Altered Expression of CXCL13 and Its Chemokine Receptor CXCR5 on B Lymphocytes during Active Graves' Orbitopathy.

PURPOSE: To characterize the phenotypic abnormalities of peripheral B cells in patients with Graves' orbitopathy (GO) and explore the role of chemokine CXC ligand 13 and its receptor type 5 (CXCL13/CXCR5) in relation to B-cell homeostasis using specific neutralizing antibodies. METHODS: Adults with active GO (n = 22), inactive GO (n = 28), and healthy control subjects (n = 28) were included in the study. Peripheral B cells and B-cell subsets were quantified and analyzed for CXCR5 expression by flow cytometry. The serum CXCL13 concentration was measured by enzyme-linked immunosorbent assays. For chemotactic experiments, Transwell plates were used, and migrating B cells were further analyzed by flow cytometry. RESULTS: Compared to healthy subjects, patients with active GO had a significantly higher number of CD19+ B cells and the CD19+CD27+ memory B-cell subset (P = .041 and P = .019, respectively), whereas a marginal increase in the number of these cells was found in patients with inactive GO (P = .062 and P = .087, respectively). Serum CXCL13 levels were significantly higher in patients with active GO (86.9 ± 30.4 pg/mL) than in those with inactive GO (41.7 ± 18.1 pg/mL; P < .001) and in healthy subjects (36.2 ± 7.8 pg/mL; P < .001). The increased CXCL13 concentration was positively and significantly correlated with the clinical activity score (r = 0.757, P < .001). Finally, serum from patients with active GO exerted a stronger chemotactic activity towards B cells and the CD19+CD27+ memory B-cell subset. Blocking CXCL13 or CXCR5 with neutralizing antibodies reduced B-cell migration by a mean of 20%. CONCLUSIONS: Our data suggest that aberrant CXCL13/CXCR5 expression may contribute to the deficits in B-lymphocyte homeostasis observed in active GO.

Hemin Promotes Corneal Allograft Survival Through the Suppression of Macrophage Recruitment and Activation.

Purpose: To explore the roles of hemin in preventing corneal allograft rejection (CGR) and the underlying mechanisms. Methods: Hemin (30 mg/kg) was intraperitoneally injected into rats with a corneal allograft on alternate days, from the day of transplantation until euthanasia. The clinical signs of the corneal allografts were evaluated and recorded according to a previously published system. Corneal edema, macrophage infiltration, and phenotype, and the expression of chemokines, cytokines, and heme oxygenase (HO)-1 were detected by histology, real-time PCR, and Western blot. The rat macrophage cell line NR8383 was used to explore the mechanisms of action of hemin in vitro. Results: Treatment with hemin significantly prolonged corneal allograft survival, with decreased corneal edema and fewer macrophages. Moreover, hemin treatment alleviated inflammation in the corneal grafts, as characterized by downregulated mRNA levels of proinflammatory mediators. In addition, hemin administration reduced the proportion of proinflammatory M1 macrophages and increased the proportion of anti-inflammatory M2 macrophages in the corneal grafts. Hemin treatment induced HO-1 expression in vivo and in vitro, whereas co-administration of zinc protoporphyrin IX (ZnPP), an HO-1 inhibitor, blocked the beneficial effects of hemin in preventing CGR. Conclusions: Our results are the first to demonstrate that hemin, a Food and Drug Administration-approved drug, promotes corneal allograft survival. These findings indicate that hemin might be a potential alternative treatment for CGR.

Baicalin modulates the Treg/Teff balance to alleviate uveitis by activating the aryl hydrocarbon receptor.

Autoimmune uveitis is a sight-threatening ocular inflammatory disorder. Immunological inflammation is regarded as the key to pathogenesis in autoimmune uveitis. Baicalin, the major bioactive component of Scutellaria baicalensis, possesses immunomodulatory properties. However, the role of baicalin in uveitis and its underlying mechanisms remain unclear. In the current study, we found that baicalin treatment obviously inhibited the intraocular inflammatory process in mice with experimental autoimmune uveitis, along with clear declines in infiltrated inflammatory cells and inflammatory cytokine transcription in the retina and draining lymph nodes. Furthermore, baicalin treatment increased the frequency and number of regulatory T cells and decreased the frequency and number of effector T cells (Th1 and Th17 cells) in the draining lymph nodes of mice with experimental autoimmune uveitis. In vitro, baicalin treatment suppressed interphotoreceptor retinoid binding protein-specific CD4+ T cell proliferation and converted CD4+ T cell differentiation. Furthermore, the expression of aryl hydrocarbon receptor was activated by baicalin treatment. Baicalin-mediated modulation of CD4+ T cell differentiation was partially abrogated by the suppression of aryl hydrocarbon receptor. These findings suggest that baicalin modulates the Treg/Teff balance and CD4+ T cell proliferation to ameliorate experimental autoimmune uveitis by activating the aryl hydrocarbon receptor.

Orbital fibroblasts of Graves' orbitopathy stimulated with proinflammatory cytokines promote B cell survival by secreting BAFF.

