WAG-F8(m1Ycb) rats harboring a factor VIII gene mutation provide a new animal model for hemophilia A.

BACKGROUND: We recently described an inherited coagulopathy arising in an inbred colony of WAG/RijYcb rats. The bleeding phenotype, demonstrated by both male and female rats, included periarticular hemorrhage, spontaneous bruising, prolonged bleeding from minor wounds and maternal peripartum deaths. Coagulation testing of affected rats revealed normal prothrombin time but prolongation of activated partial thromboplastin time to twice that of controls. OBJECTIVE: To determine the specific coagulation factor and the underlying genetic defect responsible for the inherited coagulopathy in the WAG/RijYcb rats. RESULTS: Evaluation of individual clotting factor activities revealed that the affected animals had a specific deficiency of factor (F) VIII (FVIII). The FVIII gene (F8) has an autosomal location on chromosome 18 in rats, in contrast to its location on the X chromosome in mice and humans. Sequencing of F8 cDNA led to the identification of a point mutation resulting in a substitution, Leu176Pro, in the A1 domain, that is predicted to disrupt the tertiary structure of the FVIII molecule. Administration of human plasma or human recombinant FVIII corrects the coagulation abnormality in the affected animals. CONCLUSIONS: We have now identified the genetic basis of the hemostatic defect in the WAG/RijYcb rat colony. The larger size of rats relative to mice and the presence of this coagulation defect in both sexes provide a unique model, well-suited to the development of novel therapies for acquired and hereditary FVIII deficiencies.

Morphological variability and cell-wall deficiency in Mycobacterium tuberculosis 'heteroresistant' strains.

SETTING: Sofia State Hospital for Tuberculosis Treatment, Bulgaria. OBJECTIVE: To investigate the morphology of two 'heteroresistant' clinical isolates and one non-heteroresistant isolate, all isolated from newly diagnosed tuberculosis (TB) patients, as well as the reference strain H37Rv. Heteroresistant isolates contained clonally-related sensitive and drug-resistant organisms which could subsequently be separated using drug-containing primary cultures and had been isolated from patients originally diagnosed with susceptible TB by the 1% proportion method. Mycobacterial cultures were evaluated by transmission electron microscopy after 25 days of cultivation in Dubos broth. RESULTS: In contrast to H37Rv and the non-heteroresistant isolate, the bacterial populations in both heteroresistant isolates demonstrated distinct pleomorphic variability and coexistence of both classical and cell-wall deficient forms. Electron micrographs of mutants resistant to streptomycin and isoniazid showed predominance of atypical granular L-forms, which formed L-type colonies on Dubos agar. CONCLUSION: The L-form transformation processes, observed both in clinical heteroresistant isolates containing mixed populations of Mycobacterium tuberculosis organisms with different resistance gene genotypes and in the isolated resistant (mutant) clones, indicate a possible link between resistance and cell-wall deficient L-phase states and suggest one of the possible mechanisms by which resistant mutants are able to survive in vivo.

A case-control study for multidrug-resistant tuberculosis: risk factors in four European countries.

The aim of this study was to detect risk factors for multidrug resistance in patients with pulmonary tuberculosis in four European Union countries: France, Germany, Italy, and Spain. A prospective epidemiological case control study was conducted, made up of patients with clinically diagnosed and microbiologically confirmed pulmonary tuberculosis in the four countries between 1997 and 2000. A total of 138 cases and 276 controls were studied. Considering the four countries as a whole, the most statistically significant risk factors were as follows: intravenous drug use (OR 4.68); asylum-seeker support (OR 2.55) as income factor; living in a nursing home (OR 2.05); previous tuberculosis (OR 2.03) with pulmonary location; prison (OR 2.02); known tuberculosis contacts (OR 2.01); immunosuppression other than human immunodeficiency virus (HIV) (OR 1.96); acquired immunodeficiency syndrome (AIDS) (OR 1.96); current tuberculosis with pulmonary location (OR 1.77); and health-care worker (OR 1.69). These risk factors will have to be taken into account in the European Union as a whole, as well as in each individual country, to establish a health policy of monitoring and control for these cases of multidrug resistance. Although rare, their seriousness makes them particularly important.

