Untersuchung zellulärer und humoraler Immunparameter bei Patienten mit Plattenepithelkarzinomen im Kopf-/Halsbereich in Abhängigkeit von Tumorstadium und Therapieform

Im Rahmen einer klinische Studie wurden bei Patienten mit Plattenepithelkarzinomen im Kopf­/Halsbereich Lymphozyten­Subpopulationen phänotypisch und funktionell charakteri- siert sowie deren Aktivierungszustand anhand der Bestimmung des Interleukin­2­Titers und der Konzentration von löslichem Interleukin­2­Rezeptor a (sIL­2Ra) im Plasma gemessen. Hierbei interessierte der Einfluß des Tumors bzw. der Therapieformen wie Operation, Che­ mo­, Bestrahlungstherapie und kombinierte Radio­Chemotherapie auf diese immunologischen Parameter. Die Zusammensetzung der Populationen peripherer Blutlymphozyten wird durch die Größe und Malignität des Primärtumors sowie durch Lymphknotenmetastasen beeinflußt. In der An­ fangsphase, bei einer Tumormalignität G1, ist eine starke Reduzierung der LAK­Zellaktivität zu sehen, was mit suppressiven Faktoren, wie zum Beipiel Prostaglandin­E2, abgesondert von Monozyten, in Verbindung gebracht werden könnte. Der Anteil CD3­positiver Zellen ist im G3­Stadium sehr stark erniedrigt und deutet auf eine Herunterregulierung der zu dem CD3­ Komplex gehörenden e­Kette hin. Weiterhin war bei den HNO­Tumorpatienten der Anteil der CD4­Lymphozyten auffällig erniedrigt, während die Zahl der doppeltpositiven CD4 CD8 ­ Zellen sowie die HLA­DR­Werte erhöht war. Einen Monat nach einem operativen Eingriff ließ sich eine starke Zunahme der CD25­ Expression, besonders auf T­Lymphozyten erkennen. Die Chemotherapie mit Cisplatin, Fo­ linsäure, 5­FU, Fortecortin ­ eine sogenannte Polychemotherapie ­ bewirkte eine starke Erhö­ hung der NK­Zellpopulation und eine höhere Expression des CD25­Markers auf NK­Zellen sowie eine Zunahme der LAK­Zellfunktion. Die Veränderung der NK­Zellpopulation ist wahrscheinlich auf den Einsatz von Cisplatin und 5­FU zurückzuführen. Die Bestrahlungsthe­ rapie bewirkte eine signifikante Abnahme der T­Lymphozyten, besonders der CD4­T­ Helferzellen, bei gleichzeitiger Zunahme der CD4 CD8 ­positiven Zellen. B­Zellen sind g­Strahlen sensitiv, so daß unter Bestrahlung ihr Anteil vermindert wird. Die Erhöhung der NK­Zellpopulation ist wahrscheinlich auf eine Radioresistenz dieser Lymphozytensubpopula­ tion zurückzuführen. Die Radio­Chemotherapie beinhaltet drei Zyklen mit einem Zytostatika­ gemisch: Cisplatin, 5­FU, Folinsäure, Dexametason, Rescuvolin und Fortecortin und an­ schließender Behandlung mit g­Strahlen. Die Daten der vorliegenden Studie zeigen deutlich, daß es unter dieser Radio­Chemotherapie zu einem signifikanten Abfall der CD3­ und CD2­positiven Zellen, besonders der zytotoxi­ schen T­Zellpopulation, sowie zu einer Erhöhung der CD25­exprimierenden Zellen kommt. Die Beziehungen der Zellen untereinander sind unter Operation, Betrahlung­ und Radio­ Chemotherapie nicht verändert, die Chemotherapie verursacht jedoch vermutlich ein Un­ gleichgewicht zwischen T­Helferzellen und Makrophagen bezüglich der Vermehrung von NK­Zellen. Bei Patienten ohne Therapie kam es zu einer Abnahme der CD3­positiven Zellen. Dies unterstützt die Hypothese der Herunterregulierung der e­Kette des CD3­Komplexes unter Tumoreinwirkung. Die Ergebnisse zeigen weiter, daß die alleinige Bestimmung des Interleukin­2 Proteins keine Auskunft über die Wirkung des Tumors auf die Sezernierung dieses Peptids bei einer Tumorlokalisation im Gaumen­ und Zungenbereich gibt. Erst im Verlauf der Erkrankung können Änderungen durch virale Infektionen und der Einfluß verschiedener Therapiemodali­ täten beobachtet werden. Die Konzentration von sIL­2Ra verhielt sich proportional zur Tumorgröße. Dies unterstützt die Hypothese, daß dieser Rezeptor eher vom Tumor abstammt und nicht von den infiltrierten Lymphozyten. Die Konzentration von sIL­2Ra im Plasma war abhängig von der Therapieart. Sie erreichte wesentlich höhere Werte bei Tumorpatienten im Vergleich zu den Probanden. Der niedrigste Spiegel bei Patienten fand sich bei Bestrahlungstherapie (Median = 840 U/ml), die höchsten Werte bei Patienten mit Chemotherapie (Median = 1400 U/ml). Infiltrierende CD3 ­Lymphozyten im Primärtumor, identifiziert mit einen Antikörper gegen die e­Ketten, sind im Vergleich zu metastasierten Lymphknoten signifikant erniedrigt. Unter­ suchungen der peripheren Blutlymphozyten bei Patienten mit Primärtumor ergaben eine signi­ fikante Erhöhung der CD3­Populationen bei gleichzeitiger Erniedrigung des Anteils CD45­ positiver Zellen. Es bestand für CD45­positive Zellen ein signifikanter Zusammenhang zwi­ schen peripheren Blutlymphozyten und tumorinfiltrierenden Lymphozyten

