostatic and inflammatory parameters induced by Lonomia achelous caterpillar haemolymph.

A mouse model was established to reproduce the haemorrhagic syndrome which occurs in humans after accidental contact with the hairs of the caterpillar Lonomia achelous (LA) and measures the haemostatic and inflammatory alterations that occur as a result of this contact. Mice were injected intradermally with different doses (0.4, 0.8 and 1.6 mg/animal) of L. achelous haemolymph (LAH). Haematological (haemoglobin, haematocrit, platelet count, differential leukocyte count), haemostatic (fibrinogen, plasminogen, factor XIII [FXIII], fibrinolytic activity) and inflammatory parameters (tumour necrosis factor alpha [TNF-α], nitric oxide [NO]) were measured at different times up to 48 h. C57BL/6 mice responded to LAH injection, in terms of these parameters, in a manner similar to that seen in humans, whereas the BALB/c mice were unresponsive. In C57BL/6 mice injected with LAH, time course measurements showed: a) a reduction in the haemoglobin, haematocrit, fibrinogen, FXIII and plasminogen levels, b) no effect on the platelet count and c) immediate leukocytosis and an increase in the fibrinolytic activity in plasma. An inflammatory response (TNF-α) was observed within 1 h post-injection, followed by a more persistent increase in serum NO. These findings suggest that C57BL/6 mice represent a useful model of the haemorrhagic syndrome observed in humans who have suffered contact with the caterpillar, permitting a deeper understanding of the role of the inflammatory response in the haematological and haemostatic manifestations of this syndrome.

The association of Triatoma maculata (Ericsson 1848) with the gecko Thecadactylus rapicauda (Houttuyn 1782) (Reptilia: Squamata: Gekkonidae): a strategy of domiciliation of the Chagas disease peridomestic vector in Venezuela?

OBJECTIVE: To investigate the bioecological relationship between Chagas disease peridomestic vectors and reptiles as source of feeding. METHODS: In a three-story building, triatomines were captured by direct search and electric vacuum cleaner search in and outside the building. Then, age structure of the captured Triatoma maculata (T. maculata) were identified and recorded. Reptiles living in sympatric with the triatomines were also searched. RESULTS: T. maculata were found living sympatric with geckos (Thecadactylus rapicauda) and they bit residents of the apartment building in study. A total of 1 448 individuals of T. maculata were captured within three days, of which 74.2% (1 074 eggs) were eggs, 21.5% were nymphs at different stages, and 4.3% were adults. CONCLUSIONS: The association of T. maculata and T. rapicauda is an effective strategy of colonizing dwellings located in the vicinity of the habitat where both species are present; and therefore, could have implications of high importance in the intradomiciliary transmission of Chagas disease.

Partial characterization of a basic protein from Crotalus molossus molossus (northern blacktail rattlesnake) venom and production of a monoclonal antibody.

The venom of Crotalus molossus molossus (blacktailed rattlesnake) is very basic compared to that of other Crotalinae venoms. Unlike other Crotalinae venoms that are separated by anion exchange chromatography, C. m. molossus venom must be fractionated by cation exchange chromatography. Electrophoretic titration (ET) was used to predict the isoelectric point (pI) and optimal conditions for isolation. The specific hemorrhagic activity for C. m. molossus venom was 7.5 mm/microg, making it one of the most hemorrhagic of Crotalinae venoms. Basic hemorrhagic and fibrinolytic proteins from the venom of C. m. molossus venom were further fractionated by cation exchange chromatography. A basic fibrinolytic/hemorrhagic protein (CMM4) was isolated. CMM4 has a molecular weight between 23 and 26 kDa and a pI of approximately 11.3. SDS electrophoresis revealed one band and ET curve revealed 3 bands with very similar surface charges at all pH. CMM4 did not activate plasminogen when tested with a Chrom Z-PLG assay. The proteins in CMM4 had similar N-terminal amino acid sequences to each other (D-Q-Q-N-L-P-Q-(S/A/R)-Y-(V/R/I)-E-L-V-V-V-A-D-H-R-L-F-M-K-Y-K-S-D-L- N-T). The differences in these proteins are in positions 8 and 10. CMM4 may contain isoforms that differ by minor sequence variations at their amino-termini. The amino acid sequences of CMM4 were very similar to other fibrinolytic and hemorrhagic metalloproteinases isolated from venoms of the genera Crotalus. The specific hemorrhagic activity of CMM4 decreased as the specific fibrinolytic activity increased. A monoclonal antibody (CMM1b) was produced against C. m. molossus venom that neutralized the hemorrhagic activity of some of its fractions. CMM1b also reacted with 11 of 29 venom samples tested via ELISA.

An internet database of crotaline venom found in the United States.

Many snake venoms have been shown to be complex mixtures of pharmacologically important molecules, some of which have potential therapeutic value in the treatment of clot-induced ischemia, cancer and other human disorders. The literature contains many references on how venom and/or venom components are being used in medicine. Within the United States, there are 44 subspecies of poisonous snakes. Despite this rather vast diversity, 90% of the venom-related biomedical research conducted on native snakes found in the United States has been done on a limited number of the more common species. Since the venoms from most of the native species are not available or characterized, their composition and potential usefulness in medicine and applied biomedical research has not been explored. The Natural Toxins Research Center (NTRC) at Texas A&M University-Kingsville has developed a serpentarium that presently houses a population of over 250 snakes composed of 11 species and 20 subspecies. These snakes are cataloged on the Internet database along with their geographical location data, proteolytic activities, high performance liquid chromatography (HPLC) and electrophoretic titration (ET) profiles. Many of these snake venoms have never been characterized and few locale-specific differences within a species have been examined. These venoms can be queried through an on-line search routine. The database will be a useful starting point for anyone interested in isolating fibrinolytic enzymes, specific toxins, hemorrhagins, or other pharmacologically active proteins from snake venoms.

Screening for fibrinolytic activity in eight Viperid venoms.

Snake venoms contain direct-acting fibrinolytic metalloproteinases (MMP) that could have important applications in medicine. Fibrinolytic enzymes isolated from venom can induce in vitro clot lysis by directly acting on a fibrin clot. The most ideal fibrinolytic enzyme would have high affinity for clots, dissolve clots directly without causing hemorrhage, and would not be neutralized in vivo by endogenous metalloproteinase inhibitors. The purpose of this study was to compare DEAE/HPLC venom profiles from Viperid snakes and identify fractions that contain fibrinolytic activity with no hemorrhagic activity and are not neutralized by animal sera. The sera selected were from four (Virginia opossum, Gray woodrat, Mexican ground squirrel, and Hispid cottonrat) animals known to neutralize hemorrhagic activity in snake venoms. Nineteen fractions from the Viperid venoms had fibrinolytic activity. Agkistrodon venom fractions contained the highest specific fibrinolytic activities. A. piscivorus leucostoma fraction 4 contained a high specific fibrinolytic activity, no hemorrhagic activity, and the fibrinolytic activity was not neutralized by the proteinase inhibitors of the four animal sera. A. contortrix laticinctus fraction 1 also had a high specific fibrinolytic activity and no hemorrhagic activity. However, the fibrinolytic activity was neutralized by Didelphis virginiana (Virginia opossum) serum.

The antihemorrhagic factor of the Mexican ground squirrel, (Spermophilus mexicanus).

The Mexican ground squirrel (Spermophilus mexicanus) has a natural resistance to western diamondback rattlesnake venom (Crotalus atrox). The LD50 for the Mexican ground squirrel is 53 mg/kg body weight, which is 13 times higher than that of BALB/c mice. An antihemorrhagic factor from serum of the Mexican ground squirrel was isolated using Sephadex G-200 gel filtration, ion exchange A-50, G-75 gel filtration and HPLC DEAE 5PW ion exchange chromatography. The purified factor neutralized proteolytic and hemorrhagic activity of crude C. atrox venom. The results of this research suggest that the antihemorrhagic factor in the serum of the Mexican ground squirrel is not an antibody and neutralizes hemorrhagic activity of C. atrox venom.

Natural protease inhibitors to hemorrhagins in snake venoms and their potential use in medicine.

Snake venoms are complex mixtures of many toxins and enzymes which effectively immobilize prey without a struggle and assist in digestion. Certain animals have a remarkable resistance to envenomation of snakes. Naturally occurring factors that neutralize snake venoms have been found in the sera of most snakes and a few warm-blooded animals. These antihemorrhagic and antineurotoxic factors have been purified from snake and mammalian sera. The antihemorrhagins are not immunoglobulins since they have different physical and chemical characteristics. The natural immunity to hemorrhagins is the result of tissue inhibitors of metalloproteinases (TIMP) found in animal sera of resistant animals. Most animals have matrix metalloproteinases (MMP) and TIMP that are implicated in a wide variety of normal physiological processes and pathological conditions. MMP in animals have many biological functions in embryogenesis, morphogenesis and tissue remodeling. MMP activities are precisely regulated by endogenous TIMP. Disruption of the balance between MMP and TIMP causes various diseases such as arthritis, periodontal diseases, diabetes, ophthalmologic conditions, neoplasia, metabolic bone disease, atherosclerosis and orthopedic conditions. Resistant animals that have a high titer of TIMP would have a survival advantage when bitten by poisonous snakes. Snake venoms are abundant and stable sources of MMP which are medically important. The venom MMP which cause unregulated destruction of tissue have sequences which have some degree of homology with mammalian MMP which control normal biological processes. Resistant animals are important sources of TIMP which can be used to study metalloproteinase related diseases. For these reasons the MMP in snakes and TIMP in resistant animal are excellent candidates for developing new drug therapies.

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum (Didelphis virginiana) serum.

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occurring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra (Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five-step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.

Septic arthritis due to Mycobacterium phlei presenting as infantile Reiter's syndrome.

We describe a 7-year-old Peruvian boy who presented with a 2 week history of conjunctivitis, urethritis and arthritis, in whom Mycobacterium phlei was the only organism repeatedly isolated from synovial fluid and tissue and who responded to conventional antituberculous therapy. To our knowledge this is the first documented case of human disease caused by this microorganism.