Jesper Johannesen

Suppressor of cytokine signaling 3 (SOCS-3) protects β-cells against interleukin-1β- and interferon-γ-mediated toxicity

Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-γ signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IFN-γ. IL-1β and IFN-γ alone, or potentiated by TNF-α, are cytotoxic to the insulin producing pancreatic β-cells and β-cell lines in vitro and suggested to contribute to the specific β-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat β-cell line INS-1, we demonstrate that the toxic effect of both IL-1β or IFN-γ at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-γ signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1β signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1β-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1β revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of β-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.

The significance of the prepubertal diabetes duration for the development of retinopathy and nephropathy in patients with type 1 diabetes

OBJECTIVE A Danish nationwide prospective cohort of children and adolescents with type 1 diabetes was followed for 8 years to study the effect of the prepubertal duration of diabetes on early retinopathy and elevated albumin excretion rate (AER) (>20 microg/min). RESEARCH DESIGN AND METHODS In 1989, blood glucose control (HbA(1c)) and AER was investigated in approximately 80% of all Danish children and adolescents with type 1 diabetes. A cohort of 339 young patients were restudied in 1995 including physical examination, demographic data, HbA(1c), AER, and fundus photography with central reading. Among the patients, a number of 304 had a prepubertal onset of diabetes defined as an onset age less than 11.7 years in girls and 12.9 years in boys. Microalbuminuria was defined as an AER of 20-150 microg min(-1) and macroalbuminuria as AER >150 microg min(-1) in two out of three timed overnight urine samples. RESULTS At the follow-up in 1995-1996, no patients were younger than 12 years of age. The prevalence of any level of retinopathy was 17.7% in the age group 12-15 years, 45.4% from 16 to 20 years, and increased to 67.6% in patients more than 20 years of age. Diabetic retinopathy was significantly associated to poor long-term metabolic control (HbA(1c)) (P<.0001) and to diabetes duration both in patients with a prepubertal onset of disease as well as patients with a pubertal (P<.001) onset of disease. However, the pubertal diabetes duration contributed two times more than the prepubertal diabetes duration. Mean postpubertal diabetes duration to any retinopathy was significantly shorter (9.4 years) in patients with prepubertal onset of the disease compared to patients with postpubertal onset (11.8 years) (P=.0004). In total, the prevalence of elevated AER (>20 microg/min) increased from 4% in 1989 to 13% in 1995. None of the patients younger than 15 years of age had elevated AER, while the prevalence of elevated AER was about 14% from 15 years of age and onwards. Elevated AER in 1995 was significantly related to long-term metabolic control (P<.001) and elevated AER in the preceding years (P<.001) but was not correlated to diabetes duration neither before nor after the age of 12 years. CONCLUSION The prepubertal diabetes duration is significantly associated with the development of diabetic retinopathy. The period, however, contributes less compared to the years after puberty. In concert with other studies, we found no association between raised AER and diabetes duration. This may be explained by the fact that other factors are more significant and dilute the significance of diabetes duration. Nonetheless, it seems prudent to optimise blood glucose control irrespective of age.

A 6-year nationwide cohort study of glycaemic control in young people with Type 1 diabetes: Risk markers for the development of retinopathy, nephropathy and neuropathy

