Percentage of LFA-1+ and ICAM-1+ peripheral blood mononuclear cells in children and adolescents with type 1 diabetes does not distinguish patients with vascular complications.

There are only few studies evaluating lymphocytes activation in the diabetic vascular complications. ICAM-1/LFA-1 adhesion molecules not only participate in the lymphocyte T proliferation but also mediate leukocyte migration to the site of inflammation. We assess a relationship between the percentage of ICAM-1 and LFA-1 expressing PBMCs and the evolution of vascular complications in T1D in children and adolescents. The study was carried out on 60 children and adolescents with T1D (aged 9-20): (a) T1D lasting 5 years (n=20), without complications c) T1D lasting >5 years complicated with microalbuminuria, arterial hypertension, diabetic retinopathy (20 n). 20 healthy volunteers, age and sex matched constituted the control group. The expression of adhesion molecules was evaluated by using three-color flow cytometry. In children and adolescents with T1

Increased percentage of L-selectin+ and ICAM-1+ peripheral blood CD4+/CD8+ T cells in active Graves' ophthalmopathy.

The purpose of the study was to evaluate the percentage of CD4+/CD8+ peripheral T cells expressing CD62L+ and CD54+ in patients with Graves' disease and to assess if these estimations could be helpful as markers of active ophthalmopathy. The study was carried out in 25 patients with Graves' disease (GD) divided into 3 groups: 1/ 8 patients with active Graves' ophthalmopathy (GO) (CAS 3-6, GO complaints pound 1 year), 2/ 9 patients with hyperthyroid GD without symptoms of ophthalmopathy (GDtox) and 3/ 8 patients with euthyroid GD with no GO symptoms (GDeu). The control group consisted of 15 healthy volunteers age and sex matched to groups 1-3. The expression of lymphocyte adhesion molecules was evaluated by using three-color flow cytometry. In GO group the percentage of CD8+CD54+, CD8+CD62L+, CD4+CD54+ and CD4+CD62L+ T cells was significantly higher as compared to controls (

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

CD40L and IL-4 stimulation of acute lymphoblastic leukemia cells results in upregulation of mRNA level of FLICE--an important component of apoptosis.

The use of cancer vaccines based on dendritic cells (DC) presenting tumor antigens can be a promising tool in the treatment of leukemia. The functional characteristics of leukemia derived DC is still to be elucidated. CD40 promotes survival, proliferation and differentiation of normal B cells. CD40 triggering was used to enhance the poor antigen-presenting capacity of leukemic B-cells. Since it is still unclear whether CD40 ligation drives neoplastic B-cells to apoptosis or not, we assessed the mRNA expression of FLICE, FAS, FADD and TRADD - important components of apoptosis machinery, using real-time PCR in acute lymphoblastic leukemia cells before and after CD40 and IL-4 stimulation. ALL cells stimulated with CD40L/IL-4 expressed dendritic cell phenotype at mRNA and protein levels (upregulation of main costimulatory and adhesion molecules noted in real-time RT PCR and flow cytometry); they also expressed higher amounts of mRNA for FLICE, TRADD and FADD after CD40L/IL-4 stimulation. However differences statistically significant comparing cells cultured with CD40L/IL-4 and medium alone regarded only FLICE. Concluding, we showed upregulation of important elements of apoptosis at mRNA level in ALL cells after CD40 ligation

Increased percentage of L-selectin+ and ICAM-1+ peripheral blood CD4+/CD8+ T cells in active Graves' ophthalmopathy.

The purpose of the study was to evaluate the percentage of CD4+/CD8+ peripheral T cells expressing CD62L+ and CD54+ in patients with Graves' disease and to assess if these estimations could be helpful as markers of active ophthalmopathy. The study was carried out in 25 patients with Graves' disease (GD) divided into 3 groups: 1/ 8 patients with active Graves' ophthalmopathy (GO) (CAS 3-6, GO complaints pound 1 year), 2/ 9 patients with hyperthyroid GD without symptoms of ophthalmopathy (GDtox) and 3/ 8 patients with euthyroid GD with no GO symptoms (GDeu). The control group consisted of 15 healthy volunteers age and sex matched to groups 1-3. The expression of lymphocyte adhesion molecules was evaluated by using three-color flow cytometry. In GO group the percentage of CD8+CD54+, CD8+CD62L+, CD4+CD54+ and CD4+CD62L+ T cells was significantly higher as compared to controls (p&lt;0.001, p&lt;0.05, p&lt;0.01, p&lt;0.001 respectively). The percentage of CD8+CD54+ T lymphocytes was also elevated in GO group in comparison to hyperthyroid GD patients (p&lt; 0.05). CD4+CD62L+ and CD8+CD54+ percentages were also increased in GDtox and GDeu as compared to controls. We found a positive correlation between the TSHRab concentration and the percentage of CD8+CD62L+ T cells in all studied groups (r= 0.39, p&lt;0.05) and between the TSHRab level and CAS (r= 0.77, p&lt;0.05). The increased percentage of CD8+CD54+ and CD8+CD62L+ T cells in patients with Graves' ophthalmopathy may be used as a marker of immune inflammation activity

CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;+&lt;/sup&gt; and CD4&lt;sup&gt;+&lt;/sup&gt;CD25high regulatory T cells in disseminated and localized forms of allergic contact dermatitis: relation to specific cytokines

The aim of this study was to evaluate regulatory T lymphocytes (Tregs) in the course of allergic contact dermatitis (ACD) and to elucidate the role of IL-10 and TGF-b in Tregs activity. Peripheral blood CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;+&lt;/sup&gt; and CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;high&lt;/sup&gt; cells were determined by flow cytometry in patients with acute disseminated ACD (&amp;#8216;ad&amp;#8217;, n = 36), acute localized ACD (&amp;#8216;al&amp;#8217;, n = 26), and disseminated ACD during remission (&amp;#8216;rd&amp;#8217;, n = 27) as well as in controls (n = 22). Serum levels of cytokines were measured using ELISA. The mean percentage of CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;+&lt;/sup&gt; and CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;high&lt;/sup&gt; cells in patients with ad ACD was significantly higher than in controls (p &lt; 0.01) and the remaining patients (p &lt; 0.05). Both cell populations were significantly elevated in persons with widespread skin lesions (p &lt; 0.05). In ad patients the CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;+&lt;/sup&gt; increased during three weeks of disease, although the significant increase of CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;high&lt;/sup&gt; was noted only in the third week. Patients with ad ACD showed a significantly decreased serum level of TGF-&lt;i&gt;b&lt;/i&gt;1 as compared with controls and the remaining ACD patients. IL-10 level did not differ between all groups. The elevated population of CD4&lt;sup&gt;+&lt;/sup&gt;CD25&lt;sup&gt;high&lt;/sup&gt; cells in ad ACD patients, and its dependence on the extension of skin lesions, suggest a role of Tregs in regulating the course of ACD. The growing Tregs percentages may indicate their peripheral generation during ACD. The development of lesions despite an increased population of Tregs suggests their functional defect. The role of TGF-&lt;i&gt;b&lt;/i&gt;1 in the suppressive activity of Tregs cannot be excluded. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 2, pp. 255&amp;#8211;262

CD4+CD25+ and CD4+CD25high regulatory T cells in disseminated and localized forms of allergic contact dermatitis: relation to specific cytokines

The aim of this study was to evaluate regulatory T lymphocytes (Tregs) in the course of allergiccontact dermatitis (ACD) and to elucidate the role of IL-10 and TGF-b in Tregs activity. Peripheral bloodCD4+CD25+ and CD4+CD25high cells were determined by flow cytometry in patients with acute disseminatedACD (‘ad’, n = 36), acute localized ACD (‘al’, n = 26), and disseminated ACD during remission (‘rd’, n = 27)as well as in controls (n = 22). Serum levels of cytokines were measured using ELISA. The mean percentage ofCD4+CD25+ and CD4+CD25high cells in patients with ad ACD was significantly higher than in controls(p < 0.01) and the remaining patients (p < 0.05). Both cell populations were significantly elevated in personswith widespread skin lesions (p < 0.05). In ad patients the CD4+CD25+ increased during three weeks ofdisease, although the significant increase of CD4+CD25high was noted only in the third week. Patients with adACD showed a significantly decreased serum level of TGF-b1 as compared with controls and the remainingACD patients. IL-10 level did not differ between all groups. The elevated population of CD4+CD25high cells inad ACD patients, and its dependence on the extension of skin lesions, suggest a role of Tregs in regulating thecourse of ACD. The growing Tregs percentages may indicate their peripheral generation during ACD. Thedevelopment of lesions despite an increased population of Tregs suggests their functional defect. The role ofTGF-b1 in the suppressive activity of Tregs cannot be excluded