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.     

Amnesia-producing drugs affect hippocampal frequency potentiation

The effects of intravenous injections of lorazepam, scopolamine and propranolol upon hippocampal potentiation produced by commissural stimulation have been investigated in rats anaesthetized with urethane. Administration of 250 micrograms/kg or 500 micrograms/kg lorazepam significantly delayed the onset of secondary potentiation (frequency potentiation) of the population spikes recorded in subfields CA1 and CA3 of the dorsal hippocampus. Scopolamine also delayed the onset of frequency potentiation in CA1, but only at high dose (10 mg/kg). No other measured parameters of frequency potentiation, paired-pulse potentiation or post-tetanic potentiation were affected by any of the drugs. Lorazepam (greater than or equal to 250 micrograms/kg) and propranolol (3 mg/kg) reduced the severity of hippocampal after-discharge. Rhythmic entrainment of after-discharges was occasionally observed. The results are discussed in relation to the possible link between hippocampal potentiation and memory processes.     

Comparison of two ELISAs for the determination of Hsp70 in serum

We have compared a previously developed in-house Sandwich-ELISA with a commercial kit for the determination of heat shock protein (Hsp) 70 in serum. Samples from 64 participants were tested and there was a significant correlation between results obtained using the two assays (r = 0.807, p < 0.0001). Additionally, when ranking samples on a categorical scale, the agreement was good (72%). In the commercial test system Hsp70 was detectable in 42 (66%) of the sera, compared with 61 (95%) in the in-house ELISA method. The three samples with undetectable levels of Hsp70 in the in-house ELISA were among the 22 samples with undetectable levels of Hsp70 in the commercial ELISA kit. The apparent serum concentrations detected were different in the two systems. This dissimilarity can be ascribed to differences in the matrix used. We conclude that the in-house ELISA is more economical and performs well when measuring physiologically high, as well as low, concentrations of Hsp70.     

Marginal zone and germinal center development in the spleens of neonatally thymectomized and nonthymectomized young rats

Spleens from neonatally thymectomized and nonthymectomized young rats were studied histologically and histochemically to elucidate the development of the splenic immune system with and without thymus. In intact animals primitive germinal center activity could be elicited with antigen as early as 13 days of age. More definitive germinal centers lacking tingible body macrophages were observed at 18 days of age. Germinal centers containing tingible body macrophages did not develop until 35 days of age in response to antigenic stimulation. This coincided with maximal development of the marginal zone of medium-sized lymphocytes and the mature development of nodular macrophages possessing strong acid phosphatase activity. Neonatally thymectomized rats developed marginal zones and germinal centers similar to control littermates when the young animals were maintained on tetracycline. Thymectomized animals not given tetracycline showed disturbances in splenic development. These are discussed. The results suggest that the thymus may be critical to the immune system in rats from birth to about 30 days of age but is not essential to its function beyond this period. Marginal zone lymphocytes and germinal center cells proliferate normally and mature to the plasma cell stage in the absence of a thymus if the animals are maintained on tetracycline beyond this critical age.     

Reproducibility of Immunological Tests Used To Assess Multiple Chemical Sensitivity Syndrome

Whether persons with multiple chemical sensitivity syndrome (MCS) have immunological abnormalities is unknown. To assess the reliability of selected immunological tests that have been hypothesized to be associated with MCS, replicate blood samples from 19 healthy volunteers, 15 persons diagnosed with MCS, and 11 persons diagnosed with autoimmune disease were analyzed in five laboratories for expression of four T-cell surface activation markers (CD25, CD26, CD38, and HLA-DR) and in four laboratories for autoantibodies (to smooth muscle, thyroid antigens, and myelin). For T-cell activation markers, the intralaboratory reproducibility was very good, with 90% of the replicates analyzed in the same laboratory differing by ≤3%. Interlaboratory differences were statistically significant for all T-cell subsets except CD4⁺ cells, ranging from minor to eightfold for CD25⁺ subsets. Within laboratories, the date of analysis was significantly associated with the values for all cellular activation markers. Although reproducibility of autoantibodies could not be precisely assessed due to the rarity of abnormal results, there were inconsistencies across laboratories. The effect of shipping on all measurements, while sometimes statistically significant, was very small. These results support the reliability of fresh and shipped samples for detecting large (but perhaps not small) differences between groups of donors in the T-cell subsets tested. When comparing markers that are not well standardized, it may be important to distribute samples from different study groups evenly over time.     

