Jens Mølvig

Dietary Supplementation with co‐3‐Polyunsaturated Fatty Acids Decreases Mononuclear Cell Proliferation and Interleukin‐1β Content but not Monokine Secretion in Healthy and Insulin‐Dependent Diabetic Individuals

The effects of dietary supplementation with ω‐3‐polyunsaturated fatty acids (ω‐3‐PUFA) on the proliferative response of PBMC and on the secretion of monokines and arachidonic acid metabolites from PBMC and monocytes (Mo) from healthy subjects and patients with recent‐onset insulin‐dependent diabetes mellitus (IDDM) were examined. Three groups of eight to nine healthy individuals were randomized to either 2.0 g/day or 4.0 g/day of ω‐3‐PUFA devoid of vitamins A and D, or an isocaloric amount of placebo. Furthermore, eight patients with recent‐onset IDDM received 4.0 g/day of ω‐3‐PUFA. IL‐lβ production and TNF‐α secretion was determined before and after 7 weeks of treatment, and 10 weeks after withdrawal of treatment. Significant increases in platelet and PBMC membrane eicosapentaenoic acid was found in ω‐3‐PUFA‐treated individuals. ω‐3‐PUFA treatment significantly reduced the content of IL‐Ib in lysates of PBMC, but did not affect PBMC or Mo secretion of IL‐1β, TNF‐α or prostaglandin E2 (PGE2) or PBMC leukotriene B4 (LTB4) secretion in healthy subjects or in IDDM patients. A significant inhibition of the PHA‐stimulated. but not the spontaneous or PPD‐stimulated, proliferative response of PBMC was observed in healthy and diabetic subjects treated with (o‐3‐PUFA. No correlation was found between PHA‐stimulated PBMC proliferation and PBMC secretion of TNF‐α and IL‐1β. There were no significant differences in the spontaneous or the PPD‐or PHA‐stimulated proliferative responses of PBMC between diabetic and healthy individuals at entry. We conclude that although dietary supplementation with 4.0 g/day of ω‐3‐PUFA inhibits the proliferation of PBMC and reduces IL‐I immunoreactivity in PBMC and Mo, it does not alter monokine, PGE2 or LTB4, secretion in healthy or IDDM subjects.

Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans.

SummaryHuman crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis. After 24 hours of exposure 0.5 U/ml of crude or 0.6 ng/ml of recombinant IL-1 (beta) increased the (pro)insulin biosynthesis by 42% and 58%, respectively, whereas a 10-fold greater concentration of IL-1 decreased the (pro)insulin biosynthesis by 74% and 89%, respectively. The increase in (pro)insulin biosynthesis was accompanied by an increase in total protein biosynthesis indicating a nonspecific stimulatory action of low IL-1 concentrations. In contrast, high IL-1 concentrations caused a more selective decrease of the (pro)insulin biosynthesis when compared to the total protein biosynthesis. In addition, low IL-1 concentrations were found to increase and high concentrations to decrease the relative levels of pre-proinsulin mRNA suggesting that IL-1 may act both at a pre- and post-translational level of insulin biosynthesis.

Monokine Antagonism Is Reduced in Patients With IDDM

Interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) have been implicated as immune effector molecules in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). Recently, an increased frequency of the A1/A1 genotype of an IL-1 receptor antagonist (IL-1Ra) gene polymorphism was observed in patients with IDDM. Therefore, we investigated plasma IL-1Ra and soluble TNF p55 receptor (TNFsRp55) levels in 18 men with recent-onset IDDM, 10 men with long-standing IDDM, and 35 age-matched healthy men. No differences in plasma IL-1Ra were found among the three groups. However, when the plasma IL-1Ra levels in the subjects with IDDM and the control subjects were analyzed according to IL-1Ra genotypes, we found a 30% lower level of plasma IL-1Ra in subjects with IDDM carrying the A1/A1 genotype compared with the levels in those carrying the A1/A2 genotype (372 ± 40 vs. 530 ± 54 ng/l, respectively, P = 0.025). In contrast, no significant association was seen between plasma IL-1Ra and IL-1Ra genotype in the control subjects. The TNFsRp55 level in heparinized plasma was 17% lower in patients with IDDM than in control subjects (3.93 ± 0.22 vs. 4.72 ± 0.24 μg/l, respectively, P = 0.038). These findings could not be explained by metabolic derangement in the IDDM patients. Although based on a limited number of patients, these preliminary findings suggest that μ-cells in IDDM patients may be more sensitive to the cytotoxic effects of TNF-α and IL-1 because of less production of TNFsRp55 and, in a subset of IDDM patients, of IL-IRa during the inflammatory challenge of insulitis.

