High functional levels of thrombin-activatable fibrinolysis inhibitor are associated with an increased risk of first ischemic stroke.

BACKGROUND AND OBJECTIVE: Several studies have suggested that thrombin-activatable fibrinolysis inhibitor (TAFI) levels are associated with the risk of arterial thrombosis, but results have been contradictory. We studied functional TAFI levels and TAFI gene polymorphisms in 124 patients with a recent ischemic stroke and 125 age- and sex-matched controls to establish the role of TAFI in ischemic stroke. METHODS AND RESULTS: Functional TAFI levels, defined as TAFI-related retardation (RT), the difference in clot lysis time (LT) in the absence or presence of a specific activated TAFI inhibitor (potato carboxypeptidase inhibitor [PCI]), were higher in patients than controls (19.5 +/- 4.2 vs. 17.7 +/- 3.7 min, P < 0.005). Clot LTs in the presence of PCI, which were independent of TAFI, were also increased in ischemic stroke patients. This indicates that in these patients fibrinolysis is impaired not only by high TAFI levels, but also by other mechanisms. Individuals with functional TAFI levels in the highest quartile had an increased risk of ischemic stroke compared with the lowest quartile [odds ratio (OR) 4.0, 95% confidence interval (CI): 1.6-9.8]. In an unselected group of 36 of the 125 stroke patients functional TAFI levels were also measured at 3 months, and were persistently high. This indicates that increased functional TAFI levels after stroke are not caused by an acute phase reaction. No difference was found between patients and controls with respect to TAFI genotype distribution. CONCLUSIONS: Increased functional TAFI levels, resulting in decreased fibrinolysis, are associated with an increased risk of ischemic stroke.

The -148 C/T fibrinogen gene polymorphism and fibrinogen levels in ischaemic stroke: a case-control study.

BACKGROUND: To determine whether -148 C/T fibrinogen gene promoter polymorphism increases stroke risk by modifying the fibrinogen level. DESIGN: A case-control study of patients with first ever ischaemic stroke, confirmed by computed tomography. METHODS: Venous blood samples were collected for fibrinogen and routine coagulation tests one week after the stroke, and after three months in about half the patients. Population controls were age and sex matched. -148 C/T fibrinogen polymorphism was determined by polymerase chain reaction followed by digestion with restriction enzymes HindIII/AluI. RESULTS: There were 124 patients and 125 controls, mean age 56 years (range 18 to 75); 34 patients (27%) and 41 controls (33%) were heterozygous for -148 C/T fibrinogen polymorphism; six patients (5%) and five controls (4%) had the T/T genotype. The odds ratio of ischaemic stroke associated with CC homozygotes v T carriers was 0.8 (95% confidence interval, 0.5 to 1.4). Relative risk for ischaemic stroke associated with fibrinogen levels in the highest quartile was 3.9 (1.9 to 8.4) at one week, decreasing to 1.4 (0.6 to 3.3) at three months. CONCLUSIONS: -148 C/T fibrinogen gene polymorphism was not a strong risk factor for ischaemic stroke. High fibrinogen levels early after acute stroke probably represent an acute phase response.

[From gene to disease; from mutations in the Von Willebrand factor gene to hemorrhagic diathesis and thrombocytopenia]. / Van gen naar ziekte; van mutaties in het Von Willebrand-factorgen naar hemorragische diathese en trombocytopenie.

The broad spectrum of both clinical presentation and severity of bleeding complications observed in Von Willebrand's disease is the result of a high number of mutations. Most are clustered in specific functional domains, leading to quantitative (types 1 and 3) or qualitative (types 2: A, B, M and N or Normandy) defects in the Von Willebrand factor (VWF) protein. Unlike all the other subtypes, type 2B is the result of a 'gain of function' phenotype characterised by an enhanced binding affinity of the VWF protein to the glycoprotein (Gp) Ib complex of platelets, which causes the platelets to disappear from the circulation, resulting in thrombocytopenia. The thrombocytopenia can be triggered by stress or desmopressin, among other causes, and therefore the latter is contraindicated. Type 2B is characterised in vitro by enhanced platelet agglutination with ristocetin. All 2B mutations are located in exon 28, in a small region encoding the Gp-Ib binding site within the A1 domain of VWF. These mutations inactivate a regulatory function of the A1 domain which facilitates Gp-Ib binding.