The study aimed to identify risk markers (present at the start of the study in 1989) for the occurrence and progression of microvascular complications 6 years later (in 1995) in a Danish nationwide cohort of children and adolescents with Type 1 diabetes (average age at entry 13.7 years). Probabilities for the development of elevated albumin excretion rate (AER), retinopathy, and increased vibration perception threshold (VPT) could then be estimated from a stepwise logistic regression model. A total of 339 patients (47% of the original cohort) were studied. Sex, age, diabetes duration, insulin regimen and dose, height, weight, HbA(1c), blood pressure, and AER were recorded. In addition, information on retinopathy, neuropathy (VPT), and anti-hypertensive treatment was obtained at the end of the study. HbA(1c) (normal range 4.3-5.8, mean 5.3%) and AER (upper normal limit <20 microg min(-1)) in two, timed overnight urine collections were analysed centrally. Eye examination was performed by two-field fundus photography. Determination of VPT was assessed by biothesiometry. Increased AER (> or =20 microg min(-1)) was found in 12.8% of the patients in 1995, and risk markers for this were increased AER and high HbA(1c), in 1989 (both p<0.001). Retinopathy was present in 57.8% of patients in 1995, for which the risk markers were long duration of diabetes (p<0.0001), age (p<0.01), and high HbA(1c) (p<0.0001) in 1989. Elevated VPT (>6.5 V) was found in 62.5% of patients in 1995, for which the risk markers were male sex (p<0.05), age (p<0.0001), and increased AER (p<0.05) in 1989. This study confirms that hyperglycaemia plays a major role for the development of microvascular complications in kidneys and eyes, and emphasises the need for optimal glycaemic control in children and adolescents with Type 1 diabetes.

Genetic Susceptibility Markers in Danish Patients with Type 1 (Insulin-Dependent) Diabetes-Evidence for Polygenecity in Man

Fifty-five Danish families with two offspring concordant for type 1 diabetes-identified through a nationwide population-based survey, and 57 “true sporadic” cases-matched with familial cases for age at onset, but with no IDDM-affected first-degree relatives and long disease duration, and 110 control subjects were typed for putative genetic susceptibility markers for type 1 diabetes identified from a pathogenetic model. The markers included MHC class I, II and III loci, the manganese superoxide dismutase (MnSOD) locus (chr. 6q), interleukin-1β (IL1B), the IL-1 receptor antagonist (IL1RN), and the IL-1 type 1 receptor (IL1RI) loci (each chr. 2q). No significant differences between familial and sporadic cases were found within the MHC region (including the following loci: HLA-DQ,-DR, heat shock protein (HSP) 70, tumour necrosis factor (TNF), HLA-B and-A). In both groups of patients 11% were negative for both DQA1*0301-DQB 1*0302 and DQA1*0501-DQB1*0201 genotypes, and 7% of the type 1 diabetics had genotypes ...

Improved metabolic outcome in a Danish diabetic paediatric population aged 0-18 yr: results from a nationwide continuous Registration.

The objective of the present study was to describe the changes in glycaemic control based on data from the nationwide Danish Registry of Childhood Diabetes with valid haemoglobin A1c (HbA1c) readings centrally analysed between 1996 and 2006. The glycaemic control was assessed using generalized linear mixed models. Centre, age, diabetes duration, ethnicity, sex, self‐monitoring of blood glucose, insulin regimens and hypoglycaemia was tested as explanatory variables. There were 9291 HbA1c recordings from 2705 children with T1D during the 10‐yr period. The unadjusted mean HbA1c value in 1997 was 9.05% (95% CI ± 0.82) and in 2006 was 8.20% (95% CI ± 0.06). Mean HbA1c was significantly reduced over the years with a linear decrease of 0.08% per year (95% CI ±0.011) (p < 0.0001). The decrease was unaffected by adjusting for number of injections, insulin/kg and use of insulin analogous. During the period, an increased frequency of self‐monitored blood glucose was observed that was associated with a reduction in HbA1c (p < 0.0001). The percentage of children with severe hypoglycaemia decreased from 12.2 to 7.8% in those with HbA1c between 6 and 8%. Metabolic control in diabetic children has improved on a nationwide basis from the establishment of the national registry in 1996. The reduction in HbA1c was related to an increased number of self‐monitoring of blood glucose values and a decrease in the number of hypoglycaemic events in those with the best metabolic control, whereas there were no association with the use of new analogous or insulin regimens.

