cAMP response to TSH and ¹²⁵I-binding activities of HEK 293 cells expressing wild-type TSHR and/or mutated TSHR.

<p>(a) Cyclic AMP levels in HEK 293 cells expressing TSHR(W) or TSHR(M) following TSH stimulation. TSH was added to culture medium of HEK 293 cells transfected with 0.5 µg of pcDNA3-TSHR(W) (○-○) or the same amount of pcDNA-TSHR(M) (•-•). After 30 min, cellular cAMP levels were assayed. Data are means ± S.E. of triplicate assays. (b) Cyclic AMP levels in HEK 293 cells expressing TSHR(W) and TSHR(M) following TSH stimulation. TSH was added to culture medium of HEK 293 cells transfected with 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA (□-□), 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA-TSHR(M) (▪-▪) or 0.5 µg of pcDNA-TSHR(M)+the same amount of pcDNA (•-•). After 30 min, cellular cAMP levels were assayed. Data are means ± S.E. of triplicate assays. (c) ¹²⁵I-binding activities of HEK 293 cells expressing wild-type TSHR and/or mutated TSHR. HEK 293 cells were transfected with 0.5 µg of pcDNA3-TSHR(W)+same amount of pcDNA (○-○), 0.5 µg of pcDNA3-TSHR(W)+the same amount of pcDNA-TSHR(M) (□-□) or 0.5 µg of pcDNA-TSHR(M)+the same amount of pcDNA (•-•). After 72 h of culture, TSH binding activities were assayed using ¹²⁵I-bovine TSH. Data are means ± S.E. of triplicate assays.</p

Thyroid glands from C.RF-Tshr^wild/wild and Tshr^wild/hyt mice.

<p>Macroscopic views of thyroid glands from 12-week-old C.RF-Tshr^wild/wild (a) and Tshr^wild/hyt (b) mice observed under a stereomicroscope (Olympus SZX7). Black arrows indicate the upper poles, and red arrows indicate the lower poles of the left lobe of the thyroid gland. Histology on high magnification in C.RF-Tshr^wild/wild (d) and Tshr^wild/hyt (e) mice. Tissues were fixed with 4% formaldehyde and embedded in paraffin. Sections (5 µm) were stained with hematoxylin-eosin. Bars indicate 200 µm.</p

The location and putative function of proteins unique to FT1DM as identified by LMD-LC-MS in this study (red characters) and proteins previously identified by immunohistochemistry (green characters) in FT1DM-affected pancreas tissue [10], [11].

<p>The location and putative function of proteins unique to FT1DM as identified by LMD-LC-MS in this study (red characters) and proteins previously identified by immunohistochemistry (green characters) in FT1DM-affected pancreas tissue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107664#pone.0107664-Tanaka2" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107664#pone.0107664-Aida1" target="_blank">[11]</a>.</p

Antibody-Validated Proteins in Inflamed Islets of Fulminant Type 1 Diabetes Profiled by Laser-Capture Microdissection Followed by Mass Spectrometry

<div><p>Background</p><p>There are no reports of proteomic analyses of inflamed islets in type 1 diabetes.</p><p>Procedures</p><p>Proteins expressed in the islets of enterovirus-associated fulminant type 1 diabetes (FT1DM) with extensive insulitis were identified by laser-capture microdissection mass spectrometry using formalin-fixed paraffin-embedded pancreatic tissues.</p><p>Results</p><p>Thirty-eight proteins were identified solely in FT1DM islets, most of which have not been previously linked to type 1 diabetes. Five protein-protein interacting clusters were identified, and the cellular localization of selected proteins was validated immunohistochemically. Migratory activity-related proteins, including plastin-2 (LCP1), moesin (MSN), lamin-B1 (LMNB1), Ras GTPase-activating-like protein (IQGAP1) and others, were identified in CD8⁺ T cells and CD68⁺ macrophages infiltrated to inflamed FT1DM islets. Proteins involved in successive signaling in innate/adaptive immunity were identified, including SAM domain and HD domain-containing protein 1 (SAMHD1), Ras GTPase-activating-like protein (IQGAP1), proteasome activator complex subunit 1 (PSME1), HLA class I histocompatibility antigen (HLA-C), and signal transducer and activator of transcription 1-alpha/beta (STAT1). Angiogenic (thymidine phosphorylase (TYMP)) and anti-angiogenic (tryptophan-tRNA ligase (WARS)) factors were identified in migrating CD8⁺ T cells and CD68⁺ macrophages. Proteins related to virus replication and cell proliferation, including probable ATP-dependent RNA helicase DEAD box helicase 5 (DDX5) and heterogeneous nuclear ribonucleoprotein H (HNRNPH1), were identified. The anti-apoptotic protein T-complex protein 1 subunit epsilon (CCT5), the anti-oxidative enzyme 6-phosphogluconate dehydrogenase (PDG), and the anti-viral and anti-apoptotic proteins serpin B6 (SERPINB6) and heat shock 70 kDa protein1-like (HSPA1L), were identified in FT1DM-affected islet cells.</p><p>Conclusion</p><p>The identified FT1DM-characterizing proteins include those involved in aggressive beta cell destruction through massive immune cell migration and proteins involved in angiogenesis and islet vasculature bleeding, cell repair, and anti-inflammatory processes. Several target proteins for future type 1 diabetes interventions were identified.</p></div

Protein-protein interaction networks involving 38 islet proteins unique to FT1DM-affected tissue that were identified by LMD-LC-MS in this study (yellow labeled) and proteins previously identified immunohistochemically in inflamed FT1DM-affected islets [10], [11] and classified using STRING (http://www.string-db.org).

