Zhiqiang Pan

In vitro-expanded CD4+CD25highFoxp3+ regulatory T cells controls corneal allograft rejection

AIMS Natural CD4(+)CD25(+) regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model. METHODS Natural CD4(+)CD25(+) T cells were freshly purified from naïve mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-β1). Suppression ability of iTregs was assayed by co-culturing with CD4(+)CD25(-) T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling. RESULTS Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-β1. Compared with nTregs, iTregs had similar expression of CD62L, and PD- L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4(+)CD25(-) T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression. CONCLUSION Therapeutic use of in vitro-expanded CD4(+)CD25(+) T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance.

Comparison of immunogenicity and porcine-to-rhesus lamellar corneal xenografts survival between fresh preserved and dehydrated porcine corneas

Li A, Pan Z, Jie Y, Sun Y, Luo F, Wang L. Comparison of immunogenicity and porcine‐to‐rhesus lamellar corneal xenografts survival between fresh preserved and dehydrated porcine corneas. Xenotransplantation 2011; 18: 46–55.

Evaluation of Optical Coherence Tomography Meibography in Patients With Obstructive Meibomian Gland Dysfunction.

Purpose: To evaluate optical coherence tomography meibography (OCT-M) in patients with and without obstructive meibomian gland dysfunction (MGD) and to determine the relationship between OCT-M and ocular surface clinical tests. Methods: Twenty-two patients with MGD and 16 control subjects were included. Each patient underwent an evaluation of ocular surface disease symptoms, lid margin abnormality score, lipid layer thickness evaluation, and meibomian gland (MG) morphological changes using noncontact infrared meibography and OCT-M. OCT-M scans were acquired in 4 different locations. OCT-M parameters including the MG length and width and palpebral conjunctival thickness were evaluated. Results: Within the OCT-M parameters, the mean length and width of MGs were significantly decreased in patients with MGD (310 ± 60 &mgr;m and 214 ± 30 &mgr;m, respectively) compared with the control group (361 ± 53 &mgr;m, P = 0.041 and 264 ± 41 &mgr;m, P = 0.021, respectively). The mean conjunctival thickness was also significantly increased in patients with MGD (448 ± 68 &mgr;m) than in the control group (356 ± 59 &mgr;m, P = 0.03). The mean length of MGs was correlated with symptoms (r = 0.34, P = 0.034), and the mean MG width was correlated with tear film breakup time (r = −0.412, P = 0.009) and the lid margin abnormality score (r = 0.334, P = 0.038). Conclusions: The MG length and width were significantly decreased in patients with MGD than in the control group. These parameters were correlated to both ocular surface symptoms and clinical signs. This new imaging technique may be a useful tool for MGD evaluation.

Ocular Surface Epithelial Thickness Evaluation in Dry Eye Patients: Clinical Correlations.

Purpose. To evaluate the relationship between corneal and conjunctival epithelium thickness and ocular surface clinical tests in dry eye disease (DED) patients. Patients and Methods. Fifty-four patients with DED and 32 control subjects were included. Each patient underwent an ocular surface evaluation using the ocular surface disease index (OSDI), tear film break-up time (TBUT), corneal and conjunctival staining, tear film lipid layer analysis, and Schirmer test. The central corneal (CET), limbal (LET), and bulbar conjunctival epithelium thickness (BET) were acquired using spectral-domain optical coherence tomography (SD-OCT). Results. Compared to control subjects, mean BET was significantly thicker and mean LET was significantly lower in the DED group. There was no significant difference in mean CET between the two groups. The mean LET was correlated with OSDI and TBUT. The inferior LET was correlated with OSDI, Schirmer I test, TBUT, Oxford score, and corneal sensitivity. Mean BET was correlated with OSDI and TBUT, but not with Schirmer I test and Oxford score. Conclusions. In dry eye patients, a thinner limbal epithelium and a thicker bulbar conjunctival epithelium were observed. These changes were correlated to the severity of dry eye symptoms and tear film alterations.

Effects of Adoptive Transferring Different Sources of Myeloid-Derived Suppressor Cells in Mice Corneal Transplant Survival.

