Soliman A Al-Kharashi

Deep anterior lamellar keratoplasty for keratoconus.

Purpose: To assess the endothelial toxicity and the microbiological efficacy of moxifloxacin (250 μg/mL) as an additive to Optisol-GS®. Methods: Five hundred nine donor rims were studied. One half of each donor rim was placed in standard Optisol-GS® and the other half of the rim in Optisol-GS® fortified with moxifloxacin (250 μg/mL). All rims were refrigerated for 24 hours at 3°C and placed in thioglycolate broth and incubated at 37°C for 7 days. One pair of donor buttons not used in transplantation stored in each solution was examined for endothelial changes by using electron microscopy (EM). A second pair of cornea buttons was examined for toxicity by endothelial staining with 0.3% trypan blue and 0.2% alizarin red. All endothelial cells that stained (nonviable cells) and nonstained cells (viable cells) were counted, and the ratio of nonviable cells was calculated. Results: The rate of culture-positive donor rims in the Optisol-GS® group was 11.9% (61/509) and in the moxifloxacin-fortified Optisol-GS® media was 2.5% (13/509). The difference was statistically significant (P < 0.01; χ2 test). There was no difference in the cellular morphology of the button stored in moxifloxacin-fortified Optisol-GS® compared with Optisol-GS® using EM. In the bioassay, the rate of nonviable cells in the moxifloxacin-fortified media compared with the control media was nonsignificant (P > 0.05). Conclusion: Moxifloxacin (250 μg/mL) seems to be safe as an additive agent for cornea storage media. It significantly reduces the rate of positive rim cultures and shows no signs of endothelial cytotoxicity as viewed by EM and by a bioassay of trypan blue and alizarin red.

Chemokines in the limbal form of vernal keratoconjunctivitis

BACKGROUND/AIMS Chemokines are a family of low molecular weight cytokines that attract and activate leucocytes. The CC chemokines act on eosinophils, basophils, monocytes, and lymphocytes, suggesting that they play an important part in allergic diseases. The aims of this study were to investigate the expression of the CC chemokines, RANTES, eotaxin, monocyte chemotactic protein (MCP) 1, MCP-2, and MCP-3 in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and to determine the cellular source of these chemokines. METHODS Conjunctival biopsy specimens from nine subjects with active VKC, and six control subjects were studied by immunohistochemical techniques using a panel of monoclonal and polyclonal antibodies directed against RANTES, eotaxin, MCP-1, MCP-2, and MCP-3. The phenotype of inflammatory cells expressing chemokines was examined by sequential double immunohistochemistry. RESULTS In the normal conjunctiva, superficial epithelial cells showed a constitutive, weak cytoplasmic expression of eotaxin. Few inflammatory cells in the perivascular areas expressed RANTES, MCP-1, MCP-2, and MCP-3. In VKC specimens, the epithelium showed intense cytoplasmic eotaxin staining in all cells, and cytoplasmic RANTES staining mainly in the superficial layers. Furthermore, RANTES and eotaxin were expressed on the vascular endothelium mainly in the upper substantia propria. Compared with normal controls, VKC specimens showed significantly more inflammatory cells expressing RANTES, eotaxin, MCP-1, and MCP-3 (p<0.001, 0.0028, 0.0092, and <0.001, respectively). In VKC specimens, the numbers of inflammatory cells expressing RANTES were significantly higher than the numbers of inflammatory cells expressing eotaxin, MCP-1, and MCP-2 (all p values <0.001). Colocalisation studies revealed that the majority of inflammatory cells expressing chemokines were CD68 positive monocytes/macrophages. CONCLUSIONS These results demonstrate an increase in the expression of RANTES, eotaxin, MCP-1, and MCP-3 in the conjunctiva of patients with VKC compared with control subjects. These data suggest a potential role for these chemokines in the pathogenesis of VKC. Antagonists of chemokine receptors may provide new therapeutic modalities in VKC.