["Abdominal dressing" - a new method of treatment for open abdomen following secondary peritonitis]. / "Abdominal Dressing" - Eine neue Methode in der Behandlung des offenen Abdomens bei der sekundären Peritonitis.

INTRODUCTION: Treatment of open abdomen following secondary peritonitis is a challenge for surgery and intensive care units (ICU). The aim of this study was to compare three different concurrent treatment strategies. METHODS: Patients suffering an open abdomen following surgery for secondary peritonitis at the Department of General Surgery from 01/01 to 12/03 were investigated. Factor studied: duration of open abdomen, incidence of multi-organ failure, need for surgical revisions, length of stay (LOS) in ICU, nursing requirements (change of dressing/day), survival and integrity of abdominal wall after discharge. Treatment strategies included: open packing (OP), classic vacuum assisted (V.A.C.(R))-therapy with silicone net protection for the intestine (CV) and V.A.C.(R)-therapy with "abdominal dressing" a newly developed meshed polyvinyl wrap (AD). RESULTS: 21 patients were studied: 5 patients were treated with OP, 8 patients with CV and 8 patients with AD. Mean LOS was 65 (OP) vs. 53 (CV) vs. 42 (AD) days (NS), peritonitis related death was 3 (OP) vs. 1 (CV) vs. 0 (AD) (p < 0.05 Chisquare test). Median nursing effort was 4 dressings/day (OP), 0.5 (CV) and 0.5 (AD) (p < 0.005 OP vs CV, AD Kruskal-Wallis test). CONCLUSION: The "abdominal dressing"-therapy seems to be a more efficient treatment option in patients suffering from open abdomen following secondary peritonitis. A trend towards shorter ICU-LOS, lower mortality rates and reduced nursing requirements support our hypothesis.

Validity of species status of the parasitic mite Otodectes cynotis.

A combined molecular and phenotypic approach was used to determine whether ear mites of the genus Otodectes (Acari: Psoroptidae) belong to a single species. The second internal transcribed spacer (ITS 2) of the rDNA of 16 isolates from 11 cats, two dogs, one arctic fox and two ferrets originating from four different continents was characterized. In addition, mites from dog, cat and arctic fox were investigated morphologically. Sequence comparisons revealed five different, but closely related genotypes which did not segregate according to host species or geographical origin. Morphologically, mites of the three host species did not differ significantly in their body or leg sizes. These investigations support the view that ear mites of the genus Otodectes from different hosts and geographical origins belong to a single species, Otodectes cynotis (Hering).

Detection of eight Cryptosporidium genotypes in surface and waste waters in Europe.

Cryptosporidia are important protozoan parasites of vertebrates, and a number of species and genotypes, with different host ranges, have been described. In this study a protocol was established for the detection and the genetic characterization of Cryptosporidium spp. isolated from various types of surface waters (rivers, creeks, lakes, sewage plant in- and outlets and swimming pools) from the area between Zurich (Switzerland) and Munich (Germany). Cryptosporidium oocysts were isolated by continuous-flow-centrifugation and immunomagnetic separation (IMS). A novel nested PCR combined with direct sequencing of the amplicon which spans a variable region of the 18S rRNA allowed characterization of species and genotypes. Cryptosporidium spp. were detected in 23 of the 68 water samples investigated. Almost half of these isolates represent species and genotypes known to be pathogenic to man, namely C. parvum 'bovine genotype' (from 6 samples) and C. parvum 'human genotype' (4). Furthermore, we identified C. muris 'genotype A' (3), C. muris 'genotype B' (6), C. baileyi (1) as well as 3 novel Cryptosporidium genotypes. Our results confirm the ubiquitous presence of Cryptosporidium oocysts in surface waters. The detection of a variety of species and genotypes stresses the importance that molecular characterization is indispensable before drawing conclusions of medical or epidemiological significance.