Background Adoptively transferring different sources of myeloid-derived suppressor cells (MDSCs) may assist in mice corneal transplant survival. Methods Allogeneic full thickness corneal transplantation (donor C57BL/6 to recipient Balb/c mice) was performed. Naive myeloid cells, inflammation-induced MDSCs (iMDSCs), and tumor-induced MDSCs (tMDSCs) were purified from bone marrow of naive, cecal ligation and puncture, or tumor-bearing Balb/c mice, respectively. The inhibitory abilities of myeloid cells toward CD4+ T cell proliferation were accessed by in vitro carboxyfluorescein diacetate, succinimidyl ester (CFSE) assays. Myeloid cells were adoptively transferred to corneal recipients by retroorbital injection after corneal transplantation. Corneal grafts were examined and photographed for a period of 45 days. The growth of corneal graft neovascularization was quantitatively measured by image editing software. Histopathology was performed to evaluate corneal graft inflammation. Results The iMDSCs and tMDSCs significantly inhibited T cell proliferation in vitro and significantly prolonged corneal allograft survival in vivo. Strikingly, iMDSC transferring significantly reduced neovascularization that was comparable to transferring of tMDSCs, without additional immunosuppression. However, additional adoptive transfer of MDSCs did not further ameliorate corneal survival in these allogeneic corneal transplantation mice. Conclusions Inflammation-induced MDSC transfer could reduce corneal neovascularization and prolong corneal allograft survival.

CD154 blockade modulates the ratio of Treg to Th1 cells and prolongs the survival of allogeneic corneal grafts in mice

Administration of anti-CD154 monoclonal antibody (mAb) may prolong the survival of an allograft; however, the associated therapeutic mechanisms remain poorly understood. This study aimed to evaluate the effects of anti-CD154 mAb on T-cell responses in a mouse model of corneal allograft transplantation. BALB/c mice were transplanted with corneal grafts from C57BL/6 mice and treated intraperitoneally with 250 μg anti-CD154 mAb or isotype IgG on days 0, 3 and 6 post surgery. The transparency of the corneal grafts was evaluated for potential rejection signs by slit-lamp biomicroscopy and histopathology. The percentages of CD4+ T, Tim-3+CD4+ T helper (Th) 1 and CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the spleen, ipsilateral draining lymph nodes and corneal grafts, and the frequency of splenic IFN-γ+ and IL-10+ expression in CD4+ T cells were determined by flow cytometry. Moreover, the ratio of Tregs to Th1 cells was calculated and the suppressive activity of splenic Tregs was measured. Anti-CD154 neutralization significantly prolonged the survival of the corneal allograft (P=0.0012) and reduced the numbers of inflammatory infiltrates in the corneal graft. In the spleen and lymph nodes, anti-CD154 treatment reduced the frequency of CD4+ T cells, Tregs and particularly Th1 cells. In the corneal allografts, anti-CD154 treatment downregulated graft-infiltrated CD4+ T cells and Th1 cells, but increased graft-infiltrated Tregs. Furthermore, anti-CD154 treatment increased the frequency of splenic IL-10+CD4+ T cells and decreased the concentration of splenic IFN-γ+CD4+ T cells. As a result, the ratio of Tregs to Th1 cells in the anti-CD154-treated recipients increased. Anti-CD154 treatment did not enhance the suppressive activity of Tregs in the recipients. The results indicate that the therapeutic effects of anti-CD154 mAb on prolonging the survival of the corneal allograft may be associated with an increased ratio of Tregs to Th1 cells in mice.

