Amyloid formation in human islets is enhanced by heparin and inhibited by heparinase.

Islet transplantation is a promising therapy for patients with diabetes, but its long-term success is limited by many factors, including the formation of islet amyloid deposits. Heparin is employed in clinical islet transplantation to reduce clotting but also promotes fibrillization of amyloidogenic proteins. We hypothesized that heparin treatment of islets during pre-transplant culture may enhance amyloid formation leading to beta cell loss and graft dysfunction. Heparin promoted the fibrillization of human islet amyloid polypeptide (IAPP) and enhanced its toxicity to INS-1 beta cells. Heparin increased amyloid deposition in cultured human islets, but surprisingly decreased islet cell apoptosis. Treatment of human islets with heparin prior to transplantation increased the likelihood of graft failure. Removal of islet heparan sulfate glycosaminoglycans, which localize with islet amyloid deposits in type 2 diabetes, by heparinase treatment decreased amyloid deposition and protected against islet cell death. These findings raise the possibility that pretransplant treatment of human islets with heparin could potentiate IAPP aggregation and amyloid formation and may be detrimental to subsequent graft function.

Nosocomial measles cluster in Denmark following an imported case, December 2008-January 2009.

A cluster of six confirmed cases with identical measles virus genotype was reported in Denmark between December 2008 and January 2009. The findings highlight the importance of vaccination before travelling and adherence to the routine vaccination schedule.

A foodborne outbreak of Cryptosporidium hominis infection.

Foodborne outbreaks of cryptosporidiosis are uncommon. In Denmark human cases are generally infrequently diagnosed. In 2005 an outbreak of diarrhoea affected company employees near Copenhagen. In all 99 employees were reported ill; 13 were positive for Cryptosporidium hominis infection. Two analytical epidemiological studies were performed; an initial case-control study followed by a cohort study using an electronic questionnaire. Disease was associated with eating from the canteen salad bar on one, possibly two, specific weekdays [relative risk 4.1, 95% confidence interval (CI) 2.1-8.3]. Three separate salad bar ingredients were found to be likely sources: peeled whole carrots served in a bowl of water, grated carrots, and red peppers (in multivariate analysis, whole carrots: OR 2.1, 95% CI 1.1-4.0; grated carrots: OR 2.1, 95% CI 1.2-3.9; peppers: OR 3.3, 95% CI 1.7-6.6). We speculate that a person excreting the parasite may have contaminated the salad buffet.

A role of late Epstein-Barr virus infection in multiple sclerosis.

OBJECTIVE: To assess Epstein-Barr virus (EBV) seroconversion in a high multiple sclerosis (MS) prevalence area and to evaluate the recall of diagnosed infectious mononucleosis in MS patients. METHODS: The study was based on information or blood samples, or both, from schoolchildren, young MS patients and matched controls. EBV serology was performed on 1154 blood samples. RESULTS: We demonstrate that more than one third of the population in a high MS prevalence area is seronegative to EBV at puberty. This is in contrast to the virtually complete seroconversion to EBV early in life in individuals from areas with a low prevalence of MS. Furthermore, we demonstrate that recall of diagnosed infectious mononucleosis (IM), but not recall of common childhood diseases, is significantly more frequent among MS patients than healthy controls. All MS patients, including patients without prior immunosuppressive treatment, were EBV seropositive. CONCLUSION: During or after puberty, EBV is transmitted to a major proportion of the population in an MS high-prevalence area. Together with our previous documentation of an association between late infection with EBV and an increased risk of developing MS, these data support a role of EBV infection in MS.

Changing epidemiology of HBV infection in Danish children.

