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The formation of advanced glycation end-products (AGE) was investigated in samples of isolated human glomerular basement membrane (hGBM) and human serum albumin (hSA) which had been nonenzymatically glycated in vitro. In order to measure AGE, two methods which differ in principle--the standard spectrofluorescence technique and the spectrophotometric diazonium salt reaction--have been used and compared. Samples of finely homogenized hGBM and hSA were incubated for 10 days in buffer containing 500 mmol/L (9 x 10(3) mg%) and 100 mmol/L (1.8 X 10(3) mg%) D-glucose respectively. At the end of the incubation period, the ambient glucose was removed and the samples were incubated for a further 10 days in glucose-free buffer. During this time, loosely bound sugar was released into the buffer; at the end of the incubation, the emission fluorescence at 440 nm (following continuous excitation at 370 nm) and the absorbance at 492 nm of the glycated hGBM and hSA samples were measured and found to be significantly increased by comparison with native samples (1-way ANOVA: p less than 0.05 with both techniques). Comparison of the two techniques used for AGE detection showed a positive linear correlation (Pearson's correlation coefficient r = 0.714; n = 8; p = 0.02). The released glucose probably originates from reversal of the Schiff base (the first and reversible step of the nonenzymatic glycation reaction), whereas fluorescence and photometric findings prove the presence of stable AGE on both hGBM and hSA. It is concluded that AGE can indeed be formed and detected by two different methods in hGBM (and hSA) subjected to nonenzymatic glycation in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro effect mediated by carbimazole (CBZ), a classical anti-thyroid agent on the antibody dependent cellular cytotoxicity (ADCC), low affinity E-rosetting cells (E-RFC) and high affinity E-RFC was investigated using lymphocytes from 11 normal subjects. CBZ at the doses of 250 and 500 mumol/l significantly reduced ADCC in all subjects studied. Low affinity E-RFC--mainly cells possessing receptors for the Fc portion of IgG and expressing cytotoxic properties in the ADCC system--were also significantly reduced following incubation with the same CBZ doses. These results suggest that CBZ, in addition to the known inhibitory effect on thyroid hormone synthesis, may be useful by depressing lymphocyte cytotoxicity in the treatment of autoimmune thyroid disease.

The proportion of blood mononuclear cells forming low-affinity rosettes with sheep erythrocytes (K cells) was abnormally high in 13 (57%) of 23 children with classical type-1 diabetes at diagnosis but normal in children who had had diabetes for more than a year. A raised proportion of K cells was also found in 5 out of 10 unaffected siblings with islet-cell antibodies and at least one HLA haplotype in common with the diabetic proband; and in 10 (45%) of 22 subjects with type-1 diabetes and co-existent autoimmune thyroid disease irrespective of the duration of diabetes or the presence of islet-cell antibodies. These findings may be new evidence for lymphocyte-mediated beta-cell destruction and support the idea of immunogenetic heterogeneity within type-1 diabetes.

We have investigated lymphocyte subpopulation levels with monoclonal antibodies in newly diagnosed insulin-dependent (type I) diabetics and in unaffected siblings of type I diabetic probands with islet cell antibodies. Our data show that in newly diagnosed diabetics there is 1) a decrease in T cells with suppressor phenotype, 2) an increase of T cells with cytotoxic phenotype and 3) the presence of "activated" T cells. The latter have also been found in some unaffected siblings with islet cell antibodies. These results suggest that cellular immune alterations are present, not only at diagnosis, but also in normal but "susceptible" individuals. "Activated" T cells could be a "disease" marker, but their better definition in terms of specificity should be established.

Enhanced non-enzymic glycation of proteins has been suggested to play a role in the pathogenesis of diabetic microangiopathy. Thus pharmacological inhibition of this reaction could be envisaged to delay the development of late diabetic complications. In the present study we have investigated the effect of a new compound, 1-Benzylindazole-3-oxyacetic acid, Bendazac (BDZ) on the in vitro glycation of soluble proteins (albumin and fibrinogen) and isolated glomerular basement membrane (GBM). The data obtained indicate that BDZ is capable of reducing significantly the glycation of albumin and fibrinogen (p less than 0.001). When present in concentrations usually found in patients undergoing therapy (40-80 micrograms/ml), an inhibitory effect on soluble proteins was also observed. Inhibition of glycation of GBM was found only in the presence of the active metabolite (5 hydroxy BDZ) and at high glucose concentrations. These results suggest that BDZ could interfere with protein non-enzymic glycation and its use in patients with diabetes may be then taken into consideration to evaluate the effect on late diabetic complications.

