Vitamin D effects on monocytes' CCL-2, IL6 and CD14 transcription in Addison's disease and HLA susceptibility.

Addison's disease is a rare autoimmune disorder leading to adrenal insufficiency and life-long glucocorticoid dependency. Vitamin D receptor (VDR) polymorphisms and vitamin D deficiency predispose to Addison's disease. Aim of the current study was, to investigate potential anti-inflammatory vitamin D effects on monocytes in Addison's disease, focusing on inflammatory CCL-2 and IL6, as well on monocyte CD14 markers. Addison's disease is genetically linked to distinct HLA susceptibility alleles. Therefore we analyzed, whether HLA genotypes differed for vitamin D effects on monocyte markers. CD14+ monocytes were isolated from Addison's disease patients (AD, n=13) and healthy controls (HC, n=15) and stimulated with 1,25-dihydroxyvitamin D3 and IL1ß as an inflammatory stimulant. Cells were processed for mRNA expression of CCL-2, IL6 and CD14 and DNA samples were genotyped for major histocompatibility class (MHC) class II-encoded HLA- DQA1-DQB1 haplotypes. We found a downregulation of CCL-2 after vitamin D treatment in IL1ß-stimulated monocytes both from AD patients and HC (AD p<0.001; HC p<0.0001). CD14 expression however, was upregulated in both HC and AD patients after vitamin D treatment (p<0.001, respectively). HC showed higher CD14 transcription level than AD patients after vitamin D treatment (p=0.04). Compared to IL1ß-induced inflammation, HC have increased CD14 levels after vitamin D treatment (p<0.001), whereas the IL1ß-induced CD14 expression of AD patients' monocytes did not change after vitamin D treatment (p=0.8). AD patients carrying HLA high-risk haplotypes showed an increased CCL-2 expression after IL1ß-induced inflammation compared to intermediate-risk HLA carriers (p=0.05). Also HC monocytes' CD14 transcription after IL1ß and vitamin D co-stimulation differed according to HLA risk profile. We show that vitamin D can exert anti-inflammatory effects on AD patients' monocytes which may be modulated by HLA risk genotypes.

T-lymphocyte and glycemic status after vitamin D treatment in type 1 diabetes: A randomized controlled trial with sequential crossover.

BACKGROUND: Type 1 diabetes mellitus (T1D) is mediated by autoaggressive T effector cells with an underlying regulatory T-cell (Treg) defect. Vitamin D deficiency is highly prevalent in T1D, which can aggravate immune dysfunction. High-dose vitamin D treatment may enhance Tregs and improve metabolism in T1D patients. METHODS: In a randomized double-blind placebo-controlled trial with crossover design, patients received either for 3 months cholecalciferol 4000 IU/d followed by 3 months placebo or the sequential alternative. Thirty-nine T1D patients (19 women and 20 men) completed the trial. RESULTS: Primary outcome was a change of Tregs, secondary HbA1C, and insulin demand. Effects were evaluated based on intra-individual changes between treatment and placebo periods for outcome measures. Exploratory analyses included vitamin D system variant genotyping and C-peptide measurements. Median 25(OH)D3 increased to 38.8 ng/ml with males showing a significantly stronger increase (p = .003). T-lymphocyte profiles did not change significantly (p > 2); however, the intra-individual change of Tregs between males and females was different with a significantly stronger increase in men (p = .017), as well as between genotypes of the vitamin D receptor (Apa, Taq, and Bsm: genotypes aa, TT, and bb; p = .004-0.015). Insulin demands declined significantly (p = .003-.039) and HbA1C improved (p < .001). Random C-peptide levels were low but rising (median, 0.125 ng/ml; range, 0.02-0.3) in 6 patients. No toxicity was observed. CONCLUSION: A daily vitamin D dose of 4000 IU for 3 months was well tolerated and enhanced Tregs in males. Glucometabolic control improved in all. Subsequent larger trials need to address ß-cell function and genotyping for individualized vitamin D doses.