Insulin gene polymorphisms in type I diabetes, Addison's disease and the polyglandular autoimmune syndrome type II

Background: Polymorphisms within the insulin gene can influence insulin expression in the pancreas and especially in the thymus, where self-antigens are processed, shaping the T cell repertoire into selftolerance, a process that protects from ß-cell autoimmunity. Methods: We investigated the role of the -2221Msp(C/T) and -23HphI(A/T) polymorphisms within the insulin gene in patients with a monoglandular autoimmune endocrine disease [patients with isolated type 1 diabetes (T1D, n = 317), Addison´s disease (AD, n = 107) or Hashimoto´s thyroiditis (HT, n = 61)], those with a polyglandular autoimmune syndrome type II (combination of T1D and/or AD with HT or GD, n = 62) as well as in healthy controls (HC, n = 275). Results: T1D patients carried significantly more often the homozygous genotype "CC" -2221Msp(C/T) and "AA" -23HphI(A/T) polymorphisms than the HC (78.5% vs. 66.2%, p = 0.0027 and 75.4% vs. 52.4%, p = 3.7 × 10-8, respectively). The distribution of insulin gene polymorphisms did not show significant differences between patients with AD, HT, or APS-II and HC. Conclusion: We demonstrate that the allele "C" of the -2221Msp(C/T) and "A" -23HphI(A/T) insulin gene polymorphisms confer susceptibility to T1D but not to isolated AD, HT or as a part of the APS-II

Vitamin D, FOXO3a, and Sirtuin1 in Hashimoto's Thyroiditis and Differentiated Thyroid Cancer

Background: Protective effects of vitamin D have been reported in autoimmune and malignant thyroid diseases, though little is known about the underlying mechanism. Sirtuin 1 histon deacethylase (SIRT1) links the vitamin D pathway with regulation of transcription factor FOXO3a, a key player in cell cycle regulation and apoptosis. Aim of the present study was to investigate common single nucleotide polymorphisms (SNP's) in FOXO3a gene in respect to thyroid diseases, as well as to evaluate the hypothesis of Sirtuin1-FOXO3a interaction being a mediator of anti-proliferative vitamin D effects.Methods: The SNP's FOXO3a rs4946936/rs4945816/rs9400239 were genotyped in 257 patients with differentiated thyroid carcinoma (DTC), 139 patients with Hashimoto thyroiditis (HT) and 463 healthy controls (HC). Moreover, T-helper cells of HC and papillary thyroid cancer cell line BCPAP were incubated with 1,25(OH)2D3 and/or SIRT1 inhibitor Ex-527 in order to elucidate SIRT1- dependent vitamin D effects on cell proliferation and FOXO3a gene expression in vitro.Results: Patients with DTC tended to carry more often allele C in FOXO3a rs4946936 in comparison to HC (pcorrected = pc = 0.08). FOXO3a rs9400239T and rs4945816C was more frequent in HT in comparison to HC (pc = 0.02 and pc = 0.01, respectively). In both DTC and HT, we could not find a correlation of FOXO3a SNP's with vitamin D status. However, on in vitro level, 1,25(OH)2D3 showed an anti-proliferative effect in both T-helper cells and BCPAP, that was blocked by SIRT1 inhibition (T-helper cells: p = 0.0059, BCPAP: p = 0.04) and accompanied by elevated FOXO3a gene expression in T-helper cells (p = 0.05).Conclusions:FOXO3a rs9400239T and rs4945816C may constitute risk factors for HT, independent of the vitamin D status.This indicates the implication of FOXO3a in pathogenesis of autoimmune thyroid diseases. The dependency of anti-proliferative vitamin D effects on SIRT1 activity further suggests a key role of vitamin D-SIRT1-FOXO3a axis for protective vitamin D effects

