Er-Ping Zhang

Ballistic transfer of minimalistic immunologically defined expression constructs for IL4 and CTLA4 into the corneal epithelium in mice after orthotopic corneal allograft transplantation

Abstract Background: Experiments were performed to determine whether corneal epithelium transfected with minimalistic immunologically defined expression constructs for the extracellular fragment of CTLA4 and for interleukin-4 (IL-4) or interleukin-10 (IL-10) is able to modulate an allospecific immune response after orthotopic corneal grafting in mice. Methods: Six groups of BALB/c (H-2d) mice received a C3H (H-2k) corneal graft and dexamethasone eye drops until day 11. Five groups of BALB/c mice had gold particles delivered into the corneal epithelium by Gene Gun on day 10 after transplantation. In four groups, minimalistic immunologic-ally defined gene expression (MIDGE) vectors were delivered into the corneal epithelium by ballistic transfer. The levels of expressed IL-4 and IL-10 were determined by an enzyme-linked immunosorbent assay (ELISA) in shock-frozen homogenized corneas. The expression kinetics of Gene-Gun-transfected corneas were determined by measuring luciferase in lysed whole corneas at different time intervals. Results: Luciferase expression was detectable during the first 5 days following transfection. ELISA was used to determine IL-4 and IL-10 expression in corneal tissue 36 h after transfection. Ballistic IL-4 and CTLA4 gene transfer significantly prolonged corneal graft survival in comparison with the gold-treated control group and the IL-10-treated group. Conclusion: The beneficial effect of IL-4 and CTLA4, but not IL-10 gene transfer into the corneal epithelium by MIDGE vectors was demonstrated for the first time in corneal transplantation.

Orthotopic corneal transplantation in the mouse : a new surgical technique with minimal endothelial cell loss

Abstract• Background: The murine model of orthotopic perforating keratoplasty is important for studying the allograft reaction, but the small dimensions cause technical difficulties. • Methods: The anterior chamber of the eye of the BALB/c mouse was measured with the confocal microscope and with histological methods. Ten C3H mouse donor corneas each were separated by the conventional technique and by the newly developed “underwater” technique, where the opened donor eye did not lose its shape under water. The corneal endothelium was stained with trypan blue and alizarin red S. Ten BALB/c (H-2d) mice received a corneal graft taken from a C3H (H-2k) mouse by the underwater technique. • Results: The 3.7-mm eye of the BALB/c mouse has a corneal diameter of 3.5 mm. The cornea has a central thickness of 170 μm, the epithelium comprising 30% and the stroma 70%. While none of the corneas separated by the new “underwater” technique evidenced endothelial damage, a 28 ± 17.0% defect of the endothelial surface was found with the conventional technique. All transplanted corneas were clear when the lids were opened on the 2 post-operative day and clouded between the 7th and 30th days (mean 16.5 days) due to an allograft reaction. • Conclusion: The newly developed “underwater” technique is superior to the conventional technique, since floating of the very thin donor cornea during the separation procedure prevents endothelial defects by guarding against folds. By enabling reliable keratoplasty in the mouse, this technique facilitates studies on the experimental allograft reaction.

Contribution of lymphatic drainage system in corneal allograft rejection in mice

Abstract. Purpose: To modulate aqueous outflow via the uveoscleral pathway and to determine its influence on corneal graft survival in mice. Methods: BALB/c mice received corneal transplants from C3H mice and were placed randomly in three treatment groups: saline, pilocarpine or latanoprost. Three further groups received adjuvant systemic and topical corticosteroids. The kinetics of infiltrating lymphocytes, neutrophils and macrophages in the transplants was investigated in an additional 96 animals. Cytokine expression in the submandibular lymph nodes and spleen was investigated using in-situ hybridization and RNAse protection assay. Tracer experiments were conducted using 99mTC colloidal albumin Nanocoll; count rates were determined in the submandibular lymph nodes, spleen and blood following both subconjunctival and intracameral injection. Results: Neither pilocarpine nor latanoprost had any influence on aqueous outflow or allograft survival in mice. Neutrophils and macrophages dominated the infiltrating cells 11 days postoperative in both treated and untreated grafts. On postoperative day 13, a greater increase in lymphocytes than in other cell groups was observed in allogeneic grafts. Following allogeneic transplantation, 1% of lymphocytes in ipsilateral submandibular lymph nodes were positive for IFN-γ. Tracer studies revealed a 16% aqueous outflow via the uveoscleral routes following intracameral injection of Nanocoll; this was increased by 97% with subconjunctival injection. Conclusion: Our data confirm the existence of functional lymphatic drainage via the uveoscleral pathway and conjunctiva in the mouse. Cells within the ipsilateral submandibular lymph node respond to stimuli upstream. This reaction could potentially be manipulated to improve graft survival.