Detecting and analyzing DNA sequencing errors: toward a higher quality of the Bacillus subtilis genome sequence

During the determination of a DNA sequence, the introduction of artifactual frameshifts and/or in-frame stop codons in putative genes can lead to misprediction of gene products. Detection of such errors with a method based on protein similarity matching is only possible when related sequences are available in databases. Here, we present a method to detect frameshift errors in DNA sequences that is based on the intrinsic properties of the coding sequences. It combines the results of two analyses, the search for translational initiation/termination sites and the prediction of coding regions. This method was used to screen the complete Bacillus subtilis genome sequence and the regions flanking putative errors were resequenced for verification. This procedure allowed us to correct the sequence and to analyze in detail the nature of the errors. Interestingly, in several cases in-frame termination codons or frameshifts were not sequencing errors but confirmed to be present in the chromosome, indicating that the genes are either nonfunctional (pseudogenes) or subject to regulatory processes such as programmed translational frameshifts. The method can be used for checking the quality of the sequences produced by any prokaryotic genome sequencing project.     

Immunoglobulins in the egg,embryo and young chick

Current knowledge of the immunoglobulin classes identified in some avian species is reviewed. The distribution and fate of passively acquired immunoglobulins or specific antibodies in compartments of the egg and of the developing embryo and in the newly hatched chick are described, together with the ontogeny of active Ig biosynthesis.     

Bioactivity and immunological characterization of a cholera toxin-cross-reactive cytolytic enterotoxin from Aeromonas hydrophila.

A cytolytic enterotoxin of molecular weight 52,000 was isolated and purified from culture supernatants of a human diarrheal isolate (SSU) of Aeromonas hydrophila. The toxin reacted with cholera antitoxin when tested in an enzyme-linked immunosorbent assay and by Western blot (immunoblot) analysis. The appearance of cytotoxic and hemolytic activities in culture supernatant occurred simultaneously 8 h after the initial inoculation of the culture. Loss of hemolytic activity and cholera toxin cross-reactivity was correlated with heat and pH inactivation. Homologous antibodies neutralized the cytotoxic and hemolytic activities associated with the toxin, but cholera antitoxin did not neutralize these activities. The toxin also possessed enterotoxic activity as demonstrated by fluid accumulation in rabbit ligated intestinal loops. When purified cytolytic enterotoxin was injected intravenously into mice, death occurred within 2 min, whereas mice injected with whole cells or sonicated cell fragments died after several hours or days. Results from 51Cr release experiments demonstrated that the cytolytic enterotoxin had significant membrane-damaging capability. These results indicated that the cytolytic and enterotoxic activities expressed by the described A. hydrophila toxin may contribute significantly to the pathogenesis of disease associated with A. hydrophila.     

Factors affecting transfer of experimental autoimmune thyroiditis in rats

Actively induced experimental autoimmune thyroiditis in inbred Lewis rats was comparable using standard Freund's complete adjuvant (FCA) and Perrin's modification of FCA. However, adoptively transferred disease using lymph node cells from rats immunized with Perrin's FCA was significantly more severe. With this adjuvant, and pertussis vaccine as co-adjuvant, transfer was uniformly successful when at least 480 × 10⁶ lymph node cells were taken 10 days after immunization and recipients were killed 3 days after transfer. Lymphocytic infiltrates were seen in recipient thyroids as early as 18 hours after transfer. Whole body irradiation of the recipients at 550 r reduced the severity of transferred disease. The frequency and severity of lesions were higher when the lymph node cells were first incubated with low doses of antigen. Thymectomy of the recipients decreased the severity of transferred disease. Under the conditions tested, transfer of disease could not be accomplished by antiserum alone, even using thyroidectomized donors. Administration of an early immune serum with sensitized lymph node cells significantly depressed the severity of transferred disease, while a late antiserum increased it.     

Macrophage colony-stimulating factor gene transfer into tumor cells induces macrophage infiltration but not tumor suppression

In order to analyze the effect of a high local concentration of macrophage colony-stimulating factor (M-CSF; CSF-1) on tumor growth, the plasmacytoma cell line J558L was transfected with the human M-CSF gene and injected into syngeneic BALB/c mice. In contrast to the parental tumors, M-CSF transfectants were heavily infiltrated by macrophages as evidenced by immunohistochemistry with antibodies to Mac-1 and Mac-3 and by isolation of the macrophages from the tumor. Nevertheless, tumor growth was only slightly affected by M-CSF and M-CSF-producing cells grew as tumor in all cases. The growth retardation of M-CSF-producing cells varied depending on the experiment and seemed to be due to an indirect effect because the growth rate of the cells in vitro had not changed upon gene transfer. Attempts to activate the tumor-infiltrating macrophages for tumor suppression by systemic application of interferon-γ and/or lipopolysaccharide were not successful. Altogether, our results suggest that M-CSF is a potent chemoattractant for macrophages in vivo but alone is not sufficient to activate these macrophages for tumoricidal activity.