Epidemiologic and diagnostic evaluation of a recent mumps outbreak using oral fluid samples.

OBJECTIVE: To determine the optimal strategy to investigate mumps virus infection in a partially vaccinated cohort. STUDY DESIGN: 122 oral fluid and serum samples were collected in a recent outbreak in Ireland. The largest age cohort, students aged 18-21 years old attending third level institutions, were investigated using virus isolation, detection of mumps specific IgM, IgG, RT-PCR and molecular genotyping. RESULTS: 97% of patients had both detectable serum IgM and IgG. Mumps virus RNA was detected in 17 oral fluid samples and 14 of these originated from a single geographic location. Only 6 of the IgM positive samples had detectable mumps virus RNA whereas this could be detected in 11 IgM negative samples. Genotyping studies revealed that genotypes G and J were co-circulating during this outbreak. CONCLUSIONS: The use of an oral fluid sample to detect mumps virus RNA and IgM offers a major improvement over serological diagnosis in acute infection in both non-vaccinated or partially vaccinated individuals, and has the advantage that specimens are collected non-invasively.     

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity

The integrity of sperm DNA is crucial for the maintenance of genetic health. A major source of damage is reactive oxygen species (ROS) generation; therefore, antioxidants may afford protection to sperm DNA. The objectives of the study were, first, to measure the effects of antioxidant supplementation in vitro on endogenous DNA damage in spermatozoa using the single cell gel electrophoresis (comet) assay and, second, to assess the effect of antioxidant supplementation given prior to X-ray irradiation on induced DNA damage. Spermatozoa from 150 patients were prepared by Percoll centrifugation in the presence of ascorbic acid (300, 600 microM), alpha tocopherol (30, 60 microM), urate (200, 400 microM), or acetyl cysteine (5, 10 microM). DNA damage was induced by 30 Gy X-irradiation. DNA strand breakage was measured using the comet assay. Sperm DNA was protected from DNA damage by ascorbic acid (600 microM), alpha tocopherol (30 and 60 microM) and urate (400 microM). These antioxidants provided protection from subsequent DNA damage by X-ray irradiation. In contrast, acetyl cysteine or ascorbate and alpha tocopherol together induced further DNA damage. Supplementation in vitro with the antioxidants ascorbate, urate and alpha tocopherol separately has beneficial effects for sperm DNA integrity.      

Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves

Johne's disease of cattle is widespread and causes significant economic loss to producers. Control has been hindered by limited understanding of the immune response to the causative agent, Mycobacterium avium subsp. paratuberculosis, and lack of an effective vaccine and sensitive specific diagnostic assays. The present study was conducted to gain insight into factors affecting the immune response to M. avium subsp. paratuberculosis. A persistent proliferative response to M. avium subsp. paratuberculosis purified protein derivative and soluble M. avium subsp. paratuberculosis antigens was detected in orally infected neonatal calves 6 months postinfection (p.i.) by flow cytometry (FC). CD4(+) T cells with a memory phenotype (CD45R0(+)) expressing CD25 and CD26 were the predominant cell type responding to antigens. Few CD8(+) T cells proliferated in response to antigens until 18 months p.i. gammadelta T cells did not appear to respond to antigen until 18 months p.i. The majority of WC1(+) CD2(-) and a few WC1(-) CD2(+) gammadelta T cells expressed CD25 at time zero. By 18 months, however, subsets of gammadelta T cells from both control and infected animals showed an increase in expression of CD25, ACT2, and CD26 in the presence of the antigens. Two populations of CD3(-) non-T non-B null cells, CD2(+) and CD2(-), proliferated in cell cultures from some control and infected animals during the study, with and without antigen. The studies clearly show multicolor FC offers a consistent reliable way to monitor the evolution and changes in the immune response to M. avium subsp. paratuberculosis that occur during disease progression.     

Antigen-specific B-cell unresponsiveness induced by chronic Mycobacterium avium subsp. paratuberculosis infection of cattle

Mycobacterium avium subsp. paratuberculosis infection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation with M. avium subsp. paratuberculosis antigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate upon M. avium subsp. paratuberculosis antigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne's disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne's disease in cattle.     