Zoonotic potential of Enterocytozoon bieneusi.

The reservoirs and the modes of transmission of the most frequent microsporidial species in humans, Enterocytozoon bieneusi, are still unknown. We have examined fecal samples of 26 humans and 350 animals from 37 species to find 18 samples containing this parasite from humans, cats, pigs, cattle, and a llama. Genotypic characterization of the internal transcribed spacer of the rRNA gene resulted in 14 different genotypes, 6 of them previously undescribed. Phylogenetic analysis revealed the lack of a transmission barrier between E. bieneusi from humans and animals (cats, pigs, and cattle). Thus, E. bieneusi appears to be a zoonotic pathogen.

Taxonomic reconsideration of the genus Chorioptes Gervais and van Beneden, 1859 (Acari: Psoroptidae).

The present review of the literature on mites of the genus Chorioptes Gervais and Van Beneden, 1859 argues for a support of the validity of C. bovis (Hering, 1845) and C. texanus Hirst, 1924 based on biological, morphological and molecular genetic studies. However, the validity of three further species. C. crewei Lavoipierre, 1958, C. mydaus Fain, 1975 and C. panda Fain and Leclerc, 1975, is regarded as questionable because discriminations of mites, which were described as isolated cases only, were based on morphological features while transfer or cross-breeding studies were not done.

The effects of heparin, protamine, and heparin/protamine reversal on platelet function under conditions of arterial shear stress.

UNLABELLED: Platelet dysfunction contributes to blood loss after cardiopulmonary bypass. This study examined the antiplatelet effects of heparin, protamine, and varying heparin/protamine ratios in an in vitro physiologic model and further elucidated the mechanism of the antiplatelet and anticoagulant effects of protamine. We used the Clot Signature Analyzer (CSA(TM)), a system that analyzes coagulation in flowing whole blood, to test two aspects of platelet function, with different concentrations of heparin and protamine, under conditions simulating arterial flow: collagen-induced thrombus formation (CITF) under moderate shear and high shear platelet activation, platelet hemostasis time (PHT). In addition, platelet aggregometry, celite activated clotting time (Hepcon(TM) ACT), prothrombin time (PT), and partial thromboplastin time (PTT) were measured. Both PHT and the CITF were prolonged by heparin at 20 microg/mL, protamine at 20 and 40 microg/mL, and heparin/protamine ratios of 1:1 and 1:2, but not at 1:1.5. The Hepcon ACT was prolonged by heparin 20 microg/mL and protamine alone at 20 and 40 microg/mL, was normal at a ratio of 1:1, and was prolonged at 1:1.5 and 1:2. Protamine 80 microg/mL prolonged the PT and PTT. Dependency on thrombin, protein kinase C activation, and nonspecific charge effects were examined. The direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone prolonged the PHT and ACT, but not the CITF, whereas the polycationic molecules polyarginine and polylysine prolonged the CITF, but not the PHT. The effect of protamine on the PTT, but not PT, could be shortened by the addition of excess phospholipid. Therefore, heparin inhibits both high shear collagen-independent and moderate shear collagen-dependent platelet activation; however, the latter is not mediated by its antithrombin activity. Protamine's antithrombin effect may explain its inhibition of platelet activation at high shear stress. Protamine's nonspecific charge effects are more important for inhibiting moderate shear collagen-induced platelet activation. IMPLICATIONS: This study suggests that protamine reversal of heparin's antiplatelet effect occurs within a narrow window because of the direct antiplatelet effects of protamine. Antithrombin effects may explain the inhibition of shear activation of platelets by both heparin and protamine. Nonspecific charge effects of protamine may explain the inhibition of collagen platelet activation in the presence of medium shear.

Heteroresistance in Mycobacterium tuberculosis.

