Mario Štefanić

Association of vitamin D receptor gene 3′-variants with Hashimoto's thyroiditis in the Croatian population

Hashimotos thyroiditis (HT) is the most frequent autoimmune thyroid disease with strong genetic background. Vitamin D receptor (VDR) endocrine system affects immunosuppressive, regulatory and tolerogenic decisions required for induction and maintenance of peripheral immune tolerance. With respect to the biological function of the VDR and functionally plausible gene‐expression data, we sought to test whether particular 3′‐restriction fragment length polymorphisms (RFLP) and haplotypes previously directly or indirectly associated with VDR mRNA 3′‐allelic imbalance phenotype and differences in total VDR mRNA expression are implicated in HT susceptibility. Thus, 145 Croatian HT patients and 145 age‐, sex‐ and ethnically matched euthyroid controls were genotyped for VDR rs1544410 (BsmI), rs7975232 (ApaI) and rs731236 (TaqI) polymorphisms by polymerase chain reaction‐RFLP method. Covariate‐adjusted single‐locus and haplotype–phenotype regression analyses were performed. Permutation corrections (Pc) and Akaike Information Criteria were used for model comparisons. The best‐fit [global Pc = 7.2 × 10−4]BsmI‐TaqI BT haplotype was found significantly more often in subjects without HT [12.2% vs. 3.7%; odds ratio (OR, 95% confidence intervals) = 0.28 (0.14–0.56), Pc = 8 × 10−4], whereas the bT haplotype was significantly more frequent in individuals with HT [45.7% vs. 61.8%; OR = 1.91 (1.37–2.65), Pc = 4 × 10−4]. Two extended BsmI‐ApaI‐TaqI RFLP haplotypes, the common baT [35.7 vs. 47.3%, OR = 1.63 (1.17–2.27), Pc = 0.012] and rare BaT variants [6.5 vs. 1.2%, OR = 0.17 (0.06–0.55), Pc = 1.2 × 10−3] were associated with HT, representing predisposing and protective haplotypes, respectively. In single‐RFLP association analyses, only rs1544410 polymorphism was associated with HT phenotype (allelic Pc = 0.0078) and appeared to function under the recessive model, with decreased risk of HT among the BB homozygotes [OR = 0.39 (0.21–0.7), Pc = 0.0052] when compared to the reference b+‐genotypes. These data suggest that common haplotypic variants within the VDR gene 3′‐region previously linked to VDR mRNA expression and allelic imbalance could be associated with HT in the general population, and thus, may be involved in the pathogenesis of HT.

Lack of association of vitamin D receptor gene 3′‐haplotypes with psoriasis in Croatian patients

Cis‐acting regulatory variants in biologically relevant pathways and target tissues are a common source of phenotypic variations and individual disease susceptibility. In the skin, vitamin D receptor (VDR) is a master regulator of epidermal barrier function, inflammation, stem cell proliferation and microbial defense; therefore, we tested whether VDR 3′‐regulatory haplotypes, a portion of which affect VDR transcriptional efficiency, allelic symmetry and mRNA turnover, were associated with psoriasis vulgaris. For this purpose, three VDR tag polymorphisms that capture most of the variability of the VDR 3′‐regulatory element (rs1544410, rs7975232 and rs731236) were genotyped by the polymerase chain reaction restriction fragment length polymorphism method in 180 Caucasian patients with chronic plaque psoriasis and 366 ethnically matched, healthy controls of the Croatian origin. We found no evidence of association for any of the selected polymorphisms. Similarly, none of the 3′‐VDR restriction haplotypes were associated with the risk for development of psoriasis in Croatian patients. These results show that neither VDR 3′‐restriction polymorphisms nor common 3′‐regulatory haplotypes contribute to psoriasis risk in the Croatian population.

Meta‐analysis of vitamin D receptor polymorphisms and psoriasis risk

Vitamin D receptor (VDR) gene polymorphisms have been studied as candidate variants that affect psoriasis risk. However, results have been conflicting.

