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

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

The Clinical Frailty Scale is a useful tool for predicting postoperative complications following elective colon cancer surgery at the age of 80 years and above: A prospective, multicentre observational study

Aim Identification of the risks of postoperative complications may be challenging in older patients with heterogeneous physical and cognitive status. The aim of this multicentre, observational study was to identify variables that affect the outcomes of colon cancer surgery and, especially, to find tools to quantify the risks related to surgery. Method Patients aged >= 80 years with electively operated Stage I-III colon cancer were recruited. The prospectively collected data included comorbidities, results of the onco-geriatric screening tool (G8), Clinical Frailty Scale (CFS), Charlson Comorbidity Index (CCI) and Mini Nutritional Assessment-Short Form (MNA-SF), and operative and postoperative outcomes. Results A total of 161 patients (mean 84.5 years, range 80-97, 60% female) were included. History of cerebral stroke (64% vs. 37%, p = 0.02), albumin level 31-34 g/l compared with >= 35 g/l (57% vs. 32%, p = 0.007), CFS 3-4 and 5-9 compared with CFS 1-2 (49% and 47% vs. 16%, respectively) and American Society of Anesthesiologists score >3 (77% vs. 28%, P = 0.006) were related to a higher risk of complications. In multivariate logistic regression analysis CFS >= 3 (OR 6.06, 95% CI 1.88-19.5, p = 0.003) and albumin level 31-34 g/l (OR 3.88, 1.61-9.38, p = 0.003) were significantly associated with postoperative complications. Severe complications were more common in patients with chronic obstructive pulmonary disease (43% vs. 13%, p = 0.047), renal failure (25% vs. 12%, p = 0.021), albumin level 31-34 g/l (26% vs. 8%, p = 0.014) and CCI >6 (23% vs. 10%, p = 0.034). Conclusion Surgery on physically and cognitively fit aged colon cancer patients with CFS 1-2 can lead to excellent operative outcomes similar to those of younger patients. The CFS could be a useful screening tool for predicting postoperative complications.Peer reviewe

Detection of a low-grade enteroviral infection in the islets of Langerhans of living patients newly diagnosed with type 1 diabetes

Journal ArticleThis is an author-created, uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association (ADA), publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version is available in Diabetes, May 2015, vol. 64, no. 5 pp. 1682-1687 in print and online at http://diabetes.diabetesjournals.org/content/64/5/1682.abstractThe Diabetes Virus Detection study (DiViD) is the first to examine fresh pancreatic tissue at the diagnosis of type 1 diabetes for the presence of viruses. Minimal pancreatic tail resection was performed 3-9 weeks after onset of type 1 diabetes in six adult patients (age 24-35 years). The presence of enteroviral capsid protein 1 (VP1) and the expression of class I HLA were investigated by immunohistochemistry. Enterovirus RNA was analyzed from isolated pancreatic islets and from fresh-frozen whole pancreatic tissue using PCR and sequencing. Nondiabetic organ donors served as controls. VP1 was detected in the islets of all type 1 diabetic patients (two of nine controls). Hyperexpression of class I HLA molecules was found in the islets of all patients (one of nine controls). Enterovirus-specific RNA sequences were detected in four of six patients (zero of six controls). The results were confirmed in various laboratories. Only 1.7% of the islets contained VP1(+) cells, and the amount of enterovirus RNA was low. The results provide evidence for the presence of enterovirus in pancreatic islets of type 1 diabetic patients, which is consistent with the possibility that a low-grade enteroviral infection in the pancreatic islets contributes to disease progression in humans.Academy of FinlandSouth-Eastern Norway Regional HealthAuthorityNovo Nordisk FoundationPEVNET (Persistent Virus Infection in Diabetes Network) Study GroupEuropean Union’s Seventh Framework ProgrammeSwedish Medical Research CouncilDiabetes Wellness FoundationJDR

Antibody Responses against Enterovirus Proteases are Potential Markers for an Acute Infection

Background: Enteroviruses are a group of common non-enveloped RNA viruses that cause symptoms ranging from mild respiratory infections to paralysis. Due to the abundance of enterovirus infections it is hard to distinguish between on-going and previous infections using immunological assays unless the IgM fraction is studied. Methods: In this study we show using Indirect ELISA and capture IgM ELISA that an IgG antibody response against the nonstructural enteroviral proteins 2A and 3C can be used to distinguish between IgM positive (n = 22) and IgM negative (n = 20) human patients with 83% accuracy and a diagnostic odds ratio of 30. Using a mouse model, we establish that the antibody response to the proteases is short-lived compared to the antibody response to the structural proteins in. As such, the protease antibody response serves as a potential marker for an acute infection. Conclusions: Antibody responses against enterovirus proteases are shorter-lived than against structural proteins and can differentiate between IgM positive and negative patients, and therefore they are a potential marker for acute infections

Reduced Expression of IFIH1 Is Protective for Type 1 Diabetes

IFIH1 (interferon induced with helicase C domain 1), also known as MDA5 (melanoma differentiation-associated protein 5), is one of a family of intracellular proteins known to recognise viral RNA and mediate the innate immune response. IFIH1 is causal in type 1 diabetes based on the protective associations of four rare variants, where the derived alleles are predicted to reduce gene expression or function. Originally, however, T1D protection was mapped to the common IFIH1 nsSNP, rs1990760 or Thr946Ala. This common amino acid substitution does not cause a loss of function and evidence suggests the protective allele, Ala946, may mark a haplotype with reduced expression of IFIH1 in line with the protection conferred by the four rare loss of function alleles. We have performed allele specific expression analysis that supports this hypothesis: the T1D protective haplotype correlates with reduced IFIH1 transcription in interferon-β stimulated peripheral blood mononuclear cells (overall p = 0.012). In addition, we have used multiflow cytometry analysis and quantitative PCR assays to prove reduced expression of IFIH1 in individuals heterozygous for three of the T1D-associated rare alleles: a premature stop codon, rs35744605 (Glu627X) and predicted splice variants, rs35337543 (IVS8+1) and rs35732034 (IVS14+1). We also show that the nsSNP, Ile923V, does not alter pre-mRNA levels of IFIH1. These results confirm and extend the new autoimmune disease pathway of reduced IFIH1 expression and protein function protecting from T1D

Early suppression of immune response pathways characterizes children with prediabetes in genome-wide gene expression profiling

Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic p cells in the islets of Langerhans. Although defects in various T cell subsets have been linked to the disease pathogenesis, mechanisms initiating or enhancing the autoimmunity in prediabetes remain poorly understood. To unravel genes and molecular pathways affected by the diabetes-associated autoimmunity, we investigated transcriptomic profiles of prospective whole-blood samples from children who have developed T1D-associated autoantibodies and eventually clinical T1D. Gene-level investigation of the data showed systematic differential expression of 520 probesets. A network-based analysis revealed then a highly significant down-regulated network of genes involved in antigen presentation as well as T-cell receptor and insulin signaling. Finally, detection of dynamic changes in the affected pathways at the early or late phases of autoimmunity showed down-regulation of several novel T1D-associated pathways as well as known key components of immune response. The longitudinal genome-wide data generated in the present study allows the detection of dynamic changes relevant to the disease that may be completely missed in conventional cross-sectional studies or in genome-wide association studies. Taken together, our analysis showed systemic high-level repression of immune response pathways associated with T1D autoimmunity. (C) 2010 Elsevier Ltd. All rights reserved.</p