NMR-spectroscopic screening of spider venom reveals sulfated nucleosides as major components for the brown recluse and related species

Extensive chemical analyses of spider venoms from many species have revealed complex mixtures of biologically active compounds, of which several have provided important leads for drug development. We have recently shown that NMR spectroscopy can be used advantageously for a direct structural characterization of the small-molecule content of such complex mixtures. Here, we report the application of this strategy to a larger-scale analysis of a collection of spider venoms representing >70 species, which, in combination with mass spectrometric analyses, allowed the identification of a wide range of known, and several previously undescribed, small molecules. These include polyamines, common neurotransmitters, and amino acid derivatives as well as two additional members of a recently discovered family of natural products, the sulfated nucleosides. In the case of the well studied brown recluse spider, Loxosceles reclusa, sulfated guanosine derivatives were found to comprise the major small-molecule components of the venom.     

Inactivation of complement by Loxosceles reclusa spider venom.

Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.     

The diagnosis and treatment of brown recluse spider bites

We reviewed our experience with 95 patients who carried the diagnosis of brown recluse spider bite between 1983 and 1986 and identified a reference group of 17 with confirmed bites. Eight men and seven women, average age 32 years, presented within 33 hours following the bites. The most common symptoms were pain, pruritus, malaise, chills, sweats, and rash. Patients were randomized into three treatment groups: dapsone, brown recluse spider antivenom, or combination therapy. All patients were treated with erythromycin. If two patients with very severe lesions were excluded, patients in all groups healed their wounds in an average of 20 days. A comparison of our treatment was attempted with all other bites previously confirmed in the literature, but historical data were incomplete and no conclusions could be drawn.     

A controlled trial of topical nitroglycerin in a New Zealand white rabbit model of brown recluse spider envenomation

STUDY OBJECTIVES: Topical nitroglycerin has been reported to prevent skin necrosis from brown recluse spider bites, but this has never been scientifically tested. This study attempts to assess the effects of topical nitroglycerin on experimental Loxosceles reclusa envenomations. METHODS: We performed a randomized, blinded, controlled study in an animal care facility. Twenty-four New Zealand white rabbits were experimentally envenomated by means of subcutaneous injection with 20 microg of brown recluse spider venom. Rabbits were randomized to 1 of 2 experimental groups. The treatment group received 1 in of 2% topical nitroglycerin ointment every 6 hours for 3 days applied directly to the envenomation site. The control group received the vehicle without nitroglycerin. Gross examination of the lesions and measurements of the areas of the lesions were made daily. Creatine phosphokinase (CPK), blood urea nitrogen, creatinine, hemoglobin, and hematocrit levels were measured on days 0, 5, and 10. Lesions were excised after 10 days and examined by a blinded pathologist, who measured the area of necrosis and quantified inflammation and edema using a standard wound-healing score. For all values, mean values plus SD were determined. All comparisons made over multiple time points were assessed for significance by using a repeated-measures analysis of variance followed by Fisher least significant difference and Scheffé post hoc comparisons. A P value of.05 or less was used to determine significance. The Student's t test was used to compare the means of single measures. Significance was determined by using 95% confidence intervals. Comparisons of total area of necrosis were made with the nonparametric Mann-Whitney U test because of the heavy positive skew of the data. RESULTS: Skin necrosis developed in all animals. Mean values of the lesion area were not significantly different over time between the 2 groups of animals. At day 10, the median area of necrosis was 22.3 cm2 for the treatment group and 15.4 cm2 for the control group (P =.12). The inflammation score was 3.33+/-0.78 for the treatment group and 2.79+/-1.29 for the control group (P < .01). The edema score was 1.25+/-1.28 for the treatment group and 0.98+/-1.10 for the control group (not significantly different). CPK levels increased dramatically in both groups, with the greatest increase in the treatment group. In both groups hemoglobin and hematocrit levels decreased significantly, whereas WBC counts and platelet counts increased significantly, without significant differences between the 2 groups. CONCLUSION: At the dose used in this experiment, topical nitroglycerin did not prevent skin necrosis, increased inflammation score, and increased serum CPK levels. The results of this study do not support the use of topical nitroglycerin in the treatment of L reclusa envenomation and suggest that systemic toxicity could be increased.     