The success of rituximab for the treatment of active Graves' orbitopathy (GO) suggests that B cells play a critical role in intraorbital inflammation. B cell activating factor (BAFF) and its homolog a proliferation-inducing ligand (APRIL) are critical for B cell survival. However, the contribution of BAFF/APRIL to GO remains unclear. We sought to determine the role of BAFF/APRIL in the orbits of GO, and found that BAFF was markedly upregulated, while APRIL was not. Additionally, cultured GO orbital fibroblasts (GO-OFs)2 expressing BAFF were induced to produce a large amount of BAFF. In contrast, a weak APRIL expression was detected in the OFs, and they exhibited a slight response to stimulation. Notably, pretreated GO-OFs promoted B cell survival, and this effect was significantly inhibited by a BAFF-R neutralizing antibody. This study indicates that OFs from GO can express BAFF and mediate the intraorbital survival of B cells via BAFF mechanism.

p16(INK4a) expression in retinoblastoma: a marker of differentiation grade.

BACKGROUND: The tumor suppressor protein p16(INK4a) has been extensively studied in many tumors with very different results, ranging from its loss to its clear overexpression, which may be associated with degree of tumor differentiation and prognosis. However, its expression remains unclear in human retinoblastoma (RB), a common malignant tumor of retina in childhood. The aim of this study was to explore the expression pattern of p16(INK4a) in RB, and the correlation between p16(INK4a) expression and histopathological features of RB. METHODS: Sixty-five cases of RB were retrospectively analyzed. Paraffin-embedded blocks were retrieved from the archives of ocular pathology department at Zhongshan Ophthalmic Center of Sun Yat-sen University, China. Serial sections were cut and subjected to hematoxylin and eosin staining. Immunohistochemical staining was further done with antibodies p16(INK4a), CRX and Ki67. The correlation of p16 (INK4a) expression with CRX and Ki67 and clinicopathological features of RB were analyzed. RESULTS: RB tumor histologically consists of various differentiation components including undifferentiated (UD) cells, Homer-Wright rosettes (HWR) or Flexner-Winterstein rosettes (FWR) and fleurettes characteristic of photoreceptor differentiation or Retinocytoma (RC). p16(INK4a) expression was negative in both fleurette region and the residual retinal tissue adjacent to the tumor, weakly to moderately positive in FWR, strongly positive in both HWR and UD region. However, CRX had the reverse expression patterns in comparison with p16(INK4a). It was strongly positive in photoreceptor cells within the residual retina and fleurettes, but weakly to moderately positive in UD area. Together with Ki67 staining, high p16(INK4a) expression was associated with poor histological differentiation of RB tumors, which had higher risk features with the optic nerve invasion and uveal invasion. CONCLUSIONS: p16(INK4a) expression increased with the decreasing level of cell differentiation of RBs. RB tumors extensively expressing p16(INK4a) tended to have higher risk features with poor prognosis. This study suggested that p16(INK4a) would be a valuable molecular marker of RB to distinguish its histological phenotypes and to serve as a predictor of its prognosis. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_180.

Expression levels of autophagy related proteins and their prognostic significance in retinocytoma and retinoblastoma.

AIM: To discuss the prognostic significant of autophagy related proteins (ARPs) in retinoblastoma (RB) and to find the molecular marker to distinguish retinocytoma (RC) and RB by investigating the different expression profiling of microtubule-associated protein light chain 3 (LC3B) and other ARPs in RC and RB. METHODS: Specimens with retinocytoma region (RCR) or mainly composed with Flexner-Winterstein rosettes (FWR) were screen out from 219 paraffin-embedded RB samples and respectively taken as RCR group and FWR group. Others were taken as undifferentiated (UD) group. Immunochemistry (IHC) of LC3B and electronic microscopy was used to identify autophagy. The IHC scores of LC3B and other ARPs, such as Beclin, PTEN, p27, p16(INK4a), mTOR and BCL-2 were compared and correlation analysis was applied to find potential proteins which may involve in autophagy regulation. The prognostics significance of LC3B was evaluated by comparing the high risk features (HRFs) in 3 groups of total 219 samples. RESULTS: Twenty-one specimens with RCR and 36 specimens mainly composed with FWR were screen out. RCR cell had a high level of LC3B and lots of autophagic vacuoles. Beclin, PTEN, p27 had positive correlation with LC3, and p16(INK4a) had negative correlation, while the expression of mTOR and BCL-2 in RCR and RB region did not show any difference. Cases with RCR had lower rate of HRFs than undifferentiated cases. CONCLUSION: ARPs had different expression pattern between RCR and other pathological types of RB, and could be ideal markers to distinguish RC from RB. Our finding indicated cases with RCR had favorable prognosis just like those with FWR.

Comparison of hematoxylin-eosin staining and methyl violet staining for displaying ghost cells.

PURPOSE: To compare the merits and limitations of hematoxylin-eosin (HE) and methyl violet staining for displaying ghost cells from vitreous or aqueous humor. METHODS: A specimen containing ghost cells was adjusted to five different concentrations: (12 x 10(4), 10 x 10(4), 8 x 10(4), 6 x 10(4) and 4 x 10(4) cells/ml) and subjected to smearing and methyl violet and HE staining. The staining results were observed by light microscopy. RESULTS: The ghost cells were readily observed at a cell density of > 8 x 10(4) cells/ml with methyl violet staining, but only a few cells were occasionally seen at lower cell densities. In contrast, ghost cells were seen at all cell densities with HE staining. CONCLUSION: Methyl violet staining is more rapid and simpler for the identification of ghost cells, but its staining color more readily fades, the slides cannot be stored, and it is only effective at a cell density of > 8 x 10(4) cells/ml. In contrast, HE staining is more time-consuming but it can display cell morphology and distinguish cell components more explicitly and slides can be permanently stored. HE staining has advantages over methyl violet staining in detecting the ghost cells when the concentration is < 8 x 10(4) cells/ml.