Subretinal implantation of electrospun, short nanowire, and smooth poly(ε-caprolactone) scaffolds to the subretinal space of porcine eyes.

Biodegradable scaffolds play an important adjunct role in transplantation of retinal progenitor cells (RPCs) to the subretinal space. Poly(ε-Caprolactone) (PCL) scaffolds with different modifications were subretinally implanted in 28 porcine eyes and evaluated by multifocal electroretinography (mfERG) and histology after 6 weeks of observation. PCL Short Nanowire, PCL Electrospun, and PCL Smooth scaffolds were well tolerated in the subretinal space in pigs and caused no inflammation and limited tissue disruption. PCL Short Nanowire had an average rate of preserved overlying outer retina 17% higher than PCL Electrospun and 25% higher than PCL Smooth. Furthermore, PCL Short Nanowire was found to have the most suitable degree of stiffness for surgical delivery to the subretinal space. The membrane-induced photoreceptor damage could be shown on mfERG, but the reductions in P1 amplitude were only significant for the PCL Smooth. We conclude that of the tested scaffolds, PCL Short Nanowire is the best candidate for subretinal implantation.

Requirement of B7-mediated costimulation in the induction of experimental autoimmune anterior uveitis.

PURPOSE: To study the role of costimulatory signaling through the CD28-B7 interaction in experimental autoimmune anterior uveitis (EAAU). METHODS: Naive Lewis rats were immunized with insoluble melanin-associated antigen (MAA) derived from bovine iris and ciliary body. CTLA4-Fc, a recombinant protein comprised of the extracellular domain of human CTLA4 bound to mouse IgG2a Fc, was used to block the CD28-B7 interaction. A mutant version (CTLA4-Fc-mutant) was used as a control. The effect of CTLA4-Fc on the in vivo induction of disease with MAA was studied. Subsequently, the mechanism by which CTLA4-Fc blocked the interaction of CD28 and B7 was investigated in vivo, using the adoptive transfer of T cells derived from CTLA4-Fc-treated rats, and in vitro, using the proliferative response and cytokine production of MAA-T cells in the presence of CTLA4-Fc. RESULTS: CTLA4-Fc markedly reduced the incidence and severity of EAAU in Lewis rats after sensitization with MAA. The adoptive transfer of sensitized T cells from CTLA4-Fc-treated donors did not induce EAAU in naive recipients. CTLA4-Fc inhibited the expansion of antigen-specific MAA-T cells and the production of TNF-alpha. CONCLUSIONS: The costimulatory signal delivered through CD28-B7 is required for the induction and pathogenesis of EAAU. In the absence of this signal, antigen-specific expansion of MAA reactive T cells as well as production of TNF-alpha is inhibited. Abrogation of this costimulatory signal may be an important therapeutic option for EAAU.

The association of HLA class II with pars planitis.

PURPOSE: To investigate the association of HLA class II alleles with pars planitis. METHODS: Blood samples were obtained from 28 patients with pars planitis seen in the Department of Ophthalmology and Visual Sciences and the Barnes Retina Institute at Washington University, St. Louis, Missouri. RESULTS: HLA-DR15, one of the allelic subtypes of HLA-DR2, was present in 18 (64.3%) of 28 patients vs. 10 (20%) of 50 controls (OR = 7.20, CI = 2.28--23.20, P =.0001). HLA-DR51 (HLA-DRB 5) was present in 16 (57.1%) of 28 patients vs. 6 (12%) of 50 controls (OR = 9.78, CI = 2.79--36.42, P =.0001). HLA-DR17 was present in eight (28.6%) of 28 patients vs. one (2%) of 50 controls (OR = 19.60, CI = 2.29--886.7, P =.0001). CONCLUSION: Pars planitis is associated with an increased frequency of the HLA-DR2 suballele, -DR15, HLA-DR51, and HLA-DR17. These results suggest an immunogenic predisposition exists to pars planitis.

Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation.