The Expression of T Cell FOXP3 and T-Bet Is Upregulated in Severe but Not Euthyroid Hashimoto's Thyroiditis

Hashimotos thyroiditis (HT) is an organ-specific autoimmune disorder characterized by progressive thyroid failure. Th1 and Treg subset of CD4+ cells have been implicated in the pathogenesis; however, less is known about their respective roles across the spectrum of HT clinical presentations. To shed more light on CD4+ subsets role in HT, we investigated the mRNA expression levels of several Th1/Treg-associated transcription factors (T-bet/ETS1, HIF1α/BLIMP1/FOXP3) in peripheral blood T cells of 10 hypothyroid, untreated HT patients, 10 hypothyroid patients undergoing hormone replacement therapy, 12 euthyroid HT subjects, and 11 healthy controls by the qRT-PCR. Compared to euthyroid HT patients and controls, both hypothyroid (2.34-fold difference versus controls, P < 0.01) and thyroxine-supplemented patients (2.5-fold, P < 0.001) showed an increased FOXP3 mRNA expression in T cells. Similarly, mRNA expression levels of T-bet were upregulated in severely affected but not in euthyroid HT subjects (2.37-fold and 3.2-fold, hypothyroid and thyroxine-supplemented HT patients versus controls, resp., P < 0.01). By contrast, no differences in mRNA expression levels of ETS1, BLIMP1, and HIF1α were observed across the study groups. In summary, severe but not euthyroid HT was associated with robust upregulation of T-bet and FOXP3 mRNA in peripheral T cells, independent of the thyroid hormone status but proportional to disease activity.

Estimated collective effective dose to the population from nuclear medicine diagnostic procedures in Croatia: A comparison of 2010 and 2015

Objective This study presents national surveys of patient exposure from nuclear medicine (NM) diagnostic procedures in 2010 and 2015 in the Republic of Croatia. Methods The survey was performed according to the European Commission Dose DataMed (DDM) project methodology. 28 most frequent NM diagnostic procedures were identified. Data about frequencies of procedures and average administered activities of radioisotopes used in those procedures were collected. Average administered activities were converted to effective doses according to the dose conversion coefficients. Then the collective effective dose to the population and an effective dose per capita were calculated based on the number of the most frequent NM diagnostic procedures and the average effective dose per procedure. Results In 2010, 41200 NM diagnostic procedures led to 146.7 manSv collective effective dose to the population and in 2015, 42000 NM diagnostic procedures led to 146.8 manSv collective effective dose to the population. The frequencies of NM diagnostic procedures were 9.7 and 9.8 annually per 1000 population with 34.1 μSv and 34.2 μSv effective dose per capita for 2010 and 2015, respectively. The main contributors to the annual collective dose from NM in Croatia are examinations of the bone, heart, thyroid and PET/CT tumour diagnostic. Average administered activities have not changed considerably from 2010 to 2015. Nevertheless, within the frequency of some of the procedures, significant changes were found in five-year period. Conclusions Frequencies, average administered activities and collective effective dose to the population from NM diagnostic procedures in Croatia are comparable to the values reported by other European surveys. Changes were found between 2010 and 2015 and we intend to perform this study periodically to identify possible trends, but also to raise awareness about the potential dose optimization.

Pilot study of variants of the IL-23R and STAT3 genes reveals no association with Hashimoto’s thyroiditis in the Croatian population

Abstract Interleukin-23 receptor (IL-23R) and signal transducer and activator of transcription 3 (STAT3) polymorphisms are common risk factors for a number of T helper (Th) 17-mediated autoimmune diseases. However, the importance of genetic variations in Th17 pathways to thyroid autoimmunity, and particularly Hashimoto’s thyroiditis (HT), is not fully understood. In this study, we genotyped three single nucleotide polymorphisms (SNPs) within the IL-23R (rs11209026/p.Arg381Gln, rs7530511) and STAT3 (rs744166) genes in 217 Croatian patients with HT and 161 healthy controls using fluorescence resonance energy transfer technology and melting curve analysis of polymerase chain reaction products. None of the tested SNPs or IL-23R haplotypes was associated with HT susceptibility or disease severity. These results suggest that the studied IL-23R/STAT3 polymorphisms affecting Th17 signaling efficiency are not major determinants of HT risk in the Croatian population. Further work is necessary to determine if these loci contribute modestly or conditionally to the risk of HT.