Adhesion molecules in vernal keratoconjunctivitis

AIMS/BACKGROUND Adhesion molecules play a key role in the selective recruitment of different leucocyte population to inflammatory sites. The purpose of the present study was to investigate the presence and distribution of adhesion molecules in the conjunctiva of patients with vernal keratoconjunctivitis (VKC). METHODS The presence and distribution of adhesion molecules were studied in 14 conjunctival biopsy specimens from seven patients with active VKC and in four normal conjunctival biopsy specimens. We used a panel of specific monoclonal antibodies (mAbs) directed against intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-3 (ICAM-3), lymphocyte function associated antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leucocyte adhesion molecule-1 (ELAM-1). In addition, a panel of mAbs were used to characterise the composition of the inflammatory infiltrate. RESULTS In the normal conjunctiva, ICAM-1 was expressed on the vascular endothelium only, LFA-1 and ICAM-3 on epithelial and stromal mononuclear cells , and VLA-4 on stromal mononuclear cells. The expression of VCAM-1 and ELAM-1 was absent. The number of cells expressing adhesion molecules was found to be markedly increased in all VKC specimens. This was concurrent with a heavy inflammatory infiltrate. Strong ICAM-1 expression was induced on the basal epithelial cells, and vascular endothelial cells. Furthermore, ICAM-1 was expressed on stromal mononuclear cells. LFA-1 and ICAM-3 were expressed on the majority of epithelial and stromal infiltrating mononuclear cells. VLA-4 expression was noted on stromal mononuclear cells. Compared with controls, VKC specimens showed significantly more ICAM-3+, LFA-1+, and VLA-4+ cells. VCAM-1 and ELAM-1 were induced on the vascular endothelial cells. CONCLUSIONS Increased expression of adhesion molecules may play an important role in the pathogenesis of VKC.

Immunopathogenesis of conjunctival scarring in trachoma

Purpose Trachoma, a chronic follicular conjunctivitis caused by infection with Chlamydia trachomatis, is the leading cause of preventable blindness. The blinding complications are associated with progressive conjunctival scarring that may result from immunologically mediated responses. We studied the processes involved in the regulation of inflammation and fibrosis in trachoma by investigating the expression of fibrogenic cytokines in the conjunctiva.Methods We studied conjunctival biopsy specimens obtained from nine subjects with active trachoma and from four control subjects. We used immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies directed against interleukin-1α (IL-1α), interleukin-lβ (IL-1β), tumour necrosis factor-α (TNF-α) and platelet-derived growth factor (PDGF). In addition, we characterised the composition of the inflammatory infiltrate by the use of a panel of monoclonal antibodies. Sirius red and Van Gieson stains were used to characterise the extent of fibrous tissue in the substantia propria.Results Trachoma specimens showed greater numbers of inflammatory cells than control specimens. The expression of cytokines was absent in the normal conjunctiva. Cytoplasmic IL-1α and IL-1β expression was noted in the conjunctival epithelium in all trachoma specimens. IL-1α, IL-1β, TNF-α and PDGF were detected in macrophages infiltrating the substantia propria. B lymphocytes predominated over T lymphocytes in six trachoma biopsies with fibrosis confined to the deep substantia propria, whereas T lymphocytes predominated over B lymphocytes in three biopsies with more extensive fibrosis. In all trachoma biopsies helper/inducer T lymphocytes outnumbered suppressor/cytotoxic T lymphocytes.Conclusions The upregulated local production of IL-1α, IL-β, TNF-α and PDGF might contribute to conjunctival damage and scarring in trachoma.

Full panretinal photocoagulation and early vitrectomy improve prognosis of retinal vasculitis associated with tuberculoprotein hypersensitivity (Eales’ disease)

Background/aims: Eales’ disease is an uncommon vasoproliferative retinal disease affecting otherwise healthy young men that is characterised by obliterative retinal periphlebitis, with sequelae such as recurrent vitreous haemorrhage and traction retinal detachment. This study was undertaken to determine whether visual prognosis of Eales’ disease could be improved by appropriate medical and surgical treatment. Methods: The authors retrospectively studied 30 patients (46 eyes) who were treated from 1992 to 2001. Recorded data included patient age, sex, race, medical history, medications, results of the ophthalmological examination, results of diagnostic laboratory evaluation, and details of systemic and surgical treatments. The mean follow up was 10.6 months. Results: 19 patients (23 eyes) who presented with active periphlebitis received systemic steroids and antituberculous therapy. Extensive full panretinal photocoagulation was performed in 21 eyes that presented with new vessel formation and peripheral capillary closure with or without vitreous haemorrhage. Vitrectomy and endolaser panretinal photocoagulation was necessary in 15 eyes, for severe non-clearing vitreous haemorrhage in 11 eyes and vitreous haemorrhage with traction retinal detachment in four eyes. Complete regression of the disease was achieved in all eyes. Vitrectomy resulted in a significant visual improvement with 14 of the 15 eyes (93.3%) achieving ≥20/200 visual acuity. Overall, the distribution of visual acuities among eyes improved from presentation to final follow up, with 36.4% of eyes having 20/40 or better acuity at presentation compared with 63.6% of eyes by final follow up. Conclusions: These results suggest that aggressive treatment of Eales’ disease with systemic steroids and antituberculous therapy, full panretinal photocoagulation and early vitrectomy, when necessary, may result in improving the anatomic and visual outcome.

