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.

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.

Treatment with GAD65 or BSA does not protect against diabetes in BB rats.

The M(r) 65,000 isoform of glutamic acid decarboxylase (GAD65) has been implicated as the initiating islet cell antigen in the pathogenesis of diabetes, primarily based on studies in non-obese diabetic (NOD) mice. To test the role of this islet cell autoantigen in the pathogenesis of spontaneously occurring diabetes in another animal model, purified recombinant human islet GAD65 was injected i.v. at 200 micrograms/animal into 18-day-old diabetes-prone BB rats. For controls, bovine serum albumin (BSA), which has also been implicated in the pathogenesis of diabetes, or buffer alone was injected into age matched BB rats. At 210 days of age there were no differences in diabetes incidence in the 3 groups, i.e. 73% (11 of 15) in the GAD65-treated, 81% (13 of 16) in the BSA-treated and 65% (11 of 17) in the buffer-treated animals, or in the median age at onset of disease, i.e. 79 days (range 65-111), 87 days (range 60-107) and 86 days (range 74-109), respectively. The lack of protection against diabetes following GAD65 treatment could hypothetically be explained by no or by an aberrant expression of GAD in BB-rat islet cells. However, immunohistochemistry of pancreata and immunoblotting analysis of isolated islets showed that the expression of GAD65 and GAD67 was similar in BB and Lewis rats. In conclusion, these data indicate that neither GAD65 nor BSA autoimmunity is important for the development of diabetes in BB rats, in contrast to the situation in NOD mice, and further emphasizes that extrapolation from only one animal model to autoimmune diabetes in general may not be appropriate.

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.

Cefotaxime monotherapy in septicemic patients with hematological malignancies.

Cefotaxime at a dosage of 3 gm intravenously every eight hours was administered to 80 patients with hematological malignancies and suspected septicemia. Blood samples for culturing were taken before and during antibiotic therapy. Nineteen patients had verified bacteremia and ten of them responded completely to cefotaxime. Twelve of the 19 patients had granulocyte counts of less than 0.5 X 10(9)/L. Minimal inhibitory concentrations of cefotaxime, ceftazidime, moxalactam, cefsulodin, cefoxitin, cefuroxime, and cefamandole against the pathogens were measured: cefotaxime was the best cephalosporin against gram-negative isolates and was found acceptable against gram-positive bacteria. In 61 patients no bacteremia could be demonstrated, but specific pathogens were isolated in 11 patients: from the urine in five, from the sputum in five, and from a perianal abscess in one. Complete response was obtained with cefotaxime in seven of these 11 patients. Monotherapy with cefotaxime in septicemic patients with hematological malignancies appears to be a valuable alternative to other antibiotic regimens.

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.

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.

Distribution of SCEs in lymphocytes in persons with normal, slightly increased, and heavily increased SCEs.

The distribution of SCEs in lymphocytes was examined for 165 healthy persons (58 non-smokers and 107 smokers with cigarette consumption ranging from 1 to greater than 20 per day), and for 1 patient treated with melphalan, a cytostatic drug. The data from the healthy persons did not follow a Poisson distribution. A mixed Poisson that allowed different lambda values for the 30 cells scored from each person and postulated a gamma distribution for the lambda s within the 30 cells fitted all the data examined including those from the melphalan-treated patient. In the latter case the 7 samples taken at various times after the treatment could all be represented satisfactorily with a common parameter, c, in the gamma distribution for the lambda s, even though the mean SCEs/cell varied from 9.8 to 36.8. Because the c parameter determines the spread of lambda values within the 30 cells, this suggested that the effect of the cytostatic drug was to increase all the lambda s by a constant amount. The sum of the SCEs taken over all 30 cells in a sample is a convenient summary statistic, and the transformation y = square root s + square root s + 1 behaves as a normal variate with a constant variance within a group.

[The childhood vaccination program. Background, status and future]. / Børnevaccinationsprogrammet. Baggrund, status og fremtid.