IL28B gene variants and glucose metabolism in Type 2 Diabetes.

Type 2 Diabetes (T2D) develops, when ß-cell insulin response fails to compensate for insulin resistance. Recent studies reported associations between the IL28B polymorphisms (rs12979860 and rs8099917) and T2D development in Hepatitis C virus (HCV) patients. To identify possible association with T2D independent from virus infection, we investigated both IL28B polymorphisms in T2D patients and healthy controls (HC). No association was found comparing the genotype and allele frequencies of both IL28B polymorphisms between T2D patients and HC. However, higher glucose levels were found in T2D patients carrying the IL28B CT/TT rs12979860 and GT/GG rs8099917 HCV risk genotypes compared to those with the protective CC and TT genotype (p=0.06 and p=0.02, respectively). Moreover, T2D patients with CT/TT rs12979860 HCV risk genotypes possessed significantly higher HbA1c levels than CC carriers (p=0.04). In conclusion, the IL28B HCV risk genotypes may influence glucose homeostasis in T2D patients without HCV.

Insufficient Vitamin D Response to Solar Radiation in German Patients with Type 2 Diabetes or Gestational Diabetes.

Vitamin D deficiency is highly prevalent in all forms of diabetes mellitus. Recently, we reported how ultraviolet B (UVB) radiation affected vitamin D [25(OH)D3] concentrations in patients with type 1 diabetes. Our aim was to analyze whether patients with non-autoimmune diabetes, such as type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM) also show the same vitamin D profile in relation to environmental factors including ambient temperature as an indirect parameter for outdoor activities. We analyzed 25(OH)D3 concentrations of T2DM (n=349) and GDM patients (n=327) at the University Hospital Frankfurt from 2005 to 2007. Additionally, daily UVB and monthly outside air temperature measurements for Frankfurt/Germany were obtained. We detected a positive correlation between UVB irradiation and 25(OH)D3 concentrations of T2DM and GDM patients (rho=0.50 and rho=0.63, p=0.003 and p<0.0001, respectively). UVB irradiation was in summer (April-October) higher than in winter (November-March) (5.6 kJ/m² vs. 0.5 kJ/m², p<0.0001). However, the prevalence of vitamin D deficiency in summer remained high with 76% in T2DM and 59% in GDM. In a stepwise regression analysis for the 25(OH)D3 concentration, significant predictors were outdoor temperature (estimate=0.02, p<0.0001), UVB radiation (estimate=-0.0015, p=0.02), year (2006 vs. 2005 estimate=-0.06, p>0.05, 2007 vs. 2005: estimate=-0.13, p<0.0001) and diabetes type (estimate=0.06, p=0.03). In conclusion, the strong correlation between UVB radiation and 25(OH)D3 concentrations in T2DM and GDM patients determines the seasonal variation. Additional determinants for the 25(OH)D3 concentrations were outdoor temperature, year, and diabetes type. Despite the effects of solar radiation both patients groups remain largely vitamin D deficient during summers.

Focus on vitamin D and the adrenal gland.

The main role of vitamin D is to maintain calcium and phosphorus homeostasis, thus preserving bone health. However, recent evidences have demonstrated that vitamin D may also play a role in a variety of nonskeletal disorders such as endocrine diseases and in particular type 1 diabetes, type 2 diabetes, adrenal diseases, and the polycystic ovary syndrome. Despite controversial results on an association of low vitamin D levels with cortisol and aldosterone overproduction, encouraging in vitro findings have been reported on vitamin D effects in adrenocortical cancer cells. The focus of this review is the role of vitamin D in adrenal diseases and the results of vitamin D supplementation studies in patients. Although many studies support a beneficial role of vitamin D in adrenal disease, randomized controlled trials and mechanistic studies are required to provide more insight into the efficacy and safety of vitamin D as a therapeutic tool.

