Vagn Bonnevie-Nielsen

Cytokines and bone loss in a 5-year longitudinal study : Hormone replacement therapy suppresses serum soluble interleukin-6 receptor and increases interleukin-1-receptor antagonist : The Danish Osteoporosis Prevention Study

The proinflammatory cytokines interleukin‐1β (IL‐1β) and IL‐6 may play a central role in the acceleration of postmenopausal bone loss, but observational studies have led to contradictory results. Estrogen‐dependent changes in the production of IL‐1 receptor antagonist (IL‐1ra) and the soluble IL‐6 receptor (sIL‐6R) potentially modify cytokine bioactivity. We therefore assessed the impact of menopause and hormone replacement therapy (HRT) on cytokines and activity modifiers in serum within a 5‐year longitudinal study. One hundred sixty perimenopausal women (age 50.1 ± 2.8 years) were randomized to HRT or no treatment. Serum IL‐6 increased with age (r = 0.16; p < 0.05), but cytokines did not correlate with baseline bone mineral density (BMD). HRT led to small increases in IL‐1ra (p < 0.001) and IL‐6 (p < 0.05), with a decrease in sIL‐6R (p < 0.01) and no change in IL‐1β. No changes were observed in the control group. IL‐1ra was inversely correlated with bone loss at the ultradistal forearm (r = 0.29; p < 0.05) and to a lesser degree at the spine (r = 0.20; p = 0.09). In addition, there was a weak positive correlation between sIL‐6R and bone loss at the ultradistal forearm (r = 0.26; p < 0.05). High IL‐6 levels were associated with slower bone loss (spine r = 0.31, p < 0.01) and controlling for age did not diminish this association. The percent change in sIL‐6R during HRT was correlated with the bone loss at the femoral neck (r = −0.29; p < 0.01) and weakly with bone loss in the spine (r = −0.16; p = 0.17). In conclusion, serum IL‐1ra and sIL‐6R are influenced by HRT and are associated with the rate of bone loss in perimenopausal women.

Increased concentration of the fast‐acting plasminogen activator inhibitor in plasma associated with familial venous thrombosis

Summary. We have earlier demonstrated that in a family with a tendency to recurrent venous thrombosis the release of tissue plasminogen activator (t‐PA) activity in blood after stimulation was abnormally low. This observation could be related either to an impaired release of t‐PA into the blood stream or to a masking of the released t‐PA by a high concentration of PA inhibitor(s). In order to distinguish between these two possibilities the family was reinvestigated using various newer techniques, including an ELISA for t‐PA, an assay for quantitation of the fast‐acting PA inhibitor and SDS polyacrylamide gel electrophoresis followed by fibrin‐enzymography. Hereby the family members were demonstrated to have a high concentration in plasma of the PA inhibitor. After stimulation the release of t‐PA into the blood was normal, the t‐PA activity, however. was immediately inactivated by complex formation with the fast‐acting PA inhibitor.

Evidence for a locus (IDDM16) in the immunoglobulin heavy chain region on chromosome 14q32.3 producing susceptibility to type 1 diabetes

Type 1 diabetes results from autoimmune destruction of pancreatic islet β-cells, possibly initiated or exacerbated by viral infections. Recent studies have demonstrated that antibodies towards enterovirus and autoantibodies towards islet cell components develop in the long preclinical phase of type 1 diabetes. We therefore hypothesised that susceptibility to type 1 diabetes could be influenced by genetic factors controlling production of antiviral antibodies or autoantibodies or both. To search for evidence of linkage or association (linkage disequilibrium) between type 1 diabetes and the immunoglobulin heavy chain (IGH) region, 351 North American and British families with ⩾2 diabetic children were genotyped for IGH region microsatellites. Using affected sibpair analysis, significant evidence for linkage was obtained for three markers close to the IGH gene cluster (P values 0.004, 0.002, 0.002). No evidence was found for association using family-based methods. To attempt to confirm these findings, a smaller dataset (241 families, 138 with ⩾2 diabetic children) from Denmark, a more genetically-homogeneous population, was genotyped for one marker only. These families showed no linkage, but significant evidence for association (P = 0.019). This study suggests that a locus (assigned the symbol IDDM16) in the IGH region, possibly an IGH gene, influences susceptibility to type 1 diabetes.

Effect of increased gene dosage expression on the α-interferon receptors in Down's syndrome

Abstract The gene coding for the α,β-interferon (α,β-IFN) receptor is localized to chromosome 21. Cells from patients with Downs syndrome contain an extra chromosome 21, and thereby an expected 1.5-times increase in the number of genes located to this chromosome and in consequence a 1.5-times increase in cell surface α-IFN receptors. Actual measurements of these by competition binding experiments with human recombinant α-IFN on peripheral blood mononuclear cells (PBMC) from patients with Downs syndrome resulted in a mean of 1.69, which is in accordance to the theoretical 1.50, but slightly overestimated due to the calculation method. The increased gene dosage of the α-IFN receptor was quantitatively verified by Southern blot-hybridizations. Further characterization of α-IFN receptor binding showed insignificant differences in dissociation constants among patients and healthy individuals.

