Sisko Tauriainen

LUMINESCENT BACTERIAL SENSOR FOR CADMIUM AND LEAD

A sensor plasmid was constructed by inserting the regulation unit from the cadA determinant of plasmid pI258 to control the expression of firefly luciferase. The resulting sensor plasmid pTOO24 is capable of replicating in Gram-positive and Gram-negative bacteria. The expression of the reporter gene as a function of added extracellular heavy metals was studied in Staphylococcus aureus strain RN4220 and Bacillus subtilis strain BR151. Strain RN4220(pTOO24) mainly responded to cadmium, lead and antimony, the lowest detectable concentrations being 10 nM, 33 nM and 1 nM respectively. Strain BR151(pTOO24) responded to cadmium, antimony, zinc and tin at concentrations starting from 3.3 nM, 33 nM, 1 microM, and 100 microM, respectively. The luminescence ratios between induced and uninduced cells, the induction coefficients, of strains RN4220(pTOO24) and BR151(pTOO24) were 23-50 and about 5, respectively. These results were obtained with only 2-3 h incubation times. Freeze-drying of the sensor strains had only moderate effects on the performance with respect to sensitivity or induction coefficients.

Enterovirus RNA in Blood Is Linked to the Development of Type 1 Diabetes

OBJECTIVE To assess whether the detection of enterovirus RNA in blood predicts the development of clinical type 1 diabetes in a prospective birth cohort study. Further, to study the role of enteroviruses in both the initiation of the process and the progression to type 1 diabetes. RESEARCH DESIGN AND METHODS This was a nested case-control study where all case children (N = 38) have progressed to clinical type 1 diabetes. Nondiabetic control children (N = 140) were pairwise matched for sex, date of birth, hospital district, and HLA-DQ–conferred genetic susceptibility to type 1 diabetes. Serum samples, drawn at 3- to 12-month intervals, were screened for enterovirus RNA using RT-PCR. RESULTS Enterovirus RNA–positive samples were more frequent among the case subjects than among the control subjects. A total of 5.1% of the samples (17 of 333) in the case group were enterovirus RNA–positive compared with 1.9% of the samples (19 of 993) in the control group (P < 0.01). The strongest risk for type 1 diabetes was related to enterovirus RNA positivity during the 6-month period preceding the first autoantibody-positive sample (odds ratio 7.7 [95% CI 1.9–31.5]). This risk effect was stronger in boys than in girls. CONCLUSIONS The present study supports the hypothesis that enteroviruses play a role in the pathogenesis of type 1 diabetes, especially in the initiation of the β-cell damaging process. The enterovirus-associated risk for type 1 diabetes may be stronger in boys than in girls.

Type 1 Diabetes Is Associated With Enterovirus Infection in Gut Mucosa

Enterovirus infections have been linked to type 1 diabetes in several studies. Enteroviruses also have tropism to pancreatic islets and can cause β-cell damage in experimental models. Viral persistence has been suspected to be an important pathogenetic factor. This study evaluates whether gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients. Small-bowel mucosal biopsy samples from 39 type 1 diabetic patients, 41 control subjects, and 40 celiac disease patients were analyzed for the presence of enterovirus using in situ hybridization (ISH), RT-PCR, and immunohistochemistry. The presence of virus was compared with inflammatory markers such as infiltrating T cells, HLA-DR expression, and transglutaminase 2–targeted IgA deposits. Enterovirus RNA was found in diabetic patients more frequently than in control subjects and was associated with a clear inflammation response in the gut mucosa. Viral RNA was often detected in the absence of viral protein, suggesting defective replication of the virus. Patients remained virus positive in follow-up samples taken after 12 months’ observation. The results suggest that a large proportion of type 1 diabetic patients have prolonged/persistent enterovirus infection associated with an inflammation process in gut mucosa. This finding opens new opportunities for studying the viral etiology of type 1 diabetes.

