Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite

Luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed, The expression of firefly luciferase was controlled by the regulatory unit of the ars operon of Staphylococcus aureus plasmid pI258 in recombinant plasmid pT0021, with S. aureus RN4220, Bacillus subtilis BR151, and Escherichia coli MC1061 as host strains, Strain RN4220(pT0021) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest detectable concentrations being 100, 33, and 330 nhl, respectively, Strains BR151(pT0021) and MC1061(pT0021) responded to arsenite, arsenate, antimonite, and cadmium, the lowest detectable concentrations being 3.3 and 330 mu M and 330 and 330 nM with BR151(pT0021), respectively, and 3.3, 33, 3.3, and 33 CIM with MC1061(pT0021), respectively, In the absence of the mentioned ions, the expression of luciferase was repressed and only a small amount of background light was emitted, Other ions did not notably interfere with the measurement in any of the strains tested, Freeze-drying of the cells did not decrease the sensitivity of the detection of arsenite; however, the induction coefficients were somewhat lower

Amplifying control RNA for RT-PCR applications by nucleic acid sequence based amplification (NASBA)

Control RNA for RT-PCR applications was amplified by nucleic acid sequence based amplification (NASBA) using the NucliSens (R) Basic Kit. This method was used to construct positive control RNA for enterovirus, insulin, and G-protein RT-PCR, and for interferon-alpha real-time RT-PCR. The primers were designed to amplify identical RNA from RNA templates, which differs from the usual NASBA procedure, where opposite strand RNA is amplified from the target. This "inverse NASBA" method is easy to use and it does not require any expensive special equipment. The amplification reaction is done using a water bath and detection of amplified product by agarose gel electrophoresis. Generated RNA fragments were 195-714 bases long, of positive polarity and the amount of RNA was sufficient for thousands of RT-PCR reactions depending on the sensitivity of the RT-PCR. (c) 2005 Elsevier B.V. All rights reserved

PCR inhibition in stool samples in relation to age of infants

Background: PCR is rapidly replacing traditional methods in diagnostic virus laboratories. PCR inhibitors,which are often present in clinical samples, may lead to false negative test results.Objectives: The aim was to study the presence of PCR inhibitors in stool samples collected from 3- to24-month old children.Study design: Total RNA fraction extracted from stool samples was spiked with a standardized amount ofSemliki Forest Virus RNA and amplified using specific PCR primers. The presence of PCR inhibitors wasdetected by a decrease in amplification rate compared to spiked water samples. Inhibition in differentage groups and dietary origin of PCR inhibitors were analyzed by comparing the samples taken duringexclusive and non-exclusive breastfeeding periods. The inactivation of PCR inhibitors was also assessed.Results: Complete inhibition was seen in 12% (13/108) and partial inhibition in 19% (21/108) of the samples.Inhibition was seen in none of the stool samples (0/31) taken from infants younger than 6 monthscompared to 17% of samples (13/77) taken from6 to 24 months old infants (p more common in younger age group. Addition of bovine serum albumin (BSA) into the reaction mixtureseliminated the effect of inhibitors leading to all samples being positive.Conclusions: PCR inhibitors are frequent in stool samples. They may originate from dietary componentsand can lead to false negative PCR results. The addition of BSA to the cDNA and PCR reactions proved tobe an easy and effective method for eliminating the inhibitory effect of these compounds

Molecular analysis of an echovirus 3 strain isolated from an individual concurrently with appearance of islet cell and IA-2 autoantibodies

Growing evidence has implicated members of the genus Enterovirus of the family Picornaviridae in the etiology of some cases of type I 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

Immunological changes and increased expression of myxovirus resistance protein a in thyroid tissue of patients with recent onset and untreated Graves' disease

Background: Few studies have systematically examined the immune cells that infiltrate thyroid tissue at the time of the onset of Graves&#39; disease (GD). The role of viruses in the pathogenesis of autoimmune thyroid diseases is controversial. The present study analyzed inflammatory responses with respect to signs of virus infection. Methods: Thyroid tissue was obtained from 22 patients with newly diagnosed and untreated GD, 24 patients with chronic GD, and 24 controls. Inflammation was assessed by immunostaining for CD4+ and CD8+ T cells, plasma cells (CD138+), and plasmacytoid dendritic cells (PDCs). The production of interferon-inducible myxovirus resistance protein A (MxA) was analyzed as a sign of virus infection. Results: The degree of thyroid inflammation and fibrosis was significantly higher in both patient groups compared with that in controls. The number of CD4+ T cells and plasma cells (activated B cells) was significantly higher in both patient groups. CD8+ cells were only present in patients with chronic disease. MxA expression and the number of PDCs increased only in patients with newly diagnosed GD. There was a strong positive correlation between the number of PDCs and the number of MxA+ leucocytes. Conclusion: The increase in CD8+ T cells during the chronic stage of GD suggests that they may play a role in progression of the autoimmune process from early to chronic thyroiditis. Upregulation of MxA expression during the early stages of the disease, and the positive correlation between the number of PDCs and the number of MxA+ leucocytes, suggests that activated PDCs secrete type I IFNs at the lesion site, possibly in response to viral infection. &nbsp;</p

Analysis of pancreas tissue in a child positive for islet cell antibodies

Conclusions/interpretation These observations suggest that positivity for ICA alone, even when lasting for more than 1 year, is not associated with inflammatory changes in the islets. However, it is most likely that the pancreatic islets were infected by an enterovirus in this child