Expression of T lymphocyte chemoattractants and activation markers in vernal keratoconjunctivitis

Background/aims: T lymphocytes are present in increased numbers in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and their activation has a central role in the pathogenesis of the chronic allergic inflammatory reactions seen in VKC. The aims of this study were to examine the expression of three recently described potent T lymphocyte chemoattractants, PARC (pulmonary and activation regulated chemokine), macrophage derived chemokine (MDC), and I-309, the MDC receptor CCR4, and T lymphocyte activation markers, CD25, CD26, CD62L, CD71, and CD30, and to correlate them with the counts of CD3+ T lymphocytes in the conjunctiva of patients with VKC. Method: Conjunctival biopsy specimens from 11 patients with active VKC, and eight control subjects were studied by immunohistochemical techniques using a panel of monoclonal and polyclonal antibodies directed against PARC, MDC, I-309, CCR4, CD25, CD26, CD62L, CD71, and CD30. The numbers of positively stained cells were counted. The phenotype of inflammatory cells expressing chemokines was examined by double immunohistochemistry. Results: In the normal conjunctiva, vascular endothelial cells in the upper substantia propria showed weak immunoreactivity for CD26. There was no immunoreactivity for the other antibodies. VKC specimens showed inflammatory cells expressing PARC, MDC, and I-309. The numbers of PARC+ inflammatory cells were higher than the numbers of MDC+ and I-309+ inflammatory cells and the mean values of the three groups differed significantly (17.0 (SD 10.1); 9.5 (9.9), and 4.3 (7.9), respectively, p = 0.0117, ANOVA). The numbers of PARC+ inflammatory cells had the strongest correlation with the numbers of CD3+ T lymphocytes. Few CCR4+ inflammatory cells were observed in only three specimens. Double immunohistochemistry revealed that all inflammatory cells expressing chemokines were CD68+ monocytes/macrophages. The numbers of CD25+ T lymphocytes were higher than the numbers of CD26+, CD62L+, CD71+, and CD30+ T lymphocytes and the mean values of the five groups differed significantly (46.2 (27.9), 30.7 (16.0), 20.1 (8.6), 7.8 (7.7), and 6.5 (4.0), respectively, p <0.001, ANOVA). The numbers of CD25+ T lymphocytes had the strongest correlation with the numbers of CD3+ T lymphocytes. Conclusion: These results suggest a potential role for PARC, MDC, and I-309 in attracting T lymphocytes into conjunctiva in VKC. T lymphocytes in VKC are activated and express several activation markers which might contribute to the pathogenesis of VKC.

Heparin and heparin-surface-modification reduce Staphylococcus epidermidis adhesion to intraocular lenses

Bacterial adherence to intraocular lenses (IOLs) could be the cause of endophthalmitis following cataract surgery and lens implantation. The majority of cases of postoperative endophthalmitis are caused by microflora that reside on or near the eye of the patient. Staphylococcus epidermidis commonly colonizes the eyelid margin and conjunctiva and is the most common organism causing postoperative endophthalmitis. In this study, the in vitro adherence of S. epidermidis to regular poly-methyl methacrylate (PMMA) IOLs and to heparin-surface-modified (HSM) PMMA IOLs was investigated. The effects of heparin and antibiotics in solution on the adherence of bacteria to regular PMMA IOLs were evaluated. Adhesion of bacterial cells to IOLs was determined by counting the viable cells attached to the lenses. Significantly, fewer S. epidermidis attached to HSM-PMMA IOLs and to regular PMMA IOLs treated with heparin than to PMMA IOLs (p < 0.001). Furthermore, bacteria attached in significantly lower numbers to regular PMMA IOLs treated with heparin than to HSM-PMMA IOLs (p = 0.0031). Antibiotics in solution had no significant effect on bacterial adherence to PMMA IOLs. These data indicate that the use of HSM-PMMA IOLs and treatment of PMMA IOLs with heparin could diminish the incidence of postoperative endophthalmitis and intraocular inflammation associated with IOL implantation.

