An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss

A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.     

Identification of vascular sphincters at the junction between alveolar capillaries and pulmonary venules of the mouse lung

Background: A peculiar feature of lung circulation in the lung is the pronounced variations in blood volume observed in alveolar capillaries that occur because of the changes in the conformation of the alveolar wall that are associated with the respiratory movements. This phenomenon has led to the postulate that mechanisms of postcapillary control of blood flow are to be present in the lung vessels. In the present study we searched for microanatomical evidence of vascular sphincters in the deep lung tissue of mice, namely in alveolar capillaries and pulmonary veins. Methods: We have used scanning electron microscopy (SEM) to examine two types of samples of normal lung tissue of CD-1 mice: 1) vascular corrosion casts made by vascular perfusion with Mercox® resin, and 2) routinely made gold/platinum-coated replicas of sectioned lung tissue. Results: Careful scrutiny of the vessels of the deep lung tissue led to the identification of sphincters in alveolar capillaries. These sphincters were located at the junction between capillary and pulmonary veins. They corresponded to areas to the vascular wall showing circular swellings where a radial organization was observed, since they were made up of alternating grooves and bulges. Transmission electron microscopy showed that smooth muscle cells participated in the formation of the sphincters. Conclusions: Our data reveal a new location for vascular sphincters in pulmonary vessels and, because these novel sphincters are located at the capillary-vein junction, they offer a structural setting for the existence of postcapillary control of blood flow in the pulmonary circulation of mice. © 1995 Wiley-Liss, Inc.     

Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.     

Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.     

Linomide,a novel immunomodulator that prevents death in four models of septic shock

Intravenous injections of 50 μ.g Staphylococcus aureus enterotoxin B (SEB) or bacterial lipopolysaccharide (LPS) are lethal, provided that mice are simultaneously sensitized with either N-galactosamine (GalN) or the anti-glucocorticoid RU-38486. Similar to the synthetic glucocorticoid (GC) receptor agonist dexamethasone, pharmacological doses of the immunomodulator linomide (quinoline-3-carboxamide) prevent death in all four models of lethal septic shock (LPS + GalN, LPS + RU-38486, SEB + GalN, and SEB + RU-38486) and inhibit the secretion of tumor necrosis factor, one of the major intermediate effector molecules of SEB and LPS toxicity. In this system, cyclosporine A (CsA), although effective in suppressing SEB toxicity, fails to counteract the lethal effect of LPS. This observation, together with the fact that linomide acts in the presence of excess amounts of GC receptor antagonist, indicates that linomide functions in a different way to that of known immunosuppressive agents like CsA and GC.     

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH₂ and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min–hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH₂) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.     

Human Lyme arthritis and the immunoglobulin G antibody response to the 37-kilodalton arthritis-related protein of Borrelia burgdorferi

In Borrelia burgdorferi-infected C3H-scid mice, antiserum to a differentially expressed, 37-kDa spirochetal outer-surface protein, termed arthritis-related protein (Arp), has been shown to prevent or reduce the severity of arthritis. In this study, we determined the immunoglobulin G (IgG) antibody responses to this spirochetal protein in single serum samples from 124 antibiotic-treated human patients with early or late manifestations of Lyme disease and in serial serum samples from 20 historic, untreated patients who were followed longitudinally from early infection through the period of arthritis. These 20 patients were representative of the spectrum of the severity and duration of Lyme arthritis. Among the 124 antibiotic-treated patients, 53% with culture-proven erythema migrans (EM) had IgG responses to recombinant glutathione S-transferase (GST)-Arp, as did 59% of the patients with facial palsy and 68% of those with Lyme arthritis. In addition, 75 to 80% of the 20 past, untreated patients had reactivity with this protein when EM was present, during initial episodes of joint pain, or during the maximal period of arthritis. There was no association at any of these three time points between GST-Arp antibody levels and the severity of the maximal attack of arthritis or the total duration of arthritis. Thus, after the first several weeks of infection, 60 to 80% of patients had IgG antibody responses to GST-Arp, but this response did not correlate with the severity or duration of Lyme arthritis.     

Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferon-secreting T lymphocytes of infected holstein cattle

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-gamma) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-gamma secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-gamma enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-gamma ELISPOT and in vitro by measuring T-lymphocyte IFN-gamma production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-gamma-secreting T lymphocytes in cattle with varied MHC genotypes.     

Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin‐coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml^?1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml^?1). The use of biotin‐labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml^?1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non‐biotinylated gliadin. We have found the use of the streptavidin‐biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.     

Impact of depressive symptoms on adult asthma outcomes.

BACKGROUND: Psychological disorders, including depression, are common in adults with asthma. Although depression is treatable, its impact on longitudinal asthma outcomes is not clear. OBJECTIVE: To elucidate the impact of depressive symptoms on patient-centered outcomes and emergency health care use in adults with asthma. METHODS: We conducted a prospective cohort study of 743 adults with asthma who were recruited after hospitalization for asthma. Depressive symptoms were defined as having a score of 16 or more on the Center for Epidemiologic Studies Depression Scale. We examined the impact of depressive symptoms on patient-centered outcomes (validated severity-of-asthma score, Marks Asthma Quality of Life Questionnaire, and 12-Item Short-Form Health Survey physical component summary score) and on future emergency health care use for asthma ascertained from computerized databases. RESULTS: The prevalence of depressive symptoms was 18% (95% confidence interval [CI], 15%-21%) among adults with asthma. Depressive symptoms were associated with greater severity-of-asthma scores after controlling for age, sex, race/ ethnicity, educational attainment, and cigarette smoking (mean score increment, 2.6 points; 95% CI, 1.8-3.4 points). Furthermore, depressive symptoms were associated with poorer asthma-specific quality of life (mean score increment, 19.9 points; 95% CI, 17.7-22.1 points) and poorer physical health status (mean score decrement, 3.7 points; 95% CI, 1.5-5.8 points). Depressive symptoms were associated with a greater longitudinal risk of hospitalization for asthma (hazard ratio, 1.34; 95% CI, 0.98-1.84). After controlling for differences in preventive care for asthma, the relationship was stronger (hazard ratio, 1.45; 95% CI, 1.05-2.0). CONCLUSION: Depressive symptoms are common in adults with asthma and are associated with poorer health outcomes, including greater asthma severity and risk of hospitalization for asthma.