Integrating clinical and genetic approaches in the diagnosis of 46,XY disorders of sex development.

46,XY differences and/or disorders of sex development (DSD) are clinically and genetically heterogeneous conditions. Although complete androgen insensitivity syndrome has a strong genotype-phenotype correlation, the other types of 46,XY DSD are less well defined, and thus, the precise diagnosis is challenging. This study focused on comparing the relationship between clinical assessment and genetic findings in a cohort of well-phenotyped patients with 46,XY DSD. The study was an analysis of clinical investigations followed by genetic testing performed on 35 patients presenting to a single center. The clinical assessment included external masculinization score (EMS), endocrine profiling and radiological evaluation. Array-comparative genomic hybridization (array-CGH) and sequencing of DSD-related genes were performed. Using an integrated approach, reaching the definitive diagnosis was possible in 12 children. The correlation between clinical and genetic findings was higher in patients with a more severe phenotype (median EMS 2.5 vs 6; P = 0.04). However, in 13 children, at least one variant of uncertain significance was identified, and most times this variant did not correspond to the original clinical diagnosis. In three patients, the genetic studies guided further clinical assessment which resulted in a reclassification of initial clinical diagnosis. Furthermore, we identified eight patients harboring variants in more than one DSD genes, which was not seen in controls (2.5%; P = 0.0003). In summary, taking into account potential challenges in reaching the definitive diagnosis in 46,XY DSD, only integrated approach seems to be the best routine practice.

Diagnostic significance of serum concentrations of soluble Fas ligand (sFasL) in children with autoimmune thyroid disease.

INTRODUCTION: The aim of the study was to assess serum levels of sFasL as a marker of thyroid dysfunction in children with autoimmune thyroid disease (AITD). DESIGN: The group comprised 45 newly diagnosed children with Hashimoto's thyroiditis and Graves' disease versus euthyroid control group: 11 with hypothyroidism (10 girls and 1 boy, aged 12.2 ± 1.9 years), 19 children with hyperthyroidism (15 girls and 4 boys, aged 12.4 ± 4.9 years) and 15 healthy subjects (7 girls and 8 boys, aged 10.5 ± 4.8 years). METHODS: Thyroid function (TSH, fT4, fT3), autoimmune (ATG, ATPO, TRAb) and anthropometric (weight, height, BMI, BMI-SDS, Cole index) parameters were evaluated. sFasL concentration was measured by ELISA. Nonparametric statistical test and ROC analysis were performed to assess the data. RESULTS: We found no significant differences in serum concentrations of sFasL between boys and girls in the studied groups. Significantly higher sFasL levels (median 0.26 ng/ml) were identified in children with hypothyroidism compared with the control group (median 0.06 ng/ml, p < 0.001) and in comparison to a group of children with hyperthyroidism (median 0.14 ng/ml, p < 0.05). ROC analysis indicates that sFasL effectively discriminated hypothyroid and healthy children (area under the curve/AUC = 0.897; p < 0,001; sensitivity: 100%, specificity: 73.3%), as well as both clinically opposing states: hyperthyroidism and hypothyroidism among themselves (AUC = 0.833; p= 0,003; sensitivity: 94,7%, specificity: 72.7%). CONCLUSIONS: Our work shows that sFasL may be useful marker in the assessment of thyroid dysfunction in children with autoimmune thyroid disease.

Proinflammatory cytokines in monitoring the course of disease and effectiveness of treatment with etanercept (ETN) of children with oligo- and polyarticular juvenile idiopathic arthritis (JIA).

BACKGROUND: The demonstration of the quantitative prevalence of specific cytokines in JIA formed the basis for the introduction of biological anticytokine drugs to treatment. Routine assessment of the concentration of these cytokines in blood serum may enable earlier decision making on the legitimacy of biological treatment (anti-TNF). The aim of the study was to assess the diagnostic value of TNFalpha, IL-6, and IL-1beta in monitoring the course of the disease and effectiveness of treatment with etanercept of children with oligo- and polyarticular JIA. METHODS: In a 1-year prospective study, cytokine levels were measured using ELISA in serum samples for 19 JIA patients in whom no clinical improvement was noted in spite of treatment with disease modifying antirheumatic drugs (DMARDs) and glucocorticosteroids (GCS). All the patients started treatment with ETN. The control group included healthy children (n = 18). RESULTS: Concentrations of TNF-a and IL-6 in blood serum at time 0 were significantly higher than in the control group. IL-6 concentration decreased during treatment with ETN in children in the inactive phase of the disease. Mean concentrations of TNF-a in serum increased several dozen times irrespective of clinical improvement. TNFalpha concentrations were statistically significantly higher in patients in the inactive phase of the disease in comparison with those in the active phase and correlated with the dose of ETN. Only the concentration of IL-6 correlated with the JADAS-27 value at all time points. CONCLUSIONS: We conclude that IL-6 may serve as a biomarker of activity of the disease in children with JIA treated with ETN.

