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

Somatostatin-like immunoreactivity in the amygdala of the pig.

The distribution and morphology of neurons containing somatostatin (SOM) was investigated in the amygdala (CA) of the pig. The SOM-immunoreactive (SOM-IR) cell bodies and fibres were present in all subdivisions of the porcine CA, however, their number and density varied depending on the nucleus studied. The highest density of SOM-positive somata was observed in the layer III of the cortical nuclei, in the anterior (magnocellular) part of the basomedial nucleus and in the caudal (large-celled) part of the lateral nucleus. Moderate to high numbers of SOM-IR cells were also observed in the medial and basolateral nuclei. Many labeled neurons were also consistently observed in the lateral part of the central nucleus. In the remaining CA regions, the density of SOM-positive cell bodies varied from moderate to low. In any CA region studied SOM-IR neurons formed heterogeneous population consisting of small, rounded or slightly elongated cell bodies, with a few poorly branched smooth dendrites. In general, morphological features of these cells clearly resembled the non-pyramidal Golgi type II interneurons. The routine double-labeling studies with antisera directed against SOM and neuropeptide Y (NPY) demonstrated that a large number of SOM-IR cell bodies and fibers in all studied CA areas contained simultaneously NPY. In contrast, co-localization of SOM and cholecystokinin (CCK) or SOM and vasoactive intestinal polypeptide (VIP) was never seen in cell bodies and fibres in any of nuclei studied. In conclusion, SOM-IR neurons of the porcine amygdala form large and heterogeneous subpopulation of, most probably, interneurons that often contain additionally NPY. On the other hand, CCK- and/or VIP-IR neurons belonged to another, discrete subpopulations of porcine CA neurons

Original articleAnalysis of apoptotic markers Fas/FasL (CD95/CD95L) expression on the lymphocytes in patients with acute coronary syndrome

Wstęp: Ostry zespół wieńcowy wywołany jest pęknięciem lub owrzodzeniem blaszki miażdżycowej, co inicjuje kaskadę procesów z degradacją macierzy międzykomórkowej włącznie i wydzieleniem cytokin powodujących śmierć komórek śródbłonka. Mechanizm ten odgrywa główną rolę w apoptozie. Apoptoza to jedna z postaci programowanej śmierci komórek, nazywana również śmiercią fizjologiczną. Jest mechanizmem regulacyjnym pozwalającym na usunięcie wytworzonych w nadmiarze i niepotrzebnych w danej chwili komórek. Proces apoptozy jest regulowany przez białka apoptotyczne, których ekspresja ujawnia się na limfocytach wyizolowanych z obszaru mięśnia sercowego. Główną rolę odgrywa aktywacja kompleksu Fas/FasL (przezbłonowe białka nadrodziny TNFR), inicjującego wewnątrzkomórkową kaskadę przemian. Cytoplazmatyczna cześć aktywowanego receptora Fas wiąże białko adaptacyjne FADD i powstały w ten sposób kompleks Fas-FADD aktywuje kaspazę-8, która inicjuje kilka proteaz cysteinowych kaspazy-3 uważanych za efektorowe w apoptozie. Ten cykl przemian odgrywa kluczową rolę w eliminacji obwodowych limfocytów T, komórek zapalnych i nowotworowych przy udziale komórek cytotoksycznych, CD8 oraz NK. Z kolei molekuły blokujące sygnały apoptozy to FAP-1, hamujące przekazywanie sygnałów z Fas na domeny regulatorowe, jak również FLIP, które zapobiegają tworzeniu się kompleksu FAS-FADD-FLICE tak zwanych białek adaptacyjnych, które są dalszym etapem przebiegu śmierci fizjologicznej komórek. W końcowej jej fazie dochodzi do cyklu przemian kaspaz, a regulatorami ograniczenia programowej śmierci komórki są białka inhibitorowe hamujące proces apoptozy, takie jak IAP oraz cząsteczka Bcl-2. Ta ostatnia należy do białek regulatorowych apoptozy. W jej składzie są zarówno molekuły antyapoptotyczne (Bcl-2, BAG), jak i cząsteczka proapoptyczna typu Bax. Przewaga ekspresji molekuł jednego typu nad drugim może w konsekwencji decydować o nadmiarze proliferacji nad rozpadem komórek i odwrotnie. Dane z literatury podkreślają udział zaburzeń w licznych schorzeniach. Proces ten odgrywa zasadniczą rolę w takich ostrych chorobach, jak posocznica, SIRS, ostra niewydolność wątroby, zawał mięśnia sercowego (MI), w chorobach przewlekłych, jak choroba Parkinsona, Huntingtona, reumatyczne zapalenie stawów, a także w zaburzeniach autoimmunologicznych, takich jak chociażby choroba Gravesa-Basedowa czy też zapalenie tarczycy typu Hashimoto. Cel: Ocena ekspresji markerów apoptotycznych Fas/FasL (CD95/CD95L) na limfocytach krwi obwodowej. Metodyka: Badano 3 grupy chorych &#8211; z ostrym MI (n=18, średni wiek 62&#177;8 lat), niestabilną dławicą piersiową (n=31, średni wiek 62&#177;10 lat) i grupę kontrolną (n=20, średni wiek 60&#177;9 lat) &#8211; bez towarzyszących czynników ryzyka miażdżycy i bez parametrów stanu zapalnego. Badania cząsteczek proapoptotycznych Fas/FasL przeprowadzono przy użyciu cytometrii przepływowej. Ocenę parametrów zapalnych oraz tradycyjnych czynników ryzyka wykonano, wykorzystując metodę immunoenzymatyczną ELISA. Wyniki: Przeprowadzone badania wykazały zwiększoną ekspresję cząsteczek Fas i FasL na powierzchni limfocytów krwi obwodowej u chorych z ostrym MI (pBackground: Acute coronary syndromes are caused by the rupture or erosion of an atherosclerotic plaque which by secreting a variety of proteases is capable of degrading pericellular matrix components induces death of endothelial cells. This mechanism plays the main role in apoptosis. Aim: To estimate expression of apoptotic Fas/FasL (CD95/CD95L) on lymphocytes in the peripheral blood. Methods: We examined patients with acute myocardial infarction (n=18, mean age 62&#177;8 years), in unstable angina pectoris (n=31, mean age 62&#177;10 years) and in a control group (n=20, mean age 62&#177;9 years) without coronary risk factors and inflammatory condition. All investigations of Fas/FasL were performed by flow cytometry. Inflammatory parameters and standard risk factors were investigated by standard methods (ELISA). Results: The analysis revealed a higher expression of Fas and FasL molecules on the lymphocytes from patients with acute myocardial infarction (