Tryptase compromises corneal epithelial barrier function

Corneal epithelial barrier dysfunction is harmful to corneal health; the pathogenesis is unclear. This study aims to elucidate the mechanism by which tryptase compromises corneal epithelial barrier function. Human corneal epithelial cell line (HCE cells) was cultured into monolayers using as a study platform. Quantitative reverse transcription polymerase chain reaction and Western blotting were employed to detect the expression of matrix metalloprotenases (MMP)9. The endosome/lysosome fusion was observed by confocal microscopy. The corneal epithelial barrier function was assessed in Transwell system. The results showed that HCE cells expressed proteinase‐activated receptor (PAR)2. Activation of PAR2 by tryptase induced expression of MMP9 in HCE cells, interfered with the fusion of endosome/lysosome, and compromised the epithelial barrier function, which could be prevented by pretreatment with MMP9 inhibitor. We conclude that tryptase can increase the expression of MMP9 in HCE cells and compromise the epithelial barrier function. Copyright

Adoptive transfer of donor corneal antigen-specific regulatory T cells can prolong mice corneal grafts survival.

Purpose: To explore the effects of adoptive transferring T regulatory cells (Treg cells) stimulated by donor corneal antigen (Ag) to prevent corneal transplantation immune rejection in mice. Methods: C57BL/6 mice were used as donors and BALB/c mice as recipients. Corneal Ag was harvested by homogenization and centrifugation. Bone marrow dendritic cells (DCs) from BALB/c mice were cultured with stimulation of granulocyte-macrophage colony-stimulating factor and interleukin-4 for 5 days. Then, donor corneal Ag was added to obtain donor corneal Ag-loaded DCs. The DCs were used to stimulate CD4+CD25+ and CD4+CD25− T cells from the recipient to yield Ag-stimulated Treg cells. Penetrating keratoplasty was performed in the mice. The recipients were randomly divided into 3 groups receiving 0.1 mL of phosphate-buffered saline, 1 × 106 naive Treg cells, and Ag-stimulated Treg cells, respectively, given by retroorbital injection at the end of surgery. The allografts were observed, and histopathological examination was performed 15 days after surgery. Results: The corneal Ag mainly comprised 2 proteins with molecular weight 54 and 42 kD, respectively. Corneal Ag-loaded DCs expressed higher levels of CD11c, CD80, and CD86 than bone marrow precursor cells. Both CD4+CD25+ and CD4+CD25− T cells showed vigorous proliferative responses to corneal Ag-loaded DCs. Mean survival time of the mice corneal allografts in phosphate-buffered saline, naive Treg cells, and Ag-stimulated groups was 8.1 ± 1.1, 14.3 ± 2.0, and 23.3 ± 2.6 days, respectively (P < 0.01 among groups). Histopathological examination revealed less inflammatory cells infiltration in Ag-stimulated than in naive mice. Conclusions: Adoptive transfer of donor corneal Ag-specific Treg cells prolonged survival time of corneal allografts in our mouse model, which might suggest a useful approach to cellular immunotherapy for corneal transplantation immune rejection.

Initial study of α1,3-galactosyltransferase gene-knockout/CD46 pig full-thickness corneal xenografts in rhesus monkeys.

To investigate graft survival after full‐thickness corneal xenotransplantation from α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs expressing a human complement regulatory protein (GTKO/CD46 pigs) in rhesus monkeys.

Mutation Analysis of PAX6 in a Chinese Family and a Patient with a Presumed Sporadic Case of Congenital Aniridia

Aims: Mutations in the PAX6 are the major cause of congenital aniridia. The objective of this study was to analyze genetic mutations in PAX6 in Chinese patients with congenital aniridia. Methods: Total genomic DNA was isolated from the peripheral blood of the aniridia patients, all healthy family members and 100 healthy volunteers. The 14 exons (including alternatively spliced exon 5a) of the PAX6 gene were amplified by polymerase chain reaction, and the products were sequenced to identify the mutation. Results: Two mutations of PAX6 were detected in exon 11 in the congenital aniridia patients. One mutation was caused by the duplication of the 4 nucleic acids CTCC (c.1286insCTCC), which would lead to a frameshift. The other mutation was caused by a transition from C to T (c.1311C → T), which would generate a stop codon. Neither mutation was present in the healthy family members or 100 healthy volunteers. Conclusion: We examined the exon sequence of the PAX6 gene in a Chinese family and an unrelated individual with aniridia. The predicted outcome of both mutations is premature termination. The mutation found in the unrelated individual has not previously been reported and represents a new addition to the spectrum of mutations in PAX6.