Friedrich Hoffmann

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.

Side-effects of local anesthetics on the corneal epithelium of the rabbit eye

The toxic side-effects of 0.4% Novesine and Chibro-Kerakain were investigated by scanning- and transmission electron microscopy in 15 rabbit corneae each in intervals of 3, 6, 10, 15 and 60 mins. The lesions following Kerakain were larger and occurred earlier. After 60 mins, the specimens after Novesine application showed an almost complete repair process, whereas after Kerakain the epithelial surface appeared to be functionally impaired. Raster- und transmissionselektronenmikroskopisch werden 15 Kaninchenhornhäute nach Novesin 0,4%und 15 nach Chibro-Keracain in Abständen von 3, 6, 10, 15 und 60 min auf toxische Nebenwirkungen untersucht. Die Läsionen nach Keracain waren ausgeprägter und traten rascher ein. 60 min nach Applikation sind die Reparationsvorgänge bei Novesin weitgehendst abgeschlossen. Die Präparate nach Keracain zeigen zu diesem Zeitpunkt ein in seiner Funktion noch beeinträchtigtes Oberflächenepithel.

Keratoplasty after mustard gas injury: clinical outcome and histology.

Purpose: Ocular injury by mustard gas can lead to severe eye damage with a delayed course. We report the corneal histology and follow-up after keratoplasty in a patient with mustard gas injury. Methods: The patient presented with recurrent painful corneal inflammation in both eyes not improving under local therapy. Visual acuity impaired to handmovements. A penetrating keratoplasty was performed on the left eye and afterwards an autorotation keratoplasty on the right eye with a later corneal graft. Results: After the operation of the left eye the patient was immediately painfree and the visual acuity improved to 0,4. So far there have been no signs for transplant rejection or inflammation. Histology of the cornea revealed massive stromal necrosis, and signs of chronic inflammation. Despite denervation of the cornea after autorotation keratoplasty the right eye was still painful and became only painfree after corneal transplantation. Conclusion: There has been not much experience with corneal transplanation after mustard gas injury and there is a high risk for transplant rejection due to inflammation and vascularisation of the cornea. Succesfull and painfree healing with keratoplasty seems only possible after complete removal of the necrotic material.

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.

Minkowski-type theorems and least-squares partitioning

Consider a set S of n points, called sites, in the Euclidean plane. S induces a partition of the plane into n polygonal regions in the following natural way. The region of a site s ● S, reg(s), consists of all points z which are closer to s than to the remaining n – 1 sites. This partition is known as the Voronoi diagram of S. If we fix a set X of m points in the plane, this set is partitioned by the Voronoi diagram of S into subsets. More precisely, the diagram defines an assignment function A : X + S, given by

Interleukin-2 receptor--targeted therapy by monoclonal antibodies in the rat corneal graft.

A possible selective therapeutic approach to corneal graft rejection will aim at IL–2 receptor-bearing antigenactivated T-lymphocytes with monoclonal anti IL–2R antibodies. In a rat penetrating keratoplasty model (Lewis x Lewis-BN) comparing to controls (median, 8 days), a significant delay of the allograft reaction was achieved by applying a therapeutic dose (15 mg/kg bw) of cyclosporin A (median, 18 days; p<0.01), an intraperitoneal (1.0 mg/kg bw) (median, 13.5 days; p<0.05) or a subconjunctival injection of IL–2R mab (0.5 mg/kg bw ART-18) (median, 16 days; p<0.01) with low-dose Cyclosporin A (1.5 mg/kg bw). In pharmacokinetic experiments, the corneal radioactivity 24 h after intraperitoneal injection of 125Ilabeled ART-18 was <\% (p<0.01) of the values obtained by subconjunctival injection, whereas the serum radioactivity values (p>0.05) were in the same range. The above results suggest that the onset of an allograft reaction in perforating keratoplasty seems to depend on the locally achievable antibody concentration and can be delayed with a high level of IL–2 R mab present in the immediate surrounding of the foreign antigen-expressing cells.

PRK and LASIK--their potential risk of cataractogenesis: lipid peroxidation changes in the aqueous humor and crystalline lens of rabbits.

Purpose. There are insufficient data on the possible cataractogenic side effects of excimer laser corneal surgery. Higher malondialdehyde (MDA) levels could indicate oxidative events related to the cataractogenic process. We therefore examined MDA levels after refractive laser surgery. Methods. Six white Russian rabbits received laser in situ keratomileusis (LASIK) (Schwind keratome) in the right eye and a 250-&mgr;m-deep microkeratome cut (Schwind microkeratome) in the left eye. Six others underwent photorefractive keratectomy (PRK) in the right eye; the left eye remained untreated. The 180 mJ/cm2fluence applied at a rate of 10 Hz with an optical zone diameter of 5 mm in all rabbits (438 pulses) resulted in an estimated central photoablation depth of 116 &mgr;m. Two weeks later, lenses and aqueous were taken immediately after death. MDA was detected in aqueous and homogenate of lenses after reacting with thiobarbituric acid (TBA). MDA bound to TBA (MDA–TBA) was specifically analyzed by high-performance liquid chromatography (HPLC) (excitation, 525 nm; emission, 551 nm) using phosphate-buffered methanol as eluent. Results. No significant laser-induced MDA alteration was found in either the aqueous or the lens. The microkeratome group, however, had two to three times higher MDA levels in the lenses than the control group (p = 0.12) or the PRK (p = 0.03) group. Conclusion. Elevation of MDA in the lens of the microkeratome group indicates that LASIK, but not PRK, may be a risk factor in cataractogenesis. The increased MDA levels in the LASIK group are probably caused by the microkeratome incision rather than the secondary radiation of the excimer laser. Postoperative inflammation may explain the surprising results.