VDR FokI polymorphism is associated with a reduced T-helper cell population under vitamin D stimulation in type 1 diabetes patients.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease mediated by T-helper (Th) cells. Additionally, the immune system regulator vitamin D, exerts its modulatory effects through the vitamin D receptor (VDR) expressed in Th cells. Furthermore, several genetic variants in the VDR gene including the VDR FokI (rs10735810) polymorphism have been implicated in T1D susceptibility in some Caucasian populations. Aim of the present study was to investigate the possible functional role of the VDR FokI gene polymorphism in Th cells from T1D patients and healthy controls (HC). METHODS: Isolated Th cells from 23 HC and 20 T1D patients were stimulated for 72h with 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). After in vitro culture CD3(+)CD4(+) (CD4(+)) Th cell subsets were characterized by flow cytometry and gene expression of VDR was measured by Taqman assay. Finally, the VDR FokI polymorphism was genotyped. RESULTS: Significant lower VDR gene expression was observed in non-stimulated and 25(OH)D3 stimulated Th cells from T1D compared to HC (p=0.04 and p=0.005, respectively). In addition, by stratifying subjects into VDR FokI genotypes, significant lower percentage of CD4(+) cells was observed in 25(OH)D3 and 1,25(OH)2D3 stimulated Th cells from T1D patients carrying the "FF" genotype compared to those with the genotypes "Ff/ff" (p=0.02 and p=0.05, respectively). Moreover, looking at vitamin D effects according to VDR FokI genotypes, CD4(+) cells were significantly down-regulated by 25(OH)D3 and 1,25(OH)2D3 only in T1D "FF" carriers (p=0.01 and p=0.02; respectively). CONCLUSION: According to these results, T1D patients carrying the "FF" genotype with an adequate vitamin D therapy may benefit from a more balanced T cell immunity. However, further research is needed to confirm these premilinary findings and to elucidate functional mechanisms of genetic variation in the vitamin D system. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Effect of a randomised controlled vitamin D trial on insulin resistance and glucose metabolism in patients with type 2 diabetes mellitus.

The aim of our study was to investigate the influence of a 6-month vitamin D supplementation in patients with noninsulin-requiring type 2 diabetes mellitus. We included 86 patients in a placebo-controlled, randomised, double-blind study. During 6 months patients received Vigantol oil once a week corresponding to a daily dose of 1904 IU or placebo oil, followed by 6 months of follow-up. At start and at 3-month intervals 25OHD, PTH, body mass index, HbA1c, insulin, C-peptide, and homeostasis model assessment-index were measured. The primary outcome was a change in fasting blood glucose and insulin levels. After 6 months of therapy, the verum group's 25OHD had increased to a median of 35 ng/ml in comparison to the placebo group (median 20 ng/ml, p<10-6). PTH tended to decrease in the verum group (25.5 pg/ml vs. 35.0 pg/ml, p=0.08). After 6 months of therapy, 31 patients (78%) achieved a 25OHD concentration of >20 ng/ml. Their HbA1c was significantly lower at baseline (p=0.008) and after therapy (p=0.009) than in patients with 25OHD below 20 ng/ml. C-Peptide, insulin, and HOMA-index did not change significantly in the verum group but fasting insulin was positively correlated with 25OHD concentrations after 6 months of therapy in both groups. There were no significant effects of vitamin D with a daily dose of 1904 IU on metabolic parameters in type 2 diabetes. However, the correlative findings of this study suggest a link of the 25OHD status and metabolic function in type 2 diabetes. Whether vitamin D therapy with higher doses can improve glucose metabolism needs to be investigated in follow-up trials.

Vitamin D status and gene transcription in immune cells.