Case report: dapsone hypersensitivity syndrome associated with treatment of the bite of a brown recluse spider

Dapsone (4-4-diaminodiphenyl-sulfone) is a member of the sulfone group of antibiotics used in the treatment of leprosy and various dermatitidies and more recently employed in the management of local reactions to the bite of the brown recluse spider, Loxosceles reclusa. A dapsone hypersensitivity syndrome, consisting of fever, headache, nausea, vomiting, lymphadenopathy, hepatitis, hemolysis, leukopenia, and mononucleosis, has been described in patients treated with the drug for leprosy. A case report of the hypersensitivity syndrome occurring in a patient being treated with dapsone for a brown recluse spider bite is presented.     

Detection of Loxosceles species venom in dermal lesions: a comparison of 4 venom recovery methods

STUDY OBJECTIVE: Loxosceles species spider envenomations may produce necrotic, disfiguring dermal inflammatory lesions resembling neutrophilic dermatoses. With definitive treatment options lacking, clinicians are reluctant to obtain invasive biopsy specimens for diagnostic analysis. We compared less invasive venom collection methods and determined the time limit after inoculation for feasible venom recovery in an animal model. METHODS: Nine New Zealand rabbits were randomized to 1 of 3 groups (n=3). Groups 1 and 2 were inoculated intradermally with 3 microg of L reclusa venom at 5 inoculation sites per rabbit. Albumin (3 microg) was injected intradermally in each rabbit as a negative control. Hair (group 1) and aspirate samples (group 2) were collected (1 time per site) over a 1-week period after inoculation. Group 3 was inoculated with 3 microg of Loxosceles species venom on 1 flank and 3 microg of albumin on the opposite flank. Daily serum specimens were collected over a 7-day period. On day 7, dermal punch biopsy specimens were taken from the venom and control inoculation sites. Hair, aspirate, biopsy, and serum specimens were assayed for venom by using an enzyme-linked immunosorbent assay. A generalized linear model was fit with the generalized estimating equation method to estimate the mean differences between groups. RESULTS: Venom was detected in hair, aspirate, and biopsy specimens on all days of the study period. Hair samples yielded venom recovery on day 1 (median 0.062 ng/100 microL; mean difference 0.054 ng/100 microL; 95% confidence interval [CI] 0.048 to 0.059) through day 7 (median 0.020 ng/100 microL; mean difference 0.020 ng/100 microL; 95% CI 0.013 to 0.027). Aspirates were positive for venom recovery on day 1 (median 0.275 ng/100 microL; mean difference 0.231 ng/100 microL; 95% CI 0.192 to 0.271) through day 7 (median 0.0 ng/100 microL; mean difference 0.032 ng/100 microL; 95% CI -0.18 to 0.078). The highest venom yield was from the biopsy specimens (median 1.75 ng/100 microL; mean difference 0.041 ng/100 microL; 95% CI 0.033 to 0.027). Venom was undetectable in all serum samples. CONCLUSION: Loxosceles species venom is detectable in hair, aspirate, and dermal biopsy specimens at least 7 days after venom inoculation and undetectable in serum by using the rabbit model.     

A new ImmunoCAP assay for detection of Echinococcus multilocularis-specific IgE

In alveolar echinococcosis (AE), caused by Echinococcus multilocularis (E.m.), increased levels of total and parasite-specific IgE are frequently found. These may not only have diagnostic but are also supposed to have prognostic value in the follow-up of AE patients. However, there is no commercial test available for quantification of E. m.-specific IgE (sIgE). The only commercial test available is based on E. granulosus (g.) hydatid antigen, which is not optimal for detection of E. m.-specific IgE. Therefore, a new ImmunoCAP with covalently bound crude antigen of E. m. was developed in cooperation with Pharmacia Research Forum for the analysis of E. m. sIgE. The E. m. ImmunoCAP was evaluated in 53 AE patients with different clinical disease progression and 20 healthy controls. Our data showed a higher sensitivity for sIgE determination with E. m. ImmunoCAP compared to the E. g. ImmunoCAP (73.6% vs 61.5%) and a positive correlation between total IgE and sIgE. Furthermore, there was a significant correlation between sIgE in both tests. In conclusion, the new E. m.-specific ImmunoCAP test proved to be a valuable tool for determination of sIgE. It may provide the basis for the development of further E. multilocularis-specific IgE immunotests which are essential for evaluation of sIgE during clinical course of AE.     