SETTING: Drug resistance in Mycobacterium tuberculosis is often linked to specific mutations in a limited number of resistance genes. Detection of these mutations in a cultured isolate can predict the resistant phenotype. Genotypic analysis of the mycobacteria directly in a clinical specimen would result in considerable time saving for resistance prediction. OBJECTIVE: To find out whether resistance-predicting genotypes of mycobacteria found after cultivation always give a good reflection of those in the original clinical sample. DESIGN: Restriction fragment length polymorphisms of repetitive polymerase chain reaction (PCR) amplification and cloning of PCR products were used as nonintegrative methods to describe the composition of katG, rpsL and embB genotypes involved in resistance to isoniazid, streptomycin and ethambutol, respectively, in the original sample. This result was then compared to the phenotypic resistance profile after cultivation. RESULTS: Using both methods, mixed, heteroresistant populations could be detected in almost every fifth analyzed sample (katG: 5 of 16; rpsL: 3 of 17; embB: 1 of 21). Direct sequencing, a widely used integrative method, repeatedly failed to detect heteroresistance. CONCLUSION: Heteroresistance is a valid phenomenon in clinical tuberculosis. It is not rare and not restricted to a particular resistance gene, and is obscured by cultivation as well as by some, not all, culture-independent resistance prediction tests.

Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production.

The study of megakaryocytopoiesis has been based largely on in vitro assays. We characterize an in vivo model of megakaryocyte and platelet development in which human peripheral blood stem cells (PBSCs) differentiate along megakaryocytic as well as myeloid/lymphoid lineages in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice. Human hematopoiesis preferentially occurs in the bone marrow of the murine recipients, and engraftment is independent of exogenous cytokines. Human colony-forming units-megakaryocyte (CFU-MK) develop predominantly in the bone marrow, and their presence correlates with the overall degree of human cell engraftment. Using a sensitive and specific flow cytometric assay, human platelets are detected in the peripheral blood from weeks 1 to 8 after transplantation. The number of circulating human platelets peaks at week 3 with a mean of 20 x 10(9)/L. These human platelets are functional as assessed by CD62P expression in response to thrombin stimulation in vitro. Exogenous cytokines have a detrimental effect on CFU-MK production after 2 weeks, and animals treated with these cytokines have no circulating platelets 8 weeks after transplantation. Although cytokine stimulation of human PBSCs ex vivo led to a significant increase in CFU-MK, CD34+/41+, and CD41+ cells, these ex vivo expanded cells provided only delayed and transient platelet production in vivo, and no CFU-MK developed in vivo after transplantation. In conclusion, xenogeneic transplantation of human PBSCs into NOD/SCID mice provides an excellent in vivo model to study human megakaryocytopoiesis and platelet production.

Detection of embB codon 306 mutations in ethambutol resistant Mycobacterium tuberculosis directly from sputum samples: a low-cost, rapid approach.

Substitutions of codon 306 in the gene embB are the most common mutations found in ethambutol resistant Mycobacterium tuberculosis. The characterization of these mutations has been hampered by the need for prior cultivation of the mycobacteria, or the need for DNA sequencing, or both. Here, we describe a simple and culture-independent technique to detect embB codon 306 mutations directly from sputum samples, requiring little more than a PCR machine and a simple agarose minigel. There is no need for labelled probes or DNA sequencing. In a preliminary test of feasibility, interpretable results were obtained from 21 of 24 selected sputum samples, 12 of which were determined to contain ethambutol resistant M. tuberculosis after culture. All of six samples with embB codon 306 mutations were correctly identified. Although an exact validation of this technique is beyond the scope of this technical report, we conclude from well-known embB codon 306 mutation prevalence figures that approximately one half of EMB resistant cases could already be predicted within 2 working days, with little equipment or hands-on time needed, instead of weeks required for conventional resistance testing.

Species of the genus Psoroptes (Acari: Psoroptidae): a taxonomic consideration.