Monocyte Function in IDDM Patients and Healthy Individuals

Interleukin 1β(IL‐1β) and tumour necrosis factor alpha (TNF‐α) may be pathogenetically important in insulin‐dependent diabetes mellitus (IDDM), which is associated with genes of the HLA region. Since a regulatory role of HLA region genes on monokine production may exist, we looked for an association between the monokine and prostaglandin E2 (PGE2) responses of monocytes (Mo) from 20 healthy males (18–50 years) with HLA‐DR types relevant for IDDM susceptibility and resistance (DR 1,2, DR 1,3, DR 1,4, DR 3,4). Monokine assays were established and evaluated and the secretions of IL‐1β, TNF‐α, and PGE2 measured in Mo cultures (2 h, 6 h, 20 h) prepared by endotoxin‐free techniques and stimulated by low‐dose E. coli lipopolysaccharides (LPS). There were no significant associations between Mo responses and HLA‐DR phenotype. Likewise, Mo from DR2 (n=5) and DR4 (n= 5) homozygous healthy males demonstrated no significant differences in monokine and PGE2 responses of Mo.

The susceptibility to insulin-dependent diabetes mellitus is associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3,4 heterozygotes.

In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P=6 x 10−6): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P=0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P=0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes. Since recent works have shown that the structural gene for the monokine tumor necrosis factor alpha (TNF-α) is located between the HLA-B and C4 loci and that TNF-α might be of importance in IDDM pathogenesis, the hypothesis is put forward that the C4-associated IDDM susceptibility reflects linkage dis-equilibrium between the C4 gene and a gene controlling TNF-α production. The high relative risk for IDDM in HLA-DR3,4 heterozygotes might be explained by the combined action of IDDM-specific susceptibility genes on DR4 haplotypes and DR3-linked susceptibility genes associated with predisposition to autoimmunity.

Circulating interleukin-1 receptor antagonist concentrations are increased in adult patients with thermal injury.

OBJECTIVE To investigate the balance between circulating concentrations of interleukin (IL)-1 and its natural inhibitor interleukin-1 receptor antagonist (IL-1Ra) in human inflammation. DESIGN Prospective case-control study. SETTING University hospital burn care unit. PATIENTS Fifteen patients with second- or third-degree thermal injuries of 7% to 78% of total body surface and 15 healthy age- and sex-matched control subjects. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Median plasma IL-1Ra, but not IL-1 beta or tumor necrosis factor-alpha (TNF-alpha) concentrations were markedly increased on the day of admission in patients with thermal injuries compared with controls (1615 [range 426 to 23,800] vs. 494 [range 196 to 1093] pg/mL; p < .001). In survivors, the median IL-1Ra concentration normalized 12 to 21 days after admission. The concentration of IL-1Ra on the day of admission was weakly positively correlated to the extent and degree of thermal injury (r2 = .46; p < .05). IL-1Ra on days 1 to 3 was highest in three nonsurvivors with inhalation injuries compared with survivors (2166 [range 1362 to 36,624] vs. 1344 [range 665 to 13,085] pg/mL; p < .05). IL-1Ra increased significantly after debridement and skin transplantation (preoperatively 742 [range 488 to 1506] vs. postoperatively 1431 [range 1286 to 2107] pg/mL; p < .01). In nonsurvivors, median IL-1Ra was 3.6-fold higher than IL-1 beta on days 1 to 2 and 36-fold higher than IL-1 beta in three patients with bacteremia. IL-1Ra was studied for its relationship to previously reported parameters of the acute-phase response determined in the same samples from these patients. The increased concentrations of IL-1Ra coincided with a decrease in serum albumin concentration and increases in rectal temperature. However, IL-1Ra did not correlate with rectal temperature, plasma concentrations of endotoxin, IL-1 beta, or TNF-alpha either at admission or in follow-up samples. CONCLUSIONS Thermal injury causes an increase of circulating IL-1Ra, especially in patients with inhalation injuries. With the current plasma assays for IL-1 beta, IL-1Ra may be a more sensitive marker of human inflammation than IL-1 beta or TNF-alpha.

Glutamic Acid Decarboxylase (GAD65) Autoantibodies in Prediction of β-Cell Function and Remission in Recent-Onset IDDM After Cyclosporin Treatment

We have investigated whether glutamic acid decarboxylase (GAD) autoantibodies (GAD65 Ab) were affected by cyclosporin therapy and were related to subsequent noninsulin-requiring remission and loss of glucagon-stimulated C-peptide response in 132 recent-onset insulindependent diabetes mellitus (IDDM) patients treated with cyclosporin or placebo for 12 months. GAD65 Ab were detected in a quantitative radioligand assay using as tracer recombinant, in vitro translated, human islet [35S]methionine-labeled GAD65. GAD65 Ab were found at onset in 66% (87 of 132) of IDDM patients and in 1% (1 of 100) of healthy control subjects. The prevalence of GAD65 Ab and median GAD65 Ab levels did not change in serum samples taken 3, 6, 9, and 12 months after study entry in either the cyclosporin- or the placebo-treated groups. The presence or absence of GAD65 Ab at study entry did not predict non-insulin-requiring remission in either cyclosporin- or placebo-treated patients. However, the relative (compared with 0 months) glucagon-stimulated C-peptide response was more than 30% lower in GAD65 Ab+ patients receiving placebo at 9 and 12 months compared with the GAD65 Ab− placebo patients (P < 0.035). Islet cell cytoplasmic antibody (ICA) and GAD65 Ab+ placebotreated patients showed no significant differences in stimulated C-peptide levels compared with those who were ICA− and GAD65 Ab+, suggesting that ICA was not independently associated with loss of (β-cell function. We conclude that GAD65 Ab at diagnosis may predict a more rapid loss of β-cell function, a finding of importance when selecting individuals at risk of developing IDDM or recent-onset IDDM patients for intervention therapy.