<p>The five clusters are indicated by numerals. The width of the edges depends on the confidence score for each protein association as determined by STRING analysis.</p

Immunohistochemical validation of the presence of LMD-LC-MS-identified proteins in FT1DM-affected islets.

<p>(A)–(D), Triple immunostaining for plastin-2 (LCP1) expression in FT1DM-affected pancreas (A)–(C) and non-diabetic control (D) tissues. (A), Triple immunostaining for plastin-2 (LCP1) in FT1DM-affected pancreas. LCP1 (red) was over-expressed in mononuclear cells (MNCs) that aggressively infiltrated to or around the islets (green: insulin, blue: glucagon). (B), Merged image of triple immunostaining for LCP1 (green), CD68⁺ macrophages (red), and glucagon (blue). Many MNCs are positive for both CD68 and LCP1 and appear yellow (arrowheads). (C), Merged image of triple immunostaining for LCP1 (green), CD8 (red), and glucagon (blue). Many MNCs are positive for both CD8 and LCP1 and appear yellow (arrowheads). (D), Merged image of triple immunostaining for LCP1 (red), insulin (green), and glucagon (blue) in non-diabetic pancreas tissue. Few cells are positive for LCP1. (E)–(F), Expression of Ras GTPase-activating-like protein (IQGAP1) in the islets and MNCs in FT1DM-affected (E) and non-diabetic (F) islets. IQGAP1 (brown) was highly expressed in infiltrating MNCs and some FT1DM islet cells (insulin: red, glucagon: green). (G)–(H), DEAD box helicase5 (DDX5) expression in FT1DM-affected (G) and control (H) pancreas. DDX5 (brown) was hyper-expressed in the nucleus and cytoplasm of all subsets of islet cells, including beta cells (red) and alpha cells (green). Weak expression of DDX5 was observed in the nucleus of non-diabetic pancreas cells (H). (I)–(L), Expression of thymidine phosphorylase (TYMP) in FT1DM-affected (I) and control pancreas (J) tissues. TYMP was over-expressed in MNCs infiltrated to the islets in FT1DM tissue (I). No expression of TYMP was observed in non-diabetic control pancreas tissue (J). Triple immunostaining of FT1DM pancreatic tissue for TYMP (green), CD68⁺ (red), and insulin (blue). Merged image (K) shows that TYMP is expressed on CD68⁺ macrophages and appears yellow (arrowheads). Triple immunostaining of FT1DM-affected pancreatic tissue for TYMP (green), CD8⁺ (red), and insulin (blue). Merged image (L) shows that TYMP is localized on CD8⁺ T cells (arrowheads). (M)–(N), Expression of SAM domain and HD domain-containing protein 1 (SAMHD1) in FT1DM-affected pancreas (M) and control pancreas (N) tissues. Hyper-expression of SAMHD1 (brown) in the nucleus and cytoplasm of islet beta cells (red), alpha cells (green), and infiltrating MNCs is shown. No expression of SAMHD1 was observed in non-diabetic control pancreas tissue (N). (O)–(P), 6-Phosphogluconate dehydrogenase, decarboxylating (PGD) expression in FT1DM-affected pancreas tissue (O). PDG (brown) was over-expressed in the cytoplasm of islet-cells, and non-islet cells (arrowheads). PDG was only faintly expressed in the cytoplasm of non-diabetic islet cells (P). (Q)–(R), Signal transducer and activator of transcription-1 alpha/beta (STAT1) expression in FT1DM-affected pancreas (Q) and control pancreas (R) tissues. STAT1 was over-expressed in the cytoplasm and nucleus of islet beta cells (red), alpha cells (green), and MNCs (arrowheads). No staining for STAT1 was observed in control islet tissue (R). (S)–(T), Proteasome activator complex subunit 1 (PSME1, PA28a) expression in FT1DM-affected pancreas (S) and non-diabetic pancreas (T) tissues. PSME1 (brown) was over-expressed in the nucleus and cytoplasm of beta cells (red), alpha cells (green), and infiltrating MNCs (arrowheads) (S). PSME1 was not expressed in non-diabetic control pancreas tissue (T). (U)–(V), Tryptophanyl-tRNA synthetase (WARS) expression in the islets of FT1DM-affected (U) and non-diabetic control (V) pancreas tissues. Merged image shows expression of WARS (brown) in beta cells (red) and MNCs (arrowheads) in FT1DM-affected tissue (U). No staining of WARS was observed in non-diabetic control pancreas tissue (V). (W)–(X), Heat shock protein 70 kDa protein 1-like (HSPA1L) expression in the islets of FT1DM-affected (W) and non-diabetic pancreas (X) tissues. Strong expression of HSPA1L (brown) was observed in beta cells (red), alpha cells (green), and other subsets of endocrine cells (W). Weak expression of HSPA1L was observed in non-diabetic control islets (X). Positive staining for each identified protein was significantly more frequent (<i>P</i> = 0.048) in FT1DM-affected islets than control islets.</p

Proteins identified only in islets affected by fulminant type 1 diabetes.

<p>Proteins identified only in islets affected by fulminant type 1 diabetes.</p

Additional file 1 of When face-tracking meets social networks: a story of politics in news videos

Appendix. (PDF 5520 kb

Baseline characteristics of the study patients at initiation of anti-mycobacterial treatment.

<p>Baseline characteristics of the study patients at initiation of anti-mycobacterial treatment.</p

Patient enrollment.

<p>*Both <i>M</i>. <i>avium</i> and <i>M</i>. <i>intracellulare</i> were detected in blood culture of one patient.</p