An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

Background

Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3.

Results

Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA’s performed at different times was high with Pearson correlation coefficients of 0.96-0.98.

Conclusions

The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.

     

A Double-Blind,Randomised, Crossover Trial of Two Botulinum Toxin Type A in Patients with Spasticity

Background

Botulinum toxin type A (btxA) is one of the main treatment choices for patients with spasticity. Prosigne® a new released botulinum toxin serotype A may have the same effectiveness as Botox® in focal dystonia. However, there are no randomized clinical trials comparing these formulations in spasticity treatment. The aim of our study was to compare the efficacy and safety of Prosigne® with Botox® in the treatment of spasticity.

Methodology/Principal Findings

We performed a double-blind, randomized, crossover study consisting of 57 patients with clinically meaningful spasticity. The patients were assessed at baseline, 4 and 12 weeks after Prosigne® or Botox® administration. The main outcomes were changes in the patients’ Modified Ashworth Scale (MAS), Functional Independence Measure (FIM) and Pediatric Evaluation of Disability Inventory (PEDI) scores and adverse effects related to the botulinum toxin. Both of the toxins were significantly effective in relieving the level of spasticity in adults and children. There were no significant differences found between the Prosigne® and Botox® treatments regarding their MAS, FIM and PEDI scores. Likewise, the incidence of adverse effects was similar between the two groups.

Conclusion

Our results suggest that Prosigne® and Botox® are both efficient and comparable with respect to their efficacy and safety for the three month treatment of spasticity.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00819065","term_id":"NCT00819065"}}NCT00819065.     

Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.     

Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives

To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.

Methods

We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months.

Results

The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies.

Conclusions

Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.     

How the Polls Can Be Both Spot On and Dead Wrong: Using Choice Blindness to Shift Political Attitudes and Voter Intentions

Political candidates often believe they must focus their campaign efforts on a small number of swing voters open for ideological change. Based on the wisdom of opinion polls, this might seem like a good idea. But do most voters really hold their political attitudes so firmly that they are unreceptive to persuasion? We tested this premise during the most recent general election in Sweden, in which a left- and a right-wing coalition were locked in a close race. We asked our participants to state their voter intention, and presented them with a political survey of wedge issues between the two coalitions. Using a sleight-of-hand we then altered their replies to place them in the opposite political camp, and invited them to reason about their attitudes on the manipulated issues. Finally, we summarized their survey score, and asked for their voter intention again. The results showed that no more than 22% of the manipulated replies were detected, and that a full 92% of the participants accepted and endorsed our altered political survey score. Furthermore, the final voter intention question indicated that as many as 48% (±9.2%) were willing to consider a left-right coalition shift. This can be contrasted with the established polls tracking the Swedish election, which registered maximally 10% voters open for a swing. Our results indicate that political attitudes and partisan divisions can be far more flexible than what is assumed by the polls, and that people can reason about the factual issues of the campaign with considerable openness to change.     

No Fabry Disease in Patients Presenting with Isolated Small Fiber Neuropathy

Objective

Screening for Fabry disease in patients with small fiber neuropathy has been suggested, especially since Fabry disease is potentially treatable. However, the diagnostic yield of testing for Fabry disease in isolated small fiber neuropathy patients has never been systematically investigated. Our aim is to determine the presence of Fabry disease in patients with small fiber neuropathy.

Methods

Patients referred to our institute, who met the criteria for isolated small fiber neuropathy were tested for Fabry disease by measurement of alpha-Galactosidase A activity in blood, lysosomal globotriaosylsphingosine in urine and analysis on possible GLA gene mutations.

Results

725 patients diagnosed with small fiber neuropathy were screened for Fabry disease. No skin abnormalities were seen except for redness of the hands or feet in 30.9% of the patients. Alfa-Galactosidase A activity was tested in all 725 patients and showed diminished activity in eight patients. Lysosomal globotriaosylsphingosine was examined in 509 patients and was normal in all tested individuals. Screening of GLA for mutations was performed for 440 patients, including those with diminished α-Galactosidase A activity. Thirteen patients showed a GLA gene variant. One likely pathogenic variant was found in a female patient. The diagnosis Fabry disease could not be confirmed over time in this patient. Eventually none of the patients were diagnosed with Fabry disease.

Conclusions

In patients with isolated small fiber neuropathy, and no other signs compatible with Fabry disease, the diagnostic yield of testing for Fabry disease is extremely low. Testing for Fabry disease should be considered only in cases with additional characteristics, such as childhood onset, cardiovascular disease, renal failure, or typical skin lesions.     