Hepatitis B continues to be a worldwide threat to human health, especially if infection occurs in childhood. Universal vaccination is recommended by WHO, but has not been implemented in the Scandinavian countries, Holland and UK, because of a low incidence rate. However, clinically overt infections are rare in childhood. We therefore performed a nation wide serosurvey for HBV markers in 2428 children aged primarily 6-16 years from 16 primary schools in Denmark. Anti-HBc was found in altogether 20 children (0.8%), 12 of whom were among 144 immigrant children (8.3%) compared to 8 (0.4%) in those born in Denmark. Three of the children, all immigrants, were HBsAg positive indicating chronic infection. At school level no relation of anti-HBc in Danish born children was found to schools with high number of immigrant children or schools with HBsAg positive children indicating a low risk of Hepatitis B transmission in this setting. The results do not support implementation of general vaccination, but stress the need for HBV screening in immigrants as it provides a mean for immunization of close contacts at risk and information on prevention.

Sero-epidemiology of mumps in western Europe.

Six countries (Denmark, England and Wales, France, Germany, Italy and The Netherlands) conducted large serological surveys for mumps, in the mid-1990s, as part of the European Sero-Epidemiology Network (ESEN). The assay results were standardized and related to the schedules and coverage of the immunization programmes and the reported incidence of mumps. Low incidence of disease and few susceptibles amongst adolescents and young adults was observed in countries with high mumps vaccine coverage (e.g. The Netherlands). High disease incidence and large proportions of mumps virus antibody negative samples in adolescent and young adult age groups was noted in countries with poor vaccine coverage (e.g. Italy). The build-up of susceptibles in older children and adolescents in England and Wales, France, the former West Germany and Italy indicate the possibility of further mumps outbreaks in secondary school environments. To control mumps in western Europe, current MMR immunization programmes will need to be strengthened in a number of countries. Sero-surveillance of mumps is an important component of disease control and its usefulness will be enhanced by the development of an international mumps standard.

Macrophages from high-risk HLA-DQB1*0201/*0302 type 1 diabetes mellitus patients are hypersensitive to lipopolysaccharide stimulation.

Levels of nonantigen-induced pro-inflammatory cytokines and prostaglandin in macrophages isolated from human leucocyte antigen (HLA)-matched type 1 diabetes mellitus patients, first-degree relatives and healthy controls were determined. We hypothesize that monocytes isolated from patients are sensitized or preactivated and therefore, have an altered response to in vitro stimulus compared with control groups as measured by levels of pro- and anti-inflammatory mediators. In this study, peripheral blood monocytes were differentiated to macrophages with macrophage-colony stimulating factor (M-CSF) to determine lipopolysaccharide (LPS)-stimulated tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-12 and prostaglandin E-2 (PGE-2) secretion from hetero- or homozygous HLA DQB1*0201 and *0302 type 1 diabetes mellitus patients, first-degree relatives and homozygous HLA DQB1*0602 healthy controls. LPS-stimulated secretion of TNF-alpha, IL-1beta and IL-6 was immediate and markedly higher in the HLA-DQB1*0201/*0302 type 1 diabetes patients compared with all other groups including HLA-matched healthy first-degree relatives. In DQB1*0201/*0302 diabetes patients PGE-2 secretion was delayed but increased by LPS stimulation compared with HLA-matched healthy relatives. IL-12 was not detected at any condition. These data suggest that macrophages from DQB1*0201/*0302 type 1 diabetes patients are sensitized to secrete both cytokines and PGE-2 following nonantigenic stimulation. Sensitized macrophages may be important to high-risk DQB1*0201/*0302-associated type 1 diabetes.

[Cases of measles in Denmark are caused by reintroduction of virus from abroad]. / Maeslingetilfaelde i Danmark skyldes gentagne introduktioner af virus fra udlandet.

Measles vaccination was implemented in the child vaccination programme in Denmark in 1987 and produced a rapid decline in the incidence. Few cases were recorded annually until 1999. The measles virus isolated in Denmark during 1997-1998 was compared by partial sequencing of the haemagglutinin-coding region with Danish strains from the prevaccination era collected in 1965-1983, as well as with representatives of globally circulating strains of today. The dissimilarity of the prevaccination era strains identified in Denmark in 1997-1998 along with the similarity of these five strains with globally circulating strains at present, substantiate the conclusion that there is no persistent circulation of the measles virus in Denmark.

The seroepidemiology of measles in Western Europe.