Expression of gelatinase B in trachomatous conjunctivitis

BACKGROUND/AIMS Gelatinase B is a matrix metalloproteinase involved in extracellular matrix (ECM) breakdown often associated with scarring and other pathological disorders. It was investigated whether gelatinase B is involved in the pathogenesis of ECM degradation associated with trachomatous conjunctivitis. METHODS Conjunctival biopsy specimens obtained from six patients with active trachoma, six patients with active vernal keratoconjunctivitis (VKC), and seven control subjects were studied. Immunohistochemical techniques and a specific monoclonal antibody against human gelatinase B were used, and a monoclonal antibody against macrophage CD68 to identify mononuclear cells with gelatinase B immunoreactivity. In addition, quantitative zymography was used to compare the activity of gelatinase B in conjunctival biopsy specimens from seven patients with active trachoma and seven control subjects. RESULTS Gelatinase B was detected by immunohistochemistry only in polymorphonuclear cells located in the vascular lumens in three normal conjunctival biopsy specimens. In all trachoma specimens and in five VKC specimens, gelatinase B was localised in monocyte/macrophage cells, positive for the CD68 marker, and in polymorphonuclear cells. The majority of the latter cell type was located in intravascular spaces. Compared with VKC specimens, trachoma specimens showed significantly more immunoreactive gelatinase B monocyte/macrophage cells (52.3 (21.9)v 8.2 (6.4); p <0.001) and polymorphonuclear cells (23.2 (14.2) v 6.3 (5.4); p = 0.013). Activated macrophages with giant cell morphology clearly stained with the gelatinase B specific monoclonal antibody were observed in trachoma specimens. Zymography revealed that gelatinase B levels in trachoma specimens were significantly higher than the levels found in normal conjunctiva (1739.6 (1078.3)v 609.3 (395.9) scanning units; p = 0.0127). CONCLUSIONS The increased activity of gelatinase B and numbers of inflammatory cells containing gelatinase B in trachoma specimens suggest that this enzyme plays a part in the pathogenesis of conjunctival scarring in trachoma.

An immunohistochemical study of collagens in trachoma and vernal keratoconjunctivitis.

Purpose To analyse the distribution and types of collagen in the conjunctiva of patients with trachoma and vernal keratoconjunctivitis (VKC).Methods Conjunctival biopsy specimens were collected from 9 patients with active trachoma, 9 patients with scarred trachoma, 6 patients with active VKC and 9 control subjects. The presence and distribution of collagen was assessed microscopically with immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies directed against types I, III, IV and V collagen.Results In normal conjunctiva, the staining for types I and III collagen was localised to the substantia propria. Type IV collagen was located in the epithelial, vascular endothelial and accessory lacrimal gland basement membranes. Staining for type V collagen was absent. New type V collagen deposition close to basement membranes was noted in active trachoma, scarred trachoma and VKC. The extent of deposition of type V collagen was markedly increased in scarred trachoma when compared with active trachoma. Staining for type IV collagen showed irregularly thickened epithelial basement membrane in active trachoma, and a marked increase in basement membrane type IV collagen was noted in scarred trachoma. Immunoreactivity of types I and III collagen increased in active trachoma and decreased in scarred trachoma. VKC conjunctiva contained increased amounts of types I, III and IV collagen due to marked increase in the thickness of vascular endothelial basement membrane and very prominent deposition of types I and III collagen around stromal vessels.Conclusions Our data indicate new type V collagen formation in the conjunctiva from patients with active trachoma, scarred trachoma and VKC. Increased deposition of types I, III and IV collagen is noted in VKC and active trachoma. Our findings suggest that increased deposition of type IV collagen and new type V collagen formation contributes to the development of conjunctival fibrosis in scarred trachoma.

The T-lymphocyte chemoattractant Mig is highly expressed in vernal keratoconjunctivitis

PURPOSE To examine the expression of the three interferon-gamma-inducible CXCR3-binding chemokines, CXCL10/IP-10 (interferon-gamma-inducible protein of 10 KDa), CXCL9/Mig (monokine induced by interferon-gamma), and CXCL11/I-TAC (interferon-inducible T-cell alpha chemoattractant) in the conjunctiva of patients with vernal keratoconjunctivitis (VKC). These chemokines exhibit potent T-lymphocyte chemoattractant activity. DESIGN Immunohistochemical study. METHODS Conjunctival biopsy specimens from 16 patients with active VKC and nine control subjects were studied by immunohistochemical techniques using monoclonal antibodies directed against IP-10, Mig, and I-TAC. The phenotype of inflammatory cells expressing chemokines was examined by double immunohistochemistry. RESULTS In the normal conjunctiva, very weak Mig immunoreactivity was observed on basal epithelial cells and on vascular endothelial cells in the upper substantia propria. There was no immunoreactivity for the other chemokines. In all VKC specimens, strong immunoreactivity for Mig was expressed by epithelial cells, vascular endothelial cells, and inflammatory mononuclear cells. Inflammatory mononuclear cells expressing IP-10 and I-TAC were noted in 10 and nine specimens, respectively. The numbers of Mig(+) inflammatory cells were significantly higher than the numbers of IP-10(+) and I-TAC(+) inflammatory cells (P <.001). Inflammatory cells expressing Mig were CD4(+) T-helper/inducer cells (71.6 +/- 3.2%), CD8(+) T-cytotoxic/suppressor cells (19.5 +/- 1.5%), and CD68(+) monocytes/macrophages (5.3 +/- 5%). All inflammatory cells expressing IP-10 and I-TAC were CD68(+) monocytes/macrophages. CONCLUSIONS The CXC chemokine Mig is selectively and highly expressed in VKC suggesting a pathogenic role of the chemokine receptor CXCR3 and the ligand Mig in the recruitment of activated T lymphocytes.