Increased yield of full GBA sequencing in Ashkenazi Jews with Parkinson's disease

Background

Variants in GBA are the most common genetic risk factor for Parkinson's disease (PD), and are especially prevalent in the Ashkenazi Jewish (AJ) population. However, most studies on GBA in AJ genotype only seven selected Gaucher-associated pathogenic variants rather than sequencing the whole gene, which may leave carriers of PD-associated GBA variants undiscovered.

Rapid detection and identification of human adenovirus species by adenoplex, a multiplex PCR-enzyme hybridization assay

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10(-2), 10(-1), 10(-1), 10(-2), 10(-1), and 10(-2), respectively. Similarly, the LOD for the six DNA controls ranged from 10(2) to 10(3) copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.     

An evaluation of commercial radioisotope methods for the determination of folate and vitamin B12.

Five commercial kits for the determination of folate and six kits for the determination of vitamin B12 were investigated. Their performance has been compared with microbiological methods for the two vitamins and with a non-commercial radioisotopic method for B12. The results show the importance of the determination of the reference range for an individual laboratory for each method. The precision of the kits varied appreciably, as did their performance using specimens from patients with different haematological disorders. In particular, certain kits failed to detect all patients with pernicious anaemia. The relative accuracy of the kits was assessed. Various factors which should be taken into account in the final selection of a satisfactory kit are discussed.     

Removal of GABAergic inhibition alters subthreshold input in neurons in forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) after digit stimulation

Our objective was to test the hypothesis that suppression of GABAergic inhibition results in an enhancement of responses to stimulation of the surround receptive field. Neurons in the forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) receive short latency suprathreshold input from a principal location on the forepaw and longer latency subthreshold input from surrounding forepaw skin regions. Input from principal and surround receptive field sites was examined before, during, and after administration of the GABA(A) receptor blocker bicuculline methiodide (BMI) (in 165 mM NaCl at pH 3.3-3.5). In vivo extracellular recording was used to first identify the location of the glabrous forepaw digit representation within the FBS. In vivo intracellular recording and labeling techniques were then used to impale single FBS neurons in layer IV as well as neurons in layers III and V, determine the receptive field of the cell, and fill the cell with biocytin for subsequent morphological identification. The intracellular recording electrode was fastened with dental wax to a double-barrel pipette for BMI iontophoresis and current balance. A stimulating probe, placed on the glabrous forepaw skin surface, was used to identify principal and surround components of the receptive field. Once a cell was impaled and a stable recording was obtained, a stimulating probe was placed at a selected site within the surround receptive field. Single-pulse stimulation (1 Hz) was then delivered through the skin probe and the percentage of spikes occurring in 1-min intervals before BMI onset was used as a baseline measure. BMI was then iontophoresed while the periphery was simultaneously stimulated, and spike percentage measured during and after BMI ejection was compared with the pre-BMI baseline. The major findings are: (1) suppression of GABAergic inhibition enhanced evoked responses to firing level from sites in surround receptive fields in 65% of the cells ( n=17); (2) evoked responses were rapidly elevated (within 1 min) to suprathreshold firing in the presence of BMI in 31% of the cells; (3) GABAergic inhibition was reversible [suprathreshold spiking gradually reversed to subthreshold excitatory postsynaptic potentials (EPSPs) in 45% of the cells tested]; (4) BMI altered the stimulus-evoked and non-stimulus-evoked firing pattern in SI neurons from single spikes to burst patterns in all tested cells; and (5) iontophoresis of NaCl (165 mM) without BMI was ineffective in altering evoked responses in control cells ( n=4). The present findings support the notion that subthreshold input from surround receptive fields is one possible mechanism for rapid cortical reorganization in barrel cortex and that GABAergic inhibition may regulate its expression. Possible corticocortical and thalamocortical substrates for subthreshold input to reach barrel neurons are discussed.