Hyperthyroidism in Children

Hyperthyroidism is the state of excessive synthesis and release of the thyroid hormones by thyrocytes. Graves’ disease is the most common cause of hyperthyroidism in children. The condition may occur at any age but the prevalence increases with age. According to the classical paradigm, coexistence of genetic susceptibility, environment triggers and immunological dysfunction are responsible for its development. Diagnosis of Graves’ disease is based on presence of characteristic clinical symptoms, TSH receptor antibodies and excess of thyroid hormones. The management in pediatric population involves mainly pharmacotherapy (thyrostatics, β-adrenolitics), in resistant cases radical radioiodine I131 therapy or surgical treatment is necessary

Cytometric evaluation of intracellular IFN-γ and IL-4 levels in thyroid follicular cells from patients with autoimmune thyroid diseases

<p>Abstract</p> <p>Background</p> <p>In recent few years is underlined that altered balance of pro- and anti-inflammatory cytokines play an important role in the pathogenesis of AITD.</p> <p>The aim of this study was to estimate intracellular INF-γ and IL-4 levels in thyroid-infiltrating lymphocytes and thyrocytes isolated from thyroid tissues in 54 adolescent patients aged 8-21 years, with Graves' disease (GD; n = 18), Hashimoto's thyroiditis (HT; n = 18) and non-toxic multinodular goiter (NTMG; n = 18).</p> <p>Methods</p> <p>Fresh thyroid tissues were taken on culture medium RPMI -1640, it was mechanically prepared. In next step were added cell activators -12- myristate 13- the acetate (PMA) and Ionomycin as well as the inhibitor of transportation of proteins - Breferdin A. They were cultured 24 hours in 50 ml flasks at 37°C in a 5-95% CO2-air water-saturated atmosphere. After that, thyrocytes were identified by mouse mAb directed against human TPO epitope 64 conjugated with rabbit anti-mouse antibodies IgG (Fab')₂labeled by FITC. After incubation at room temperature to each of samples added reagent A fixative the cellular membrane. In next step into the cell suspensions were added reagent B to permeabilization of cellular membrane and specific anti-IL-4-PE or anti-IFN-γ-PE mAbs. Identification of intracellular cytokines in T lymphocytes was performed in the same procedure with application of anti-CD4-PerCP and anti-CD8-PerCP mAbs specific for T lymphocytes. The cells were analyzed in a flow cytometry (Coulter EPICS XL).</p> <p>Results</p> <p>In examined group of patients with GD we observed statistically significant higher mean percentage of cells with phenotype CD4+IL-4 (p < 0.05; p < 0.025), CD8+IL-4 (p < 0.033; p < 0.01) and TFCs-IL-4+ (p < 0.05; p < 0.01) in comparison to patients with HT and NTMG. The analysis of mean percentages of positive TILs and TFCs with intracellular INF-g levels in patients with HT revealed statistically significant increase percentage of CD4+INF-γ (p < 0.04; p < 0.001), CD8+ INF-γ (NS; p < 0.025), TFCs+INF-γ (p < 0.03; p < 0.001) cells in comparison to the percentage of positive cells from patients with GD and NTMG.</p> <p>Conclusions</p> <p>We conclude that human thyrocytes in autoimmune thyroid disorders could be a source of cytokine production and that their activation influences local interaction with T lymphocytes inflowing to the thyroid gland.</p

The activity of gastric ghrelin positive cells in obese patients treated surgically.

Ghrelin is a 28 amino acid peptide hormone regulating food intake and stimulating releasement of growth hormone. It is produced in a distinct endocrine call known as X/A - like cells. The most abundant source of this very important factor in energy homeostasis is gastric fundus. Regulatory mechanisms of ghrelin synthesis and secretion in physiological and pathological states are not discovered completely. The aim of our study was evaluation of the activity of gastric X/A-like cells in obese patients before and after the most popular surgical bariatric procedures - Roux - Y Gastric Bypass (RYGB) and Laparoscopic Adjustable Gastric Banding (LAGB). Obese patients in number 18 took part in the study. LAGB was performed in 7 patients and RYGB in 11 patients. Peripheral blood was taken from each patient before operation and first day, seventh day, one month and three months after surgery. Ghrelin level was determined by RIA technique. The specimen of stomach was taken from circular stapler after gastrojejunostomy during RYGB and immunohistochemical study of gastric mucosa, using the EnVision method and specific monoclonal antybodies against ghrelin was performed. The intensity of ghrelin-immunoreactivity in X/A-like cells was analyzed using Olympus Cell D image analysis system. Efficiency of bariatric procedures was estimated by EWL- excess weight loss. We observed very strong immunohistochemical reactions of gastric X/A-like cells, accompanied by lower ghrelin plasma concentration, in comparison to the control group. LAGB procedure induced increase of ghrelin plasma level while RYGB procedure induced decrease of this hormone. The main finding of the present study is the hypoactivity of gastric X/A-like cells in obese patients in comparison to the control group

