Xiangkun Huang

T-bet expression is upregulated in active Behçet’s disease

Aim: To investigate T-bet mRNA and protein expression on peripheral blood mononuclear cells (PBMC) in patients with Behçet’s disease with active uveitis. Methods: Blood samples were taken from 24 patients with Behçet’s disease who had active uveitis and 16 healthy individuals. PBMC were subjected to analysis of T-bet mRNA and protein expression using semiquatitative reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, respectively. The products from PCR were sequenced. In order to determine the influence of activation on T-bet expression, the phytohaemagglutinin (PHA) stimulated PBMC from each sample were also evaluated for expression of T-bet mRNA and protein. Results: A significantly increased T-bet mRNA accumulation was detected in the samples from patients with active Behçet’s disease compared with that in controls. A 62 kDa band was detectable in patients with active Behçet’s disease, but not in controls. No difference was found between patients with Behçet’s disease who had active uveitis and normal controls concerning the expression of either T-bet mRNA or its protein after stimulation with PHA for 72 hours. Conclusion: Behçet’s disease is associated with an upregulation of T-bet expression, which supports a role for the Th1 subset of T cells in the pathogenesis of this disease.

Longitudinal quantification of aqueous flare and cells in Vogt–Koyanagi–Harada disease

Aims: To quantitatively evaluate the changes of aqueous flare and cells in eyes with Vogt–Koyanagi–Harada (VKH) disease. Methods: This prospective study included 35 initial-onset VKH patients (70 eyes) and 46 recurrent VKH patients (92 eyes) following immunotherapy. Aqueous flare and cells were quantified using the laser flare-cell meter before treatment, 2 weeks, 1, 3, 6 and 9 months after treatment. Results: Before treatment, mean aqueous flare (ph/ms) in initial-onset and recurrent VKH eyes were 8.1 (SD 4.1) vs 43.6 (20.7) (pu200a=u200a0.000). Following treatment, recurrent VKH eyes showed a significantly higher flare value than initial-onset VKH eyes at 2 weeks, 1, 3 and 6 months. Prior to treatment, mean cell counts (cells/0.5 mm3) in initial-onset and recurrent VKH eyes were 2.0 (1.9) vs 39.4 (23.1) (pu200a=u200a0.000). Following treatment, recurrent VKH eyes showed significantly higher cell counts than initial-onset VKH eyes at 2 weeks, 1 and 3 months. Conclusions: Our study shows that recurrent VKH patients displayed a more striking and long-lasting breakdown of the BAB and more severe inflammation than initial-onset VKH patients. Our study also indicates that the disruption of BAB lasted longer than aqueous cells either in initial-onset or in recurrent VKH patients.

Upregulation of T-bet expression in peripheral blood mononuclear cells during Vogt-Koyanagi-Harada disease

Aim: To test the hypothesis that T-bet expression is altered in patients with Vogt-Koyanagi-Harada (VKH) disease. Methods: Peripheral blood was withdrawn from 16 VKH patients before and after immunosuppressive treatment and from 16 healthy individuals. IFN-γ, IL-2, and IL-4 in the serum and the supernatants of peripheral blood mononuclear cells (PBMC) cultured with or without phytohaemagglutinin (PHA) were measured by ELISA. T-bet mRNA and protein expression in PBMC cultured with or without PHA was detected by RT-PCR and western blot, respectively. Results: The level of IFN-γ, but not IL-2 and IL-4, was significantly higher in the supernatants of stimulated PBMC in patients than in controls. A significantly increased T-bet mRNA was found in VKH patients during an active uveitis episode, but not in quiescent patients, compared to controls. T-bet protein was detectable in VKH patients during an active uveitis episode, but not in quiescent patients nor in the healthy controls. Stimulation of PBMC with PHA resulted in a marked upregulation of T-bet mRNA and protein expression for both patients and controls with no significant difference between the two groups. Conclusions: Upregulation of T-bet may be associated with the development of a Th1 mediated immune response in VKH disease.

