Urinary tract infections in women with type 1 diabetes mellitus: survey of female participants in the epidemiology of diabetes interventions and complications study cohort.

PURPOSE: We determined the prevalence of and risk factors for urinary tract infection in women with type 1 diabetes, and compared the prevalence of cystitis to that in nondiabetic women. MATERIALS AND METHODS: Women enrolled in the Epidemiology of Diabetes Interventions and Complications study were surveyed at year 10 as part of the Uro-EDIC study to assess the prevalence of cystitis and pyelonephritis in the preceding 12 months. Multivariate logistic regression models including measures of glycemic control and vascular complications of type 1 diabetes were used for risk factor analyses. The prevalence of cystitis in Uro-EDIC women was compared to that in a nondiabetic subset of women participants in the National Health and Nutrition Examination Survey III (NHANES III). RESULTS: A total of 550 women participated in the Uro-EDIC survey. The prevalence of cystitis and pyelonephritis in the preceding 12 months was 15% and 3%, respectively. Duration of diabetes, hemoglobin A1C, retinopathy, neuropathy, nephropathy, composite vascular complication score and intensive glycemic therapy during the Diabetes Control and Complications Trial, and Diabetes Control and Complications Trial cohort were not associated with cystitis or pyelonephritis. Sexual activity was associated with increased cystitis risk (adjusted OR 8.28; 95% CI 1.45, 158.32; p = 0.01). The adjusted prevalence of cystitis was 19.1% in Uro-EDIC women and 23.1% in NHANES III participants (adjusted OR 0.78; 95% CI 0.51, 1.22; p = 0.28). CONCLUSIONS: In Uro-EDIC women sexual activity rather than measures of diabetes control and complications was the main risk factor for urinary tract infection. The prevalence of cystitis was similar to that in nondiabetic women participants in NHANES III.

Comparative evaluation of colorimetric microtiter plate systems for detection of herpes simplex virus in cerebrospinal fluid.

In the past few years, application of the PCR to the detection of herpes simplex virus (HSV) DNA in the cerebrospinal fluid (CSF) from patients with encephalitis and meningitis has become standard laboratory practice. However, from an operational perspective, the true diagnostic value of PCR in this setting is yet to be realized because most laboratories subject the amplification products to lengthy probe hybridization procedures by Southern blotting. As alternatives to Southern blotting, we evaluated colorimetric microtiter plate (MTP) systems from ViroMed Laboratories, Inc. (PrimeCapture), CPG, Inc. (Quanti-PATH), and Incstar Corp. (GEN-ETI-K), in addition to a system developed at the Mayo Clinic with the PCR ELISA system (Boehringer Mannheim Corp.). We tested PCR products from 86 clinical CSF specimens submitted to our Molecular Microbiology Laboratory. The CSF specimens used had to have sufficient volume for comparative analysis. By conventional Southern blotting methods, 54 were positive and 32 were negative for HSV DNA. Compared with Southern blotting, the sensitivity and specificity were 63.0 and 100.0%, respectively, for the PrimeCapture system, 98. 2 and 96.9%, respectively, for the Quanti-PATH system, 98.2 and 100. 0%, respectively, for the GEN-ETI-K system, and 100.0 and 96.9%, respectively, for the Mayo system. All four MTP systems had turnaround times 12 to 24 h less than that for Southern blotting. There were no significant differences in costs or technologist time between the Mayo system and Southern blotting. Other features of the Mayo system include type-specific genotypic identification of HSV and the potential for determination of drug resistance by DNA sequencing. Overall, we found that colorimetric MTP systems were likely to improve test turnaround times and patient care at no additional cost.

The progressive outer retinal necrosis syndrome: successful treatment with combination antiviral therapy.