Simple and noninvasive method for assessment of digestive efficiency: Validation of fecal steatocrit in greenfinch coccidiosis model

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Barefoot Plantar Pressure Indicates Progressive Neurological Damage in Patients with Human T-Cell Lymphotropic Virus Type 1 Infection

Background

The human T-Cell Lymphotropic Virus Type 1 (HTLV-1) is a retrovirus associated with neurological alterations; individuals with HTLV-1 infection may develop HTLV-1 associated myelopathy / tropical spastic paraparesis (HAM/TSP). Frequent neurological complaints include foot numbness and leg weakness. In this study, we compared the distribution of the body weight on different areas of the foot in HTLV-1 patients with HAM/TSP, asymptomatic HTLV-1 patients, and healthy individuals.

Methodology

We studied 36 HTLV-1 infected patients, who were divided in two groups of 18 patients each based on whether or not they had been diagnosed with HAM/TSP, and 17 control subjects. The evaluation included an interview on the patient’s clinical history and examinations of the patient’s reflexes, foot skin tactile sensitivity, and risk of falling. The pressure distribution on different areas of the foot was measured with baropodometry, using a pressure platform, while the patients had their eyes open or closed.

Main Findings

The prevalence of neurological disturbances—altered reflexes and skin tactile sensitivity and increased risk of falling—was higher in HTLV-1 HAM/TSP patients than in HTLV-1 asymptomatic patients. The medium and maximum pressure values were higher in the forefoot than in the midfoot and hindfoot in both HTLV-1 groups. In addition, the pressure on the hindfoot was lower in HAM/TSP patients compared to control subjects.

Conclusions

The neurological disturbances associated with HTLV-1 infection gradually worsened from HTLV-1 asymptomatic patients to HAM/TSP patients. Baropodometry is a valuable tool to establish the extent of neurological damage in patients suffering from HTLV-1 infection.     

Regulation of Gag- and Env-Specific CD8+ T Cell Responses in ART-Na?ve HIV-Infected Patients: Potential Implications for Individualized Immunotherapy

Strategies to develop a functional cure for HIV infection will likely require boosting of effector T cell responses to eliminate reactivated, latently infected cells. We have recently explored an assay for assessing antigen-specific regulation of T cell proliferation, which was related to clinical progression in untreated patients and to vaccine efficacy in two trials of therapeutic Gag-based vaccines. We here expand the same assay to further investigate regulation mediated by various inhibitory pathways. Peripheral blood mononuclear cells from 26 asymptomatic HIV-infected, antiretroviral therapy-naïve patients were stimulated with Gag and Env overlapping peptide panels for 5 days. Monoclonal antibodies (mAbs) blocking inhibitory mediators interleukin (IL) 10, transforming growth factor (TGF) β, programmed death ligand (PD–L) 1 and herpes virus entry mediator (HVEM) were added to parallel cultures. Functional T cell regulation (FTR) was defined as the difference in proliferation between stimulated cultures with and without blocking mAbs. FTR was detected in 54% of patients. Blockade of IL-10/PD-L1 and IL10/TGF-β detected all cases with Gag- and Env-associated FTR, respectively. In accordance with previous findings, isolated Env FTR was associated with higher plasma HIV RNA and lower CD4 counts, while patients with both Gag and Env FTR also had higher Gag- and Env-specific proliferative CD8⁺ T cell responses. There was no association between FTR and frequencies of activated regulatory T cells. In conclusion, we observed substantial heterogeneity in FTR between patients, inhibitory pathways and HIV antigens. FTR may help to individualize immunomodulation and warrants further assessment in clinical immunotherapy trials.     

A high-throughput method for liquid chromatography–tandem mass spectrometry determination of plasma alkylresorcinols,biomarkers of whole grain wheat and rye intake

Plasma alkylresorcinols are increasingly analyzed in cohort studies to improve estimates of whole grain intake and their relationship with disease incidence. Current methods require large volumes of solvent (>10 ml/sample) and have relatively low daily sample throughput. We tested five different supported extraction methods for extracting alkylresorcinols from plasma and improved a normal-phase liquid chromatography coupled to a tandem mass spectrometer method to reduce sample analysis time. The method was validated and compared with gas chromatography–mass spectrometry analysis. Sample preparation with HybridSPE supported extraction was most effective for alkylresorcinol extraction, with recoveries of 77–82% from 100 μl of plasma. The use of 96-well plates allowed extraction of 160 samples per day. Using a 5-cm NH₂ column and heptane reduced run times to 3 min. The new method had a limit of detection and limit of quantification equivalent to 1.1–1.8 nmol/L and 3.5–6.1 nmol/L plasma, respectively, for the different alkylresorcinol homologues. Accuracy was 93–105%, and intra- and inter-batch precision values were 4–18% across different plasma concentrations. This method makes it possible to quantify plasma alkylresorcinols in 100 μl of plasma at a rate of at least 160 samples per day without the need for large volumes of organic solvents.