The measurement of Fc+ (killer or K) cells in insulin dependent (Type 1) diabetes may be of considerable value in monitoring activity of immune disturbance. K cells were investigated in Type 1 diabetics, their relatives and normal controls employing two methods based upon different principles. A close positive linear correlation was observed between the two methods in all subjects. Type 1 diabetic patients showed an increased level of K-cells with both techniques. In addition there was a good correlation between raised levels of K-cells and the occurrence of organ specific autoantibodies.

We have previously shown that YC8, a Moloney virus-induced BALB/c lymphoma, is susceptible to lysis by BALB/c anti-DBA/2 effector cells. To further evaluate the relationship between non-H-2 antigens of DBA/2 background and tumor-associated determinants, we investigated the pattern of cytotoxic and proliferative responses induced in BALB/c mice by immunization with YC8 lymphoma. Spleen cells from tumor-immunized animals were restimulated in vitro with YC8 cells and tested for cytotoxicity on target cells of different strains and haplotypes. Cytotoxicity was observed only against YC8, DBA/2 blasts, and P815 (a DBA/2 mastocytoma). BALB/c anti-YC8 effectors were equally blocked by unlabeled YC8 and P815 cells when assayed on YC8 labeled targets. A clonal analysis, however, revealed the existence of at least two types of effectors, one lytic for both P815 and YC8 cells, the other lytic for YC8 cells only. BALB/c anti-YC8 cells proliferate when stimulated both with YC8 and DBA/2 cells in the presence of accessory cells. When tested in an adoptive transfer assay, anti-YC8 cells given i.v. cured 100% of i.v. and 75% of i.p. tumor-injected mice, respectively. When given i.p. anti-YC8 effectors cured 100% of i.p. tumor-injected animals. These results confirm the expression of non-H-2, DBA/2-like antigens on the BALB/c lymphoma YC8 and reveal the presence of additional tumor-associated determinants; both sets of antigens may induce a cellular immune response and elicit immune lymphocytes which can eradicate YC8 cells in an adoptive immunotherapy assay.

In search of a marker for monitoring the progression of diabetic nephropathy from the stage of charge- to that of size-selectivity loss, attention has been focused on the evaluation of immunoglobulin G (IgG) subclasses in the urine. We have developed a new sensitive enzyme immunoassay for the quantitation of urinary IgG1 and IgG4. Mouse monoclonal antibodies specific for each subclass were bound to microtitre wells precoated with rabbit anti-mouse immunoglobulin antibody. IgG1 and IgG4 of standard preparations (or of samples to be tested) were revealed using peroxidase-conjugated rabbit anti-human IgG. The procedure was carried out at 4 degrees C. This method can detect about 2 ng/ml of IgG4 and 20 ng/ml of IgG1. The monoclonal antibodies used were shown to be highly subclass-specific. IgG1 and IgG4 have a similar molecular weight but a different pH (about 9 and 4.6 respectively); a change in their ratio in the urine of diabetic patients may indicate a progressive deterioration of kidney function at the stage of incipient diabetic nephropathy.

The chronic hyperglycemia in diabetes mellitus enhances the nonenzymic glycation of structural proteins possibly increasing the formation of highly reactive advanced glycation end products (AGE). These protein changes might be involved in tissue-damaging mechanisms leading to diabetic complications, including diabetic nephropathy. To simulate these events, an in vitro model, based on isolated human glomerular basement membrane (hGBM), has been developed. In this study we have investigated the extent of AGE formation and the binding changes induced by the nonenzymic glycation of hGBM. An enriched fraction of hGBM was isolated from normal human kidneys and glycated in vitro by incubation with glucose (500 mmol/l) at 37 degrees C for 10 days. The presence of AGE was investigated by two methods - spectrofluorescence and the diazonium salt reaction - both specific for this type of chemical entity. The binding capacity of glycated hGBM was tested by a 10-day incubation with human insulin, albumin, immunoglobulin G and fibrinogen. Higher relative spectrofluorescence values at 440 nm emission (20.0 +/- 2.0 vs. 12.5 +/- 5.0) and higher absorbance values at 492 nm (0.798 +/- 0.063 vs. 0.429 +/- 0.228) indicated the presence of increased levels of AGE in glycated vs. native hGBM. Insulin and the three proteins were bound to hGBM in increased amounts after its glycation (p less than 0.05). The results obtained in this in vitro model confirm that enhanced nonenzymic glycation of hGBM induces the formation of AGE and possibly, through these compounds, alters its physicochemical and binding properties. This reaction might contribute to the mechanisms eventually leading to diabetic nephropathy.

BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct 51Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1+ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.