Maternal Antibodies in Breast Milk Protect the Child From Enterovirus Infections

OBJECTIVE. Enterovirus infections are frequent in infants and may cause severe complications. We set out to assess whether breastfeeding can protect against these infections and whether such an effect is related to maternal antibodies in breast milk or in the peripheral circulation of the infant. METHODS. One hundred fifty infants who were prospectively followed up from birth were monitored for enterovirus infections. The duration of breastfeeding was recorded, and maternal breast milk and blood samples were regularly taken at 3-month intervals for the detection of enterovirus antibodies and RNA. Maternal serum was available from early pregnancy, delivery, and 3 months postpartum. RESULTS. Enterovirus infections were frequent and were diagnosed in 43% of infants before the age of 1 year and in 15% of the mothers during pregnancy. Infants exclusively breastfed for >2 weeks had fewer enterovirus infections by the age of 1 year compared with those exclusively breastfed for ≤2 weeks (0.38 vs 0.59 infections per child). High maternal antibody levels in serum and in breast milk were associated with a reduced frequency of infections. This effect was seen only in those infants breastfed >2 weeks, indicating that breast milk antibodies mediate this effect. Enterovirus RNA was not found in any of the breast milk samples. CONCLUSIONS. These results suggest that breastfeeding has a protective effect against enterovirus infections in infancy. This effect seems to be mediated primarily by maternal antibodies in breast milk.

Virus Antibody Survey in Different European Populations Indicates Risk Association Between Coxsackievirus B1 and Type 1 Diabetes

Enteroviruses (EVs) have been connected to type 1 diabetes in various studies. The current study evaluates the association between specific EV subtypes and type 1 diabetes by measuring type-specific antibodies against the group B coxsackieviruses (CVBs), which have been linked to diabetes in previous surveys. Altogether, 249 children with newly diagnosed type 1 diabetes and 249 control children matched according to sampling time, sex, age, and country were recruited in Finland, Sweden, England, France, and Greece between 2001 and 2005 (mean age 9 years; 55% male). Antibodies against CVB1 were more frequent among diabetic children than among control children (odds ratio 1.7 [95% CI 1.0–2.9]), whereas other CVB types did not differ between the groups. CVB1-associated risk was not related to HLA genotype, age, or sex. Finnish children had a lower frequency of CVB antibodies than children in other countries. The results support previous studies that suggested an association between CVBs and type 1 diabetes, highlighting the possible role of CVB1 as a diabetogenic virus type.

DETECTING BIOAVAILABLE TOXIC METALS AND METALLOIDS FROM NATURAL WATER SAMPLES USING LUMINESCENT SENSOR BACTERIA

Abstract We have generated microbial sensors for analyzing the presence of various metals or metalloids by recombinant DNA technology. The strains are based on strictly regulated promoters controlling the expression of the firefly luciferase gene in microbial cells. The regulator-reporter constructs are located in shuttle plasmids capable of replicating in gram-negative or -positive microbial organisms. The sensors developed are real-time indicators of metal responsive gene expression giving results in approximately 30xa0min, with optimal induction times ranging from 60 to 240xa0min. We describe here the performance of these metal sensing bacteria for the assessment of different water samples spiked with lead, arsenic, mercury or cadmium. We show that these bacteria are sensitive detectors of metal bioavailability, which is difficult or even impossible to measure by traditional analytical chemistry methods. All measurements were done using freeze-dried bacteria, which makes these sensors reagent-like and also easy to use in field conditions.

Molecular Analysis of an Echovirus 3 Strain Isolated from an Individual Concurrently with Appearance of Islet Cell and IA-2 Autoantibodies

ABSTRACT Growing evidence has implicated members of the genus Enterovirus of the family Picornaviridae in the etiology of some cases of type 1 diabetes (T1D). To contribute to an understanding of the molecular determinants underlying this association, we determined the complete nucleotide sequence of a strain of echovirus 3 (E3), Human enterovirus B (HEV-B) species, isolated from an individual who soon after virus isolation developed autoantibodies characteristic of T1D. The individual has remained positive for over 6 years for tyrosine phosphatase-related IA-2 protein autoantibodies and islet cell autoantibodies, indicating an ongoing autoimmune process, although he has not yet developed clinical T1D. The sequence obtained adds weight to the observation that recent enterovirus isolates differ significantly from prototype strains and provides further evidence of a role for recombination in enterovirus evolution. In common with most HEV-B species members, the isolate exhibits 2C and VP1 sequences suggested as triggers of autoimmunity through molecular mimicry. However, comparisons with the E3 prototype strain and previously reported diabetogenic and nondiabetogenic HEV-B strains do not reveal clear candidates for sequence features of PicoBank/DM1/E3 that could be associated with autoantibody appearance. This is the first time a virus strain isolated at the time of commencement of beta-cell damage has been analyzed and is an invaluable addition to enterovirus strains isolated previously at the onset of T1D in the search for specific molecular features which could be associated with diabetes induction.