The rs1990760 polymorphism within the IFIH1 locus is not associated with Graves' disease, Hashimoto's thyroiditis and Addison's disease

Background: Three genes have been confirmed as major joint susceptibility genes for endocrine autoimmune disease:human leukocyte antigen class II, cytotoxic T-lymphocyte antigen 4 and protein tyrosine phosphatase non-receptor type 22. Recent studies showed that a genetic variation within the interferon induced helicase domain 1 (IFIH1) locus (rs1990760 polymorphism) is an additional risk factor in type 1 diabetes and Graves' disease (GD). Methods: The aim of the present study was to investigate the role of the rs1990760 polymorphism within the IFIH1 gene in German patients with GD (n=258), Hashimoto's thyroiditis (HT,n=106), Addison's disease (AD,n=195) and healthy controls (HC,n=227) as well as in 55 GD families (165 individuals, German) and 100 HT families (300 individuals, Italian). Furthermore, the interaction between rs1990760 polymorphism with human leukocyte antigen (HLA) risk haplotype DQ2(DQA*0501-DQB*0201), the risk haplotypes DQ2/DQ8 (DQA*0301-DQB*0302) and the status of thyroglobulin antibody (TgAb), thyroid peroxidase antibody (TPOAb) and TSH receptor antibody (TRAb) in patients and families were analysed. Results:No significant differences were found between the allele and genotype frequencies for rs1990760 IFIH1 polymorphism in patients with GD, HT, AD and HC. Also no differences were observed when stratifying the IFIH1 rs1990760 polymorphism for gender, presence or absence of thyroid antibodies (GD:TRAb and HT:TPOAb/TgAb) and HLA risk haplotypes (DQ2:for GD and HT, DQ2/DQ8:for AD). Furthermore the transmission analysis in GD and HT families revealed no differences in alleles transmission for rs1990760 IFIH1 from parents with or without HLA risk haplotype DQ2 to the affected offspring. In contrast, by dividing the HT parents according to the presence or absence of thyroid Ab titers, mothers and fathers both positive for TPOAb/TgAb overtransmitted the allele A of IFIH1 rs1990760 to their HT affected offspring (61.8% vs 38.2%;p=0.05;corrected p [pc]=0.1). However, these associations did not remain statistically significant after correction of the p-values. Conclusion: In conclusion, our data suggest, no contribution from IFIH1 rs1990760 polymorphism to the pathogenesis of either Graves' disease, Hashimoto's thyroiditis or Addison's disease in our study populations. However, in order to exclude a possible influence of the studied polymorphism in specified subgroups within patients with autoimmune thyroid disease, further investigations in larger populations are needed

Monocytic cytokines in autoimmune polyglandular syndrome type 2 are modulated by vitamin D and HLA-DQ

Context: Autoimmune polyglandular syndrome (APS-2: autoimmune Addison’s disease or type 1 diabetes) is conferred by predisposing HLA molecules, vitamin D deficiency, and heritable susceptibility. Organ destruction is accompanied by cytokine alterations. We addressed the monocytic cytokines of two distinct APS-2 cohorts, effects of vitamin D and HLA DQ risk. Methods: APS-2 patients (n = 30) and healthy controls (n = 30) were genotyped for HLA DQA1/DQB1 and their CD14+ monocytes stimulated with IL1β and/or 1,25(OH)2D3 for 24 h. Immune regulatory molecules (IL-6, IL-10, IL-23A, IL-15, CCL-2, PD-L1), vitamin D pathway gene transcripts (CYP24A1, CYP27B1, VDR), and CD14 were analyzed by enzyme-linked immunosorbent assay and RTqPCR. Results: Pro-inflammatory CCL-2 was higher in APS-2 patients than in controls (p = 0.001), whereas IL-6 showed a trend – (p = 0.1). In vitro treatment with 1,25(OH)2D3 reduced proinflammatory cytokines (IL-6, CCL-2, IL-23A, IL-15) whereas anti-inflammatory cytokines (IL-10 and PD-L1) rose both in APS-type 1 diabetes and APS-Addison´s disease. Patients with adrenal autoimmunity showed a stronger response to vitamin D. Expression of IL-23A and vitamin D pathway genes VDR and CYP27B1 varied by HLA genotype and was lower in healthy individuals with high-risk HLA (p = 0.0025; p = 0.04), while healthy controls with low-risk HLA showed a stronger IL-10 and CD14 expression (p = 0.01; p = 0.03). Conclusion: 1,25(OH)2D3 regulates the monocytic response in APS-2 disorders type 1 diabetes or Addison´s disease. The monocytic cytokine profile of individuals carrying HLA high-risk alleles is proinflammatory, enhances polyglandular autoimmunity and can be targeted by vitamin D