[Intermittent thrombocytopenia as a manifestation of Von Willebrand's disease]. / Intermitterende trombocytopenie als uiting van de ziekte van Von Willebrand.

A 38-year-old man with Von Willebrand's disease type 2 came to us for treatment advice in relation to a trip abroad, and also a 53-year-old woman with bleeding treated as idiopathic thrombocytopenic purpura (ITP). In the man, a trial dose of desmopressin led to severe thrombopenia, and in the woman, treatments, such as splenectomy and prednisone in the past, had been ineffective. In both patients further investigations led to the diagnosis 'Von Willebrand-disease type 2B'. Despite the fact that Von Willebrand's disease type 2B has distinctive laboratory characteristics, such as thrombocytopenia, a positive Ristocetin Induced Platelet Agglutination (RIPA) test at a low ristocetin concentration, and an abnormal multimer pattern, some cases have incomplete or atypical presentations which can be misleading for the diagnosis. A wrong diagnosis can lead to ineffective and potentially dangerous therapeutic interventions. Molecular genetic analysis of type 2B mutations is simple and can help in making the correct diagnosis in cases of familial thrombocytopenia or for a further characterisation of Von Willebrand type 2.

Elevated prothrombin is a risk factor for cerebral arterial ischemia in young adults.

The prevalence of elevated prothrombin (PT) in the absence of the G20210A mutation has not been studied in patients with cerebral ischemia. We carried out a case-control study of PT G20210A and PT activity in 49 adult patients aged 45 years or less, with TIA or ischemic stroke without cardiac embolism or large vessel disease, and 87 controls from a group of blood donors. Five patients were heterozygous for PT 20210A (OR=2.3, 95% CI: 0.6-8.0). Even after exclusion of individuals with the PT gene variant, the PT activity was significantly higher in patients than in controls (1.11 vs. 0.97, P=0.0003). The relative risk of cerebral ischemia in patients within the fourth quartile of PT activity (1.10 U/ml or higher), was 3.2 fold (95% CI: 1.03-9.96), than in patients whose level of PT activity was in the second or third quartile. We conclude that, although PT 20210A may be a weak risk factor for TIA and ischemic stroke in young patients, increased PT activity, which is more frequent than the mutation, appears to be more strongly related to cerebral ischemia.

cumA, a gene encoding a multicopper oxidase, is involved in Mn2+ oxidation in Pseudomonas putida GB-1.

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designated cumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putida wild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designated cumB) is located downstream from cumA. Both cumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 of Bradyrhizobium japonicum. A mutation in orf74 resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumA mutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.

The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1.

A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn2+ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.

High sensitivity, homogeneous particle-based immunoassay for thyrotropin (Multipact).

We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).

[Influenza in piglets, back again?]. / Biggengriep, terug van weggeweest?

A diagnosis of Mycoplasma hyopneumoniae infection in coughing piglets was verified using an ELISA technique by the Animal Health Service of the province of Limburg, the Netherlands.

Influence of a crown-gall tumor initiation enhancer on bacterial attachment to the host plant cell wall.

The competitive activities of different plant cell walls upon Agrobacterium tumefaciens attachment have been studied in vitro by means of two crown-gall tumor initiation assays. The low or high susceptibility of different plant species is independent of their capacity to cause bacterial cells to adhere to specific sites on the plant cell walls. However, the attachment properties of cell wall fragments derived from Helianthus cotyledons seem to be age-dependent. It is found that a tumor initiation enhancer, present in extract fractions derived from highly susceptible plants and closely related with the competence for tumor formation, does not influence bacterial adherence. The two steps, attachment and the step by which the tumor initiation enhancer is involved, clearly differ in the processes leading to the transformation of a normal cell into a tumor cell.