Human parechovirus seroprevalence in Finland and the Netherlands

BACKGROUNDnHuman parechoviruses (HPeVs) are RNA viruses associated with mild gastrointestinal and respiratory infections in children, but may also cause neonatal sepsis and CNS infections in infants. While the prevalence of HPeVs is known mostly among hospitalized populations, the knowledge of HPeV seroprevalence in the general population is poor.nnnOBJECTIVESnThe aim of this study was to identify and compare the HPeV1-6 seroprevalence in Finnish and Dutch populations.nnnSTUDY DESIGNnA type specific microneutralization assay was set up for detecting neutralizing antibodies (nABs) against HPeV types 1-6. Altogether 616 serum samples from Finnish and Dutch population were analyzed for antibodies against HPeVs. The samples were collected from Finnish children aged 1, 5 or 10 years, Finnish adults, 0- to 5-year-old Dutch children, Dutch women of childbearing age and Dutch HIV-positive men.nnnRESULTSnIn both adult populations, seropositivity was high against HPeV1 (99% in Finnish and 92% in Dutch samples) and HPeV2 (86% and 95%). Against HPeV4, the seropositivity was similar (62% and 60%). In Dutch adults, nABs against HPeV5 and 6 (75% and 74%) were detected more often than in Finnish adults (35% and 57%, respectively). In contrast, seropositivity against HPeV3 was as low as 13% in the Finnish and 10% in the Dutch adults. The seroprevalence of all HPeV types increased with age.nnnCONCLUSIONSnThe seroprevalence of HPeVs is high in Finnish and Dutch populations and HPeV type 2 and types 4-6 are significantly more prevalent compared to earlier reports. The seroprevalence of antibodies observed against HPeV3 was low.

Maternal Enterovirus Infection as a Risk Factor for Type 1 Diabetes in the Exposed Offspring

OBJECTIVE Maternal enterovirus infections during pregnancy have been linked to an increased risk of type 1 diabetes in the offspring. The aim of this study was to evaluate this association in a unique series of pregnant mothers whose child progressed to clinical type 1 diabetes. RESEARCH DESIGN AND METHODS Maternal and in utero enterovirus infections were studied in 171 offspring who presented with type 1 diabetes before the age of 11 years and in 316 control subjects matched for date and place of birth, sex, and HLA-DQ risk alleles for diabetes. Acute enterovirus infections were diagnosed by increases in enterovirus IgG and IgM in samples taken from the mother at the end of the first trimester of pregnancy and cord blood samples taken at delivery. RESULTS Signs of maternal enterovirus infection were observed in altogether 19.3% of the mothers of affected children and in 12.0% of the mothers of control children (P = 0.038). This difference was seen in different HLA risk groups and in both sexes of the offspring, and it was unrelated to the age of the child at the diagnosis of diabetes or the age of the mother at delivery. CONCLUSIONS These results suggest that an enterovirus infection during pregnancy is not a major risk factor for type 1 diabetes in childhood but may play a role in some susceptible subjects.

Detection of enteroviruses in stools precedes islet autoimmunity by several months: possible evidence for slowly operating mechanisms in virus-induced autoimmunity

Aims/hypothesisThis case–control study was nested in a prospective birth cohort to evaluate whether the presence of enteroviruses in stools was associated with the appearance of islet autoimmunity in the Type 1 Diabetes Prediction and Prevention study in Finland.MethodsAltogether, 1673 longitudinal stool samples from 129 case children who turned positive for multiple islet autoantibodies and 3108 stool samples from 282 matched control children were screened for the presence of enterovirus RNA using RT-PCR. Viral genotype was detected by sequencing.ResultsCase children had more enterovirus infections than control children (0.8 vs 0.6 infections per child). Time-dependent analysis indicated that this excess of infections occurred more than 1xa0year before the first detection of islet autoantibodies (6.3 vs 2.1 infections per 10 follow-up years). No such difference was seen in infections occurring less than 1xa0year before islet autoantibody seroconversion or after seroconversion. The most frequent enterovirus types included coxsackievirus A4 (28% of genotyped viruses), coxsackievirus A2 (14%) and coxsackievirus A16 (11%).Conclusions/interpretationThe results suggest that enterovirus infections diagnosed by detecting viral RNA in stools are associated with the development of islet autoimmunity with a time lag of several months.