Inhibition of corneal allograft reaction by CTLA4-Ig

Abstract• Background: Activation of T cells requires both the interaction of T-cell receptor with major histocompatibility complex on the antigen-presenting cell and costimulatory signals, for instance the B7 antigens expressed on antigen-presenting cells and the CD28 molecule expressed on T cells. A recombinant fusion protein, CTLA4-Ig, has been produced that contains the extracellular domain of human CTLA4 fused to IgGl constant region and that binds the B7 molecule with high affinity. Blocking the CD28/B7 interaction with CTLA4-Ig inhibits T cell activation in vitro and in vivo. • Methods: We used CTLA4-Ig in a fully MHC-mismatched mouse keratoplasty model. The animals were divided into four groups: (1) no treatment, (2) intraperitoneal treatment with 130 μg CTLA4-Ig, (3) intraperitoneal treatment with 300 μg CTLA4-Ig, (4) subconjunctival treatment with 290 μg CTLA4-Ig. • Results: The allograft reaction occurred in untreated animals between days 12 and 16 (mean 13.5). While topical application of CTLA4-Ig seemed to shorten the graft survival (mean 11.6 days) and systemic application of 130 μg had no influence (mean 14.0), only intraperitoneal injection of 300 μg of CTLA4-Ig prolonged the survival of allografts (mean >20 days) (P<0.01). • Conclusion: CTLA4-Ig prolonged significantly the survival of corneal allografts in a fully MHC-mismatched mouse keratoplasty model, but the small antigen load of the corneal transplant and the anterior chamber-associated immune deviation (ACAID) may have a disadvantage to induce tolerance in this model of CTLA4-Ig therapy.

The effect of corticosteroid and cyclosporin A on murine corneal allograft rejection

Abstract Background: The immunomodulatory T-helper type 1 (Th1) cytokine interferon-γ (IFN-γ) was measured in serum and cornea to ascertain its general contribution to corneal graft rejection and to establish a rational basis for the decision for or against systemic therapy. Methods: Eight groups of differently treated BALB/c (H-2d) mice received a C3H (H-2 k) corneal graft. There was one saline-treated control group and two groups that received intramuscular cyclosporin A (CsA) for 14 or 40. Three groups received systemic or topical, systemic plus topical corticosteroid treatment, which was combined with CsA in two further groups. To measure the IFN-γ level by enzyme-linked immunosorbent assay (ELISA), blood was taken by heart puncture and corneae were excised at the limbus. Results: Five days of systemic corticosteroid and 14 days of CsA had no significant effect on graft survival. A 40-day CsA treatment and a 40-day combined corticosteroid treatment significantly prolonged graft survival. An 80-day topical corticosteroid treatment produced additional prolongation. IFN-γ could not be detected (limit of detection 25 pg/ml) in any of the serum samples, while significantly increased amounts of IFN-γ were detected in the supernatants of the corneal tissue 13 or 14 days after allogeneic but not syngeneic corneal graft, corresponding to 9.5 pg, 5.1 pg and 1.8 pg per cornea. Conclusion: The detection of Th1 cytokines in the cornea but not the serum of mice at the time of allograft rejection is in accordance with the finding of long-lasting dose-dependent immunosuppression of topical steroids and the inefficacy of short-term systemic CsA and corticosteroids.

Significant prolongation of orthotopic corneal-graft survival in FTY720-treated mice.

Background. The novel immunomodulator, FTY720, mainly acts through sequestering of lymphocytes to secondary lymphatic tissue, thereby suppressing their infiltration into grafted organs. This study aimed to investigate its influence on corneal-graft survival. Methods. Sixteen BALB/c mice (H-2d) received corneal transplants from C3H (H-2k) mice. Eight mice were treated with FTY720 (10 mg/kg per day) orally from day −1 to day 11, and all animals received 0.1% dexamethasone eye drops for the same time. In addition, eyes and regional lymph nodes from similarly treated animals were subjected to immunohistochemistry and proliferation assays. Results. FTY720 significantly prolonged graft survival from 28±8.1 to 36.5±7.1 days (P =0.021). In treated animals, corneal infiltration by CD4+ and F4/80+ cells was reduced from 70.8±60.3 to 7.0±9.0 (P =0.004) and from 97.5±30.7 to 44.8±24.9 (P =0.01) cells, respectively, and allogeneic T-cell proliferation was decreased. Conclusions. FTY720 treatment substantially protects corneal allografts and may provide an immunomodulatory strategy in clinical corneal transplantation.