The clinical role of serum concentrations of selected cytokines: IL-1ß, TNF-α and IL-6 in diagnosis of autoimmune thyroid disease (AITD) in children.

Chronic autoimmune thyroiditis (cAIT) leads to hypothyroidism due to T cell-mediated cytotoxicity in most cases. By contrast, Graves' disease (GD) with thyrotropin receptor stimulatory autoantibodies cause hyperthyroidism. Cytokines play a crucial role in modulating immune response in both disorders. The aim of study was to evaluate the concentrations of cytokines: IL-1ß, TNF-α and IL-6 in these two opposite clinical and hormonal thyroid diseases. The study group consisted of 64 children, 44 newly diagnosed, untreated children with cAIT (n = 22; with hypothyroidism) and GD (n = 22; hyperthyroidism), and the control group of 20 healthy children. Cytokine concentrations were evaluated using the ELISA technique. The studied groups of children did not differ significantly in concentrations of IL-6 (p = 0.48) and TNF-α (p = 0.067). In children with hypothyroidism, we found significantly higher concentrations of IL-1ß (median 2.16 pg/ml, IQR 0.87) compared to hyperthyroidism (median 1.39 pg/l, IQR 1.27) (p < 0.01) and the control group (median 1.88 pg/ml, IQR 1.04) (p < 0.05). The results of ROC curve analysis demonstrated the usefulness of IL-1ß (AUC = 0.77, p = 0.003) and TNF-α (AUC = 0.691, p = 0.034) as diagnostic parameters in cAIT which enable discrimination of children with autoimmune thyroid disease from healthy individuals. Concentrations of these markers are increased in autoimmune hypothyroidism. We found no significant sex differences in the tested parameters. In conclusion, IL-1ß and TNF-α may be considered as markers of hypothyroidism, and could efficiently discriminate between healthy and autoimmune hypothyroid children. Significantly higher concentrations of IL-1ß in children with hypothyroidism may be used to distinguish children with cAIT from GD patients.

The role of the immune system and cytokines involved in the pathogenesis of autoimmune thyroid disease (AITD).

Autoimmune thyroid disease (AITD) is the most common organ-specific autoimmune disorder. AITD development occurs due to loss of immune tolerance and reactivity to thyroid autoantigens: thyroid peroxidase (TPO), thyroglobulin (TG) and thyroid stimulating hormone receptor (TSHR). This leads to infiltration of the gland by T cells and B cells that produce antibodies specific for clinical manifestations of hyperthyroidism in Graves' disease (GD) and chronic autoimmune thyroiditis (cAIT). In addition, T cells in Hashimoto's thyroiditis induce apoptosis in thyroid follicular cells, leading ultimately to the destruction of the gland. Cytokines are involved in the pathogenesis of thyroid diseases working in both the immune system and directly targeting the thyroid follicular cells. They are involved in the induction and effector phase of the immune response and inflammation, playing a key role in the pathogenesis of autoimmune thyroid disease. The presence of multiple cytokines has been demonstrated: IL-1alpha, IL-1b, IL-2, IL-4 , IL-6, IL-8, IL-10, IL-12, IL-13, IL-14, TNF-alpha and IFN-gamma within the inflammatory cells and thyroid follicular cells. Finally, cytokines derived from T cells can directly damage thyroid cells, leading to functional disorders and may also stimulate the production of nitric oxide (NO) and prostaglandin (PG), thus increasing the inflammatory response in AITD. Immunological mechanisms involved in the pathogenesis of AITD are strongly related to each other, but differences in the image of cAIT and GD phenotype are possibly due to a different type of immune response observed in these two counteracting clinical thyroid diseases. This article describes the potential role of cytokines and immune mechanisms in the pathogenesis of AITD.

Serum TNF-α levels and indicators of disease activity in children with oligoarticular juvenile idiopathic arthritis (oJIA) in the first year of the disease.

BACKGROUND: Juvenile idiopathic arthritis is the most common arthropathy in childhood. Clinical assessment in JIA patients is based on clinical examination and conventional parameters of inflammation. Regardless of the JIA form, a distinctive characteristic of JIA is joint inflammation, which is sustained by an imbalance between pro- and anti-inflammatory factors. A significant amount of research has confirmed elevated levels of TNF-alpha in the serum and synovial fluid of JIA patients. The aim of the study was to compare serum TNF-alpha levels and indicators of disease activity in children with newly diagnosed oJIA in the first year of the disease and to assess the diagnostic value of TNF-alpha. METHODS: In a 1-year prospective study, TNF-alpha levels were measured using ELISA in serum samples for 22 oJIA patients. The control group consisted of 16 healthy children. The data were correlated with disease activity indicators and CHAQ score. RESULTS: Concentrations of TNF-alpha were significantly higher in the study group than in the control group at time 0 [10.03 pg/mL (2.16 - 15.53) vs. 0.00 pg/mL (0.00 - 0.12); p < 0.001] and at time 2 [0.00 pg/mL (0.00 - 9.26) vs. 0.00 pg/mL (0.00 - 0.12); p = 0.014]. The analysis of changes in TNF-alpha concentrations in the study group over time showed no statistically significant differences. No correlation between concentrations of TNF-alpha and any of the analyzed indicators of disease activity and CHAQ was found. CONCLUSIONS: Assessment of TNF-alpha concentration in blood serum in children with oJIA has no diagnostic value in monitoring the severity of the disease and the effectiveness of treatment.