The European Regional Office of WHO has targeted measles for elimination from the region in 2007. Large national, age and sex stratified serological surveys of measles antibody were conducted in seven Western European countries from 1994-8 as part of the European Seroepidemiology Network. Three patterns were observed in the country-specific measles seroprofiles, ranging from (very) low susceptibility (four countries) to high susceptibility (one country). Susceptibility levels amongst 2-4-year-olds ranged from 2.9 to 29.8%, in 5-9-year-olds from 2.5 to 25% and 10-19-year-olds from 2.1% to 13.9%. A country's susceptibility profile was highly associated with vaccine coverage for the first dose. First dose coverage ranged from 91 to 97.5% for low susceptibility countries, 75 to 85% for intermediate susceptibility countries and 55% for the high susceptibility country. Only the high susceptibility country still reports epidemic measles. In low susceptibility countries, which have achieved or are very close to measles elimination, the priority will be to maintain high MMR vaccine coverage in all geopolitical units for both vaccine doses. In moderate susceptibility countries there is still some endemic transmission, but also risk of outbreaks as pools of susceptibles accumulate. In the high susceptibility country the priority will be to increase infant vaccine coverage and reduce regional variation in coverage levels.

Low serum conditions for in vitro generation of human macrophages with macrophage colony stimulating factor.

Animal serum is often used to generate human macrophages in vitro. Since fetal calf serum (FCS) may complicate antigen uptake, processing and presentation on HLA molecules, we tested the ability of M-CSF to generate macrophages at low fetal calf serum conditions. Peripheral blood monocytes from 12 individuals were cultured 1-4 days with 0-100 ng/ml macrophage colony stimulating factor (M-CSF) at either 1 (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50-100 ng/ml) M-CSF stimulation and low FCS induced 65+/-5% esterase positive cells in all individuals compared to 52+/-7% without M-CSF (P<0.001). M-CSF increased the mean proportion of esterase positive cells after 24 or 96 h by 13% (P<0.005) and 13% (P<0.005), respectively, in 1% FCS, and 8% (P<0.05) and 2% (NS), respectively, in 5% FCS, indicating a slight negative interaction between 5% FCS and M-CSF (P<0.05). All cells were positive for CD14 and HLA class II, but cell number did not increase, confirming that M-CSF promote macrophage differentiation also at low FCS. M-CSF increased the average cell size after 24 or 96 h by 5.9+/-1.0 (P<0.05) and 8.6+/-0.5 (P<0.001) microm, respectively, without an increase in 5% FCS, further demonstrating the efficiency of M-CSF to promote macrophage generation at low FCS. The culture supernatants were negative for IL-1beta and TNF-alpha, which demonstrates that M-CSF did not activate the macrophages. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.

Preservation of enzyme activity and antigenicity after mutagenesis of the membrane anchoring domain of GAD65.

The smaller isoform of glutamic acid decarboxylase, GAD65, is an important autoantigen implicated in the pathogenesis of type 1 diabetes whereas the larger isoform, GAD67 appears to play no major role. The primary difference between the two isoforms resides in the N-terminal part of the molecule including the GAD65 membrane-anchoring domain. The aim of this study was to generate mutants of the membrane targeting domain spanning amino acids 24 to 31 of GAD65 to determine effects on enzyme activity and antibody recognition. Three GAD65 mutants were generated by substituting two, nine or eleven nucleotides coding for the membrane targeting with the corresponding bases of GAD67. SDS-PAGE and Western blotting wildtype (wt) and mutated GAD65 ascertained that they were of similar size and recognized GAD65-specific antibodies. No difference in enzymatic activity was found between the mutants and wt GAD65. GAD65 antibody positive sera from type 1 diabetes patients immunoprecipitated mutated GAD65 whether two, nine or eleven nucleotides were replaced. Mono-or polyclonal antibodies to the N-terminal region demonstrated that the mutated GAD65 with two or nine nucleotides replaced was immunoprecipitated markedly better than wt whereas no difference was detected using antibodies specific for the PLP-binding site in the middle part of GAD65 or the C-terminal region. Taken together, these data suggest that no major conformational changes have been introduced by mutating the membrane-anchoring domain of GAD65.