IDDM12 (CTLA4) on 2q33 and IDDM13 on 2q34 in Genetic Susceptibility to Type 1 Diabetes (Insulin-dependent)

Type 1 diabetes (insulin-dependent) is a multifactorial disease with polygenic susceptibility. The major genetic component (IDDM1) resides within the HLA region, but several non-HLA loci have been implicated in the genetic susceptibility. In the present study, we have analysed two such loci, IDDM12 (CTLA4) on 2q33 and IDDM13 on 2q34, in Danish (n = 254) and Spanish (n = 39) type 1 diabetic multiplex families. No significant evidence of linkage of IDDM12 was observed in any of the two studied data sets. However, when the present data were combined with previously published data, they strengthened the evidence of linkage at this locus, p = 0.00002. For the IDDM13 region, we found some positive evidence of linkage of the D2S137-D2S164-D2S1471 markers (p-values 0.007, 0.02, and 0.007, respectively) using transmission disequilibrium testing (TDT) and the Tsp version of the TDT. Importantly, random transmission of all tested alleles was observed in unaffected offspring (p > 0.3). Stratification for HLA (high risk and non-high risk genotypes) in the Danish families did not reveal heterogeneity at IDDM12 or IDDM13. In conclusion, our data on an entirely new family data set did not support the existence of IDDM12 as a type 1 diabetes susceptibility locus in the Danish population. In addition, we found support for evidence of linkage and association of the IDDM13/D2S137-D2S1471 region (approximately 3.5 cM) to type 1 diabetes, however, further studies are needed to substantiate this observation.

Predictors of IDDM recurrence risk in offspring of Danish IDDM patients

Summary It has previously been observed that offspring of mothers with insulin-dependent diabetes mellitus (IDDM) have a lower risk of IDDM than offspring of IDDM affected fathers. To assess the offspring IDDM recurrence risk in a Danish population-based study and to investigate parental and offspring-related biological variables that might influence this risk, we identified 2726 IDDM probands and their 2826 offspring from a background population of 1.725 million people (33 % of the Danish population). Current age of probands was 20–65 years and their age at IDDM onset was 30 years or less. Sixty-nine offspring (2.4 %) were affected with IDDM. The sex difference in the parental-offspring IDDM transmission rate was confirmed. The cumulative IDDM risk up to age 30 years was found to be significantly decreased in maternal offspring compared to paternal offspring (2.3 ± 0.6 and 5.7 ± 0.9 %, RR = 2.40, 95 % CI 1.30–4.47; p = 0.004) only if parents were diagnosed with IDDM before birth of the offspring. However, due to the low number of diabetic offspring of probands diagnosed with IDDM after offspring birth, this observation needs to be confirmed in a larger population. In a subpopulation of the 2380 offspring, whose parents were all diagnosed with IDDM before offspring birth, the recurrence risk was significantly increased in offspring of male probands diagnosed up to age 17 years compared to offspring of fathers diagnosed at older ages (8.5 ± 1.8 and 3.6 ± 1.0 %; RR = 2.27, 95 % CI 1.21–4.25; p = 0.006). No such relation was found in maternal offspring. Using the Cox proportional hazards model on this offspring subpopulation we found that paternal age at IDDM onset was the only statistically significant predictor of IDDM recurrence risk. Our findings may be important for counselling families in which one parent has IDDM. [Diabetologia (1998) 41: 666–673]

Zoledronic acid improves bone mineral density, reduces bone turnover and improves skeletal architecture over 2 years of treatment in children with secondary osteoporosis

There are limited data on the use of bisphosphonate therapy for secondary osteoporoses in childhood, and no previous reports of the use of zoledronic acid in this group. We report 20 children with a variety of underlying primary diagnoses with associated secondary osteoporosis, who were treated with 3 monthly zoledronic acid for 2 years (annualised dose 0.1mg/kg/year). There was a significant improvement in lumbar spine (by 1.88 SD±1.24 over first 12 months, p<0.001) and total bone mineral density as assessed by dual energy absorptiometry (DXA) scans, with a similar increase in bone mineral content for lean tissue mass (mean increase 1.34 SD in first 12 months, p<0.001). Bone turnover was reduced with a suppression of both osteocalcin and alkaline phosphatase in the first 12 months of treatment. Skeletal architecture was improved, with increased second metacarpal cortical thickness from 2.44mm to 2.72mm (p<0.001) and improved vertebral morphometry, with 7 patients who had vertebral wedging at baseline showing improved anterior (p=0.017) and middle (p=0.001) vertebral height ratios. Aside from well reported transient side effects with the first dose, there were no adverse effects reported. No adverse effects on anthropometric parameters were seen over the course of the study. Despite all patients having sustained fragility fractures prior to treatment, no fractures were reported during the study period. Further evidence is required to confirm efficacy, with long term follow up required to assess the impact of treatment on fracture risk.