The insulin-like growth factor-1 and expression of its binding protein­3 in chronic hepatitis C and hepatocellular carcinoma.

The role of growth factors produced by the liver, including insulin-like growth factor-1 (IGF-1) and its main binding protein, IGF binding protein-3 (IGFBP-3), in hepatitis C virus (HCV)-associated carcinogenesis has only partially been recognized and there is not much data available on the local expression of IGF-1 and IGFBP-3 in chronic hepatitis C (CH­C). Therefore, the aim of the present study was to evaluate the IGF­1 and IGFBP­3 serum levels and tissue expression in liver biopsies of CH­C patients (n=37) and hepatocellular carcinoma (HCC) samples (n=61) as related to age- and gender-matched control serum samples (n=15) and healthy liver samples (n=10). Serum concentrations of IGF-1 (S-IGF-1) and IGFBP­3 (S-IGFBP­3) were measured by the ELISA method. Tissue expression of proteins was detected using ABC immunocytochemistry and evaluated applying a spatial visualization technique. Concentrations of S-IGF-1 and hepatic expression of IGF-1 (H-IGF-1) proved to be lower in CH-C compared to the controls. No significant differences were detected in the concentration of S-IGFBP-3 between the studied groups but the S-IGF-1/IGFBP-3 ratio in the CH-C group was significantly lower compared to the control. H-IGFBP-3 was higher in CH-C compared to those in the control and HCC. In HCC, lower expression of H-IGF-1 was detected compared to the control and a higher H-IGF-1/IGFBP-3 ratio compared to CH-C. A negative correlation was detected between S-IGF-1 and S-IGF-1/IGFBP-3 ratio, on the one hand, and age, grading and concentration of α-fetoprotein (AFP) on the other, while H-IGFBP-3 was negatively correlated with BMI in the CH­C group. In patients with CH­C, the H­IGF­1/IGFBP­3 ratio was higher compared to that of the S­IGF­1/IGFBP­3 ratio. The studies documented a disturbed H­IGF­1 and H­IGFBP­3 in CH­C, which may be of significance in carcinogenesis. Examination of serum concentration and tissue expression of the two proteins and, first of all, estimation of the IGF­1/IGFBP­3 ratio may provide additional (to the estimation of IGF­1 and AFP) non-invasive markers in HCV­related liver injury.

Alterations of insulin-like growth factor I (IGF-I) and estradiol serum levels in chronic hepatitis C.

AIM OF THE STUDY: Deregulation of insulin-like growth factor I (IGF-I) production and decreased hepatic estrogen levels were associated with development of hepatocellular carcinoma (HCC) in hepatitis C virus (HCV) infected cirrhotic patients. The aim of our study was to determine serum levels of IGF-I, insulin and 17-ß estradiol (17-ßE) in relation to other markers of liver injury in chronic hepatitis C (CHC) patients. MATERIAL AND METHODS: Thirty anti-viral treatment-naïve CHC patients and 10 healthy subjects were examined. HCV infection was confirmed by presence of anti-HCV and HCV-RNA in serum. Serum levels of IGF-I, insulin and of 17-ßE were evaluated using ELISA methods. RESULTS: Serum levels of IGF-I and 17-ßE were significantly lower in CHC patients than in controls while insulin levels were similar in both groups. A lower IGF-I level (but not the level of 17-ßE) was observed in cirrhotic CHC patients in comparison to non-cirrhotic ones. Decreased serum level of IGF-I was associated with more advanced staging and liver steatosis, higher levels of alpha-fetoprotein (AFP) and gamma globulin levels, and higher aspartate transaminase (AST) activity in CHC patients. Insulin and 17-ßE levels positively correlated with patient's age. A positive correlation was observed between insulin level on one hand and staging, liver steatosis and levels of gamma globulins in CHC patients on the other. A negative correlation between IGF-I and insulin levels was noted only in HCV infected patients. CONCLUSIONS: Decreased IGF-I levels and increased levels of insulin better than estradiol serum levels characterize staging and liver steatosis in CHC patients. The lower serum level of 17-ßE in the CHC group than in control patients suggests that CHC patients carry higher risk of liver injury and of HCC development.