miR-29a-3p/T-bet Regulatory Circuit Is Altered in T Cells of Patients With Hashimoto’s Thyroiditis

Objective Hashimoto’s thyroiditis (HT) is a common autoimmune thyroid disorder that frequently evolves from asymptomatic, T-cell mediated chronic inflammation toward overt hypothyroidism. Previously, we have demonstrated a role for T-bet, a T helper 1/CD8+ T cell transcription factor (TF), and FoxP3, a regulatory T cell TF, in disease progression and severity, but the basis behind their altered mRNA expression remains unknown. In this study, we aimed to leverage the role for microRNAs, representing negative transcriptional regulators, across the spectrum of HT clinical presentations using the same, well-characterized RNA sample cohort. Method Ten hypothyroid, untreated patients (hypoHT), 10 hypothyroid cases rendered euthyroid by l-thyroxine therapy (substHT), 11 spontaneously euthyroid HT subjects (euHT), and 10 healthy controls (ctrl) were probed for three candidate immunoregulatory miRNA (miR-9-5p, miR-29a-3p, and miR-210-3p) using quantitative real-time PCR measurements. Data were normalized to U6snRNA and fold difference in expression calculated by the efficiency corrected 2−ΔΔCt model. Results Compared to healthy controls, peripheral blood (PB) T cells of HT patients exhibited significantly diminished miR-29a-3p expression levels [median expression levels (IQR), HT vs CTRL, 0.62 (0.44–1.01) vs 1.373 (0.63–2.7), P = 0.046], and a similar, but not significant decline in miR-210-3p abundance [HT vs CTRL, 0.64 (0.39–1.31) vs 1.2 (0.5–2.56), P = 0.24, Wilcoxon test]. A significant inverse correlation was observed between the two differentially expressed transcripts, T-bet mRNA and miR-29a-3p. Moreover, altered miR-29a-3p/T-bet expression in T cells of untreated HT patients was related to low serum FT4, high serum thyrotropin, and decreased thyroid volumes. Of note, miR-210-3p expression was positively correlated to HIF1α, and inversely to FoxP3 mRNA levels, but no evidence of differential expression for any of these miRNA–mRNA pairs was observed. Finally, miR-9-5p expression levels were no different in HT vs control comparisons, or related to clinicopathological features. Conclusion T cell miR-29a-3p is downregulated in HT patients and associated with clinical and biochemical parameters of progressive thyroid injury, plausibly subsequent to altered control of T-bet expression in PB T cells. As such miR-29a-3p/T-bet axis should be further explored as a biomarker or as a plausible target for therapeutic interventions in HT.

Expression of TIGIT and FCRL3 is Altered in T Cells from Patients with Distinct Patterns of Chronic Autoimmune Thyroiditis

BACKGROUND Co-inhibitory receptors (IR), such as TIGIT and FCRL3, provide a checkpoint against highly destructive immune responses. Co-expression of TIGIT and FCRL3, in particular, has been linked to the HELIOS+ subset of regulatory CD4+FOXP3+T-cells. Of these, CD4+FOXP3-exon(E)2+ cells have higher expression of IR and exhibit strongest suppressive properties. Nevertheless, how the expression of TIGIT, FCRL3, HELIOS, and FOXP3E2 is regulated in chronic autoimmune thyroiditis (AT), is not known. METHODS Thirty patients with AT [encompassing spontaneously euthyroid (euAT), hypothyroid-untreated and L-thyroxine-treated cases)] and 10 healthy controls (HC) were recruited. FCRL3, TIGIT, HELIOS and FOXP3E2 mRNA expression levels in peripheral blood (PB) T cells were measured via quantitative real-time PCR and compared to clinicopathological factors. RESULTS The TIGIT and FCRL3 expression levels from T cells of AT cases were inversely related to the thyroid volume, and were significantly increased in hypothyroid patients (on+off L-thyroxine), but not euAT cases. The FCRL3 expression in PB T cells positively correlated with thyroid-peroxidase autoantibody levels; by contrast, T cells from aged AT patients and combined samples (AT+HC) accumulated more TIGIT mRNA. The patients with higher TIGIT mRNA levels had a greater prevalence of hypothyroidism, showing higher peak thyrotropin levels at diagnosis or at follow-up. CONCLUSIONS Multiple IR, namely FCRL3 and TIGIT, but not the transcription factors HELIOS and FOXP3E2, showed increased mRNA levels in PB T cells from end-stage, long-standing and/or more aggressive AT, in proportion to disease severity. A relation with major clinical subphenotypes was observed, thereby identifying IR as potentially important players in AT.