Prolongation of corneal allograft survival by an interleukin-2-immunoglobulin fusion protein in mice

Abstract · Background: Interleukin 2 (IL2) production by activated T-helper cells leads to activation and proliferation of cytotoxic T cells. Recently, an IL2-IgG fusion protein was found to suppress cell-mediated and humoral immune responses in mice. · Methods: We used the genetically engineered murine IL2-IgG2b fusion protein in a fully MHC-mismatched mouse keratoplasty model. The DTH reaction against sheep red blood cells was investigated as a measure of IL2-IgG2b-mediated immunosuppression. The animals were divided into three control groups (n≥6) [no treatment, subconjunctival (SQ) treatment with saline or mouse serum], two IL2 SQ-treated groups (14 μg or 140 μg), and four IL2-IgG2b-treated groups (14 μg, 140 μg or 280 μg SQ or 280 μg IP). · Results: Administration of 20 μg of IL2-IgG2b twice daily from the time of immunization until the time of challenge resulted in almost complete prevention of footpad swelling. The 140 μg SQ application of IL2 (allograft reaction on day 20.5 ± 4.04) and the 280 μg SQ (day 19.2 ± 2.48) or IP (day 19.7 ± 1.5) application of IL2-IgG2b fusion protein significantly prolonged the corneal graft survival in comparison to the untreated group (day 13.4 ± 1.35) (P<0.01) or saline control group (P<0.01) and the mouse-serum-treated group (day 14.7 ± 3.5) (P<0.05). · Conclusion: Our results indicate that, at a total dose of 280 μg, the fusion protein IL2-IgG2b causes no detectable side effects and very effectively suppresses the immune response of the corneal allograft in mice. This fusion protein could prove useful in the treatment of allograft rejections and autoimmune diseases.

Topische Dexamethason-Therapie

ZusammenfassungUntersuchungsziel. Ermittlung der Wirkung von Dexamethason auf antigenpräsentierende Zellen (APCs) der Hornhaut und deren Einfluss auf die Überlebenszeit MHC-differenter Hornhauttransplantate der Maus. Methoden. Mittels EDTA präparierte epitheliale Flatmounts und tangentiale Gefrierschnitte des Hornhautstromas von unbehandelten und 7 Tage mit Dexamethason Augentropfen vorbehandelten BALB/c Mäusen (n=6) wurden immunhistologisch auf F4/80+- und MHC II+-Zellen untersucht. Weiterhin wurden an unbehandelten und entsprechend vorbehandelten Mäusen (n=8) allogene (C3H) Keratoplastiken durchgeführt. Ergebnisse. Im Hornhautepithel, nicht aber im Hornhautstroma reduzierte Dexamethason drastisch die F4/80+- und die MHC II+-Zellen (p<0,01). Die Transplantate der unbehandelten Tiere überlebten 16±4 Tage, die der Dexamethason-vorbehandelten 16±3 Tage.AbstractObjective. To determine the influence of dexamethasone treatment on APCs and the time of graft survival of MHC-disparate grafts. Methods. Flatmounts of the ocular surface prepared with EDTA and tangential frozen sections of the remaining corneal stroma from untreated eyes of normal mice (n=6) and from eyes treated for 7 days with dexamethasone (n=6) were immunohistologically examined for content of F4/80+ and MHC II+ cells.Furthermore, corneas of C3H mice without and with 7-day dexamethasone eye drop treatment (n=8) were grafted into BALB/c mice receiving the same treatment. Results. The number of positive cells within the epithelial flatmounts showed a dramatic reduction in the dexamethasone-pretreated group (p<0.01 compared to the untreated control group).The number of positive cells in the corneal stroma remained unchanged. The grafts of untreated control mice survived 16±4 days, the treated grafts 16±3 days. Conclusions. Most investigators assume that normal murine corneas contain no APCs such as macrophages and Langerhans cells. For the first time we were able to detect APCs in flatmounts of the ocular surface and frozen sections of corneal stroma.Our investigations show that, in contrast to the ocular surface, the number of F4/80+ cells in the corneal stroma is not influenced by dexamethasone treatment.Transplantation of corneas containing donor-derived APCs promotes acute rejection (direct pathway of allorecognition).Thus,dexamethasone treatment did not prolong the time of allograft survival.