The seroepidemiology of rubella in western Europe.

Most of the countries in western Europe have now implemented mass infant rubella immunization programmes, instead of or in addition to selective vaccination in order to achieve the elimination of congenital rubella syndrome. The European countries Denmark, England and Wales, Finland, France, Germany, Italy and the Netherlands undertook large, national serological surveys collecting several thousand serum specimens during 1994-8. Antibodies against rubella virus were detected by a variety of enzyme immuno-assays. Comparability of the assay results was achieved by a standardized methodology. The age- and sex-stratified serological results were related to the schedules, coverage of rubella vaccination and the incidence in these countries. The results show widely differing levels of immunity to rubella both in the general population and in the specific age groups of males and females. A low rate (< 5%) of susceptibles in childhood and adolescents of both sexes was obtained only in Finland and the Netherlands. Countries such as Italy with only moderate coverage for the infant immunization programme currently have both high susceptibility levels in the general population and in the at-risk population. The likelihood is of continued epidemics of rubella with cases of congenital rubella syndrome. The continued implementation of selective vaccination will help to offset the impact of this ongoing transmission and to protect women on reaching childbearing age.

Sequence analysis of the hemagglutinin gene of measles virus isolates in Denmark 1997-1998: no evidence of persistent circulation of measles virus in Denmark.

The hemagglutinin-coding region of 17 virus samples from 12 measles cases in Denmark during 1997-1998 was analysed by partial nucleotide sequencing. The cases appeared as three sporadic cases and two epidemics, both with a limited time course and geographical distribution. The measles strains identified from the three sporadic cases and two epidemics could be allocated to five different previously well-defined sequence groups consistent with the assumption that cases of measles in Denmark are due to repeated introduction from abroad rather than persistent circulation of strains in the population.

Case-control study of allergic reactions to Japanese encephalitis vaccine.

Japanese encephalitis (JE) vaccine is widely used in Asia for childhood immunizations, but the vaccine is also used for travellers to Asia from other parts of the world. In Denmark, more than 400,000 doses have been distributed from Statens Serum Institut since 1982. In 1989, the first allergic mucocutaneous reactions after JE vaccination were registered in Denmark and, although the number of reactions have decreased since 1992, reactions are still observed. No explanation of these reactions have been found. The present case-control study, including 49 travellers with allergic reactions and 148 travellers without similar reactions after JE vaccination was performed in order to clarify any possible risk factors. About one third of the adverse reactions to the vaccine could be attributed to an allergic predisposition in the vaccinees. The main risk factors were young age, female gender and previous allergic skin reactions or hayfever. The study also indicated that cases more often reacted to nickel and more often had severe edema after mosquito or other insect bites. Hormone intake was more often spontaneously reported by females in the case group. Accordingly, information on any history of allergy in young adults should be given before JE vaccination, the vaccination should be carried out more than a week before departure and antihistamine treatment should be available if a reaction occurs.

Gait disturbance interpreted as cerebellar ataxia after MMR vaccination at 15 months of age: a follow-up study.

Measles, mumps and rubella (MMR) vaccination was included in the Danish childhood vaccination programme in 1987. During the following 10-y period, 550 notification records of adverse events after MMR vaccination at 15 mo of age have been registered, and a total of 41 notifications have included "gait disturbance". This corresponds to a frequency of 8 per 100,000 doses of MMR vaccine used for 15-mo-old children. The symptoms and signs are characteristic of cerebellar ataxia. In 28 notifications, the descriptions by the doctors included only "gait disturbance", while in 13 an additional interpretation was included. Thirty-two parents (78%) filled in a questionnaire and 26 (63%) agreed to participate in a clinical follow-up study. The gait disturbance symptoms mainly occurred 7-14 d after the vaccination, and the duration was median 1-2 wk (range 1 d to more than 4 mo). One-third of the children had symptoms lasting more than 2 wk. Significantly more children with long duration of symptoms had some kind of complaint or clinical signs at the follow-up in 1997. Gait disturbance registered after MMR vaccination seems to be more frequent than hitherto reported. Most cases are mild and short-lasting and a longer duration of symptoms seems to be predictive of late sequelae. A clinical diagnosis of cerebellar ataxia after MMR and the exact frequency of this adverse event remains to be tested in prospective studies.