Genetic and functional evaluation of an interleukin-12 polymorphism (IDDM18) in families with type 1 diabetes.

Type 1 (insulin dependent) diabetes mellitus (T1DM) [MIM 222100] is an autoimmune disease with both genetic and environmental components. The HLA region is the major genetic susceptibility region, but in addition several minor susceptibility loci have been suggested. Their identification is, however, difficult because of the complexity of the disease.1 Recently, a new susceptibility locus, IDDM18 [MIM 605598], was reported and mapped to chromosome 5q31.1-q33.1, close to the IL12B gene, which encodes the p40 subunit of interleukin (IL) 12.2–4 IL-12p40 production influences T cell response, and may therefore be important in T1DM pathogenesis.5 IL-12 drives the differentiation of T lymphocytes towards the Th1 subset, characterised by production of cytokines leading to cell mediated immunity.6–8 Furthermore, in the non-obese diabetic (NOD) mouse, IL-12 has been shown to play a primary role in T1DM induction.5,9,10 In addition, IL-12 is important in immune reactivity against infections, but it has been shown that in the absence of infection, IL-12 induced autoreactive T cell responses might predispose to self destructive immunity.11,12 IL-12 is a disulphide linked heterodimer composed of a heavy chain of 40 kDa, p40, and a light chain of 35 kDa, p35, encoded by two separate genes.2,13 The gene for the p35 subunit, IL12A , is located on chromosome 3p12-q13.2. The two subunits of IL-12 are in contrast to most cytokines, which possess only one polypeptide chain. Many cell types express the p35 chain, while the p40 subunit is expressed principally by activated macrophages and B cells.2 The heterodimer, p70 or p75, is the biologically active IL-12.13 IL12B is a candidate for IDDM18 , as linkage disequilibrium is confined to a 30 kb region on chromosome 5, in which IL12B is the only known gene.4 Several polymorphisms …

Characterization of new polymorphisms in the 5' UTR of the human interleukin-1 receptor type 1 (IL1R1) gene: linkage to type 1 diabetes and correlation to IL-1RI plasma level.

The human interleukin-1 type I receptor (IL-1RI) is the signal transducing receptor for IL-1, a principal proinflammatory cytokine, which is cytotoxic to pancreatic islet beta cells. The IL-1RI gene, IL1R1, maps to chromosome 2q12. We have previously examined part of the IL1R1 promoter region and in the present study we further characterized the promoter region demarcating exon 1B and 1C by sequencing and mutation scanning. New sequence was obtained 1762 bp upstream and 1609 bp downstream the known region. Within this sequence, we identified four frequent single nucleotide polymorphisms (SNPs). PCR-based RFLP assays were established and three of the polymorphisms were typed in a Danish Type 1 (insulin-dependent) diabetes mellitus (T1DM) family collection comprising 103 simplex and 150 sib-pair affected families. Linkage was evaluated by the sib-TDT (transmission disequilibrium test). One of the polymorphisms, defined by a HinfI RFLP assay, demonstrated linkage to T1DM, P(sTDT) = 0.026. Random transmission was observed to unaffected offspring from heterozygous parents, P = 0.87. No evidence for positive linkage was seen for the other tested polymorphisms, P = 0.14 and P = 0.21, respectively. To evaluate the possible functional significance of the HinfI polymorphism, we measured circulating IL-1RI plasma level in 30 T1DM patients and in 30 control subjects – 10 with each genotype in both groups. Significant differences in plasma levels in relation to genotype – independent of disease status – were found (P = 0.04). In both diabetic and non-diabetic subjects, the wt/wt genotype correlated with the highest IL-1RI plasma level, whereas the plasma levels were lowest for the mt/mt genotype.