Morphological and biochemical evidence of blood vessel damage and fibrinogenolysis triggered by brown spider venom.

The venom of the brown spider is remarkable because it causes dermonecrotic injury, hemorrhagic problems, hemolysis, platelet aggregation and renal failure. The mechanism by which the venom causes hemorrhagic disorders is poorly understood. Rabbits intradermally exposed to the venom showed a local hemorrhage starting 1 h after inoculation and reaching maximum activity between 2 and 3 days. Biopsies examined by light and transmission electron microscopy showed subendothelial blebs, vacuoles and endothelial cell membrane degeneration in blood vessels, plasma exudation into connective tissue, and fibrin and thrombus formation within blood vessels. Loxosceles intermedia venom incubated with fibrinogen partially degrades Aalpha and Bbeta chains of intact fibrinogen, and significantly cleaves all Aalpha, Bbeta and gamma chains when they were separated or when fibrinogen is denatured by boiling. Proteolytic kinetic studies showed that the Aalpha chain is more susceptible to venom hydrolysis than the Bbeta chain. The fibrinogenolysis is blocked by ethylenediamine tetraacetic acid and 1,10-phenanthroline, but not by other protease inhibitors. Human plasma incubated with the venom had coagulation parameters such as prothrombin time, activated partial thromboplastin time and thrombin time increased. Through molecular sieve chromatography, we isolated a venom toxin of 30 kDa with fibrinogenolytic activity. We propose that the local and systemic hemorrhagic disorders evoked in loxoscelism are consequences of direct venom fibrinogenolysis together with cytotoxicity to subendothelial structures and endothelial cells in blood vessels.     

PTH assay: whole PTH assay (new IRMA assay)

The common intact-PTH assay detects not only PTH (1-84) but also PTH (7-84) fragment. Recently, it is reported that PTH (7-84) fragment is the antagonist to PTH (1-84) biological action. Thus, conventional intact-PTH assay might mislead the overestimation of parathyroid function in uremic patients. Whole PTH assay, which detect only PTH (1-84) fragments may be useful for more precise evaluation of PTH activity in uremic patient.     

A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths

Soil-transmitted helminth infections remain a major public health burden in low- and middle-income countries. The traditional diagnosis by microscopic examination of fecal samples is insensitive and time-consuming. In this study, a pentaplex real-time polymerase chain reaction (PCR) was evaluated for the simultaneous detection of Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis. The results were compared with those obtained by conventional parasitological diagnostic methods. Real-time PCR was positive in 48 of 77 samples (62.3%) and microscopic examination was positive in six samples (7.8%) only (P < 0.05). In conclusion, the real-time PCR assay described in this study provides a specific and sensitive diagnostic tool for the detection of these four helminth species in epidemiological studies and monitoring of treatment programs.     

等位基因特异性多重聚合酶链反应方法用于快速检测结核分枝杆菌利福平耐药株

目的建立检测耐利福平(RFP)结核分枝杆菌的等位基因特异性多重聚合酶链反应(multiplex allele specific polymerase chain reaction,MAS-PCR)方法,快速、特异地检测rpoB基因核心突变区的主要突变,用于快速诊断结核分枝杆菌对利福平的耐药性。方法根据结核分枝杆菌的rpoB序列,分别设计出3对特异性寡聚核苷酸引物,采用MAS-PCR技术,分别检测rpoB基因上531、526、516这3个最常见的突变位点的突变。结果对临床分离的利福平敏感株(15株)及利福平突变株(81株)进行检测,以直接测序结果为参照,对利福平耐药株的总检出率为81.5%(66/81)。结论MAS-PCR方法敏感、特异,可快速、简便地检测结核分枝杆菌rpoB基因突变,有利于耐药结核分枝杆菌的快速检测。     

A comparison between a new serological method, thin layer immunoassay (TIA), and the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in schistosomiasis

A comparison has been made of a new serological method, thin layer immunoassay (TIA), and an established method, enzyme-linked immunosorbent assay (ELISA), in the detection and quantification of antibodies in schistosomiasis. Using sera from known S. haematobium and S. mansoni cases, the performance of the two tests was almost identical; 95.6% positive for TIA and 96.4% by ELISA. TIA however produced a small number of false positives with sera from other helminth infections whereas ELISA gave none. There was excellent correlation between the tests in the quantification of anti-S. haematobium antibodies, both in human cases and in infected baboons. TIA has the advantage of being extremely simple to perform, but has the disadvantage of requiring a higher concentration of antigens.     