BACKGROUND AND OBJECTIVE: To assess a two-drug combination of antiviral therapy for the progressive outer retinal necrosis syndrome (PORN), given the current poor outcome with acyclovir alone. PATIENTS AND METHODS: A retrospective review was performed on six consecutive patients who were diagnosed with PORN and were treated with various combinations of intravenous or oral plus intravenous antiviral therapy. The relative efficacies of these modalities were compared. RESULTS: Six eyes of six patients showed active retinitis at the time of presentation. Three patients had unilateral retinitis, and the remaining patients had necrotic, end-stage disease in their fellow eye. All the patients were treated with combination therapy, consisting of either ganciclovir and acyclovir (three patients), foscarnet and ganciclovir (two patients), or foscarnet and acyclovir (one patient). Standard induction doses were employed. During the combination therapy, all six eyes showed resolution of the retinitis, manifested by complete fading of the original retinal lesions and an absence of new lesion formation. At the final follow-up, the areas of prior active retinitis had resolved and remained quiescent. A mild recurrence developed in one eye when ganciclovir and foscarnet were both tapered to a single daily dose. This recurrence promptly resolved with reinduction (twice daily) dosing. Two patients maintained a visual acuity of 20/50 or better in their involved eye for the duration of follow-up (38 and 27 weeks, respectively). One patient maintained a visual acuity of 20/40 for 14 weeks. The remaining three patients had macula-off retinal detachments despite resolution of active retinitis. In addition, for the duration of follow-up, one of the three patients with unilateral disease had retinitis in the uninvolved eye; all three uninvolved fellow eyes maintained a visual acuity of 20/20. One patient had progressive optic atrophy. CONCLUSIONS: Prolonged combination antiviral therapy for PORN may successfully arrest the progression of retinitis, maintain remission, and prevent involvement of the fellow eye. Furthermore, if aggressive therapy is begun early, good vision may be preserved.

Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice.

Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1(-/-) mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1(-/-) mice, as was expression of IL-4, IL-5, and interferon gamma in splenocytes. In contrast, MCP-1(-/-) mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.

In vivo properties of monocyte chemoattractant protein-1.

Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes, memory T lymphocytes, and natural killer (NK) cells in vitro. Its expression has been documented in disorders characterized by mononuclear cell infiltrates, suggesting that it may contribute to the inflammatory component of such diseases as atherosclerosis, multiple sclerosis, or rheumatoid arthritis. To prove a causal association, the in vivo properties of MCP-1 must be understood. Several lines of transgenic mice have been constructed to address this question. A transgenic line in which MCP-1 expression is controlled by the MMTV-LTR expressed high levels of MCP-1 in multiple organs but showed no evidence for monocyte infiltration. Instead, these mice were more susceptible to infection by the intracellular pathogens, Listeria monocytogenes and Mycobacterium tuberculosis. These mice had high serum levels of MCP-1, suggesting that their circulating monocytes may have been desensitized or that MCP-1 stimulated a Th2-dominant response. In contrast, another model in which MCP-1 expression was controlled by the insulin promoter demonstrated a monocytic infiltrate in pancreatic islets. These results indicate that MCP-1 expression at low levels in an anatomically confined area results in monocyte infiltration, suggesting that when properly expressed, MCP-1's in vitro properties are reproduced in vivo. This justifies the examination of MCP-1-deficient mice in disease models in order to explore MCP-1's role in pathogenesis.

No evidence for Borrelia burgdorferi infection in lesions of morphea and lichen sclerosus et atrophicus in Spain. A prospective study and literature review.

The possible association of Borrelia burgdorferi with morphea and lichen sclerosus et atrophicus has been the focus of research and discussion in dermatology during the last 10 years. To investigate the etiopathogenic role of B. burgdorferi in morphea and lichen sclerosus et atrophicus lesions in Spain, we studied 14 cases: 8 patients with lichen sclerosus et atrophicus and 6 with morphea. For the whole group, a prospective study was performed, including serologic studies by indirect immunofluorescence, histologic evaluation of skin biopsy specimens, culture studies, and polymerase chain reaction with different primers sensitive for detecting virtually all B. burgdorferi strains tested to date. Although one patient with morphea had positive serologic findings at low titer, we were not able to culture or detect borrelial DNA in any of the specimens. These findings do not confirm an association between B. burgdorferi and morphea and lichen sclerosus et atrophicus.

Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1.

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, the ability of MCP-1 to elicit monocytic infiltration depends on its being expressed at low levels in an anatomically restricted area.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in cerebrospinal fluid in Lyme neuroborreliosis.

A polymerase chain reaction (PCR) assay that detects Borrelia burgdorferi DNA in cerebrospinal fluid (CSF) was evaluated as a diagnostic test for acute or chronic Lyme neuroborreliosis. In one laboratory, 102 samples were tested blindly, and 40 samples were retested in a second laboratory. In the first laboratory, B. burgdorferi DNA was detected in CSF samples in 6 (38%) of 16 patients with acute neuroborreliosis, 11 (25%) of 44 with chronic neuroborreliosis, and none of 42 samples from patients with other illnesses. There was a significant correlation between PCR results and the duration of previous intravenous antibiotic therapy. The overall frequency of positive results was similar in the second laboratory, but concordance between the laboratories and among primer-probe sets was limited because many samples were positive with only one primer-probe set. Thus, PCR testing can sometimes detect B. burgdorferi DNA in CSF in patients with acute or chronic neuroborreliosis, but with current methods, the sensitivity of the test is limited.

Optical coherence tomography of macular lesions associated with optic nerve head pits.

PURPOSE: Although optic pits were described more than a century ago, the pathogenesis and pathologic nature of the associated macular lesions remain controversial. The authors used the technique of optical coherence tomography (OCT) to further define the anatomic relation that exists between optic pits, macular schisis-like spaces, and macular detachments. METHODS: Four eyes of three consecutive patients with optic pit-related macular pathology were evaluated. Cross-sectional OCT images were correlated with findings from slit-lamp biomicroscopy and stereo fundus photography. All eyes previously had undergone unsuccessful photocoagulation to the temporal juxtapapillary retina. One eye had undergone vitrectomy and intraocular gas tamponade, resulting in partial resorption and displacement of the submacular fluid. RESULTS: Retinal edema and cystic degeneration were present, overlying macular neurosensory detachments in all four eyes. The most prominent edema was present in the outer retina at the level of the outer plexiform layer. This mimicked a true retinoschisis cavity, although bridging retinal elements were identifiable. A lesser degree of edema was present in the inner retina, predominantly located between the disc and fovea. In one eye, a lamellar hole was shown to be a defect in the outer neurosensory retina. In another eye, a macular detachment developed under a pre-existing schisis-like cavity. The schisis-like cavity or edematous retina communicated with the optic disc in all eyes, whereas none of the eyes demonstrated a direct connection between the macular detachment and optic pit. CONCLUSION: These findings support the concept of a bilaminar structure in which a macular detachment develops secondarily to a pre-existing schisis-like lesion consisting of severe outer retinal edema. Fluid may enter from the optic pit into the retinal stroma and not directly into the subretinal space, explaining the prolonged recovery and frequency of treatment failure after photocoagulation to the juxtapapillary retina.

High level monocyte chemoattractant protein-1 expression in transgenic mice increases their susceptibility to intracellular pathogens.