Cytokiny w chorobie niedokrwiennej serca i zawale mięśnia sercowego

Background: Cytokines are responsible for the modulation of immunological and inflammatory processes as well as proliferative responses and apoptosis. It has been recently suggested that such cytokines as interleukin 6 (IL-6), soluble interleukin 6 receptor (sIL-6R) and anti-inflammatory cytokines such as interleukin 10 (IL-10) may play a significant role in the pathogenesis of acute coronary syndromes.Aim: To assess serum concentration of IL-6, sIL-6R and IL-10 in patients with ischaemic heart disease or acute myocardial infarction (AMI).Methods: The study group consisted of 74 patients (25 females, 49 males, aged 40-69 years) divided into three groups; group I - 18 patients with AMI (up to 12 hours from the onset of symptoms), group II - 31 patients with unstable angina and group III - 25 patients with stable angina. The control group consisted of 20 healthy subjects.Results: The IL-6 and sIL-6R serum levels were significantly higher in patients from groups I and II compared with patients from group III and controls, whereas the IL-10 serum concentration was similar in all studied groups. In patients with acute coronary syndromes serum concentrations of examined cytokines were positively correlated with acute inflammatory phase parameters and classical risk factors such as body mass index, blood pressure and lipid levels.Conclusions: IL-6 and sIL-6R are markers of acute coronary syndromes and may be used for the identification of high-risk patients with unstable angina or AMI

Analysis of chosen polymorphisms rs2476601 a/G – PTPN22, rs1990760 C/T – IFIH1, rs179247 a/G – TSHR in pathogenesis of autoimmune thyroid diseases in children

