Aberrant activation of CD8+ T-cell and CD8+ T-cell subsets in patients with newly diagnosed IDDM.

Two- and three-color cytofluorimetric techniques were used to study the expression patterns of the activation antigen HLA-DR on peripheral blood immunoregulatory T-cells from 25 patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 14 age- and sex-matched control subjects. The mean percentage of total activated (CD3+HLA-DR+) T-cells was significantly elevated in the IDDM group compared with the control group (P < 0.001). In control subjects, basal activation of CD4+ and CD8+ lymphocytes accounted for the low percentage levels of activated T-cells. In contrast, the majority of IDDM patients showed an unbalanced activation of CD4+ and CD8+ lymphocytes with predominant activation of the CD8+ lymphocyte subset. The composition of the activated T-cell fraction was dependent on the composition of the total (activated + nonactivated) T-cell population, as indicated by the positive correlation between the CD4+/CD8+ T-cell ratios in these two cell populations (r = 0.714; P < 0.001). Excessive activation of CD8+ T-cells was attributable to similar increases in the proportions of CD8+ CD45RA+HLA-DR+ (naive) and CD8+CD45RA-HLA-DR+ (memory) cells. Analysis of the CD11b-defined subsets revealed predominant activation of CD8+ CD11b- (cytotoxic) T-cells; CD8+ CD16+ HLA-DR+ natural killer cells were unchanged. The distribution of HLA-DR+ cells among subsets of CD4+ T-cells differed from the pattern in the CD8+ population in that selective activation of CD4+ CD45RA- (memory, helper-inducer) cells accounted for the small increase in activated CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxic effects of cyclosporine on the endocrine pancreas of Wistar rats.

The widely used immunosuppressive drug cyclosporine exerts toxic effects on various parenchymal organs including the liver and kidney. This study was performed with the aim of testing whether cyclosporine also affects the endocrine pancreas. Daily cyclosporine doses of 50 mg/kg body weight over 3 weeks in rats enhanced the serum bilirubin and creatinine concentrations, led to light-microscopic destruction in the liver and kidneys, and resulted in the development of an impaired glucose tolerance--and, later on, of hyperglycemia. The pancreatic insulin content decreased to 33% of values observed in vehicle-treated controls, which can be ascribed to a 50% decrease of beta-cell volume and a slightly smaller reduction of islet insulin content. The reduction of the cyclosporine dose to 15 mg/kg body weight daily, which also reduced the popliteal lymph node weight gain after allogeneic stimulation, was not accompanied by serochemical or morphological alterations of livers or kidneys in the rats when treated for 3 weeks. However, the animals had already developed an impaired glucose tolerance, accompanied by a decrease in pancreatic insulin content (to 50% that of controls), a decrease of islet insulin content (to 70%) and a reduced pancreatic beta cell volume (to 72%). The findings let us conclude that pancreatic beta cells are sensitive to toxic effects of cyclosporine in vivo. We suggest that the measurement of glucose tolerance, as a sensitive parameter of a toxic cyclosporine action, should be included in the monitoring of grafted patients under cyclosporine treatment.

Spin-triplet superconductivity in Sr2RuO4 probed by andreev reflection

The superconducting gap function of Sr2RuO4 was investigated by means of quasiparticle reflection and transmission at the normal conductor-superconductor interface of Sr2RuO4-Pt point contacts. We found two distinctly different types of dV/dI vs V spectra either with a double-minimum structure or with a zero-bias conductance anomaly. Both types of spectra are expected in the limit of high and low transparency, respectively, of the interface barrier between a normal metal and a spin-triplet superconductor. Together with the temperature dependence of the spectra this result strongly supports a spin-triplet superconducting order parameter for Sr2RuO4.

Hybridoma cells producing antibodies to cathepsin L have greatly reduced potential for tumour growth.

