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.     

Expression of L-Selectin (CD62L), CD44, and CD25 on activated bovine T cells

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.     

Induction of Th1 cytokine responses by mycobacterial antigens in leprosy.

Twelve mycobacterial antigens were compared for induction of gamma interferon (IFN-gamma) secretion by human blood mononuclear cells of patients with leprosy. Fractionated Mycobacterium leprae antigens containing cell wall proteins or cytosolic and membrane proteins induced good IFN-gamma responses in tuberculoid leprosy patients. Lipoarabinomannan from M. tuberculosis Erdman and M. leprae mycolylarabinogalactan peptidoglycan were the poorest IFN-gamma inducers.     

Estradiol effects on the growth hormone/insulin-like growth factor-1 axis in amenorrheic athletes.

Disruption of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis has been reported and studied in menopause, hypothalamic amenorrhea, and anorexia nervosa, but not in weight-stable amenorrheic athletes. We investigated the effects of short-term transdermal estradiol on basal and exercise-stimulated serum GH, IGF-1, and associated binding proteins (IGFBP-1 and IGFBP-3) in seven weight-stable female amenorrheic athletes with percentage body fats greater that 12%. Each subject received a 72 h placebo patch followed by 144 h of transdermal estradiol. Serum samples for GH, IGF-1, IGFBP-1, and IGFBP-3 were obtained at baseline (t1), 72 hr (t2), 144 hr (t3), and during three 90-minute trials of aerobic exercise. Basal, and exercise GH, IGF-1, and IGFBP-1 were not different between trials. Baseline IGFBP-3 decreased from t1 to t2 (p = 0.04) and serum free fatty acids increased from t1 to t2, and t1 to t3 (p = 0.04, and 0.02 respectively). These findings differ from postmenopausal women, and women having weightloss-associated amenorrhea, suggesting that estrogen, exercise, and nutritional deficiencies may have independent effects on the GH/IGF-1 axis.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.