The biosystematic status of mite species belonging to the genus Psoroptes Gervais, 1841 is difficult to determine by phenotypic methods and has been subject to taxonomic revisions and ongoing debate. At present, the existence of five species, P cuniculi (Delafond, 1859), P. ovis (Hering, 1838). P. equi (Hering, 1838), P. cervinus Ward, 1915 and P. natalensis Hirst, 1919, is generally accepted. This classification is based mainly on the host species, the localization of the mites on their hosts and morphological characters of male mites. However, a critical review of the literature indicates that the features used to discriminate between the five species are not unequivocal: (a) the localization of mite populations on host animals is not completely strict, (b) the lengths of the outer opisthosomal setae of male mites, which are the main morphological features used for species discrimination, overlap between the five postulated species, and (c) host specificity cannot be deduced from results of transfer experiments. Rather, conspecificity of the members of the genus Psoroptes has to be presumed which is supported by molecular genetic analyses. On these grounds and on rules of priority P. cervinus Ward, 1915, P. cuniculi (Delafond, 1859), P. natalensis Hirst, 1919 and P. ovis (Hering, 1838) are seen as synonyms of P. equi (Hering, 1838).

Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands.

The prevalence of mutations at amino acid (aa) position 315 in the katG gene of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in The Netherlands and the mutation's association with the level of INH resistance, multidrug resistance, and transmission were determined. Of 4288 M. tuberculosis isolates with available laboratory results, 295 (7%) exhibited INH resistance. Of 148 aa 315 mutants, 89% had MICs of 5-10 microg/mL, whereas 75% of the other 130 INH-resistant strains had MICs of 0.5-1 microg/mL. Of the aa 315 mutants, 33% exhibited monodrug resistance, compared with 69% of other INH-resistant strains (P<.0001). Multidrug resistance was found among 14% of the aa 315 mutants and 7% of the other INH-resistant strains (P>.05). The probability of being in an IS6110 DNA restriction fragment length polymorphism cluster was similar for aa 315 mutants and INH-susceptible strains, but the probability was reduced in other INH-resistant strains. Thus, aa 315 mutants lead to secondary cases of tuberculosis as often as INH-susceptible strains do.

Cocaine, HIV, and their cardiovascular effects: is there a role for ACE-inhibitor therapy?

Cocaine abuse and HIV disease each have potentially adverse effects upon the heart and cardiovascular system which may be exacerbated when these risk factors are combined. The development of a safe and effective agent to treat both cocaine addiction and its cardiovascular sequelae, that is well-tolerated by HIV patients, would thus be of considerable clinical utility. In this article we discuss the rationale for the investigation of angiotensin converting enzyme (ACE) inhibitors, commonly used to treat hypertension, for treatment in cocaine-abusing populations, based on their potential to reduce cocaine use by modulating levels of dopamine and corticotropin releasing factor in the brain, and on their ability to reverse cardiovascular and platelet abnormalities. We present preliminary findings from echocardiographic and platelet activation studies in 16 HIV-positive, cocaine abusing patients, as well as tolerability and efficacy studies of the ACE-inhibitor, fosinopril, for the treatment of cocaine abuse in both HIV-positive (n=6) and HIV-negative (n=5) methadone-maintained cocaine abusers. Findings suggest that HIV-positive cocaine-abusing patients possess abnormalities of diastolic heart function and platelet activation that are potentially reversible with ACE-inhibitor therapy. Findings also suggest that fosinopril is well-tolerated regardless of HIV serostatus, does not appear to cause hypotension, and may possess effectiveness for reducing cocaine use. We conclude that ACE-inhibitor therapy may offer a new pharmacologic approach to the treatment of cocaine abuse and its complications, and that controlled research of this class of agents may be promising.

Genotypic status of Babesia microti within the piroplasms.