Lack of Predictive Value of Islet Cell Antibodies, Insulin Antibodies, and HLA-DR Phenotype for Remission in Cyclosporin-Treated IDDM Patients

The effect of immunosuppression on the humoral immune response to islet autoantigens and exogenously administered insulin and the predictive value of islet cell cytoplasmic antibodies (ICAs), insulin antibodies (IAs), and HLA-DR phenotype for remission during immunosuppression were studied in a prospective randomized double-blind trial of cyclosporin administration in 98 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients. HLA-DR phenotype and glycosylated hemoglobin were determined at study entry, and insulin requirement, glucagon-stimulated C-peptide, ICAs, and IAs were measured at entry and after 1, 3, 6, 9, and 12 mo of follow-up. Cyclosporin therapy caused significant suppression of the prevalence and serum concentrations of ICAs and lAs. Cyclosporin-treated IDDM patients ICA+ at study entry had higher levels of stimulated C-peptide after 1 mo of study, but the increased (β-cell function was not associated with a higher frequency of insulin-free remission at 1 mo. ICA and IA status at entry did not predict cyclosporininduced remission as assessed by the prevalence of insulin-free remission or β-cell function at 3–12 mo of study, and significant decrements in the titers or total disappearance of ICAs were not associated with an increased prevalence or duration of non-insulinrequiring remission or higher stimulated C-peptide values. There was no correlation between the serum levels of ICAs and lAs at entry and β-cell function at 12 mo of follow-up. Although patients who were HLADR3/X seemed to respond better to cyclosporin therapy at 6 mo than patients who were HLA-DR3/4 or -DR4/X when analyzed cross-sectionally, a split-plot analysis of variance with time as the split-plot factor showed that the HLA-DR phenotype failed to identify patients with an overall better response to cyclosporin therapy. These data indicate that, although cyclosporin therapy inhibits the humoral immune response to islet components and exogenous insulin, significant decrements in ICA titers are not useful in monitoring efficacy of immunosuppression with cyclosporin, and the determination of ICA and IA status and immune response phenotype at diagnosis is of no predictive value for remission in selecting recent-onset IDDM patients for cyclosporin immunointervention.

On the Pathogenesis of Insulin-Dependent Diabetes Mellitus — A Discussion of Three Recently Proposed Models

Only in 1974 was it unequivocally shown that insulin-dependent (IDDM) and noninsulin-dependent (NIDDM) diabetes mellitus are two distinct disease entities [1]. In their paper Nerup et al. presented the first attempt to formulate a fully comprehensive model to explain the etiology and pathogenesis of IDDM: Histocompatibility complexes like HL-A contain, in addition to genes controlling serologically detectable antigens, so-called immune-response genes which control the development of cell-mediated immunity to certain antigens. One or more immune-response genes associated with HL-A8 and/or W15 might be responsible for an altered T-lymphocyte response. The genetically determined host response could fail to eliminate an infecting virus (Coxsackie B4 and others) which in turn might destroy the pancreatic beta-cells or trigger an autoimmune reaction against the infected organ.

HLA Haplotype Analysis in Danish HLA-DR3,4-Positive Insulin-Dependent Diabetics and Controls

The association of insulin-dependent diabetes mellitus (IDDM) with HLA-DR3 and 4 is well established, and the increased risk of DR3,4 heterozygotes compared with DR3 or 4 homozygotes has been confirmed by numerous studies. Some reports have indicated that HLA factors other than DR might confer an increased risk for the disease, and that genetic factors not coded by the genes of the HLA region might be associated with IDDM. We wanted to apply a number of techniques to compare the distribution of HLA and non-HLA markers in a sample of DR3,4 heterozygous IDDM patients and controls from a homogeneous population. We chose to study a sample of unrelated HLA-DR3,4 heterozygous individuals from the homogeneous Danish population: 27 unrelated IDDM patients who had been HLA-A,B and DR typed in Copenhagen (included in the study by Svejgaard et al. (1), and 19 unrelated controls typed in Copenhagen and Aarhus. (The contribution of normal DR3,4-positive individuals by Dr. L.U. Lamm and Dr. L.S. Nielsen is gratefully acknowledged.) None of the controls had a family history of diabetes or other autoimmune disease.