Immunomodulatory effects of 25-hydroxyvitamin D3 on monocytic cell differentiation and influence of vitamin D3 polymorphisms in type 1 diabetes

BACKGROUND Preventive measures and a causal therapy for type 1 diabetes (T1D) remain elusive. An imbalance between different dendritic cells (DC) with increased immunogenic DC and decreased tolerogenic DC (tDC) may lead to T1D. Furthermore, 25(OH)D3 is associated with less adverse effects than 1,25(OH)2D3. PURPOSE The present study was performed to clarify the remaining issues about the cellular effects of 25(OH)D3 in patients with T1D and the role of genetic polymorphisms of the vitamin D3 (VD3) metabolism on a functional cellular level. MATERIALS AND METHODS Twelve patients with T1D were case-matched to twelve healthy controls (HC). Monocytes (MC) were either not supplemented or supplemented with 25(OH)D3 in vitro and phenotyped with fluorescence-activated cell sorting. In vitro synthesis and plasma levels of 25(OH)D3 and 1,25(OH)2D3 were analyzed as well as twelve gene polymorphisms of the VD3 metabolism. RESULTS 25(OH)D3 significantly inhibited differentiation of MC into DC and led to an increase of intermediate cells (IC), which show a similar phenotype as tDC. The patient with a recent onset of T1D showed a higher increase in MC and IC compared to patients with long-standing T1D. There were significant differences for the increase of IC with supplementation of 25(OH)D3 between different genotypes within the polymorphisms of VDR-BsmI-rs1544410, VDR-TaqI-rs731236 and CYP24A1-rs927650. CONCLUSION This study suggests that 25(OH)D3 shows immunomodulatory effects on a cellular level in patients with T1D and HC by inhibiting the differentiation of MC into DC and promoting the formation of IC, which are similar to tDC, thereby shifting immunity to self-tolerance. The potency of 25(OH)D3 did not differ between patients with T1D and HC. Increased plasma levels of 25(OH)D3 may inhibit a proinflammatory cell milieu. Despite of the limited patient number, this study generates the hypothesis that the immunmodulatory effects may be influenced by genotypes of the VDR and CYP24A1 illustrating their functional role in T1D susceptibility, which is worth further investigation

Vitamin D supplementation enhances C18(dihydro)ceramide levels in type 2 diabetes patients

Sphingolipids are characterized by a broad range of bioactive properties. Particularly, the development of insulin resistance, a major pathophysiological hallmark of Type 2 Diabetes mellitus (T2D), has been linked to ceramide signaling. Since vitamin D supplementation may slow down T2D progression by improving glucose concentrations and insulin sensitivity, we investigated whether vitamin D supplementation impacts on plasma sphingolipid levels in T2D patients. Thus, plasma samples of 59 patients with non-insulin-requiring T2D from a placebo-controlled, randomized, and double-blind study were retrospectively analyzed. Once per week, patients received either 20 drops of Vigantol oil, corresponding to a daily dose of 1904 IU/d vitamin D (verum: n = 31), or a placebo oil consisting of medium chain triglycerides (placebo: n = 28). Blood samples were taken from all of the participants at three different time points: 1) at the beginning of the study (baseline), 2) after 6 months supplementation, and 3) after an additional 6 months of follow-up. Plasma sphingolipids were measured by high-performance liquid chromatography tandem mass spectrometry. At baseline and 6 months follow-up, no significant differences in plasma sphingolipid species were detected between the placebo and verum groups. After 6 months, vitamin D supplementation significantly enhanced plasma C18dihydroceramide (dhCer; N-stearoyl-sphinganine (d18:0/18:0)) and C18ceramide (Cer; N-stearoyl-sphingosine (d18:1/18:0)) levels were observed in the verum group compared to the placebo group. This was accompanied by significantly higher 25-hydroxyvitamin D3 (25(OH)D3) blood levels in patients receiving vitamin D compared to the placebo group. Taken together, vitamin D supplementation induced changes of the C18 chain-length-specific dhCer and Cer plasma levels in patients with T2D. The regulation of sphingolipid signaling by vitamin D may thus unravel a novel mechanism by which vitamin D can influence glucose utilization and insulin action. Whether this acts favorably or unfavorably for the progression of T2D needs to be clarified

HLA-DQB1 pos. 57 defines susceptibility to isolated and polyglandular autoimmunity in adults: interaction with gender

We define the amino acids of high risk HLA DQB1 alleles in position 57 in cohorts of<br>isolated and polyglandular disorders revealing gender dependent increased<br>susceptibilit