Immunmodulation nach Keratoplastik durch CTLA4-Ig und anti-CD154-Antikörper

ZusammenfassungHintergrund. Die Immunreaktion nach Hornhauttransplantation erfordert die Interaktion des T-Zell-Rezeptors mit dem Major-Histokompatibilitätskomplex- (MHC-)Rezeptor der antigenpräsentierenden Zelle. Das Signal wird durch den CD4-Rezeptor und durch die kostimulatorischen Signalinteraktionen CD28-B7 und die CD40-CD154 verstärkt. Wir wollten den Einfluss der Blockierung der kostimulatorischen Signale auf das Überleben der Hornhauttransplantate der Maus untersuchen. Methoden. Sieben Gruppen von je 6 BALB/c-Mäusen erhielten orthotop ein Hornhauttransplantat von Minor- und Major-MHC-differenten C3H-Mäusen und wurden wie folgt nachbehandelt: (1) 6 Tage je 80 μg CTLA4-Fusionsprotein intraperitoneal (i.p.); (2) 6 Tage je 50 μl PBS/Tag i.p.; (3) 5 Tage je 1 mg Solu-Decortin H i.p. + 35 Tage Dexamethason AT 0,1%; (4) Therapie (3) + 6 Tage je 50 μg CTLA4-Fusionsprotein i.p.; (5) CTLA4 wie (1) + je 15 μg anti-CD154 subkonjunktival (s.c.) am Tag 0, 2, 4, 6, und 8; (6) CTLA4 wie (1) + 9 Tage je 25 μg anti-CD154 s.c.; (7) 9 Tage je 25 μl PBS/Tag s.c. Ergebnisse. Bei allen Tieren trat eine Immunreaktion an folgenden Tagen auf: (1) Tag 18±3,1; (2) Tag 13,6±1,6; (3) Tag 48±6,6; (4) Tag 65±4,1; (5) Tag 23,5±8,5; (6) Tag 16,2±3,6; (7) Tag 13,8±2,7. Schlussfolgerung. Kortikosteroide verlängern signifikant (p<0,001) das Transplantatüberleben, gemeinsam mit CTLA4-Ig kommt es zu einer weiteren signifikanten Verlängerung des Transplantatüberlebens (p<0,001). Die Kombination der spezifischen Immuntherapie und der unspezifischen Steroidtherapie könnte auch klinisch die Ergebnisse von Hornhauttransplantationen verbessern. CTLA4-Ig und anti-CD154 hatten nur bei niedriger Dosierung gegenüber den beiden Kontrollgruppen einen Einfluss auf das Überleben der Transplantate (p<0,001).AbstractBackground. The immunoreaction after corneal transplantation is caused by the T cell receptor interacting with the major histocompatibility complex (MHC) receptor of the antigen-presenting cell. The signal is amplified by the CD4 receptor and the costimulatory signal interactions of CD28-B7 and CD40-CD154. We investigated the influence of costimulatory signal blocking on corneal transplant survival in mice. Methods. Seven groups of 6 BALB/c mice received an orthotopic corneal transplant from C3H mice differing in minor and major MHC and were postoperatively treated as follows: (1) 80 μg of CTLA4 fusion protein intraperitoneally (i.p.) for 6 days; (2) 50 μl of PBS i.p. for 6 days; (3) 1 mg of Solu-Decortin H i.p. for 5 days+dexamethasone AT 0.1% for 35 days; (4) therapy (3)+50 μg of CTLA4 fusion protein i.p. for 6 days; (5) CTLA4-Ig as in (1)+15 μg of anti-CD154 subconjunctivally (s.c.) on days 0, 2, 4, 6, and 8; (6) CTLA4-Ig as in (1)+25 μg of anti-CD154 s.c. for 9 days; and (7) 25 μl of PBS s.c. for 9 days. Results. All animals had an immunoreaction on the following days: (1) day 18±3.1; (2) day 13.6±1.6; (3) day 48±6.6; (4) day 65± 41; (5) day 23.5±8.5; (6) day 16.2±3.6; (7) day 13.8±2.7. Conclusion. The significant prolongation of transplant survival achieved by corticosteroids alone (P<0.001) is again significantly increased by combining them with CTLA4-Ig (P<0.001). Specific immunotherapy combined with nonspecific steroid therapy may also improve clinical corneal transplantation results. Compared to the two control groups, CTLA4-Ig and anti-CD154 only influenced transplant survival at a low dosage (P<0.001).