Surveillance of the Danish childhood immunization programme has taken place at Statens Seruminstitut since 1980. A description of the prevalence of the diseases, which are included in the programme, is presented. The Danish childhood immunization programme has for many years been one of the best in the world although it differs markedly from other countries. The polio immunization programme with inactivated polio vaccine given first and then later live attennuated vaccine is probably the optimal polio immunization programme. The childhood immunization programme began in 1943 with free diphtheria vaccination, and tetanus immunization was added in 1949. There was a big polio epidemic in 1952/53 and the polio vaccine was introduced in 1955. All three vaccines have markedly reduced the prevalence of these diseases. Pertussis vaccine was introduced in 1961 and measles, mumps and rubella vaccination in 1987. Vaccination against Haemophilus Influenzae type b was introduced with success in 1993. In the future several changes will probably be made in the programme because of the possibility using new combined vaccines.

[The AIDS epidemic in Denmark 1980-1990]. / AIDS-epidemien i Danmark 1980-1990.

By 31.12.1990, a total of 726 patients with AIDS were diagnosed and notified including 679 men (94%) and 47 women (6%). The first diagnosis was made in 1980. Since then, an annual increase in the number of notified AIDS patients has been observed although the increase has declined from 1989 to 1990. During the years, homo/bisexual men have constituted a decreasing proportion of the annual number of notified AIDS patients (p less than 0.001): from 87% in 1980-1986 to 72% in 1989-1990. The proportion of heterosexually infected patients and intravenous drug users increased during the period of study from 3% and 0.7%, respectively, during the period 1980-1986 to 11% and 7%, respectively, during the period 1989-1990. The number of women with AIDS also increased during the period and constituted 9% of notified patients in 1990. The figures are, however, limited. AIDS patients have been notified from all counties but 72% of the patients live in the Copenhagen region. During the period 1987-1990, approximately 2/3 of the notified AIDS patients had been recognized as HIV-positive for more than one year before the diagnosis of AIDS was established. A stable proportion of approximately 1/4 were HIV tested for the first time and found positive around the time the diagnosis of AIDS was established.(ABSTRACT TRUNCATED AT 250 WORDS)

[Markedly increasing incidence of apoplexy among men. A clinical, epidemiologic study of apoplexy incidence between 1972 and 1990 in the municipality of Frederiksberg]. / Markant stigning i apopleksiincidensen blandt maend. En klinisk, epidemiologisk undersøgelse af apopleksisygdommens incidens mellem 1972 og 1990 i Frederiksberg Kommune.

The purpose of this study was to determine possible temporal changes of stroke incidence in a European community. All cases of first-ever stroke (n = 927) were prospectively recorded in the municipality of Frederiksberg, Denmark, in the two periods 1972-1974 and 1989-1990. Complete case ascertainment was ensured by registration of both hospitalized and nonhospitalized patients. Death certificates were also scrutinized. The annual stroke incidence rate per 1000 increased by 18% from 2.6 in 1972-1974, to 3.1 in 1989-1990 (p < 0.01). This increase was due solely to a 42% increase in men, for whom stroke incidence rose from 2.1 to 3.0 (p < 0.0005). Incidence was unchanged in women, 3.0 and 3.1 respectively. In the second study period 85% had CT or necropsy; 91% had cerebral infarction; 8% had intracerebral haemorrhage and 1% had subarachnoid haemorrhage. In a period when decline in stroke incidence has stopped in USA and has continued in Japan, a marked increase in stroke incidence in European men was observed.

[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.

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.

Allergic mucocutaneous reactions to Japanese encephalitis vaccine.

Japanese encephalitis (JE) vaccine has been used in childhood immunization programmes in Asia for many years. Also travellers from other parts of the world have been vaccinated before travelling in Asian countries. Recommendations for the use of JE vaccine are still debated because severe cases of adverse reactions have been reported. In Denmark an inactivated JE vaccine derived from infected mouse brain has been used and 350000 doses have been distributed from the Statens Serum Institut since 1983. In the period 1983-1995, 101 adverse reactions after JE vaccination have been registered including 73 allergic mucocutaneous reactions. These reactions have occurred each year since 1989 with frequencies varying from 1-17 per 10,000 vaccinees. The highest frequencies and the most serious reactions acquiring hospitalization were seen in the period 1989-1992. Three batches (EJN 012, 033 and 048) distributed before 1992 caused the highest number of reactions but all batches delivered in Denmark since 1989 have caused reactions. Therefore, it is still very important to collect information on adverse events as well as information of the risk of acquiring JE infection in order to give sound recommendations to travellers.