Measurement of antigen-specific mouse IgE by a fluorometric reverse (IgE-capture) ELISA

A reverse, or IgE-capture, enzyme-linked immunosorbent assay (ELISA) for measuring ovalbumin-specific IgE antibody in the serum of immunized mice has been developed. Microplate wells were first coated with a commercial anti-mouse IgE rat monoclonal antibody, and then incubated with two-fold serial dilutions of test sera with 10% normal mouse serum as diluent for the capturing of only IgE class molecules. Biotinylated ovalbumin and then beta-D-galactosidase-conjugated streptavidin were added and, finally, 4-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate. The fluorescence intensity of the reaction product (4-methylumbelliferone) was determined on a microplate fluorescence reader. The sensitivity of this assay was equal to that of passive cutaneous anaphylaxis (PCA). In contrast to indirect ELISAs this IgE-capture assay is free from competition by non-IgE antibodies. Furthermore, it requires much less antigen than the PCA assay.     

Detection of alternatively spliced variant messages of Fas gene and mutational screening of Fas and Fas ligand coding regions in peripheral blood mononuclear cells derived from silicosis patients

Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities such as the appearance of autoantibodies and complications of autoimmune diseases. Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, has been considered to play a role in the pathogenesis of autoimmune diseases. It has been found that serum soluble Fas (sFas) levels are elevated in silicosis patients (SIL) and the sFas message is dominantly expressed in peripheral blood mononuclear cells (PBMC) derived from these individuals. In the present study, one tried to detect alternatively spliced variant messages including typical sFas message and found four that were highly and frequently expressed, and which possess a signal peptide domain, but not transmembrane and signal transducing domains, in PBMC derived from SIL. Functional mutations were not detected in Fas and FasL genes in silicosis PBMC. Still, alternative spliced variants of the Fas gene including typical sFas message appear to play an important role in the immunological dysregulation in SIL.     

Structure-activity relationship of mycoloyl glycolipids derived from Rhodococcus sp. 4306

Novel mycoloyl glycolipids with short carbon chains were isolated and purified from Rhodococcus sp. 4306, a soil origin of Actinomycetales. Their chemical structures were identified as trehalose 6,6'-dimycolate (TDM), trehalose 6-monomycolate, glucose 6-monomycolate, mannose 6-monomycolate and fructose 6-monomycolate. The length of carbon chains and number of double bonds of mycolic acids were C(34), C(36)and C(38)saturated, monoenoic and dienoic molecular species, which were much shorter than those of Mycobacterium tuberculosis (C(78-88)monoenoic and dienoic). Among them, only TDM could induce prominent granulomatous inflammation of the lung and spleen in mice. By contrast, other mycoloyl glycolipids induced mild lesions. The small-sized TDM of Rhodococcus possessed granulomatogenic activity, however, the toxicity was much lower than that of M. tuberculosis. Rhodococcal TDM was composed of mycolic acid with the shortest carbon chains, when compared to granulomatogenic TDM of Mycobacterium, Nocardia and Rhodococcus reported previously. Our results imply that rhodococcal TDM is a pathogenetic factor similar to that of M. tuberculosis, although rhodococcal TDM exhibits low toxicity.     

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.     

Characterization of Aeromonas sobria hemolysin by use of monoclonal antibodies against Aeromonas hydrophila hemolysins.

Aeromonas sobria produces hemolysin in a form activable with trypsin under defined cultural conditions. In immunoblotting analyses with the culture supernatant of A. sobria, the monoclonal antibody reacting specifically to Aeromonas hydrophila CA-11 hemolysin bound to the 53,000- and 49,000-dalton bands before and after trypsinization, respectively. The monoclonal antibody reacting to A. hydrophila AH-1 hemolysin did not bind either band. A. sobria hemolysin is, therefore, related antigenically to CA-11 hemolysin, while the molecular weights before and after activation differ from those of A. hydrophila hemolysins, being 54,000 and 51,000, respectively. The hemolytic and enterotoxigenic activities of A. sobria hemolysin were both neutralized by the monoclonal antibody against CA-11 hemolysin. It seems, therefore, that the same site on A. sobria hemolysin is responsible for both biological activities.     

Characterization of CD4+ T helper cells in patients with Kawasaki disease (KD): preferential production of tumour necrosis factor-alpha (TNF-alpha) by V beta 2- or V beta 8- CD4+ T helper cells.