PURPOSE: To report the histopathology after retinal pigment epithelial cell transplantation and subfoveal membranectomy in age-related macular degeneration. METHODS: An 85-year-old white woman with bilateral choroidal neovascularization underwent subfoveal membranectomy combined with transplantation of a sheet of human adult retinal pigment epithelium (retinal pigment epithelium) under the foveal center in the right eye. The patient was immunosuppressed postoperatively with prednisone, cyclosporine, and azathioprine. The patient died from congestive heart failure 114 days after surgery. RESULTS: A patch of hyperpigmentation was visible at the transplant site under the foveola after surgery. Mound-like clusters of individual round, large densely pigmented cells were present in the subretinal space and outer retina in this area. There was loss of the photoreceptor outer segments and native retinal pigment epithelium in the center of the transplant bed, with disruption of the outer nuclear layer predominantly over regions of multilayered pigmented cells. Cystic spaces were present in the inner and outer retina. A residual intra-Bruchs membrane component of the original choroidal neovascular complex was present under the transplant site. CONCLUSIONS: The transplant site contained clusters of round, pigmented cells that did not form a uniform monolayer in most areas. The morphology at the transplant site is consistent with the lack of visual improvement seen after surgery in this patient.

Herpes simplex virus type 2 as a cause of acute retinal necrosis syndrome in young patients.

PURPOSE: To determine the causative virus in acute retinal necrosis (ARN) syndrome in a series of patients by calculation of modified Witmer coefficients. DESIGN: Noncomparative case series. PARTICIPANTS: Ten patients with ARN syndrome from four medical centers. METHODS: Aqueous samples, vitreous samples, or both were collected prospectively during surgery from patients with a clinical diagnosis of ARN syndrome. Serologic measures of intraocular and serum antibodies to potentially causative viruses were measured by enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Modified Witmer coefficients (immunoglobulin G and immunoglobulin A) for herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV), and cytomegalovirus (CMV), as well as adenovirus type 2, were calculated from aqueous or vitreous samples, or both. RESULTS: Intraocular antibody measurements were strongly suggestive of a single diagnosis in 9 of 10 patients tested. Modified Witmer coefficients demonstrated intraocular antibody production to HSV in five patients and antibodies to VZV in four patients, and the measurement was inconclusive in one patient. No patients were positive for adenovirus or CMV. Strain-specific antibody titers demonstrated that all HSV-positive patients were reactive only to HSV-2. Herpes simplex virus type 2 was found predominantly in younger patients with ARN syndrome (mean age, 21.2 +/- 10 years; range, 17-39 years), whereas VZV was more commonly seen in older patients (mean age, 40.8 +/- 12.2 years; range, 29-58 years; P = 0.033). Immunoglobulin A testing confirmed immunoglobulin G testing in all patients examined. CONCLUSIONS: Although VZV is thought to be the most common cause of ARN syndrome, HSV-2 is an important cause of ARN syndrome, particularly in younger patients. Because infection with HSV-2 has important medical ramifications, these results suggest that determination of a causal agent should be considered in some cases of ARN syndrome.

Outcome of vitrectomy for retained lens fragments after phacoemulsification.

PURPOSE: To evaluate the incidence of complications and the visual outcome of pars plana vitrectomy in patients with retained lens fragments in the vitreous cavity after phacoemulsification. METHODS: A retrospective chart review of 85 patients who underwent vitrectomy for removal of retained lens fragments at the Barnes Retinal Institute/Washington University Medical Center between 1990 and 1998. RESULTS: At the time of presentation, uveitis (n = 57, 67.1%), increased intraocular pressure >25 mmHg (n = 44, 51.8%), and corneal edema (n = 42, 49.4%) were frequently observed. The initial visual acuity was 20/200 or worse in 61 (71.8%) eyes. However, the final visual acuity after vitrectomy, with 10.1 months follow-up, was 20/40 or better in 44 (51.8%) eyes. The major complication observed was retinal detachment, which was present in seven (8.2%) eyes: four before vitrectomy and three after vitrectomy. Visual outcome after vitrectomy and cataract extraction was compared among three groups based on the timing of the second surgery: < or =7 days postcataract extraction; 8-30 days postcataract extraction; and >30 days postcataract extraction. No statistically significant difference in final visual acuity was observed between the three intervals. CONCLUSIONS: The major complication associated with vitrectomy for retained lens fragments in the vitreous cavity after phacoemulsification was retinal detachment. The timing of vitrectomy did not affect the final visual acuity outcome. Visual prognosis was most closely related to the presence of age-related macular degeneration and cystoid macular edema. The type of intraocular lens did not influence the visual outcome. Management with vitrectomy yielded favorable visual results in most patients with retained lens fragments.