Several tumour-forming cell lines are known to secrete the precursor of a lysosomal cysteine proteinase, procathepsin L. The function in tumour growth and proliferation of this neutral-pH-labile proteinase or its precursor outside lysosomes is as yet unknown. Murine myeloma cells (P3X63Ag8.653) secrete procathepsin L and exhibit a high potential for malignant tumour growth and metastasis. Such cells were fused with spleen cells of mice immunized with cathepsin L. Clones of the resulting hybridoma cells continued to secrete procathepsin L, but also secreted the antibody to cathepsin L. Here we show that the hybridoma cells producing an antibody to cathepsin L have, to a great extent, lost the potential that they otherwise exhibit for inducing solid tumours after implantation into mice.

Concentrations of lysosomal cysteine proteases are decreased in renal cell carcinoma compared with normal kidney.

Renal cell carcinoma contains significantly lower concentrations of the lysosomal cysteine proteases, cathepsins B, C, H, L and S, than does normal kidney, as shown by several methods, such as activity determination, enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry. The same low levels of enzyme activity and concentration have been determined in renal cell carcinoma metastases in the lung. Our results on the decreased concentration of cysteine peptidases at the protein level would seem to conflict with earlier results on an increased concentration of the cathepsin L mRNA in renal cell carcinoma.

Co-localization of CD44 and urokinase-type plasminogen activator on the surface of human melanoma cells.

Adhesion and proteolysis are basic reactions of tumor growth and metastasis. During these complex processes malignant cells change their adhesion behaviour and proteolytic capacity. Therefore, an extensive characterization of tumor cells is necessary if results of functional assays e.g., tests for tumor cell invasion are to be correlated with the presence of tumor antigens. This paper describes the detection of CD44 variant sequences, urokinase-type plasminogen activator (uPA) and uPA-receptor (uPAR) by immunoluminescence and activity measurements. For these investigations the melanoma cell line IGR 1 was used. The expression of CD44 (v5), uPA and uPAR on the cell surface was shown by indirect labelling with monoclonal antibodies (mAb). The marker enzyme horseradish peroxidase (HRP) of the secondary Ab was used to release luminescence and fluorescence with suitable substrates. The enhanced luminescent assay was superior to fluorescence analysis. uPA-activity in intact cells was examined with the substrates plasminogen, Z-Gly-Gly-Arg-AMC and Z-Lys-SBzl including selective inhibitors. The immunoluminescent assay can be alternatively used with well-tried immunofluorescent methods e.g. flow cytometry, for the detection of cellular cancer markers (1).

Active fragments of native streptokinase obtained by partial tryptic degradation.

By partial tryptic digestion of streptokinase, degradation products of the molecular weight range 27 000--42 000 were obtained. Among these, fibrinolysis activating components could be demonstrated.

Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis.

Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anti-catalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid.

[Secondary failures in modern therapy of diabetes mellitus with blood glucose lowering sulfonamides (author's transl)]. / Neue Untersuchungen zum Sekundärversagen der Therapie mit blutglucosesenkenden Sulfonamidderivaten.

The analysis of the case histories of 914 diabetics exclusively treated with blood glucose lowering sulfonamides in a university clinic outpatient station yielded an annual secondary failure rate of 5-10 p.c. Cumulative incidence calculations showed a continuous increase of secondary failure depending on the duration of treatment. Thus less than 20 p.c. of the diabetics have a chance of a satisfactory long-term therapy after 10 years of treatment. Any dependency of secondary failure from age at onset of diabetes could not be proved. Both "old" and "new" sulfonamide derivatives were equally subject to secondary failure. Changing the treatment from a therapy using grammscale derivatives to any of the modern milligramm-scale substances has been more or less equally "successfull" but lastly unsatisfactory. Diabetics with secondary failure show more overweight than patients with positive long-term treatment

Screening for hemoglobin C using Technicon H-6000: the effect of formalin.

It has recently been shown that the automated H-6000 cell counter is unable to correctly process blood samples containing hemoglobin C. In this paper, we report that formalin is responsible for the failure of red blood cell lysis in the H-6000.