BACKGROUND: Vitamin D is a modulator of the immune system. Its insufficiency has been implicated in type 1 diabetes (T1D) and studies showed significant associations with polymorphisms of vitamin D genes. Aim of the study was to investigate whether gene expression in immune cells, vitamin D status and genetic variants are correlated in healthy controls (HC). METHODS: From 23 HC monocytes (Mo), T-helper cells (Th) and natural killer cells (NK) were isolated. In all immune cells gene expression of vitamin D receptor (VDR), 25-vitamin-D-hydroxylase (CYP2R1) and 25-hydroxyvitamin-D3-1a-hydroxylase (CYP27B1) were measured by Taqman assay. Furthermore, CYP2R1 (rs10741657), CYP27B1 (rs10877012) and the VDR-FokI (rs10735810) polymorphisms in HC were genotyped. Finally, 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plasma levels in HC were measured by radioimmunoassay. RESULTS: All studied immune cells showed a significantly different gene expression of CYP2R1 and CYP27B1 (p=1×10(-6), respectively). When stratifying the HC according to vitamin D deficiency and vitamin D sufficiency, within the 25(OH)D3 deficient group significantly lower 1,25(OH)2D3 plasma levels (p=0.02) in HC and a significant down-regulation of the VDR expression only in Mo were observed (p=0.04). Furthermore, a significant correlation between CYP2R1 gene transcription and 1,25(OH)2D3 plasma levels in Th cells was found (p=0.04). No associations between the gene expression levels and the investigated polymorphism in all different immune cells were detected. However, vitamin D deficiency in combination with the "AC" CYP27B1 genotype appeared to inhibit the CYP27B1 expression in NK cells (p=0.03). CONCLUSION: both 25(OH)D3 deficiency and low 1,25(OH)2D3 levels appear to interact with its system gene transcription illustrating the relevance for targeted vitamin D therapy. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Influence of the vitamin D plasma level and vitamin D-related genetic polymorphisms on the immune status of patients with type 1 diabetes: a pilot study.

Vitamin D (VD) has been implicated in type 1 diabetes (T1D) by genetic and epidemiological studies. Individuals living in regions with low sunlight exposure have an increased T1D risk and VD supplementation reduced the risk in human individuals and mouse models. One possibility of how VD influences the pathogenesis of T1D is its immunomodulatory effect on dendritic cells (DC), which then preferentially activate regulatory T cells (T(regs) ). In the present pilot study, we collected blood samples from a small cohort of patients with T1D at baseline and months 6 and 12. VD-deficient patients were advised to supplement with 1000 IU/day VD. We found a considerable variation in the VD plasma level at baseline and follow-up. However, with higher VD plasma levels, a lower frequency of interleukin (IL)-4-producing CD8 T cells was observed. We further performed a comprehensive genotyping of 13 VD-related polymorphisms and found an association between VD plasma level and the genotype of the VD binding protein (DBP). The frequency of DC and T cell subsets was variable in patients of all subgroups and in individual patients over time. Nevertheless, we found some significant associations, including the 1,25-dihydroxyvitamin D(3) hydroxylase (CYP27B1) genotype with the frequency of DC subtypes. In summary, our preliminary results indicate only a limited influence of the VD plasma level on the immune balance in patients with T1D. Nevertheless, our pilot study provides a basis for a follow-up study with a larger cohort of patients.

What affects the quality of life in autoimmune Addison's disease?

INTRODUCTION: Several studies have shown a reduced quality of life in patients with Addison's disease, but little is known about the potential influences. METHODS: We determined the quality of life in 200 patients with Addison's disease using an Addison's disease-specific quality-of-life questionnaire. Data about first symptoms, time to diagnosis and current medication were collected by questionnaires. RESULTS: With increasing latency between first symptoms and diagnosis of adrenal insufficiency, the quality of life decreased in highly significant manner (p<0.001). Age at manifestation correlated negatively with quality of life (p=0.01). Significantly lower scores were observed in females versus males (141 vs. 159, p<0.001). Quality of life decreased significantly with increasing autoimmune comorbidity (p=0.01). Coeliac disease (p=0.05), atrophic gastritis (p=0.01) and primary ovarian failure (p=0.01) were highly correlated with reduced scores. CONCLUSIONS: Quality of life was significantly lower in female patients and in those with manifestation at older ages. With more autoimmune comorbidities, the quality of life scores dropped. The most important factor, however, was latency between first symptoms and diagnosis that affected patients' quality of life even years after manifestation of the disease. These results confirm and extend previous observations and emphasize the importance of a timely diagnosis. Therefore, medical awareness for this rare but easily treatable disorder needs to be sharpened.