Pubovaginal sling technique utilizing a unique bone anchor instrumentation system.

INTRODUCTION: In the past, the pubovaginal sling (PVS) technique was originally delegated for the treatment of intrinsic sphincter deficiency syndrome (ISD). Today, it has not only undergone a revitalization, but is being recommended for the treatment of all forms of stress urinary incontinence (SUI) as well as the ISD syndrome. In an attempt to combine the best features of the traditional approach plus add the benefits of simplicity, reduction of costs, morbidity, and rapid return to patient normality, a new variation of the PVS has been developed. The technique utilizes pre-threaded bone anchors to which either a natural fascia or pre-prepared cadaveric fascia can be anchored. MATERIAL AND METHODS: This study consists of 78 female patients treated between September 1997 and December 1998 with our PVS procedure. The patient population spans the spectrum of pure stress incontinence, with or without associated pelvic relaxation defects, pure ISD group and lastly, those individuals who suffered from both anatomical incontinence and overactive bladder syndrome. In our 72 evaluable patients, the results as of this publication are: an overall cure rate of 86% with an additional 11% improved and 3% failure. The following text describes in detail the patient population, the surgical technique, the final results, complications, and patient satisfaction scores. Also included is a short review of the literature documenting several other techniques utilizing bone anchoring fixation devices. CONCLUSION: A simplification of the true-and-tried PVS is described which provides the surgeon with a new and exciting methodology for the treatment of all forms of hypermobility, i.e. stress incontinence, as well as the intrinsic sphincter deficiency syndrome. At the same time the surgical learning curve, patient morbidity, and hospital stay are decreased; without compromising total surgical outcome.

Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59.

PURPOSE: To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system. METHODS: Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH(50) and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity. RESULTS: Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH(50)). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an M(r) >/= 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (>/=19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M(r) >/= 3 kDa also had an inhibitory effect on the alternative pathway activity (AH(50)). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid. CONCLUSIONS: Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59.

Chronic low level complement activation within the eye is controlled by intraocular complement regulatory proteins.

PURPOSE: To explore the role of the complement system and complement regulatory proteins in an immune-privileged organ, the eye. METHODS: Eyes of normal Lewis rats were analyzed for the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay-acceleration factor (DAF), membrane inhibitor of reactive lysis (MIRL, CD59), and cell surface regulator of complement (Crry), using immunohistochemistry, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR). Zymosan, a known activator of the alternative pathway of complement system was injected into the anterior chamber of the eye of Lewis rats. Animals were also injected intracamerally with 5 microl (25 microg) of neutralizing monoclonal antibody (mAb) against rat Crry (5I2) or CD59 (6D1) in an attempt to develop antibody induced anterior uveitis; control animals received 5 microl of sterile phosphate-buffered saline (PBS), OX-18 (25 microg), G-16-510E3 (25 microg), or MOPC-21 (25 microg). The role of complement system in antibody-induced uveitis was explored by intraperitoneal injection of 35 U cobra venom factor (CVF), 24 hours before antibody injection. Immunohistochemical staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blot analysis were used to detect the presence of membrane attack complex (MAC) and C3 activation products, respectively, in normal and antibody-injected rat eyes. RESULTS: Complement activation product MAC was present in the normal rat eye, and intraocular injection of zymosan induced severe anterior uveitis. The complement regulatory proteins, MCP, DAF, CD59, and Crry, were identified in the normal rat eye. Soluble forms of Crry and CD59 were also detected in normal rat aqueous humor. Severe anterior uveitis developed in Lewis rats injected with a neutralizing mAb against Crry, with increased formation of C3 split products. Systemic complement depletion by CVF prevented the induction of anterior uveitis by anti-Crry mAb. Intracameral injection of anti-rat CD59 (6D1), anti-rat MHC class I antigen (OX-18), anti-rat Ig (G-16-510E3), or MOPC-21 caused no inflammatory reaction. CONCLUSIONS: The results suggest that the complement system is continuously active at a low level in the normal eye and is tightly regulated by intraocular complement regulatory proteins.

