J.F. Bach

Detection of circulating immune complexes in human sera by simplified assays with polyethylene glycol

The search for circulating immune complexes by precipitation tests using polyethylene glycol (PEG) was performed on a series of normal and pathological sera. Various factors affecting PEG precipitation were studied. Immunoglobulins and complement factors percipitated by PEG (3.5%) were quantified and their significance was discussed in relation to serum levels. The PEG test was compared to labeled C1q binding test with a fairly good correlation. The direct evaluation of the amount of C4 precipitated with IgG by 3% PEG (C4 test) provided a simpler routine assay than the C1q binding test for detecting complement-fixing immune complexes. The direct PEG test and the C4 tests gave positive results in patients with diseases generally presumed to be associated with immune complexes including systemic lupus erythematosus, acute glomerulonephritis, bacterial sub-acute endocarditis and chronic acitve hepatitis. The demonstration of HBs antigen and antibody after acid dissociation of PEG precipitates from hepatitis B seronegative sera illustrated the fact that PEG does precipitate and thus concentrates circulating immune complexes.

Evidence of CD4+ regulatory T cells in the non-obese diabetic male mouse

SummaryThe NOD mouse, which shows many features of human IDDM, is extensively used to evaluate the role of T lymphocytes in the pathogenesis of autoimmune diabetes. The development of diabetes in this model appears to be controlled by a finely tuned immunoregulatory balance between autoaggressive T cells and regulatory immune phenomena, the disruption of which may result in destruction of insulin-secreting cells. The absolute requirement of sublethal irradiation to permit transfer of the disease to non-diabetic adult syngeneic mice provides indirect evidence for the presence of regulatory T cells in non-diabetic NOD mice. We have previously reported that the reconstitution of irradiated recipients by CD4 + T cells from nondiabetic female NOD mice blocks the transfer of diabetes by spleen cells from diabetic donors. We now report evidence that anti-CD4 monoclonal antibodies can substitute for irradiation in rendering adult NOD male mice susceptible to diabetes transfer by diabetogenic spleen cells. Efficient diabetes transfer can be achieved in non-irradiated adult NOD recipients provided they are thymectomized and CD4 + T-cell depleted prior to the transfer. The role of thymectomy is to limit T cell regeneration after anti-T cell monoclonal antibody challenge. Our data confirm that regulatory CD4 + T-cells, which efficiently counterbalance diabetogenic cells, are present in adult NOD male animals.

Resistance of T-cells to apoptosis in autoimmune diabetic (NOD) mice is increased early in life and is associated with dysregulated expression of Bcl-x

Summary Activated lymphocytes of autoimmune non-obese diabetic (NOD) mice exhibit an increased resistance to programmed cell death (PCD) following withdrawal of interleukin-2 (IL-2). In the present study, we found that resistance of NOD T lymphocytes to PCD was increased as early as 1 week of age, hence several weeks before the invasion of the pancreas by inflammatory cells, which is compatible with a role of the NOD apoptotic phenotype in the autoimmune susceptibility of this strain. In the thymus, mature single positive but not double positive or double negative thymocytes were more resistant to PCD in NOD compared to B6 mice. Moreover, in both NOD and B6 mice, CD4+ T cells were more resistant to PCD induced by IL-2 deprivation than CD8+ cells. As a result, NOD CD4+ T cells were remarkably resistant to cell death induced in this manner. In relation with this increased resistance to apoptosis, expression of the anti-apoptotic Bcl-x protein was upregulated in activated T cells of NOD mice, most notably after 24 h of IL-2 deprivation. These results should help us to understand the relationship of the NOD apoptotic phenotype to the emergence of the NOD mouse autoimmune disease. [Diabetologia (1998) 41: 178–184]

An enzyme immunoassay for synthetic thymulin

Thymulin, a metallononapeptide with the following aminoacid sequence: pyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-AsnOH is a thymic hormone involved in T cell differentiation requiring zinc to express biological activity as measured by the rosette assay. We established an enzyme immunoassay (EIA) for synthetic zinc-free thymulin with a thymulin-acetylcholinesterase conjugate as tracer and specific polyclonal rabbit antithymulin antibodies. The assay is performed as a classical competition assay in microtiter plates previously coated with mouse monoclonal IgG to rabbit IgG. A quantitative thymulin assay more sensitive than radioimmunoassays (RIAs) previously described was obtained with a sensitivity (IC50) of 32.5 +/- 5 pg/ml and a detection limit of 5 pg/ml. Analysis in the EIA of synthetic thymulin analogs showed that the minimal peptidic structure necessary for enzymatic tracer competition is the C-terminal part Lys3 to Asn9. It was also shown that the biologically active form of thymulin (zinc-bound) has the same immunoreactivity as zinc-free thymulin and that other thymic hormones, thymosin alpha 1 and thymopoietin II (or TP5) and unrelated short peptides do not cross-react with thymulin. These data demonstrate the specificity of this EIA for thymulin and show its suitability for application in biological fluids.

A new radioimmunoassay for the thymic peptide thymulin, and its application for measuring thymulin in blood samples.

A new, specific and sensitive radioimmunoassay, using a polyclonal antiserum raised in rabbits, is described for quantitating plasma thymulin. As little as 300 fg thymulin can be measured in one assay tube. The method has been used to measure thymulin in human blood (umbilical vessel blood, 2191 +/- 123 fg/ml; children and adults up to the age of 20 years, 1499 +/- 119 fg/ml; and adults between 21-65 years, 371 +/- 18 fg/ml). There is a highly significant decrease within these three groups (P less than 0.001 by one way analysis of variance). Also plasma thymulin levels were determined in rats (601 +/- 127 fg/ml) and in pooled plasma samples from mice (638 +/- 56 fg/ml). No thymulin was detected in plasma obtained from nude rats, nude mice and thymectomised mice. These results show that the radioimmunoassay described here is a useful quantitative tool for measuring plasma thymulin that will have applications in basic, applied and clinical research.