Background: Autoimmune thyroid diseases are multifactorial diseases with a genetic susceptibility and environmental factors. A potential role of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, the interferon-induced helicase domain 1 (IFIH1) gene, the thyroid-stimulating hormone receptor (TSHR) gene polymorphisms on autoimmune thyroid diseases (AITDs) in adults has been established unequivocally, but there is still lack of research articles including group of children. Objective and hypotheses: To estimate the association of polymorphisms of PTPN22, IFIH1 and TSH-R genes with the pre-disposition to Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) in children. Methods: The study was performed in 142 patients with GD, 57 with HT and 160 healthy volunteers. The three single-nucleotide polymorphisms (SNPs): rs2476601 – PTPN22, rs1990760 – IFIH1 and rs179247 – TSHR were genotyped by TaqMan SNP genotyping assay using the real-time PCR. Results: Rs2476601 A alleles were more frequent in patients with GD in comparison to healthy subjects (p = .009 with odds ratio [OR] = 2.13). Rs2476601 A alleles were more frequent in patients with HT in comparison to healthy subjects (p = .008, OR = 2.48). Rs1990760 T alleles were more frequent in male patients with GD in comparison to healthy males (p = .003, OR = 3.00). In case of HT patients, rs1990760 T alleles were also more frequent in males compared to healthy subjects (p = .086, OR =2.47). Rs179247 A alleles were more frequent in patients with GD in comparison to healthy subjects (p = 0.039, OR = 1.51). Conclusions: Rs2476601 A/G, Rs1990760 C/T and Rs179247 A/G polymorphisms could contribute to the development of AITDs in children. The main risk factor for rs2476601 and rs179247 is allele A. In case of rs1990760, the main risk factor is allele T

Somatostatin-like immunoreactivity in the amygdala of the pig.

The distribution and morphology of neurons containing somatostatin (SOM) was investigated in the amygdala (CA) of the pig. The SOM-immunoreactive (SOM-IR) cell bodies and fibres were present in all subdivisions of the porcine CA, however, their number and density varied depending on the nucleus studied. The highest density of SOM-positive somata was observed in the layer III of the cortical nuclei, in the anterior (magnocellular) part of the basomedial nucleus and in the caudal (large-celled) part of the lateral nucleus. Moderate to high numbers of SOM-IR cells were also observed in the medial and basolateral nuclei. Many labeled neurons were also consistently observed in the lateral part of the central nucleus. In the remaining CA regions, the density of SOM-positive cell bodies varied from moderate to low. In any CA region studied SOM-IR neurons formed heterogeneous population consisting of small, rounded or slightly elongated cell bodies, with a few poorly branched smooth dendrites. In general, morphological features of these cells clearly resembled the non-pyramidal Golgi type II interneurons. The routine double-labeling studies with antisera directed against SOM and neuropeptide Y (NPY) demonstrated that a large number of SOM-IR cell bodies and fibers in all studied CA areas contained simultaneously NPY. In contrast, co-localization of SOM and cholecystokinin (CCK) or SOM and vasoactive intestinal polypeptide (VIP) was never seen in cell bodies and fibres in any of nuclei studied. In conclusion, SOM-IR neurons of the porcine amygdala form large and heterogeneous subpopulation of, most probably, interneurons that often contain additionally NPY. On the other hand, CCK- and/or VIP-IR neurons belonged to another, discrete subpopulations of porcine CA neurons

Functional TSH receptor antibodies in children with autoimmune thyroid diseases

Introduction: The diagnostic value of the level of TSH receptor antibodies (TSHR-Ab) in the population of children with autoimmune thyroid diseases (AITDs) is still unknown. The aim of this cross-sectional study was to investigate the prevalence of TSHR-Ab in a paediatric cohort with AITD and healthy controls. Materials and methods: A total of 240 serum samples were obtained from 205 patients with AITD, type 1 diabetes (T1D), juvenile arthritis (JA), and healthy controls (C). TSHR stimulating (TSI) and -blocking (TBI) immunoglobulins were measured in cell-based bioassays using CHO cells expressing a chimeric TSHR and a c-AMP response-element-dependent luciferase. TSI was reported as percentage of specimen-to-reference ratio (cutoff 140SRR%). Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (40% inhibition). Results: C as well as children with JA and T1D were both TSI and TBI negative. In contrast, children with Graves’ disease (GD) were positive for TSI in 47/53 samples (88.7%) while those with thyroidal and orbital GD showed TSI positivity in 95.8% (23/24 samples). Serum TSI levels were SRR% 320 ± 157 and 417 ± 135 in GD and GD + orbitopathy, respectively (p = .02). Children with Hashimoto’s thyroiditis (HT) were TSI positive in 4/83 (4.8%) samples, including two with orbital involvement. TSI levels were increased in HT children with vs. those without eye disease (SRR% 177 vs. 51, p < .01). In comparison, TBI were negative in all tested samples of children with GD but positive in one HT sample. Conclusions: In conclusion, TSI is prevalent in children with GD while the highest serum TSI levels were noted in children with AITD and orbitopathy