Time trend of UVB irradiation and the vitamin D concentration in German type 1 diabetes patients.

BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease which is characterised by the destruction of insulin-producing beta cells in human pancreas leading consequently to a hyperglycaemic metabolism. Recent studies have shown that low cholecalciferol (25(OH)D3) concentrations may contribute to the development of T1D. The 25(OH)D3 status depends mostly on human skin production influenced by exposure to UVB radiation. Our intention was to examine whether there was a change in UVB radiation in the past years and if this has an impact on patients' vitamin D status. METHODS: We analysed the 25(OH)D3 concentration of blood samples from 287 T1D patients in the years 2004-2007 at the University Hospital Frankfurt. Moreover, daily UVB irradiation data of this time were received. Wilcoxon-Mann-Whitney test and Spearman correlation test were used for statistical analyses. RESULTS: We observe a strong correlation between UVB irradiation and the 25(OH)D3 concentration of German T1D patients (correlation coefficient=rho=0.56, p=7×10(-3)). Moreover, 25(OH)D3 blood levels obtained in summer (Apr-Oct) were significantly higher than in the winter season (p=8×10(-3)). In the years 2004-2007 there was a significant decline of UVB radiation in the summers (rho=-0.21, p<10(-6)) but no change was found in (rho=-0.07, p=0.12). This corresponds to a significant decrease of 25(OH)D3 levels in T1D patients over the summers (rho=-0.24, p=2×10(-3)) but not in winters (rho=-0.03, p=0.73). CONCLUSION: Our results reveal a significant correlation of UVB irradiation and the vitamin D concentration of German T1D patients. A decrease of UVB irradiation over the summers 2004-2007 is accompanied by a decline of 25(OH)D3 levels observed in those summer months which may indicate a local time trend requiring further investigation into the environmental factors of vitamin D deficiency. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Isolation of thyroid cells obtained by fine-needle aspiration biopsy.

Usually thyroid cells isolated from tissue obtained by surgery or thyroid cell lines are used to investigate the pathogenesis of autoimmune thyroid diseases. Isolation and cultivation of thyrocytes from fine-needle aspiration biopsy (FNAB) has not yet been published. The aim of this study was to isolate and cultivate thyrocytes from samples of FNAB. FNAB samples were obtained from nine adults and nine children with Hashimoto's thyroiditis (HT). The aspiration material was filtered resulting in small samples of tissue on the surface of the filter membrane. These tissue fragments were digested by collagenase I and dispase II. The yielding cells were cultivated for 3 weeks in Ham's F12 Kaighn's Modification medium in presence of 1 mU/mL bovine thyrotropin (TSH), 10 microg/mL human insulin, 6 microg/mL transferrin, and 10(-8) M hydrocortisone. Finally, isolated thyroid cells were characterized by determination of gene expression of thyrotropin receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) using a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Thyroid cells obtained by FNAB can be maintained over a time period of approximately 3 weeks. Depending on the sample size a final number of 1000-14,000 cells was gained per FNAB. In addition, all cells isolated by the described method expressed TPO mRNA. TSHR mRNA was found in 4 samples, whereas 15 samples were Tg mRNA-positive. There were no differences with respect to the expression TSHR and TPO mRNA between samples from adults and children. The isolation and cultivation of thyroid cells obtained by FNAB has been established. In contrast to surgical specimen, this technique provides an easy access to thyrocytes derived from individual patients allowing repeated sampling to investigate the time progression of the chronic disease or the effect of treatment over time.