Zmiana ekspresji mRNA dla CTLA-4, CD28, VDR i CD45 w limfocytach T u osób z chorobą Hashimoto — badanie pilotowe

INTRODUCTION CD28/T-cell receptor (TCR)/cytotoxic T-lymphocyte antigen 4 (CTLA4) complex controls T-cell tolerance and autoimmunity in Hashimotos thyroiditis (HT). In addition, CD45 protein tyrosine phosphatase (PTPase) and vitamin D receptor (VDR) cooperatively interact with the TCR complex to affect autoimmune processes central to the pathogenesis of HT. Nevertheless, their role in HT aetiology has been less well established. In this study, we aimed to explore mRNA expression levels of CTLA4, CD28, CD45, and VDR in T-cells, across different outcomes of HT. MATERIAL AND METHODS The study included 45 HT patients and 13 euthyroid, healthy controls. T-lymphocytes were isolated from peripheral blood mononuclear cells, total mRNA was extracted from T-cells, and gene expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) and ImageQuant method relative to glyceraldehyde-3-phosphate dehydrogenase RT-PCR products. RESULTS Nominally higher expression levels of VDR, CTLA4, CD28, and CD45RAB mRNA were found in unsorted T-lymphocytes of healthy controls when compared to the HT patients. No difference was observed between hypothyroid/untreated, spontaneously euthyroid and LT4-treated HT patients. VDR mRNA expression was linked to both T3 levels and CTLA4 gene expression, whilst CD45RB mRNA expression coincided with CTLA4 and CD28 transcript levels. Conversely, older age and lower T3 levels were associated with increased abundance of CD45R0 isoform in HT patients. CONCLUSIONS The results suggest a cross talk between endocrine and immune functions in HT pathology: an altered peripheral T cell mRNA profile with reduced VDR, CTLA4, CD28, and CD45RAB transcript levels is accompanied by age-related shift from naive to memory/late-differentiated T cell CD45R mRNA signature and associated with thyroid hormone status in the HT patients.

Measurable and Unmeasurable Features of Ultrasound Lymph Node Images in Detection of Malignant Infiltration

The aim of the study was to assess diagnostic value and utility of selected morphological features in predicting lymph node (LN) malignancy using B-mode, Doppler ultrasonography and multivariate settings in a tertiary radiological referral center. The study included 123 patients having undergone ultrasound-guided fine-needle aspiration and cytologic analysis (FNAC) of cervical, axillary and inguinal LNs. Each LN was characterized by long/L and short/T-axis, shape, margins, echogenicity, cortical thickness, vascularization, and examiners subjective impression. Within the limitations of FNAC, altered shape and vascularization had relatively high specificity and positive predictive value (>80%), whereas subjective impression had high sensitivity and negative predictive value (100%) for malignancy. The cut-off levels for different features of LN by ROC analysis were as follows: long-axis 23 mm, short-axis 11 mm, L/T ratio 2.19, and maximal cortical thickness 5.1 mm. On multivariate analysis (adaptive regression splines, n=108), the addition of long-axis, L/T ratio, age and sex considerably improved diagnostic accuracy (88%), sensitivity (margins + vascularization) and specificity (subjective impression) of the diagnostic model. The combination of morphological and demographic features could improve diagnostic accuracy, usually with a trade-off between the sensitivity and specificity of the predictive model. The performance may depend on the level of expertise and institutional settings.