P C Maudgal

Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation

Biological functions of proteins are influenced by posttranslational modifications such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttranslational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1–77), generating CXCL8(1–77)Cit5. Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD efficiently and site-specifically citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1β. In comparison with CXCL8(1–77), CXCL8(1–77)Cit5 had reduced affinity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1–77), CXCL8(1–77)Cit5 was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6–77). Upon intraperitoneal injection, CXCL8(6–77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1–77). Despite its retained chemotactic activity in vitro, CXCL8(1–77)Cit5 was unable to attract neutrophils to the peritoneum. Finally, in the rabbit cornea angiogenesis assay, the equally potent CXCL8(1–77) and CXCL8(1–77)Cit5 were less efficient angiogenic molecules than CXCL8(6–77). This study shows that PAD citrullinates the chemokine CXCL8, and thus may dampen neutrophil extravasation during acute or chronic inflammation.

Inhibition of Vascular Endothelial Growth Factor Reduces Scar Formation after Glaucoma Filtration Surgery

PURPOSE Filtration failure due to excessive postoperative scarring remains a major problem after glaucoma surgery. The authors have investigated whether glaucoma and filtration surgery are associated with increased levels of vascular endothelial growth factor (VEGF), and whether a humanized monoclonal antibody against VEGF, bevacizumab, can reduce postoperative scar formation and improve surgical outcome. METHODS The levels of VEGF in samples of aqueous humor were measured by ELISA. The expression of the VEGF receptors Flt-1 and KDR was analyzed in cultured Tenon fibroblasts by real-time RT-PCR and Western blotting. The effect of VEGF and bevacizumab on Tenon fibroblasts in vitro was determined using a proliferation assay. The in vivo effect of the antibody was investigated in a rabbit model of trabeculectomy by measuring the intraocular pressure (IOP) and bleb area, and by immunohistological analysis of angiogenesis, inflammation, and fibrosis. RESULTS VEGF levels were increased significantly in the aqueous humor of glaucoma patients and rabbits that had undergone surgery. Both VEGF receptors were expressed on Tenon fibroblasts. Fibroblast proliferation in vitro was stimulated by delivery of VEGF, and was inhibited by administration of bevacizumab. The antibody also reduced angiogenesis and collagen deposition significantly, and improved the outcome of glaucoma surgery in rabbits. CONCLUSIONS VEGF was upregulated in the aqueous humor of glaucoma patients and in the rabbit model, and it stimulated fibroblast proliferation in vitro. This suggests that it is involved in the scarring process after filtration surgery. Bevacizumab reduced the proliferation of fibroblasts in vitro and improved surgical outcome.

Efficacy of (E)-5-(2-bromovinyl)-2′-deoxyuridine in the topical treatment of herpes simplex keratitis

BVDU [(E)-5-(2-bromovinyl)-2′-deoxyuridine] has a potent and selective activity against herpes simplex (type 1) in both cell culture systems and animal models. The efficacy of topical BVDU treatment (0.1% eye drops) has been evaluated in 37 patients with different forms of herpes simplex keratitis. Of these patients, 35 were followed for 2–9 months (average 6.5 months). Most of the patients had first been treated with topical IDU (idoxuridine) or ara-A (adenine arabinoside), albeit unsuccessfully, before BVDU treatment was started. Upon BVDU treatment, dendritic corneal ulcers healed in 7.8 days (on average) and the geographic corneal ulcers in 10.8 days. BVDU also exerted a pronounced healing effect on stromal keratitis, whether it was used alone or in combination with topical corticosteroids. No early recurrences were observed. Late recurrences were seen in four patients who again responded quickly to BVDU treatment. No toxic side effects, whether local or systemic, were noted in any of the patients treated with BVDU. These results establish the efficacy of BVDU in the local treatment of herpetic keratitis in man.

Corneal epithelial dysplasia after trifluridine use

Prolonged topical trifluridine treatment of herpes simplex keratitis in three elderly patients produced slightly raised dysplastic corneal epithelial lesions. The involved epithelium had a ground-glass appearance and exhibited opaque cells, edema, and spindle-shaped surface cells. Histopathology demonstrated severe cellular atypism, loss of cell polarity, dyskeratosis, parakeratosis, and a few mitotic figures. The pathological change was apparently intraepithelial. After scraping, the edema and opaque cells in the regenerated epithelium gradually disappeared in 3–4 months on cessation of trifluridine therapy. In one patient recurrence of dendritic keratitis, 5 weeks after scraping, was treated by topical bromovinyldeoxyuridine eye drops. Since severe epithelial dysplasia may represent a precancerous condition, prolonged use of trifluridine should be avoided.

Effects of phosphonylmethoxyalkyl-purine and -pyrimidine derivatives on TK+ and TK- HSV-1 keratitis in rabbits.

The phosphonylmethoxyalkyl derivatives HPMPA [(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine], HPMPC [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] and PMEA [9-(2-phosphonylmethoxyethyl)adenine] were evaluated as 0.2% eyedrops for their efficacy in the treatment of experimental herpes simplex virus type 1 (HSV-1) keratitis in the rabbit model. BVDU 0.2% eyedrops were used as the reference treatment. HPMPA, HPMPC, PMEA and BVDU eyedrops showed a rapid and highly significant healing effect (P less than 0.005) on keratitis caused by TK+ HSV-1 (McIntyre strain) when compared with placebo eyedrops, whereas BVDU treatment did not affect the course of TK- HSV-1 (VMW-1837) keratitis. HPMPA and HPMPC treatment again caused a highly significant healing (P less than 0.005, compared with placebo eyedrops). Although PMEA eyedrops were less effective than HPMPA or HPMPC eyedrops, the effect of PMEA eyedrops was significantly (P less than 0.05) different from the effect of either BVDU or placebo eyedrops.

