Addison's Disease Revisited in Poland: Year 2008 versus Year 1990

This study aimed at comparing two groups of patients with Addison's disease: A, including 180 patients described in 1991 and B, consisting of 138 patients registered since 1991. The incidence of coexisting autoimmune disorders was evaluated and etiological factors were analyzed. Immunological and imaging studies (computed tomography in group B) were performed. Adrenal autoantibodies were examined by an indirect immunofluorescence technique in group A, and by the assay measuring autoantibodies against steroid 21-hydroxylase in group B. Adrenal autoantibodies were revealed in 37% of patients examined by the immunofluorescence method and in 63% investigated by the modern technique. Tuberculosis was found in 52 patients in the group A and in two patients in the group B; metastatic infiltrations of the adrenals in CT were detected in 16 patients. Probable autoimmune Addison's disease was diagnosed in 125/180 patients (69%) in the group A and in 116/138 patients (84%) in the group B

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

Localization of key amino acid residues in the dominant conformational epitopes on thyroid peroxidase recognized by mouse monoclonal antibodies.

Autoantibodies to thyroid peroxidase (TPO), the major target autoantigen in autoimmune thyroid diseases, recognize conformational epitopes limited to two immunodominant regions (IDRs) termed IDR-A and -B. The apparent restricted heterogeneity of TPO autoantibodies was discovered using TPO-specific mouse monoclonal antibodies (mAbs) and later confirmed by human recombinant Fabs. In earlier studies we identified key amino acids crucial for the interaction of human autoantibodies with TPO. Here we show the critical residues that participate in binding of five mAbs to the conformational epitopes on the TPO surface. Using ELISA we tested the reactivity of single and multiple TPO mutants expressed in CHO cells with a panel of mAbs specifically recognizing IDR-A (mAb 2 and 9) and IDR-B (mAb 15, 18, 64). We show that antibodies recognizing very similar regions on the TPO surface may interact with different sets of residues. We found that residues K713 and E716 contribute to the interaction between mAb 2 and TPO. The epitope for mAb 9 is critically dependent on residues R646 and E716. Moreover, we demonstrate that amino acids E604 and D630 are part of the functional epitope for mAb 15, and amino acids D624 and K627 for mAb 18. Finally, residues E604, D620, D624, K627, and D630 constitute the epitope for mAb 64. This is the first detailed study identifying the key resides for binding of mAbs 2, 9, 15, 18, and 64. Better understanding of those antibodies' specificity will be helpful in elucidating the properties of TPO as an antigen in autoimmune disorders

Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula

Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA−). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1–MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines - Fig 3

<p><b>The expression of lactoperoxidase (LPO) in breast tissues (A and B) and cell lines (C and D) derived from normal (184A1) and cancerous mammary tissues (MCF-7 and MDA-MB-231).</b> (A) Representative immunohistochemical staining of human LPO in breast cancer (upper panel) and peritumoral tissues (lower panel) (magnification: 100×). (B) Human LPO expression in breast cancer and peritumoral tissues detected by immunoblotting with the 10376-1-AP antibody. Human lung and thyroid tissue (Graves’ disease case) lysates were used as a negative and positive control, respectively. For each lane, 50 μg of crude protein extract were loaded. A β-actin-specific antibody was used as a loading control. (C) LPO expression in cell lines as shown by immunofluorescent staining. Positive immunofluorescent signal (red) was detected in MDA-MB-231 and, to a lesser extent, in MCF-7 cells. No staining was detected when pre-immune rabbit IgG was used (insets). Nuclei were counterstained with DAPI (blue). Magnification: 630×. (D) LPO protein expression in cell lines analyzed by Western blot. LPO was detected in breast cancer cells, while no band was observed in normal 184A1 cells. For each lane, 10 μg of crude protein extract were loaded. A β-actin-specific antibody was used as a loading control.</p

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines - Fig 1

<p><b>The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C).</b> (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.</p

Expression Analysis of PIN Genes in Root Tips and Nodules of Lotus japonicus

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical &beta;-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root&rsquo;s stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines - Fig 2

<p><b>Representative TPO immunostaining results obtained with a panel of monoclonal antibodies (mAbs) against human thyroid peroxidase (TPO) (A) and human serum pools (B) in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231).</b> A normal human thyroid cell line, NTHY, was used as a positive control. (A) Positive signal (red) was detected with all mAbs except the isotype control (negative control). In the cells incubated with TPOAbs obtained from autoimmune thyroid disease (AITD) patients (TPOAbs(+)), a positive signal (green) was observed but this staining was not observed when TPOAb-free serum (TPOAbs(-)) was used (negative control). Nuclei (blue) were counterstained with DAPI. Magnification: 630×. IIAb: secondary antibody; DAPI: 4′,6-diamino-2-phenylindole; NTHY: NTHY-ori 3–1 cell line.</p