Progressive changes in the fluorescein and indocyanine green angiogram in acute idiopathic maculopathy.

AIMS/BACKGROUND: To report progressive changes in the fluorescein and indocyanine green angiograms of a patient with acute idiopathic maculopathy (AIM). METHODS: Over a two-year period, the patient underwent repeated ophthalmoscopic examinations and fluorescein (FA) and indocyanine green (ICG) angiography. RESULTS: The patient presented with subretinal neovascularization in his right eye. He developed recurrences after laser photocoagulation and surgical removal of the neovascular complex. One year later, he experienced a sudden loss of vision in his left eye with a maculopathy consistent with AIM. The maculopathy resolved after two weeks with poor vision. During the acute stage, FA showed lobular hyperfluorescence in the early phase and pooling in the late phase of the angiogram. In the resolved stage of the disease, FA showed irregular window defects and blockage. ICG revealed late hyperfluorescence of the macula in the acute stage. In the resolved stage of the disease, early hypofluorescence was noted in the ICG, which persisted throughout the late phase. CONCLUSION: This patient had poor vision in his right eye as a result of subretinal retinal neovascularization and poor vision in his left eye from a severe form of AIM. FA and ICG differed markedly during the acute and resolved stages of AIM. All cases of idiopathic subretinal neovascularization should be carefully evaluated to exclude AIM as the primary disease.

Fas ligand (CD95 ligand) controls angiogenesis beneath the retina.

A principal cause of blindness is subretinal neovascularization associated with age-related macular degeneration. Excised neovascular membranes from patients with age-related macular degeneration demonstrated a pattern of Fas+ new vessels in the center of the vascular complex, surrounded by FasL+ retinal pigment epithelial cells. In a murine model, Fas (CD95)-deficient (Ipr) and FasL-defective (gld) mice had a significantly increased incidence of neovascularization compared with normal mice. Furthermore, in gld mice there is massive subretinal neovascularization with uncontrolled growth of vessels. We found that cultured choroidal endothelial cells were induced to undergo apoptosis by retinal pigment epithelial cells through a Fas-FasL interaction. In addition, antibody against Fas prevented vascular tube formation of choroidal endothelial cells derived from the eye in a three-dimensional in vitro assay. Thus, FasL expressed on retinal pigment epithelial cells may control the growth and development of new subretinal vessels that can damage vision.

Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.

PURPOSE: To determine the fate of human retinal pigment epithelial (RPE) cells seeded onto different layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from 16 human cadaver eyes (7 eyes age <50 years; 9 eyes >50 years) by removing native RPE cells with ammonium hydroxide to expose the RPE cell basal lamina (BL). The inner collagenous layer (ICL) and elastin layer (EL) were exposed by removing apical layers sequentially by mechanical and enzymatic means. Synchronized first passage human RPE cells (15,000 cells/(6-mm-diameter explant) were plated onto each layer of human BM. The RPE cell reattachment and apoptosis rates at 24 hours, proliferation rates and mitotic index 24 hours after growth stimulation, and the ability of RPE cells to repopulate the explant surface were determined on each layer. RESULTS: RPE cell reattachment was highest on BL but decreased on deeper layers of BM. The apoptosis rate of attached cells increased as deeper layers of BM were exposed. The proliferation rate and mitotic index of the grafted cells were higher on BL than on deeper layers. RPE cells plated onto BL repopulated the explant surface within 14 +/- 3 days, whereas cells plated onto the ICL and EL eventually died and never reached confluence. CONCLUSIONS: The fate of RPE cells seeded onto BM depends on the ultrastructural layer of BM available for reattachment. These findings suggest that the ability of transplanted RPE cells to repopulate bare BM will depend on the layer of BM available for RPE cell reattachment.