Splenic CD8+ T cells secrete TGF-β1 to exert suppression in mice with anterior chamber-associated immune deviation

BackgroundCD8+ regulatory T cells (Treg) have been considered to be involved in a model of ocular-induced tolerance, known as anterior chamber-associated immune deviation (ACAID). The mechanisms of suppression by CD8+ T cells in ACAID remain only poorly understood. TGF-β1 is considered as an inhibitory cytokine for immunosuppression in some models. The production of TGF-β1 by CD8+ T cells in ACAID, and whether CD8+ T cells exert suppression through TGF-β1, is unknown.MethodsThe suppressive effect of CD8+ T cells in ACAID mice was determined by a local adoptive transfer (LAT) assay. The production of TGF-β1 by CD8+ T cells was measured by enzyme-linked immunosorbent assay (ELISA). Anti-TGF-β1 antibodies were used in the LAT assay to test if they could block the inhibitory effect of CD8+ T cells.ResultsCD8+ T cells from ACAID mice were shown to block the delayed-type hypersensitivity (DTH) response in an antigen-specific manner in a LAT assay. These CD8+ T cells secreted TGF-β1, and their suppression could partially be blocked by anti-TGF-β1 antibodies.ConclusionsOur study confirms that CD8+ T cells from ACAID mice possess inhibitory properties. This population exerts part of its suppressive function via the production of TGF-β1.

MALDI-TOF/TOF-MS Reveals Elevated Serum Haptoglobin and Amyloid A in Behcet’s Disease

Behcets disease (BD) is a multisystemic autoimmune disease with unclear etiology and pathogenesis. To screen aberrant serum proteins in BD, serum samples were obtained from eight male BD patients with active uveitis and eight male healthy volunteers with informed consent. The serum samples from active BD patients and normal controls were pooled. Highly abundant serum proteins (albumin and IgG) were depleted from these two samples using an affinity capture based kit. The obtained samples were subjected to two-dimensional gel electrophoresis (2-DE). Protein spots were visualized with the blue silver staining. Differently expressed proteins were subsequently identified by matrix-assisted laser desorption /ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Western blot and enzyme-linked immunosorbent assay (ELISA) were performed using the serum samples from 18 patients with active BD, 6 patients with inactive BD, 22 patients with Vogt-Koyanagi-Harada (VKH) syndrome, and 20 healthy volunteers to validate the results of 2-DE and MS. Proteomic profiles of the pooled samples were compared, and approximately 800 protein spots were observed in each of the gels. Expression levels of four of the protein spots in active BD were significantly higher than those in the normal controls. Mass spectrometric protein identification revealed that the four protein spots corresponded to two proteins: haptoglobin (Hp) and serum amyloid A (SAA). Western blot and ELISA showed that Hp was only overexpressed in active BD but not in inactive BD, VKH syndrome, or healthy controls. An obvious band of SAA was detected in 72.2% of the serum samples from BD patients, whereas a vague band of this protein was found in 10.0% of the tested normal samples and 9.1% of VKH samples. Our results revealed a significantly increased expression of Hp and SAA in serum of active BD patients. These two proteins may be involved in the development of BD.

Disturbed expression of Fas/FasL on CD4(+) and CD8(+)T cells in Behcet's disease, Vogt-Koyanagi-Harada syndrome, and idiopathic anterior uveitis.

Aims: To evaluate the expression of Fas/FasL antigen on peripheral blood T lymphocytes in patients with Behcets disease, Vogt-Koyanagi-Harada (VKH) syndrome, and idiopathic anterior uveitis. Methods: The expression of Fas and FasL on peripheral blood T lymphocytes was determined using flow cytometry in 26 patients with Behcets disease (BD), 17 patients with VKH syndrome, 25 patients with idiopathic anterior uveitis, and 43 healthy individuals (controls). Results: A higher proportion of CD4 + T cells expressing Fas was noted in patients with Behcets disease (25.70 ± 7.32%), VKH syndrome (19.60 ± 11.02%), and idiopathic anterior uveitis (20.81 ± 7.40%) compared with controls (14.02 ± 6.30%). The expression of Fas on CD8 + cells from patients with Behcets disease (9.47 ± 6.97%) and VKH syndrome (6.84 ± 5.5%) was also higher than that seen in controls (3.47 ± 2.75%). There was no difference in FasL expression on T cells between patients and controls except that a lower expression of FasL on CD8+ T cells was noted in patients with idiopathic anterior uveitis. Conclusion: A disturbed expression of Fas and FasL on T cells is present in patients with Behcets disease, VKH syndrome, and idiopathic anterior uveitis, which may be involved in the perpetuation and recurrence of uveitis.