空肠弯曲菌hipO基因的Real-time PCR检测研究

目的建立基于新型Taqman荧光探针的荧光实时(Real-time)PCR方法用于空肠弯曲菌的筛选检测与快速鉴定。空肠弯曲菌(Campylobacter jejuni)是近十几年来在世界范围内广泛重视的人畜共患病原菌,可以导致人类的急性肠炎与食物中毒,并能引发格林-巴利综合征等并发症。为更好地利用分子生物学技术对空肠弯曲菌进行快速、准确的基因检测,本研究从样品增菌液与单菌落中提取细菌DNA,建立了针对空肠弯曲菌特异的hipO(hippuricase,马尿酸酶)基因的Real-time PCR方法,用于空肠弯曲菌的快速筛选检测与可疑菌落快速鉴定。试验结果表明,该方法检测细菌的灵敏度为1~10CFU,人工布菌鸡肉的检测灵敏度为1~10CFU。本研究所建立的空肠弯曲菌荧光实时PCR方法具有准确、可靠、快速的特点。     

CSTX-13, a highly synergistically acting two-chain neurotoxic enhancer in the venom of the spider Cupiennius salei (Ctenidae)

The survival of the spider Cupiennius salei depends on its hunting success, which largely relies on its immediately paralyzing multicomponent venom. Here, we report on the isolation and characterization of CSTX-13, a neurotoxic enhancer in the spider venom. De novo elucidation of the disulfide bridge pattern of CSTX-13 and the neurotoxin CSTX-1 by tandem MS revealed an identical arrangement. However, in contrast to CSTX-1, CSTX-13 is a two-chain peptide with two interchain and two intrachain disulfide bridges. Furthermore, the insecticidal activity of CSTX-13 is synergistically increased in the presence of K+ ions as well as of the cytolytic peptide cupiennin 1a. We demonstrated that the weakly neurotoxic CSTX-13 enhances the paralytic activity of the neurotoxin CSTX-1 by 65% when it is administered with the latter at its entirely nontoxic physiological concentration, which is 440 times below its LD50 concentration.     

Application of the enzyme linked immunospecific assay (ELISA) for the detection of platelet antibodies

Many methods have been described to identify platelet antibody, but they are either not very sensitive or too complex for general use. Therefore, we have developed an enzyme immunoassay for the detection of platelet antibodies in serum. The method involves incubating platelets with serum antibody; any attached antibody is shown by the addition of an enzyme (alkaline phosphatase) labeled anti-human IgG, followed by assay of the enzyme reaction with its substrate. The reaction product is indicated by a color change, which is proportional to the antibody concentration. Assay conditions such as the use of paraformaldehyde fixed versus unfixed platelets, conjugate dilutions, and substrate concentration and incubation time were investigated. Positive results were obtained in 16 of 19 sera of patients with various diseases including 2 of 4 patients with idiopathic thrombocytopenic purpura, 2 of 2 with post-transfusion purpura, 2 of 3 with neonatal purpura, and all 9 polytransfused patients. Sensitivity and specificity were 84% and 98%, respectively. Also, enzyme linked immunospecific assay (ELISA) was found to be superior to the lymphocytotoxicity (LCT) and platelet immunofluorescence test (PIIFT) for platelet antibody identification.     

Enzyme-linked immunosorbent assay (ELISA) screening test for detection of rheumatoid factor

Summary A new enzyme-linked immunosorbent assay (ELISA) screening test for total rheumatoid factor (RF) activity is described. Rabbit IgG was used as antigen and enzyme-conjugated monoclonal anti-kappa antibody as third layer. Of 183 samples measured for RF isotype levels, 60 were found to have one or more raised. In terms of raised isotypes the ELISA screening test had a sensitivity of 97% (58/60) while the Rheumaton had a sensitivity of only 75% (45/60). Nearly all discordant false-negative samples had only one RF isotype raised. The ELISA test gave 29% (53/183) and the Rheumaton 34% (63/183) false-positive results. Thus the ELISA test was more specific and sensitive for the detection of raised single RF isotypes than the Rheumaton and Rose-Waaler tests. Moreover, approximately 30% of RA patients were seronegative according to the conventional RF tests but only 8% in the new ELISA system.