No Association Between Ljungan Virus Seropositivity and the Beta-Cell Damaging Process in the Finnish Type 1 Diabetes Prediction and Prevention Study Cohort

BACKGROUND: Ljungan virus (LV) has not confirmed to associate with any human disease, but a possible connection with type 1 diabetes (T1D) has been suggested. Ljungan virus (LV) is a rodent-borne picornavirus that induces a diabetes-like condition in rodents. Approximately 30% of adults and 60% of children are seropositive in Finland. The Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study enabled the use of very well characterized sample panels from children seroconverted to positivity for multiple islet autoantibodies during their prospective observation from birth, in addition samples from age, sex, HLA and residence area matched control children.METHODS: We analyzed LV IgG seroprevalence in 102 case children (65 had also developed T1D), in addition to non-diabetic control children. LV and human parechovirus (HPeV) immunofluorescence assays were used to analyze LV and HPeV specific IgG from 102 plasma samples taken at the time of islet autoantibody appearance, and from 204 samples from the matched control children.RESULTS: Altogether 46.1% of the case and 50.7% of the control children were positive for LV IgG (odds ratio 0.8, 95% CI 0.47-1.36, p=0.416), and 67.6% vs. 79.8% were positive for HPeV IgG, respectively (odds ratio 0.49, 0.27-0.9, p=0.023).CONCLUSIONS: Thus, no risk associations between LV or HPeV -specific IgG, and islet autoimmunity were observed. However, a trend for significantly higher prevalence of HPeV antibodies in control children (p=0.023) suggests a possible protective association of this virus with islet autoimmunity.</p

Co-Morbidity between Early-Onset Leukemia and Type 1 Diabetes – Suggestive of a Shared Viral Etiology?

Background: Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are common early-onset malignancies. Their causes are largely unknown but infectious etiology has been implicated. Type 1 diabetes (T1D) is an autoimmune disease for which infectious triggers of disease onset have been sought and increasing pointing to enteroviruses. Based on our previous results on co-morbidity between leukemia and T1D, we updated the Swedish dataset and focused on early onset leukemias in patients who had been hospitalized for T1D, comparing to those not hospitalized for T1D. Methods and Findings: Standardized incidence ratios (SIRs) were calculated for leukemia in 24,052 patients hospitalized for T1D covering years 1964 through 2008. T1D patients were included if hospitalized before age 21 years. Practically all Swedish children and adolescents with T1D are hospitalized at the start of insulin treatment. SIR for ALL was 8.30 (N = 18, 95% confidence interval 4.91-13.14) when diagnosed at age 10 to 20 years after hospitalization for T1D and it was 3.51 (13, 1.86-6.02) before hospitalization for T1D. The SIR for ALL was 19.85 (N = 33, 13.74-27.76) and that for AML was 25.28 (8, 10.80-50.06) when the leukemias were diagnosed within the year of T1D hospitalization. The SIRs increased to 38.97 (26, 25.43-57.18) and 40.11 (8, 17.13-79.42) when T1D was diagnosed between ages 10 to 20 years. No consistent time-dependent changes were found in leukemia risk. Conclusion: A shared infectious etiology could be a plausible explanation to the observed co-morbidity. Other possible contributing factors could be insulin therapy or T1D related metabolic disturbances

COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants

As SARS-CoV-2 has been circulating for over a year, dozens of vaccine candidates are under development or in clinical use. The BNT162b2 mRNA COVID-19 vaccine induces spike protein-specific neutralizing antibodies associated with protective immunity. The emergence of the B.1.1.7 and B.1.351 variants has raised concerns of reduced vaccine efficacy and increased re-infection rates. Here we show, that after the second dose, the sera of BNT162b2-vaccinated health care workers (n=180) effectively neutralize the SARS-CoV-2 variant with the D614G substitution and the B.1.1.7 variant, whereas the neutralization of the B.1.351 variant is five-fold reduced. Despite the reduction, 92% of the seronegative vaccinees have a neutralization titre of >20 for the B.1.351 variant indicating some protection. The vaccinees' neutralization titres exceeded those of recovered non-hospitalized COVID-19 patients. Our work provides evidence that the second dose of the BNT162b2 vaccine induces cross-neutralization of at least some of the circulating SARS-CoV-2 variants. Emerging SARS-CoV-2 variants contain mutations in the spike protein that may affect vaccine efficacy. Here, Jalkanen et al. show, using sera from 180 BNT162b2-vaccinated health care workers, that neutralization of SARS-CoV2 variant B.1.1.7 is not affected, while neutralization of B.1.351 variant is five-fold reduced.Peer reviewe

COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants

As SARS-CoV-2 has been circulating for over a year, dozens of vaccine candidates are under development or in clinical use. The BNT162b2 mRNA COVID-19 vaccine induces spike protein-specific neutralizing antibodies associated with protective immunity. The emergence of the B.1.1.7 and B.1.351 variants has raised concerns of reduced vaccine efficacy and increased re-infection rates. Here we show, that after the second dose, the sera of BNT162b2-vaccinated health care workers (n = 180) effectively neutralize the SARS-CoV-2 variant with the D614G substitution and the B.1.1.7 variant, whereas the neutralization of the B.1.351 variant is five-fold reduced. Despite the reduction, 92% of the seronegative vaccinees have a neutralization titre of >20 for the B.1.351 variant indicating some protection. The vaccinees’ neutralization titres exceeded those of recovered non-hospitalized COVID-19 patients. Our work provides evidence that the second dose of the BNT162b2 vaccine induces cross-neutralization of at least some of the circulating SARS-CoV-2 variants