For resolution of the controversial taxonomic status of Babesia microti in relation to other Babesia and Theileria spp. a phylogenetic analysis of an American and a German B. microti strain was performed on the basis of sequences of the small-subunit rRNA gene (rDNA) using distance-matrix, maximum-parsimony, and maximum-likelihood algorithms. Both B. microti isolates clearly separated from a group containing other Babesia spp. as well as from a second group consisting of Theileria spp. Interestingly, the B. microti isolates clustered in a monophyletic group together with other piroplasm species of unclear taxonomic status, B. rodhaini, and a recently described small canine piroplasm species. These results support the existence of a third taxonomic entity of equal rank besides the Babesiidae and Theileriidae.

Sensitizing capacity of azole derivatives: Part III. Investigations with anthelmintics, antimycotics, fungicides, antithyroid compounds, and proton pump inhibitors.

BACKGROUND: More than hundred azole derivatives are used today for different purposes. The majority possess antimycotic, antibacterial, anthelmintic, and antiprotozoal properties. They are used as agricultural fungicides; topical antimycotics, and, for example, in antidandruff cosmetics. More recently the antithyroid and antiulcerative activity of certain imimidazoles and benzimidazoles also has been proven, which led to the introduction of proton pump inhibitors and antithyroid drugs to the market. OBJECTIVE: Case reports from the literature and experimental studies suggest that some of the azole derivatives possess a distinct sensitizing potency. Occupational exposure either in the pharmaceutical manufacture or in the pesticide's application by farmers caused allergic contact dermatitis (ACD). However, experimental studies in guinea pigs to determine their sensitizing capacity have been performed only sporadically. METHOD: Guinea pigs were sensitized by a modified Freund's complete adjuvant (FCA) method, with 35 azoles used as agricultural fungicides, proton pump inhibitors, antimycotics, antithyroid agents, antiprotozoals, antimicrobials, anthelmintics, and wood preservatives. RESULTS: Four azoles exhibited a strong sensitizing capacity, 3 a moderate, and 11 a weak sensitizing capacity. Seventeen derivatives remained negative. Among the strong sensitizers were the 3 proton pump inhibitors omeprazole, pantoprazole, and rabeprazole, as well as the antithyroid drug carbimazole. The latter displayed the highest sensitizing power (mean response = 2.50) of all 62 azole derivatives investigated in the present and the 2 previous experimental studies. CONCLUSION: As long as the strong sensitizing azoles are used only systemically the risk of acquiring contact hypersensitivity is low. However, if the idea should arise to use them topically, for example in ointments, tinctures, or lotions against fungal infections, skin problems will probably be observed abundantly.

'Babesia gibsoni' of dogs from North America and Asia belong to different species.

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0.5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related species.

Detection of a new pathogenic Babesia microti-like species in dogs.

Small babesiae in dogs are generally considered to belong to Babesia gibsoni. Here we describe the genotypic characterisation of small piroplasms found in the blood of a dog which suffered from clinical babesiosis. Pairwise identities as well as distance, parsimony and maximum likelihood analyses of the 18S rDNA clearly demonstrated that this isolate was only distantly related to the other canine piroplasms characterised genetically so far, including B. gibsoni. It was more closely related to B. microti, B. rodhaini, and Theileria equi. It is concluded that the small canine piroplasms described in this study represent a hitherto unknown species and that the fauna of piroplasms occurring in dogs is more diverse than assumed so far.

Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle.

The reservoirs and the routes of transmission of Enterocytozoon bieneusi are still unknown. In humans, it is the most commonly found microsporidial species. It has also been found repeatedly in pigs, too. The first detection of E. bieneusi in cattle is reported herein. Two distinct genotypes were characterized and compared with 4 other genotypes from humans, 6 from pigs, and 1 from a cat. From these 13 E. bieneusi genotypes known to date, 25 polymorphic sites could be identified in the internal transcribed spacer of the rRNA gene. The spectrum of polymorphisms within and between each of the 4 host species indicates a close relationship between E. bieneusi strains from humans and pigs, whereas those from cattle are more distantly related. The data suggest the absence of a transmission barrier between pigs and humans for this pathogen.