The action of cyclosporin A on the endocrine pancreas in vitro--functional and morphological studies.

We investigated the effect of Cyclosporin A (100 ng/ml - 12000 ng/ml) on functional parameters of the endocrine pancreas in vitro. The insulin secretion of isolated islets in response to a variety of secretory stimulators is characterized by minimal alteration occasionally observed in the presence of very high Cyclosporin A concentrations. Cyclosporin A did not alter the glucose-induced biphasic insulin secretion, nor the arginine-stimulated glucagon secretion of the perfused rat pancreas. Exposure of isolated islets to 2000 ng Cyclosporin A/ml for 3 weeks resulted in a minimal decrease of insulin content and a marked reduction of glucagon content, which were not accompanied by drastic alterations of insulin secretion. Scanning electron micrographs of these Cyclosporin A-treated islets did not differ from cultured control islets. The results let us assume that Cyclosporin A exerts no toxic effects on the endocrine pancreas in vitro, when exposed to concentrations greater than or equal to 2000 ng/ml.

Reversibility of the acute toxic effect of cyclosporin A on pancreatic B cells of Wistar rats.

This study investigated if and to what extent the acute toxic effect of Cyclosporin A on pancreatic Wistar rat B cells is reversible. After 2 weeks of treatment rats developed marked glucose intolerance accompanied by reduced pancreatic insulin content due to a loss of B cells, diminished islet DNA synthesis and decreased B-cell insulin content. Cyclosporin A had accumulated in the pancreas. Three weeks after withdrawal of Cyclosporin A, pancreatic tissue concentrations of Cyclosporin A were still 100 times larger than in serum. Glucose tolerance, however, had already improved, associated with an increase of B-cell insulin content and apparent islet replication, and the insulin response of isolated islets was reduced. Five weeks after the withdrawal of Cyclosporin A, glucose tolerance was normal, but pancreatic insulin content and relative B-cell volume were still diminished in comparison to vehicle-treated controls. Eight weeks after withdrawal, the morphometric parameters had also been normalized. The results suggest that the loss of pancreatic B cells is caused by a toxic destruction, possibly combined with an apparent decrease of replicatory activity. The acute toxic effects of Cyclosporin A in pancreatic B cells are stepwise reversible.

Antibody response to islet antigens in anti-CD4/prednisolone immune intervention of type 1 diabetes.

Cytoplasmic islet cell antibodies, glutamic acid decarboxylase autoantibodies, spontaneous insulin autoantibodies, and insulin-induced antibodies were analyzed in a 1-year follow-up study of 12 newly diagnosed patients with insulin-dependent diabetes mellitus aged 14 +/- 2 years (range 7-20 years) who had been initially treated with either multiple injections of insulin alone (control group) or, in addition, anti-CD4 monoclonal antibody/prednisolone (treatment group). Despite individual variations in islet cell antibody titers, there were no significant differences in the prevalence or changes in the mean titers between the two groups. Glutamic acid decarboxylase autoantibodies remained almost unchanged, but correlated with levels of islet cell antibodies. While at initiation of treatment only 50% of the patients from both groups had spontaneous insulin autoantibodies, all patients developed insulin-induced antibodies upon conventional insulin therapy during the course of follow-up. This was not related to islet cell antibody or glutamic acid decarboxylase antibody levels. The insulin requirement was markedly reduced through the period of follow-up, but did not significantly differ between the two groups. A correlation between islet cell antibody levels and insulin requirement was observed in the control group but not in the treatment group. Plasma levels of the antibodies were not associated with changes in stimulated C-peptide or hemoglobin A1 concentrations. Activated T-lymphocytes persisted in both groups of patients, but their mean levels were not significantly different. The reason for the absence of statistically significant differences between treatment and control groups could be due to the small number of patients in the study. In conclusion, short-term immune intervention with anti-CD4 monoclonal antibody in addition to insulin therapy did not suppress autoimmune reactions towards the beta cells.