Immune response to diphtheria booster vaccine in the Baltic states.

A study was done to measure baseline levels of immunity to diphtheria and antibody responses to different doses of diphtheria vaccine in study participants in the three Baltic states. Diphtheria booster vaccines containing either 3 (Estonia and Lithuania), 6 (Latvia), or 12 (Latvia) limit of flocculation units of diphtheria toxoid were administered to 2315 adults. Diphtheria antibody levels were tested before and 1-2 months after vaccination. Before vaccination, 40% of the participants in Estonia, 32% in Lithuania, and 38% in Latvia had antibody levels <0.01 IU/mL, the level for minimum protection. After vaccination, 79% of the participants in Estonia, 83% in Lithuania, and 81% in Latvia had antibody levels >0. 1 IU/mL, the minimum level for full protection. However, in each of the countries, about one-third of the 40- to 49-year-old participants would have benefited from additional doses of vaccine. There was not a significantly different antibody response among persons receiving the three different doses. Age and the level of prevaccination immunity had a modifying effect on the response to vaccination; however, sex did not.

Infant BCG vaccination study in Lithuania.

SETTING: Infants identified in maternity hospitals in Vilnius, Lithuania. OBJECTIVES: To test the capacity of the BCG vaccine, Danish strain 1331 (Danish vaccine), to induce tuberculin reactivity and scar formation in neonates compared to the WHO International Reference Preparation of BCG (IRP vaccine), and to study the effect of dose and of age at vaccination. DESIGN: A randomized four-armed study: 1) normal dose, 0.05 ml Danish vaccine given to neonates at birth, 2) half the normal dose of Danish vaccine given at birth, 3) IRP vaccine given at birth at normal infant dose, and 4) the normal infant dose of Danish vaccine given at 3 months of age. RESULTS: Larger tuberculin reactions, as well as an increased frequency and larger scars, were seen when Danish vaccine was given at 3 months of age in comparison to neonatal vaccination. Halving the dose resulted in smaller reactions, but the difference was not significant. The IRP vaccine resulted in borderline significantly larger reactions in comparison to the Danish vaccine. The number of infants receiving very early vaccination (0-2 days) was not evenly distributed in all groups, however, which is believed to explain the observed difference.

Immunization of diabetes-prone or non-diabetes-prone mice with GAD65 does not induce diabetes or islet cell pathology.

Glutamic acid decarboxylase autoimmunity was investigated by immunizing female BALB/c, C57B1/6, National Marine Research Institute (NMRI) and non-obese diabetic (NOD) mice once or twice with glumatic acid decarboxylase, GAD65, bovine serum albumin, or phosphate-buffered saline in incomplete Freunds adjuvant, or not treating. Mice immunized with GAD65, showed splinic T-cell reactivity to GAD 65 in vitro assessed by cytokine secretion. However untreated NOD mice did not. NOD mice showed a vigorous IFN-gamma response after one immunization, whereas NMRI mice showed a lower response. IL-4 and IL-10 were only detected after two immunizations with higher levels in BALB/c, NMRI and NOD mice, compared to C57B1/6 mice. High levels of GAD65 antibodies were detected in all mice immunized with GAD65, though lower levels were found in C57B1/6 mice. Histological analysis of pancreata revealed that no control mice, regardless of treatment, had mononuclear cell infiltration in the islets. In NOD mice, peri-insulitis was detected in all groups, but less so in GAD65 and bovine serum albumin (BSA) immunized animals. These data demonstrate that NOD mice respond more vigorously to immunization with GAD65 than non-diabetic mice strains. Furthermore, immunization with GAD65 is not sufficient to provoke onset of diabetes in NOD mice or induce islet cell pathology in non-diabetes prone mice.