Kinetics of cytokine production in experimental autoimmune anterior uveitis (EAAU).

PURPOSE: Experimental autoimmune anterior uveitis (EAAU) can be induced in Lewis rats with bovine melanin associated antigen (MAA) extracted from the iris/ciliary body (CB) and does not require adjuvant. The present investigation was undertaken to study the expression of various cytokines in EAAU. METHODS: Lewis rats were immunized with bovine MAA and sacrificed at various time points. The iris/CB and popliteal lymph nodes were harvested, and total RNA isolated. The reverse transcription polymerase chain reaction (RT-PCR) was utilized to determine the mRNA expression of IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6 and IL-10. RESULTS: TNF-alpha mRNA levels in iris/CB paralleled the course of EAAU and increased dramatically at the peak of disease. However, mRNA levels of TNF-alpha demonstrated little change in the popliteal lymph node. IFN-gamma mRNA was barely detectable in the iris/CB and increased only slightly at the peak of disease. In contrast, IFN-gamma mRNA levels in the popliteal lymph node paralleled the course of disease and increased during the peak of disease. IL-10 mRNA did not change in the iris/CB but increased modestly in the popliteal lymph node. IL-2, IL-4, and IL-6 mRNA levels did not change during the course of EAAU in either tissue. CONCLUSIONS: Our study reveals an interesting correlation between the expression of TNF-alpha, IFN-gamma and disease progression in EAAU. Furthermore, they suggest that TNF-alpha is an important cytokine in the target tissue, while IFN-gamma is in the draining lymph node.

Harvest and storage of adult human photoreceptor cells: the vibratome compared to the excimer laser.

PURPOSE: To develop a method using the vibratome and the excimer laser to harvest a sheet of human photoreceptor cells from the retinas of cadaveric donors. METHODS: Adult human photoreceptor cells were harvested as intact sheets from the retinas of cadaver eyes using a vibratome or excimer laser. The sheets were embedded in 50% gelatin (in minimum essential medium and 300 mM sucrose) and stored at 4 degrees C. The morphology, integrity, viability and sterility of the harvested photoreceptor cells was studied. RESULTS: Light and scanning electron microscopy demonstrated sheets of adult human photoreceptor cells with an outer nuclear layer and inner and outer segments with either method of harvest. The initial viability of the outer nuclear layer, harvested an average of 28.2 h after death, was > or =94.7%. Sheets stored up to 72 h after harvest maintained a viability of > or =86.5%. The sheet of cells harvested with the vibratome frequently fragmented (n = 25, 35%) during passage through the delivery cannula in contrast to the excimer laser. Harvested sheets were sterile when the gelatin powder was irradiated prior to reconstitution. CONCLUSIONS: Intact, viable adult human photoreceptor cell sheets can be isolated from the retina of a cadaver using either the vibratome or the excimer laser and stored up to 72 h at 4 degrees C. With the vibratome, there is damage to the outer segments of the photoreceptors, the sheets are fragile, and the harvest of specimens is time-consuming as only one or two specimens can be harvested from a single donor retina. These technical limitations are avoided with the excimer laser.

Spontaneous resolution of retinal detachment occurring after macular hole surgery.

OBJECTIVE: To document the spontaneous resolution of retinal detachment developing after macular hole surgery. METHODS: We identified all patients who developed a postoperative retinal detachment after undergoing macular hole surgery at Washington University School of Medicine, St Louis, Mo; the surgery was performed by one of us (L.V.D.P. or H.J.K.) between 1991 and 1996. RESULTS: Six of 73 eyes developed a postoperative retinal detachment; the retinal detachment was inferior in all cases. Two eyes that had inferior retinal breaks underwent further surgery to repair the retinal detachment. Retinal breaks could not be identified in the other 4 eyes; the retinal detachment resolved without further surgery in all 4 of these eyes. CONCLUSION: The recognition that retinal detachment occurring after macular hole surgery can resolve without additional surgery may result in the avoidance of further surgical intervention in some eyes.