The heterogeneity of type IIA von Willebrand's disease: studies with protease inhibitors

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.     

Serological cross-sectional studies on salmonella incidence in eight European countries: no correlation with incidence of reported cases

ABSTRACT: BACKGROUND: Published incidence rates of human salmonella infections are mostly based on numbers of stool culture-confirmed cases reported to public health surveillance. These cases constitute only a small fraction of all cases occurring in the community. The extend of underascertainment is influenced by health care seeking behaviour and sensitivity of surveillance systems, so that reported incidence rates from different countries are not comparable. We performed serological cross-sectional studies to compare infection risks in eight European countries independent of underascertainment. METHODS: A total of 6,393 sera, mostly from existing serum collections representative of the adult population in Denmark, Finland, France, Italy, Poland, Romania, Sweden, and The Netherlands were analyzed. Immunoglobulin A (IgA), IgM, and IgG against salmonella lipopolysaccharides were measured by in-house mixed ELISA. We converted antibody concentrations to estimates of infection incidence ('sero-incidence') using a Bayesian backcalculation model, based on previously studied antibody decay profiles in persons with culture-confirmed salmonella infections. We compared sero-incidence with incidence of cases reported through routine public health surveillance and with published incidence estimates derived from infection risks in Swedish travellers to those countries. RESULTS: Sero-incidence of salmonella infections ranged from 56 (95% credible interval 8-151) infections per 1,000 person-years in Finland to 547 (343-813) in Poland. Depending on country, sero-incidence was approximately 100 to 2,000 times higher than incidence of culture-confirmed cases reported through routine surveillance, with a trend for an inverse correlation. Sero-incidence was significantly correlated with incidence estimated from infection risks in Swedish travellers. CONCLUSIONS: Sero-incidence estimation is a new method to estimate and compare the incidence of salmonella infections in human populations independent of surveillance artefacts. Our results confirm that comparison of reported incidence between countries can be grossly misleading, even within the European Union. Because sero-incidence includes asymptomatic infections, it is not a direct measure of burden of illness. But, pending further validation, it is a promising and cost-effective approach to assess infection risks and to evaluate the effectiveness of salmonella control programmes across countries.     

Assessing potential introduction of universal or targeted hepatitis A vaccination in the Netherlands

In many industrialized countries, hepatitis A incidence rates have declined steadily in the past decades. Since future cohorts of non-vaccinated elderly will lack protection against disease and the burden of hepatitis A is higher with increasing age, this could be an argument in favour of taking preventive measures such as including hepatitis A vaccine into the National Immunisation Program, or offering hepatitis A vaccine to the elderly only. Using a vaccination evaluation scheme, we assessed the potential benefits and drawbacks of introducing hepatitis A vaccine in the National Immunisation Program in the Netherlands. The average number of annual hepatitis A notifications is declining, from 957 in the period 1991 to 1995 to 211 over the period 2006 to 2010. The direct health care costs and costs due to productivity losses per patient are rising, because the age at infection increases and older patients require a relatively higher number of hospitalizations. Initiating a vaccination program would most likely not be cost-effective yet. The annual costs of mass-vaccination are large: about €10 million for infants and €13 million for older people (and only in the first year €210 million), based on current retail prices. The annual effects of mass-vaccination are small: the cost-of-illness in recent years attributed to hepatitis A infection is estimated to be €650,000 per year, and the disease burden is on average 17 DALYs. Given the current low hepatitis A incidence, and the continuing decline in incidence, targeted preventive measures such as vaccinating travellers and other high-risk groups and timely vaccination of close contacts of hepatitis A patients are adequate. However, because susceptibility to hepatitis A is increasing in the group with the highest risk of developing severe complications upon infections, careful monitoring of the epidemiology of hepatitis A remains important.     

The burden of infectious intestinal disease (IID) in the community: a survey of self-reported IID in The Netherlands

In 2009, a 1-year retrospective survey was performed in The Netherlands to estimate the incidence and the disease burden of infectious intestinal disease (IID) in the community, to study the selection of patients consulting a general practitioner and to identify potential risk factors for IID in the community. A questionnaire was sent to 6000 persons selected at random from the population registries of 28 municipalities, with 500 persons being approached per month. A total of 1975 (33%) persons participated. The incidence rate of IID was 964/1000 person-years. Potential risk factors associated with IID in the community were young age (0-4 years) [odds ratio (OR) 3.9, 95% confidence interval (CI) 1.5-10.5], having asthma as a child (OR 3.4, 95% CI 1.1-10.3) and use of gastric acid suppressive medication by persons aged ≥ 45 years (OR 2.8, 95% CI 1.4-5.6). Of the 146 cases with IID, 11 (8%) consulted a physician. Cases with a long duration of symptoms, blood in the stool, children with IID and cases with a low level of education were more likely to consult a physician. Two cases had a stool sample taken and one was admitted to hospital. In conclusion, IID is common and has a significant burden of illness in The Netherlands. Our data indicate that about 15.9 million episodes of IID occur in The Netherlands per year. The incidence rate is substantially higher than the rate of 283/1000 person-years as estimated in 1999 in The Netherlands. This is probably largely due to the retrospective nature of the present study and, to a lesser extent, to differences in case definitions.     

A large increase of Salmonella infections in 2003 in The Netherlands: hot summer or side effect of the avian influenza outbreak?

In June 2003, the Dutch National Salmonella Centre reported a significant excess isolation rate of Salmonella Enteritidis when compared with earlier years in most regional public health laboratories. By the end of 2003, this amounted to an extra 540 laboratory confirmed cases for the whole of the Netherlands, which implies an estimated 7500 extra cases of gastroenteritis caused by S. Enteritidis in the general population, an increase of 50% on previous years. The hot summer could not explain the findings. Strong evidence has been found to suggest that the increase in importation of salmonella contaminated eggs, as a side effect of a concurrent avian influenza outbreak, was the most probable reason for this excess.     

Isovaleric acidaemia presenting with dwarfism,cataract and congenital abnormalities

Isovaleric acidaemia was diagnosed in a 9-year-old girl with an unusual clinical presentation. She was severely mentally retarded, had an extreme growth retardation, bilateral cataracts, multiple fractures of the long bones, vitium cordis and malformations of the head. Frequent infections occurred since early childhood. The relevance of these findings is discussed.     

Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.