KD is an acute febrile illness in children characterized by coronary arteritis accompanied by aneurysm and thrombotic occlusion. The etiology of KD is unknown. It has been recently reported that KD is associated with the selective expansion of V beta 2+ and V beta 8.1+ T cells in peripheral blood lymphocytes (PBL), by studying the T cell receptor (TCR) repertoire of in vitro activated T cells. KD may therefore be caused by a superantigen [1-3]. To understand better the immunopathology of KD, we investigated TCR V beta 2 and V beta 8.1 expression on both the T cells of freshly isolated PBL and T cell clones (TCC) from patients with KD. Cytokine production by TCC was also studied. Blood samples were obtained from patients with acute (n = 20) and convalescent (n = 20) KD, age-matched children with non-infectious diseases (n = 18), and healthy adults (n = 20). Among these four groups, there were no significant differences in the percentages of either V beta 2+ or V beta 8.1+ T cells of freshly isolated PBL. The same was true for the CD4+ or CD8+ T cell subsets. One hundred and five TCC (98 CD3+ CD4+ CD8- and seven CD3+ CD4- CD8+) established from the affected skin, lymph node or PBL of six patients with KD were also negative for either V beta 2 or V beta 8.1 TCR. Sixty-eight of 105 TCC (65%) produced detectable levels (> 5 pg/ml) of TNF-alpha (6-1016 pg/ml), in the absence of any stimuli. In contrast, only 11 (10%) of 105 TCC or 7 (7%) of 97 TCC produced detectable levels of IL-2 or IL-6, respectively, in the absence of any stimuli. Stimulation with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) induced most TCC to produce higher amounts of TNF-alpha, IL-2 and IL-6. These results suggest that CD4+ T helper cells expressing TCR-beta other than V beta 2 or V beta 8 receptor, primarily through TNF-alpha production, are involved in the immunopathology of KD.     

Induction Of Megamitochondria In The Rat Liver By N-Propyl Alcohol And N-Butyl Alcohol

Propyl alcohol and butyl alcohol had similar effects to ethyl alcohol on ultrastructure of liver mitochondria. Rats were given 32% ethyl alcohol, 32% n-propyl alcohol, and 6.9% n-butyl alcohol in drinking water for up to three months. After one month, mitochondria in hepatocytes obtained from the experimental animals became elongated, constricted or cup-shaped with scanty cristae. After two months, mitochondria in some hepatocytes became gigantic. In extreme cases, the megamitochondria exceeded 10 μm in diameter. Coupling efficiencies of hepatic mitochondria obtained from alcohol-fed animals were well preserved despite their drastic morphologic changes. ACTA PATHOL. JPN. 34: 471–480, 1984.     

Adriamycin-induced DNA strand breaks in HeLa and in P388 leukaemia cells detected using in situ nick translation

DNA strand breaks produced by adriamycin (ADR) were measured in HeLa cells and ADR-sensitive and -resistant P388 leukaemia cells, using the in situ nick translation method. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labelled dTTP, and were visualized by autoradiographic observation of the grains. The DNA strand breaks in the HeLa cells increased in a dose-dependent manner, compared with findings in the untreated control cells, i.e., 15.2 fold at 20 micrograms/ml of ADR for 1 h. This level correlated with DNA single-strand breaks detected by the alkaline elution method. DNA breaks were also noted in the ADR-sensitive P388 cells, but in the ADR-resistant cells the level of DNA strand breaks was low. The enhanced cytotoxicity is apparently the consequence of the enhanced potential of ADR to cause breaks in the DNA strands. Our findings show that the survival response of the cells decreases and the level of DNA strand breaks increases following exposure to ADR. ADR resistance may be mediated by a reduction in the level of DNA strand breaks.     

Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state

Elimination of CD25+ T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, leads to spontaneous development of various autoimmune diseases. These immunoregulatory CD25+CD4+ T cells are naturally unresponsive (anergic) in vitro to TCR stimulation, and, upon stimulation, suppress proliferation of CD25-CD4+ T cells and CD8+ T cells. The antigen concentration required for stimulating CD25+CD4+ T cells to exert suppression is much lower than that required for stimulating CD25-CD4+ T cells to proliferate. The suppression, which results in reduced IL-2 production by CD25-CD4+ T cells, is dependent on cellular interactions on antigen-presenting cells (and not mediated by far-reaching or long-lasting humoral factors or apoptosis-inducing signals) and antigen non-specific in its effector phase. Addition of high doses of IL-2 or anti-CD28 antibody to the in vitro T cell stimulation culture not only breaks the anergic state of CD25+CD4+ T cells, but also abrogates their suppressive activity simultaneously. Importantly, the anergic/suppressive state of CD25+CD4+ T cells appeared to be their basal default condition, since removal of IL-2 or anti-CD28 antibody from the culture milieu allows them to revert to the original anergic/suppressive state. Furthermore, transfer of such anergy/suppression-broken T cells from normal mice produces various autoimmune diseases in syngeneic athymic nude mice. These results taken together indicate that one aspect of immunologic self-tolerance is maintained by this unique CD25+CD4+ naturally anergic/suppressive T cell population and its functional abnormality directly leads to the development of autoimmune disease.