BACKGROUNDGraves' ophthalmopathy/orbitopathy (GO) patients often suffer ocular surface damages and tear fluid proteins play a significant role in maintaining healthy ocular surfaces, while changes in tear protein components reflect the changes ocular surface abnormalities. In this study proteomics techniques were used to investigate tear protein compositions in GO patients.

METHODSWe carried out a case-control study by comparing tear fluid contents of GO patients with that of healthy subjects. In the first step the tears were subjected to SDS-PAGE electrophoresis and then single protein bands were analyzed by to in-gel trypsin digestion and nano-flow liquid mass spectrometry (LC-MS/MS) using a MS software.

RESULTSIn tear samples of GO subjects, the protein fractions of inflammation-related protein immunoglobulin kappa chain C region (IgKC) and serum albumin were essentially reduced, whereas a novel isoform of complement component 3 (C3), which we detected in control subjects, was completely absent in the GO patients' tears.

CONCLUSIONSReduced protein concentrations of particularly IgKC and complement C3 as well as albumin in the tears of GO patients may contribute to changes in their ocular surfaces via diminished reactive oxygen species (ROS) depletion and adaptive immune responses. The completely absent of C3 in the GO patients' tears, may imply that an important inflammatory signaling pathway is affected, which needs further investigation.

Adolescent ; Adult ; Case-Control Studies ; Chromatography, Liquid ; Eye Proteins ; analysis ; metabolism ; Female ; Graves Ophthalmopathy ; metabolism ; Humans ; Male ; Middle Aged ; Tandem Mass Spectrometry ; Young Adult

The aim of the present study was to identify a new candidate anti-inflammatory compound for use in the active stage of thyroid-associated ophthalmopathy (TAO). Benzylideneacetophenone compound JC3 [(2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one] was synthesized based on a structural modification of yakuchinone B, a constituent of the seeds of Alpinia oxyphylla, which belongs to the ginger family (Zingiberaceae), has been widely used in folk medicine as an anti-inflammatory phytochemical. Orbital fibroblasts were primarily cultured from patients with TAO, and the potential of JC3 to suppress the interferon (IFN)-gamma-induced protein (IP)-10/CXCL10 production in these cells was determined. IFN-gamma strongly increased the level of IP-10/CXCL10 in orbital fibroblasts from patients with TAO. JC3 exerted a significant inhibitory effect on the IFN-gamma-induced increase in IP-10/CXCL10 in a dose-dependent manner; its potency was greater than that of an identical concentration of yakuchinone B with no toxicity to cells at the concentration range used. Moreover, the constructed dimer and trimer polystructures of JC3, showed greater potency than JC3 in suppressing the IFN-gamma-induced production of IP-10/CXCL10. JC3 significantly attenuated the IP-10/CXCL10 mRNA expression induced by IFN-gamma, and a gel-shift assay showed that JC3 suppressed IFN-gamma-induced DNA binding of signal transducer and activator of transcription-1 (STAT-1) in TAO orbital fibroblasts. Our results provide initial evidence that the JC3 compound reduces the levels of IP-10/CXCL10 protein and mRNA induced by IFN-gamma in orbital fibroblasts of TAO patients. Therefore, JC3 might be considered as a future candidate for therapeutic application in TAO that exerts its effects by modulating the pathogenic mechanisms in orbital fibroblasts.
Cells, Cultured ; Chalcone/chemical synthesis/*pharmacology ; Chemokine CXCL10/genetics/*metabolism ; Diarylheptanoids/chemistry/pharmacology ; Fibroblasts/*drug effects/metabolism ; Graves Ophthalmopathy/*metabolism ; Humans ; Interferon-gamma/*metabolism ; Orbit/cytology ; RNA, Messenger/genetics/metabolism ; STAT1 Transcription Factor/genetics/*metabolism

OBJECTIVE: To study the cell surface antigen expression of orbital preadipocytes between patients with thyroid-associated ophthalmopathy (TAO) and healthy adults. METHODS: Tissue culture Methods were used in the cell culture of orbital preadipocytes in patients with TAO and healthy adults. Flow cytometry was used to analyze the expression of CD29, CD44, CD49d, and HLA-DR in these 2 cells. RESULTS: Positive expressions were examined in orbital preadipocytes of patients with TAO and healthy adults. The orbital preadipocytes of patients with TAO had a higher expression of HLA-DR than that of healthy adults. CONCLUSION The orbital preadipocytes of patients with TAO and healthy adults cultured by tissue cultured Methods both belong to mesenchymal stem cells, and the former may participate in the orbital inflammation.
Adipocytes ; immunology ; Adult ; Antigens, Surface ; immunology ; metabolism ; Cells, Cultured ; Female ; Graves Ophthalmopathy ; immunology ; metabolism ; HLA-DR Antigens ; immunology ; Humans ; Hyaluronan Receptors ; immunology ; Integrin beta1 ; immunology ; Male ; Middle Aged ; Orbit ; immunology ; metabolism

OBJECTIVE: To investigate the effect of mevastatin (Mev) on the expression of peroxisome-proliferator-activated receptor-gamma (PPAR-gamma) mRNA and differentiation of Thyroid-associated ophthalmopathy (TAO) derived orbital preadipocytes in vitro. METHODS: Orbital adipose tissues were obtained from TAO patients undergoing orbital decompression surgery. The orbital preadipocytes cultured from the orbital adipose tissues were divided into Group A (a control group) and Group B (an intervention group). Group B was subdivided into Group B1-B5, all groups were stimulated to differentiate into mature adipocytes with cocktail differentiation medium.The entire course of differentiation was 10 d. The differentiation of orbital preadipocytes in Group A was induced with routine inducer,while at in Group B1,B2, and B3 was interfered with 5 micromol/L (B1), 10 micromol/L(B2),20 micromol/L (B3) mevastatin respectively during the whole process of differentiation. The differentiation of orbital preadipocytes in Group B4 and B5 was interfered with 10 micromol/L mevastatin day 4 (B4) or day 8 (B5) of the differentiation process until the entire course was over. Intracellular fat accumulation in differentiated adipocytes was determined by oil red O staining. The value of optical absorption was measured at 492 nm with enzyme-linked immunosorbent assay. The expression of PPAR-gamma mRNA was detected by reverse transcription polymerase chain reaction. RESULTS: The light absorption value (A) and PPAR-gamma mRNA expression of differentiated cells in Group A,B1,B2,and B3 decreased successively,and there was significant difference in any of the 2 groups among Group A, B1 and B2, and B3 (P<0.05). The value A and PPAR-gamma mRNA expression of differentiated cells in Group A, B4, and B2 decreased successively, and the difference in any of the 2 groups among these 3 groups was significant. However, there were no significant difference between Group A and B5. CONCLUSION Mevastatin inhibits the differentiation of TAO derived orbital preadipocytes by blocking PPAR-gamma mRNA expression. The degree of inhibition is not only concentration-dependent but also associated with the stage of differentiation. The earlier the differentiation, the stronger the inhibition.
Adipocytes ; pathology ; Adipose Tissue ; pathology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Depression, Chemical ; Graves Ophthalmopathy ; pathology ; Humans ; Lovastatin ; analogs & derivatives ; pharmacology ; Orbit ; PPAR gamma ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism