Double vital staining using trypan blue and infracyanine green in macular pucker surgery.

AIMS: To study the clinical properties of double vital staining in premacular fibrosis, facilitating complete removal of all epiretinal tissue. METHODS: In a two step surgery, the epiretinal pucker was removed after staining with trypan blue, whereafter the inner limiting membrane was peeled after staining with infracyanine green. RESULTS: In all 30 patients, a separate epiretinal layer and inner limiting membrane were removed from the macular area. Pathological examination showed different histological properties of the removed layers. An increased visual acuity was measured in 26 patients, and a slightly decreased visual acuity in one patient. CONCLUSION: The described double staining technique could be a novel valuable tool that may help to achieve optimal anatomical and functional recovery after surgery for premacular fibrosis

Characterization of Toxoplasma gondii-specific T cells recovered from vitreous fluid of patients with ocular toxoplasmosis.

PURPOSE: The mechanisms involved in reactivations of latent ocular Toxoplasma gondii (Tg) infections in immunocompetent patients are poorly understood. In view of the possible role of T cells in the immunopathogenesis of the disease, ocular infiltrating T cells obtained from patients with recurrent ocular toxoplasmosis were characterized phenotypically and functionally. METHODS: Ocular infiltrating T cells were recovered from vitreous fluid (VF) samples of 10 patients with active recurrent ocular toxoplasmosis. Two patients with uveitis of other origins were included as control subjects. T-cell lines (TCLs) were generated by mitogenic stimulation and tested for reactivity to Tg and human retinal protein extracts. The TCLs of three patients were cloned by limiting dilution. Tg-reactive T-cell clones (TCCs) were characterized with respect to their phenotype, T-cell receptor variable (TCR V)-beta gene usage, HLA restriction, and cytokine secretion profile. RESULTS: Reactivity to Tg could be detected only in the TCLs of patients with ocular toxoplasmosis. None of the TCLs showed reactivity to human retinal antigens. All tested intraocular Tg-specific TCCs (n = 23) were CD3+CD4+ and displayed differential TCR Vbeta usage. Twenty-one TCCs were HLA-DR restricted and two TCCs were restricted by HLA-DP. The majority of the intraocular Tg-specific TCCs showed a bias toward a T-helper (Th)0-Th2 cytokine profile. CONCLUSIONS: The data indicate that T cells specific for the triggering microorganism infiltrate the eye of patients with recurrent ocular toxoplasmosis. The functional characteristics of the VF-derived Tg-specific T cells and their presence at the site of inflammation suggest their involvement in the local inflammatory response of ocular toxoplasmosis.

ICG staining of the inner limiting membrane facilitates its removal during surgery for macular holes and puckers.

Formation of a macular hole is an uncommon idiopathic disease whereby a disruption of the neural retinal layer is formed at the foveal area. It has been suggested that tangential traction of the inner limiting membrane (ILM) on the neuroretina may contribute to the formation of a macular hole. Removal of the ILM has been associated with very high closure rates in macular hole surgery. However, since the inner limiting membrane is a transparent layer, it can be difficult to achieve complete removal from the underlying neural retina. Also in surgery for macular pucker formation, whereby growth of epiretinal tissue induces metamorphopsia, removal of the ILM may play a beneficial role. This paper describes a method facilitating the removal of the inner limiting membrane by staining it with indocyanine green.

High concentration of dexamethasone in aqueous and vitreous after subconjunctival injection.

PURPOSE: To determine the dexamethasone concentration in aqueous, vitreous, and serum of patients after a subconjunctival injection with dexamethasone disodium phosphate and to compare the effectiveness of a subconjunctival injection as a method of delivering dexamethasone into the vitreous with that of two previously tested routes: peribulbar injection and oral administration. METHODS: In a prospective study, 50 phakic patients who underwent a pars plana vitrectomy received a single subconjunctival injection with 2.5 mg of dexamethasone disodium phosphate, aqueous solution (after topical anesthesia and a subconjunctival injection with lidocaine) at varied intervals before surgery. An aqueous and a vitreous sample were taken from each patient, and serum samples were collected at multiple time points from nine of 50 patients. Dexamethasone concentrations were measured by radioimmunoassay. RESULTS: The estimated maximum dexamethasone concentration in the aqueous was 858 ng per ml at 2.5 hours after injection, and in the vitreous, 72.5 ng per ml at 3 hours. In serum, a mean maximum concentration of 32.4 ng per ml was measured at approximately 30 minutes after injection. CONCLUSIONS: Subconjunctival injection of 2.5 mg of dexamethasone disodium phosphate resulted in an estimated vitreous dexamethasone peak concentration three and 12 times higher, respectively, than after a peribulbar injection of 5 mg of dexamethasone disodium phosphate and an oral dose of 7.5 mg of dexamethasone. Thus, a subconjunctival injection is the most effective method of delivering dexamethasone into both the anterior and posterior segments of the eye. Systemic drug absorption is considerable and is of the same order of magnitude as after peribulbar injection.

Follow-up of functionally lost eyes after vitrectomy and silicone oil tamponade for tractional retinal detachment.

BACKGROUND: Is there any association between, on the one hand, retention or removal of silicone oil or any specific ocular finding in patients with functionally lost eyes after vitrectomy and silicone oil tamponade for tractional retinal detachment and, on the other, a greater chance of preservation of the eye? This information is important in deciding whether to remove silicone oil, as well as in counseling patients about their individual chances of preserving their eye. METHODS: Seventy-three consecutive patients with a functionally lost eye with a minimum follow-up of 3 years were retrospectively studied. The relation between the variables at study entry or the removal of silicone oil during the follow-up period and a subsequent intervention (enucleation, evisceration or conjunctival cover with a scleral shell) were tested for statistical significance with Cox proportional hazards analysis. RESULTS: The absence or removal of silicone oil was not associated with a greater chance of finally preserving the eye. Nor could we identify other factors which predicted better chances of preservation. CONCLUSION: The notion that functionally lost eyes after treatment with vitrectomy and silicone oil tamponade for complicated tractional retinal detachment have better chances of preservation of the eye without silicone oil is not supported by our study.

Analysis of immunoregulatory cytokines in ocular fluid samples from patients with uveitis.

PURPOSE: To investigate the T-helper cell cytokine profiles in two well-defined clinical uveitis entities caused by an infectious mechanism. METHODS: Cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and interferon [IFN]-gamma) were measured in ocular fluid samples obtained from patients with herpes simplex- or varicella-zoster virus-induced acute retinal necrosis (ARN; n = 17) and toxoplasma chorioretinitis (n = 27) using enzyme-linked immunosorbent assay techniques. The data were compared with data for 51 control samples taken during cataract surgery (n = 10), vitrectomy in diabetic retinopathy (n = 10), eye bank eyes (n = 10) and with samples from patients with "autoimmune" uveitis (n = 21). RESULTS: Interleukin-6 was detected in 44 of 51 control samples and 43 of 44 eyes of patients with uveitis. The highest levels in the control samples were detected in 9 of 10 vitreous samples from patients with diabetic retinopathy (mean, 648 pg/ml). In 8 of 10 samples taken from patients during cataract surgery and in 7 of 10 eye bank eyes the amount of IL-6 was significantly lower (mean, 10 pg/ml and 136 pg/ml, respectively). Interleukin-6 levels in patients with ARN (mean, 1436 pg/ml) were significantly higher than in those with toxoplasma chorioretinitis (mean, 272 pg/ml). Interleukin-2 was detected in one of the samples from patients with toxoplasma chorioretinitis (1105 pg/ml) and in three samples from the control subjects suffering from Fuchs' heterochromic anterior uveitis (mean, 752 pg/ml). No IL-4 (<2 pg/ml) was detected either in patient or control samples. Interferon-gamma could be detected in 7 of 17 ARN patients (range, 277-3483 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 12-250 pg/ml), and in 1 of 21 of the samples from control subjects with uveitis (31 pg/ml) but was absent in nonuveitic control samples. Interleukin-10 was detected in 10 of 17 ARN patients (range, 29-3927 pg/ml), in 13 of 27 samples from patients with toxoplasma chorioretinitis (range, 4-67 pg/ml), and in only 3 of 51 control samples (6 pg/ml, 16 pg/ml, and 20 pg/ml). CONCLUSIONS: Various immunoregulatory cytokines (IL-6, IL-10, and IFN-gamma) were detected in ocular fluid samples from patients with uveitis. A separate role for either a T-helper type 1 or T-helper type 2 response in the pathogenesis of clinical uveitis could not be proven.

Differences between intraocular and serum antibody responses in patients with ocular toxoplasmosis.

PURPOSE: To investigate the antigen specificity of the intraocular anti-Toxoplasma gondii antibody response in patients with ocular toxoplasmosis. METHODS: Paired ocular fluid and serum samples were collected from 13 patients with active ocular toxoplasmosis. Serum IgM anti-T. gondii antibodies were tested to distinguish recently-acquired from chronic infection. Anti-T. gondii IgG specificity was analyzed by immunoblotting using a crude T. gondii extract. RESULTS: Two of the 13 patients tested were IgM positive and considered to have acquired ocular toxoplasmosis. The antibody specificity in ocular fluid and serum of these two patients was similar, whereas in the patients with presumed chronic disease, marked differences could be observed. Most ocular fluid samples contained antibodies that stained a 28-kD antigen more intensely than did antibodies from paired serum samples. Using absorption and elution experiments, we demonstrated that this 28-kD protein was identical to the GRA-2 antigen, which is expressed in both the tachyzoite and the bradyzoite stages of the parasite. CONCLUSIONS: Our results show that the intraocular T. gondii antibody response of patients with recurrent ocular toxoplasmosis differs from the systemic response. This finding may have implications for our understanding of the immunopathogenesis of ocular toxoplasmosis and could be employed to improve diagnosis of the disease.

T cells specific for the triggering virus infiltrate the eye in patients with herpes simplex virus-mediated acute retinal necrosis.

Acute retinal necrosis (ARN) is a rare, potentially blinding retinal disease resulting from ocular infections with herpes simplex virus (HSV) or varicella-zoster virus (VZV). To determine the antigen specificity and functional characteristics of ocular infiltrating T cells in ARN, T cells were isolated and expanded nonspecifically from intraocular fluid (IOF) samples from 2 patients with HSV-1- and 3 with VZV-mediated ARN. HSV-specific T cell reactivity could be detected only in the IOF-derived T cell lines (TCLs) of the 2 patients with HSV-mediated ARN. These TCLs consisted of both HSV type-common and type-specific CD4+ and CD8+ T cell clones (TCCs) with differential T cell receptor usage. Irrespective of their phenotype, the TCCs were cytolytic and secreted interferon-gamma, tumor necrosis factor-alpha, interleukin-4, and interleukin-5. In both patients, the antigen specificity of a substantial number of HSV-1-specific TCCs could be mapped to approximately 0.67-0.73 HSV-1 map units. The data presented suggest the contribution of T cells, specific for the triggering virus, to the pathogenesis of ARN.

Increased presence of Epstein-Barr virus DNA in ocular fluid samples from HIV negative immunocompromised patients with uveitis.

AIMS: To investigate whether routine testing for Epstein-Barr virus (EBV) is necessary in the examination of a patient with uveitis. METHODS: Intraocular EBV DNA was determined in 183 ocular fluid samples taken from patients with AIDS and uveitis, HIV negative immunocompromised uveitis, acute retinal necrosis, toxoplasma chorioretinitis, intraocular lymphoma, anterior uveitis, and miscellaneous uveitis of unknown cause. In 82 samples from this group of patients paired serum/ocular fluid analysis was performed to detect local antibody production against EBV. Controls (n = 46) included ocular fluid samples taken during surgery for diabetic retinopathy, macular pucker, or cataract. RESULTS: Serum antibody titres to EBV capsid antigen proved to be significantly increased in HIV negative immunocompromised patients with uveitis (p < 0.01) compared with controls. Local antibody production revealed only three positive cases out of 82 patients tested, two results were borderline positive and one patient had uveitis caused by VZV. EBV DNA was detected in three out of 46 control ocular fluid samples. In the different uveitis groups EBV DNA was noted, but was not significantly higher than in the controls, except in six out of 11 HIV negative immunocompromised patients (p = 0.0008). In four out of these six cases another infectious agent (VZV, HSV, CMV, or Toxoplasma gondii) had previously been identified as the cause of the uveitis. CONCLUSIONS: When comparing various groups of uveitis patients, EBV DNA was found more often in HIV negative immunocompromised patients with uveitis. Testing for EBV does not have to be included in the routine management of patients with uveitis, since indications for an important role of this virus were not found in the pathogenesis of intraocular inflammation.

Oligoclonal activation of CD4+ T lymphocytes in posterior uveitis.

Several lines of evidence support an important role for activated T lymphocytes in the perpetuation of autoimmune intraocular inflammatory disease (posterior uveitis). In this study peripheral blood lymphocytes (PBL) were examined by three-colour flow cytometry to assess the distribution of IL-2 receptors (IL-2R) among CD4+ and CD8+ T cell subsets in patients with active posterior uveitis and control subjects. Patients with uveitis (n = 70) showed a significant increase in PBL expressing the alpha-chain (Tac) of the IL-2R compared with controls (n = 28) (34.2% versus 29.6%) (P < 0.05). This increased Tac expression was present on both the CD4+ subset (25.7% versus 20.9%) (P < 0.05) and the CD8+ subset (2.5% versus 1.8%) (P < 0.05) of lymphocytes. We also examined whether the activated CD4+ PBL from uveitis patients (n = 30) showed a dominant pattern of T cell receptor (TCR) gene rearrangement, suggestive of an oligoclonal response to a small number of antigenic peptides. A significant increase in the usage of the V alpha 2.3 TCR family by activated but not by non-activated CD4+ PBL was detected in patients (3.9% versus 3.4%) (P < 0.05) compared with controls. There was evidence of oligoclonal activation of CD4+ PBL in 11/30 patients (36.7%) but in none of the controls (n = 10). However, different V alpha or V beta TCR families were selectively activated among and even within individual patients. The heterogeneity in TCR expression among patients with active intraocular inflammatory disease is discussed.

Distribution of IL-2R and CD45Ro expression on CD4+ and CD8+ T-lymphocytes in the peripheral blood of patients with posterior uveitis.

Different lines of evidence support a major role for activated T-lymphocytes in the pathogenesis of posterior uveitis. The initial site of activation of these autoreactive T-cells, either locally in the eye or in the peripheral immune compartment, is still unknown. This study was undertaken to investigate whether with currently available techniques, it is possible to detect alterations in the levels and subsets of activated T-cells in the peripheral blood of patients with posterior uveitis. For this reason, 3-colour immunofluorescent staining was performed to assess the distribution of IL-2 receptors (IL-2R) and the CD45RO-antigen on CD4+ and CD8+ subsets of peripheral blood lymphocytes (PBLs) from patients with posterior uveitis (n = 29). Only the subgroup of patients with posterior uveitis as part of a systemic immune-mediated disease (sarcoidosis, Behçet's disease) (n = 9) showed a significant increase in IL-2R expression on peripheral blood lymphocytes (p less than 0.005) when compared to normals (n = 12). This increased expression was reflected much more significantly in the CD4+ (p less than 0.0005) rather than in the CD8+ subset (p less than 0.05) of lymphocytes. In contrast, no significant increase in CD45RO expression on either subset of T lymphocytes was found in any subgroup of posterior uveitis in comparison with normals.