Evidence from twins for acquired cellular immune hyperactivity in type 1 diabetes

Type 1 diabetes has been associated with an increased frequency of activated T cells and T‐cell hyperactivity to non‐specific and disease‐specific stimuli including the islet autoantigen glutamic acid decarboxylase 65 (GAD). To address whether T‐cell hyperactivity is genetic or acquired we measured whole blood cytokines in vitro in response to GAD or tetanus in 18 identical twin pairs, nine discordant for type 1 diabetes. In addition, the activity of 2′, 5′ oligoadenylate synthetase (OAS) in blood mononuclear cells was measured as a marker of viral infection. Interleukin‐2 (IL‐2) basally and IL‐2 and interferon‐γ (IFN‐γ) in response to GAD, were detected more frequently and at higher levels in diabetic compared to non‐diabetic twins. IL‐10 was not different between groups. OAS activity was increased in diabetic compared to non‐diabetic twins and showed a correlation with basal IL‐2 and GAD‐stimulated IFN‐γ and IL‐10. These findings suggest that T‐cell hyperactivity in type 1 diabetes is an acquired trait and could reflect persisting virus expression.

Differential Responsiveness to Interferon-α in β-Cells and Non-β-Cells

Interferon-α (IFN-α) is important in the innate immune defense, particularly in viral infections. IFN-α induces 2ʹ,5ʹA synthetase, the products of which, 2ʹ,5ʹ-oligoadenine nucleotides, activate mRNA degrading enzymes. IFN-α is the first detectable cytokine in the insulitis lesion seen in recent-onset IDDM, and insulin promoter directed expression of IFN-α in transgenic mice leads to development of IDDM. Here, we demonstrate that IFN-α induces 2ʹ,5ʹA synthetase activity only in insulin-producing βTC3 cells and in isolated single rat β-cells but not in αTC3 cells or in isolated rat non-β-cells. The increased responsiveness of β-cells but not non-β-cells to IFN-α with the ensuing activation of the mRNA-degrading 2ʹ,5ʹA synthetase system suggests why only the β-cells are destroyed in the diabetogenic process.

An H-2 Alloantiserum Preserves β-Cell Function in Mice Made Diabetic by Low-Dose Streptozocin

The pancreatic β-cell mass and function in C57BL/KsJ mice is markedly reduced the day after the last injection of five daily injections of a subdiabetogenic, 40 mg/kg, dose of streptozocin (STZ). In this study, we prepared an H-2 alloantiserum by injecting C57BL/6J mice (H-2b) with spleen lymphocytes from C57BL/KsJ (H-2d) mice. The alloantiserum given on five consecutive days, 5 h before each injection of STZ, did not prevent the initial β-cytotoxic effect of STZ detected by perfusion of the pancreas and subsequent morphometric analysis of in situ dithizone-perfused pancreas. However, 12 days after the first injection of STZ, total insulin release in response to D-glucose, total pancreatic insujin, and pancreatic glucagon was greater in the alloantiserum-treated mice compared with controls receiving normal mouse serum. It is concluded that an H-2 alloantiserum may protect the function and amounts of β-cells remaining after the initial five lowdose STZ injections.

Induction and activation of antiviral enzyme 2′,5′-oligoadenylate synthetase by in vitro transcribed insulin mRNA and other cellular RNAs

Double-stranded RNA (dsRNA) can induce antiviral enzyme 2′,5′-oligoadenylate synthetase (2′5′AS) expression and activate latent 2′5′AS. Our previous data have shown pancreatic β cells are sensitive to dsRNA-induced 2′5′AS expression, and constitutive high basal 2′5′AS expression is associated with susceptibility to developing type 1 diabetes (T1D), a disease due to pancreatic β cell loss. Here we report that in vitro transcribed human insulin mRNA induces the activation of human OAS gene promoter sequences, and specifically and dose-dependently induces 2′5′AS expression in murine pancreatic βTC3 cells. Over-expression of dsRNA receptor retinoic acid-inducible gene-1 enhances insulin mRNA-induced 2′5′AS expression. In vitro transcribed insulin and other mRNAs, as well as total cellular RNAs, activate latent 2′5′AS in vitro with activation ability likely associated with the sequence and length of individual mRNAs or the sample source of total cellular RNA. Insulin mRNA does not show any specificity to activate 2′5′AS, but total cellular RNA from βTC3 cells has high activation ability. Constitutive 2′5′AS expression in βTC3 cells leads to cell proliferation inhibition and apoptosis. Our study suggests the possibility of cellular RNA-regulated 2′5′AS expression and activation, and the potential risk of high insulin gene transcription in pancreatic β cells, and may help explain genetic predisposition to T1D associated with INS VNTR class I alleles.

Acute Platelet Activation Induced by Smoking Cigarettes: In Vivo and Ex Vivo Studies in Humans

The epidemiological and pathological evidence for a relationship between cigarette smoking and atherosclerosis is considerable (1). In particular, a strong correlation between cigarette smoking and acute cardiac events in those with an already compromised coronary circulation is evident (2). As platelets seem to play a central role for the development of atherosclerosis and its thromboembolic complications (3), it is natural that a vivid interest has been taken in the effects of cigarette smoking on platelet function.

Separate estimation of biological and analytical variance components when quantities and reagents are unstable

A model for reliable estimation of variance components for biological within- and between-subject variation as well as for analytical variation when both the quantity and the reagents are unstable has been established. This model was applied to the alpha-interferon receptor on human leucocytes which involves two major problems. First, the receptor has to be quantified within a few hours after blood sampling, and second, the reagents for the measurement procedure must be used within 2 weeks. For the number of receptors per cell the biological estimates of coefficients of variation were 14% for within-subject variation and 20% for between-subject variation, respectively. For the dissociation constant both estimates were zero as expected. The model is robust and applicable to other systems with unstable quantities and reagents.