Analiza ekspresji cząsteczek Fas, FasL oraz kaspazy 8 w tkance gruczołu tarczowego u młodych pacjentów z chorobami immunologicznymi i nieimmunologicznymi gruczołu tarczowego

Introduction: Apoptosis, programmed cell death is a regulating mechanism enabling the removal of superabundantly produced and unnecessary at the certain moment cells. Disturbances of the apoptosis regulation contribute to the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of this study was to estimate expression of proapoptotic Fas/FasL and caspase-8 in thyroid tissues in patients with Graves&#8217; disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto&#8217;s thyroiditis (HT). Material and methods: Inclusion criteria of Graves&#8217; patients were: large goiter, ophthalmopathy, TRAb > 5 U/L, positive titre of anti-TPO and anti-TG antibodies and concentration of TSH < 0.45 mIU/mL for more the 2&#8211;3 months from an onset of the disease. Isolated thyrocytes were identified by indirect method: in the first stage mouse monoclonal antibodies (mAbs) anti-TPO were bound to rabbit anti-mouse antibodies IgG (Fab&#8217;)2 labeled FITC. To obtained cellular suspension mAbs directed against apoptotic Fas/FasL molecules labeled with PE (Phycoerythrin) was added. All investigations were performed onCoulter EPICS XL flow cytometer. Detection of apoptotic proteins was confirmed by Western Blot and immunohistochemistry methods using mAbs in DAB chromogene visuality and marked by Mayer&#8217;s haematoxylin. Evaluation of caspase-8 expression in thyroid follicular cells was performed by Western Blot test. Results: The analysis of Fas and FasL expression on surface of thyroid follicular cells was higher in patients with Hashimoto&#8217;s thyroiditis (38%, 26%) in comparison with patients with Graves&#8217; disease (18%, 14%). In case of patients with Hashimoto&#8217;s thyroiditis significantly lowerpercentage of thyroid tissue infiltrating immune Fas+ (13%) and FasL+ (22%) T cells in comparison with Graves&#8217; patients (33%, 43% respectively) was observed . Identification of proapoptotic Fas and FasL molecules in the thyroid follicular cells revealed higher expression of both proteins in patients with GD (++,++) and HT (+++; +++, respectively) in comparison with NTNG patients (+/0; +/0). Caspase-8 expression was detected in band 55 kDa using Western Blot test in patients with thyroid autoimmune diseases. Conclusions: We conclude that alteration in the expression of proapoptotic proteins in thyroid follicular cells may play a role in pathogenesis of thyroid autoimmune disorders. In addition, suppression of apoptosis in Graves&#8217; disease led to increased proliferation of thyroid follicular cells which is responsible for goiter formation. (Pol J Endocrinol 2007; 58 (4): 303-313)Wstęp: Apoptoza to programowe obumieranie komórki. Jest to mechanizm regulacyjny pozwalający na usunięcie wytworzonych w nadmiarze i niepotrzebnych w danej chwili komórek. Zaburzenia w procesie apoptozy mogą uczestniczyć w rozwoju schorzeń autoimmunologicznych tarczycy. Celem pracy była ocena ekspresji cząsteczek Fas/FasL i kaspazy 8 w tkance gruczołu tarczowego u pacjentów z chorobą Gravesa-Basedowa (GB, Graves' disease), wolem guzkowym nietoksycznym (NTNG, non-toxic nodular goiter) oraz zapaleniem tarczycy typu Hashimoto (HT, Hashimoto's thyroiditis). Materiał i metody: Do kryteriów kwalifikacji pacjentów z chorobą GB należą: wole II°, obecność oftalmopatii, przeciwciała przeciw receptorom TSH (TRAb, anti-TSH receptor antibodies) wyższe niż 5 j./l, dodatnie stężenia przeciwciał anty-TPO i anty-TG oraz utrzymujące się ponad 2-3 miesiące od początku rozpoznania stężenie hormonu tyreotropowego (TSH, thyroid stimulating hormone) niższe niż 0,45 mmj./ml. Wyizolowane tyreocyty znakowano metodą pośrednią: początkowo komórki łączono z przeciwciałami monoklonalnymi mysimi anty-TPO, następnie z przeciwciałami króliczymi IgG (Fab&#8217;)2 anty-mysimi znakowanymi izotiocytrynianem fluoresceiny (FITC, fluorescein isothiocyanate). Do tak uzyskanej zawiesiny komórkowej podawano przeciwciała monoklonalne anty-Fas i anty-FasL znakowane PE (Phycoerythrin). Odczytu dokonywano w cytometrze przepływowym (Coulter EPICS XL). Analizę ekspresji Fas/FasL uzupełniono badaniami Western Blot i immunohistochemicznym z wizualizacją DAB-em i barwieniem hematoksyliną Mayera. Oznaczenie ekspresji kaspazy 8 w komórkach pęcherzykowych tarczycy przeprowadzono za pomocą metody Western Blot. Wyniki: W analizie ekspresji molekuł apoptozy Fas oraz FasL na powierzchni komórek tarczycy wykazano jej istotnie wyższy odsetek u pacjentów z zapaleniem tarczycy typu Hashimoto (HT, Hashimoto's thyroiditis) (ok. 38%, 26%) w porównaniu z pacjentami z chorobą Gravesa-Basedowa (18%, 14%). U pacjentów z HT wykazano znacznie niższy odsetek limfocytów napływających do gruczołu tarczowego z ekspresją molekuł Fas (13%) i FasL (22%) w porównaniu z grupą osób z chorobą GB (odpowiednio: 33%, 43%). W identyfikacji białek proapoptotycznych FasL i Fas stwierdzono znamienną ich ekspresję w komórkach tarczycy u pacjentów z chorobą GB (++; ++) i HT (+++, +++) w porównaniu z ekspresją w grupie osób z NTNG (0/+; 0/+). W analizie ekspresji kaspazy 8 w badanych grupach wykazano jej obecność u pacjentów ze schorzeniami autoimmunologicznymi tarczycy w prążku p55 (kDa) za pomocą metody Western Blot.Wnioski: Podsumowując, można stwierdzić, że przewaga ekspresji markerów proapoptotycznych w komórkach pęcherzykowych tarczycy w zapaleniu Hashimoto może świadczyć o wzroście eliminacji tych komórek i w konsekwencji wytworzeniu niedoczynności tarczycy. Odmienna sytuacja ma miejsce w chorobie GB, gdzie mniejsza aktywność apoptozy i tym samym przewaga proliferacji nad eliminacją komórek tarczycy w rezultacie prowadzi do rozwoju wola