Immunocytological study of phlyctenular eye disease

Scrapings from phlyctens and conjunctiva of 12 patients with phlyctenular keratoconjunctivitis were studied using OKT4-Leu3a, OKT8, B1, BA1, S-100 and HLA-DR monoclonal antibodies. T-lymphocytes were present in both conjunctival and phlyctenular scrapings. OKT4-Leu3a positive cells outnumbered the OKT8 positive cells in both conjunctival and phlyctenular scrapings. B1 and BA1 positive cells were absent from the conjunctival scrapings, but were present in the phlyctenular scrapings. S-100 positive cells were present in both conjunctival and phlyctenular scrapings. However, they were very few in the conjunctival scrapings. Most of the cells in both conjunctival and phlyctenular scrapings were HLA-DR positive. These findings support the hypothesis that cell mediated immunity is responsible for the pathogenesis of phlyctenular eye disease.

Topical Bromovinyldeoxyuridine Treatment of Herpes Simplex Keratitis

Bromovinyldeoxyuridine ((E)-5-(2-bromovinyl)-2′-deoxyuridine, BVDU) is a newly synthesized thymidine analogue (1), structurally related to the classical antiherpes agents idoxuridine (5-iodo-2′-deoxyuridine, IDU) and trifluridine (5-trifluoromethyl-2′-deoxyuridine, TFT). These compounds are 5-substituted analogues of 2′-deoxythymidine (dThd), the natural precursor of DNA synthesis.

Bromovinyldeoxyuridine and interferon treatment in ulcerative herpetic keratitis: a double masked study.

Bromovinyldeoxyuridine is a potent and safe antiherpes compound that in combination with a placebo treatment promoted the partial and complete healing of herpetic epithelial disease in 22 patients in average times of 4-6 days and 8.5 days respectively. However, when BVDU was combined with 1.5 X 10(6) IU of recombinant a 2C interferon, partial and complete healing times for keratitis in 19 patients were reduced to 2-6 days and 4-6 days respectively. No toxic effects of the medications were observed in any patient.

Cytological and immunohistochemical study of the limbal form of vernal keratoconjunctivitis by the replica technique.

The cellular composition of the inflammatory infiltrate present in 13 patients with the limbal form of vernal keratoconjunctivitis was examined in the conjunctival scrapings and the limbic replicas by means of Giemsa stain and immunohistochemistry. Conjunctival scrapings showed the presence of mast cells, lymphocytes, plasma cells, polymorphonuclear leucocytes, and very few basophils in all the specimens. Eosinophils were present in only four scrapings. The superficial epithelium of the limbic lesion and the adjacent cornea and conjunctiva was studied by the replica technique. The limbic lesion area showed the presence of necrotic epithelial cells mixed with inflammatory cells, including eosinophils, mast cells, lymphocytes, plasma cells, and polymorphonuclear leucocytes and very few basophils. Most of the inflammatory cells were HLA-DR+. Many OKT6+ cells were present, indicating the presence of Langerhans cells. T-lymphocytes including a few helper/inducer cells and many suppressor/cytotoxic cells, were detected in the infiltrate. In addition many B-lymphocytes were observed. These findings suggest that other immune mechanisms in addition to type 1 reaction are involved in the pathogenesis of the disease.

Effects of commercial ophthalmic drugs on rabbit corneal epithelium

Surface changes on rabbit cornea were studied by scanning electronmicroscopy after instillation of commercial corticosteroid and antibiotic collyria extensively used in ophthalmic practice. All the collyria caused loss of surface microvilli and cell damage. The collyrium containing dexamethasone, neomycin, and benzalkonium chloride caused less damage than similar collyrium with polymyxin, but without dexamethasone. On the other hand the collyrium containing dexamethasone and neomycin was still less damaging than similar collyrium with benzalkonium chloride as preservative. Chloramphenicol with dexamethasone caused damage similar to collyria containing dexamethasone, neomycin, polymyxine B sulphate, and benzalkonium chloride. Mit Hilfe der Raster-Elektronenmikroskopie wurden Oberflächenveränderungen in der Kaninchenhornhaut untersucht nach Instillation von handelsüblichen Cortison- und Antibiotica-Augentropfen, wie sie in großem Stil in der täglichen Praxis verwendet werden. Alle Augentropfen verursachten Verlust von Oberflächen-Mikrovilli und Zellschädigungen. Die Tropfen, welche Dexamethason, Neomycin und Benzalkonium-Chlorid enthielten, verursachten mehr Schädigung als ähnliche Tropflösungen mit Polymyxin, aber ohne Dexamethason. Andererseits waren die Tropfen mit Dexamethason und Neomycin immer noch weniger schädigend als ähnliche Tropflösungen mit Benzalkonium-Chlorid als Präservierungsmittel. Chloramphenikol mit Dexamethason verursachte ähnliche Schädigungen als Tropflösungen, welche Dexamethason, Neomycin und Polymyxin-B-Sulfat und Benzalkonium Chlorid enthielten.