Monoclonal antibody against the serum thymic factor (FTS)

Hybrid cell lines secreting antibodies specific for synthetic serum thymic factor (FTS) were prepared by cell fusion and cloning techniques. Spleen cells from BALB/c mice immunized with BSA-coupled FTS were fused with P3-x63-Ag8.653 myeloma cells. Antibodies produced by these hybrids were screened in vitro for their ability to absorb the activity of synthetic FTS in a rosette assay and in vivo for their capacity to induce the disappearance of endogenous FTS. Subsequently, the clones selected were transferred intraperitoneally into BALB/c mice. Ascitic fluid was produced and used as a source of antibody. The monoclonal antibody was shown to bind specifically to thymic reticulo-epithelial cells, using an indirect immunofluorescence technique. Furthermore, once injected into normal mice, the antibody induced the disappearance of FTS from the serum and modified the azathioprine sensitivity of spleen rosette-forming cells for more than 3 weeks.

Immune lysis of hepatocytes in culture: accurate detection by aspartate aminotransferase release measurement

Determination of the immune lysis of epithelial cells, especially of hepatocytes, in short term culture is dealt with inadequately because of the lack of accuracy inherent in most classical methods of measurement of cell lysis or because of the high spontaneous release of the lytic marker. We have studied different methods of detection of lysis of rat hepatocytes cultured for a short term (24-48 h) at a concentration of 10 000 cells/50 microliters. The determination of aspartate aminotransferase (ASAT) release, measured with a centrifugal analyser parallels lactate dehydrogenase (LDH) release and trypan blue dye staining which are indisputable markers of cell death, but ASAT release is a more sensitive determination. Surprisingly, 51chromium release (1.72%/h) is much higher than ASAT release (0.51%/h) for an experimental period of 24 h. In cell mediated cytotoxicity tests, the ASAT content of lymphocytes, in contrast to that of LDH, is much lower than that of hepatocytes and this makes determination of ASAT release a sensitive marker of cytotoxicity under these conditions.

Antagonistic analogue of Serum Thymic Factor (FTS) interacting with the FTS cellular receptor

Abstract An analogue of facteur thymique serique (FTS), which was inactive in the rosette assay used to characterize FTS, has been shown to inhibit FTS effects on rosette-forming cells and to displace [3H]FTS from its specific cellular receptor.

Islet cell antibody heterogeneity among Type 1 (insulin-dependent) diabetic patients

SummaryConventional detection of islet cell antibodies is based on indirect immunofluorescence performed on frozen human pancreas sections. The number and nature of epitopes recognized by antibodies detected by such techniques are unknown. To determine the existence of heterogeneous fluorescence patterns of islet cell antibodies on pancreatic sections, we selected two sera showing a distinctive granular fluorescence. We then tested random sera from patients with Type 1 (insulin-dependent) diabetes mellitus for their ability to block ultimate binding of fluorescein isothiocyanate-labelled immunoglobulins purified from these two sera with a characteristic granular pattern. Among 102 subjects with recent-onset Type 1 diabetes, 79 had detectable anti-islet cell antibodies; 21 showed complete blockade of the binding to islets of granular fluorescein isothiocyanate-labelled immunoglobulins. The majority of these 21 patients were women carrying a DR3 non-DR4 DQB1*0201 allele, with under-representation of DRB1*0402 and 0405. Discrimination between islet cell antigenic specificities may help in identifying islet cell autoantibodies in autoimmune Type 1 diabetes.

Anti-bovine serum albumin antibodies: genetic heterogeneity and clinical relevance in adult-onset IDDM.

OBJECTIVE To evaluate the prevalence of anti-bovine serum albumin (BSA) antibodies in patients with adult-onset insulin-dependent diabetes mellitus (IDDM) and investigate a possible link between their presence and genetic susceptibility or resistance determined by human leukocyte antigen (HLA) complex. RESEARCH DESIGN AND METHODS Sera from 60 recent-onset diabetic patients, 5 prediabetic subjects, and 102 healthy control subjects were tested using a radioimmunoprecipitation assay. HLA-DRB and -DQB alleles were determined by means of allele-specific oligonucleotide typing. Islet cell antibodies (ICAs) were assayed by indirect immunofluorescence. RESULTS Levels of anti-BSA antibodies were significantly higher in IDDM patients (18.1 ± 3.5%, n = 60) than in healthy control subjects (7.5 ± 1.2%, n = 102) (P < 0.001), but in only 16.6% of IDDM patients (10 of 60) were the titers above the 95th percentik of control values. Anti-BSA antibody titers were higher in HLA-DR3 and/or -DR4 patients (23.4±4.9%, n = 41) compared with DR3 and/or DR4 control subjects (3.1 ± 1.0%, n = 10) (P < 0.001). DR3 IDDM patients showed higher levels of anti-BSA antibodies (26.3 ± 6.3%, n = 30) than non-DR3 patients (9.9 ± 2.6%, n = 30) (p < 0.01) and healthy control subjects. Only two out of five prediabetic subjects had significant anti-BSA levels before clinical onset of diabetes. CONCLUSIONS Our data confirm that antibodies to BSA are present in adult-onset IDDM patients, particularly in HLA-DR3-positive patients. However, the prevalence of anti-BSA antibodies was lower than previously reported in children, and there was a considerable overlap with healthy control subjects. Only two out of the five prediabetic patients demonstrated anti-BSA antibodies. Taken together, these results do not bring strong support to the clinical usefulness of anti-BSA antibodies as a relevant marker in diabetes prediction or diagnosis.