The mRNA expression of pro- and anti-inflammatory cytokines in T regulatory cells in children with type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune-mediated destruction of insulin-producing beta cells in the pancreas. T regulatory cells (Tregs) represent an active mechanism of suppressing autoreactive T cells that escape central tolerance. The aim of our study was to test the hypothesis that T regulatory cells express pro- and anti-inflammatory cytokines, elements of cytotoxicity and OX40/4-1BB molecules. The examined group consisted of 50 children with T1DM. Fifty two healthy individuals (control group) were enrolled into the study. A flow cytometric analysis of T-cell subpopulations was performed using the following markers: anti-CD3, anti-CD4, anti-CD25, anti-CD127, anti-CD134 and anti-CD137. Concurrently with the flow cytometric assessment of Tregs we separated CD4+CD25+CD127dim/- cells for further mRNA analysis. mRNA levels for transcription factor FoxP3, pro- and anti-inflammatory cytokines (interferon gamma, interleukin-2, interleukin-4, interleukin-10, transforming growth factor beta1 and tumor necrosis factor alpha), activatory molecules (OX40, 4-1BB) and elements of cytotoxicity (granzyme B, perforin 1) were determined by real-time PCR technique. We found no alterations in the frequency of CD4+CD25highCD127low cells between diabetic and control children. Treg cells expressed mRNA for pro- and anti-inflammatory cytokines. Lower OX40 and higher 4-1BB mRNA but not protein levels in Treg cells in diabetic patients compared to the healthy children were noted. Our observations confirm the presence of mRNA for pro- and anti-inflammatory cytokines in CD4+CD25+CD127dim/- cells in the peripheral blood of children with T1DM. Further studies with the goal of developing new strategies to potentiate Treg function in autoimmune diseases are warranted

Frequency and Predictive Factors of Hypoglycemia in Patients Treated With rhIGF-1: Data From the Eu-IGFD Registry

The European Increlex® Growth Forum Database (Eu-IGFD) is an ongoing surveillance registry (NCT00903110) established to collect long-term safety and effectiveness data on the use of recombinant human insulin-like growth factor-1 (rhIGF-1, mecasermin, Increlex) for the treatment of children/adolescents with severe primary insulin-like growth factor-1 deficiency (SPIGFD). Objective: This analysis of Eu-IGFD data aimed to identify the frequency and predictive factors for hypoglycemia adverse events (AEs) in children treated with rhIGF-1. Methods: Data were collected from December 2008 to May 2021. Logistic regression was performed to identify predictive risk factors for treatment-induced hypoglycemia AEs. Odds ratios (ORs) are presented with 95% CIs for each factor. Results: In total, 306 patients were enrolled in the registry; 84.6% were diagnosed with SPIGFD. Patients who experienced ≥ 1 hypoglycemia AE (n = 80) compared with those with no hypoglycemia AEs (n = 224) had a lower mean age at treatment start (8.7 years vs 9.8 years), a more frequent diagnosis of Laron syndrome (27.5% vs 10.3%), and a history of hypoglycemia (18.8% vs 4.5%). Prior history of hypoglycemia (OR 0.25; 95% CI: [0.11; 0.61]; P = .002) and Laron syndrome diagnosis (OR 0.36; 95% CI: [0.18; 0.72]; P = .004) predicted future hypoglycemia AEs. Total hypoglycemia AEs per patient per treatment year was 0.11 and total serious hypoglycemia AEs per patient per treatment year was 0.01. Conclusion: Hypoglycemia occurs more frequently in patients with prior history of hypoglycemia and/or Laron syndrome compared with patients without these risk factors, and these patients should be carefully monitored for this AE throughout treatment