We have constructed transgenic mice in which the mouse mammary tumor virus long terminal repeat controls the expression of murine monocyte chemoattractant protein-1 (MCP-1). Several independently derived lines of transgenic mice constitutively expressed MCP-1 protein in a variety of organs. Protein extracts from these organs had substantial in vitro monocyte chemoattractant activity that was neutralized by an anti-MCP-1 Ab, indicating that transgenic MCP-1 protein is biologically active. However, no transgenic mouse at any age displayed monocyte infiltrates in MCP-1-expressing organs. Two transgenic lines had circulating MCP-1 levels of 13 to 26 ng/ml, which is a concentration sufficient to induce maximal monocyte chemotaxis in vitro. These transgenic lines showed a 1 to 1.5 log greater sensitivity to infection with Listeria monocytogenes and Mycobacterium tuberculosis. A third transgenic line had lower serum levels of MCP-1 and was resistant to L. monocytogenes. The results suggest that this transgenic model is one of monocyte nonresponsiveness to locally produced MCP-1 due to either receptor desensitization or neutralization of a chemoattractant gradient by high systemic concentrations of MCP-1. Regardless of the mechanism, the data indicate that constitutively high levels of MCP-1 expression do not induce monocytic infiltrates, and that MCP-1 is involved in the host response to intracellular pathogens.

Quantitative assessment of macular edema with optical coherence tomography.

OBJECTIVE: To evaluate optical coherence tomography, a new technique for high-resolution cross-sectional imaging of the retina, for quantitative assessment of retinal thickness in patients with macular edema. DESIGN: Survey examination with optical coherence tomography of patients with macular edema. SETTING: Referral eye center. PATIENTS: Forty-nine patients with the clinical diagnosis of diabetes or diabetic retinopathy and 25 patients with macular edema secondary to retinal vein occlusion, uveitis, epiretinal membrane formation, or cataract extraction. MAIN OUTCOME MEASURES: Correlation of optical coherence tomograms with slit-lamp biomicroscopy, fluorescein angiography, and visual acuity. RESULTS: Optical coherence tomograms of cystoid macular edema closely corresponded to known histopathologic characteristics. Quantitative measurement of retinal thickness is possible because of the well-defined boundaries in optical reflectivity at the inner and outer margins of the neurosensory retina. Serial optical coherence tomographic examinations allowed tracking of both the longitudinal progression of macular thickening and the resolution of macular edema after laser photocoagulation. In patients with diabetic retinopathy, measurements of central macular thickness with optical coherence tomography correlated with visual acuity, and optical coherence tomography was more sensitive than slit-lamp biomicroscopy to small changes in retinal thickness. CONCLUSIONS: Optical coherence tomography appears useful for objectively monitoring retinal thickness with high resolution in patients with macular edema. It may eventually prove to be a sensitive diagnostic test for the early detection of macular thickening in patients with diabetic retinopathy.

Detection of Borrelia burgdorferi DNA in museum specimens of Peromyscus leucopus.

To determine whether Borrelia burgdorferi was enzootic within the United States at the beginning of the 20th century, ear skin samples taken from museum specimens of the white-footed mouse (Peromyscus leucopus) were examined for evidence of spirochetal DNA. In total, 280 samples from mice collected between 1870 and 1919 were analyzed by a nested polymerase chain reaction protocol. Of these, 2 specimens from the vicinity of Dennis, Massachusetts, during 1894 were reproducibly positive for B. burgdorferi OspA sequences. The remaining 278, representing both currently endemic and nonendemic sites, were negative for spirochetal DNA. These studies suggest that the agent of Lyme disease was present in a suitable reservoir host in the United States before the turn of the century and provide evidence against a hypothesis of recent introduction of this zoonotic agent to North America.

Structure/activity analysis of human monocyte chemoattractant protein-1 (MCP-1) by mutagenesis. Identification of a mutated protein that inhibits MCP-1-mediated monocyte chemotaxis.

Monocyte chemoattractant protein-1 (MCP-1) is a monocyte-specific chemoattractant and activator and is a member of the chemokine-beta family of cytokines. To identify regions of MCP-1 which are required for its biological activity, we constructed human MCP-1 mutants that were expressed in eukaryotic cells and tested for their ability to attract monocytes in vitro. Deletion of amino acids 2-8 destroyed activity, suggesting that the amino-terminal region is necessary for activity. Within the deleted region, mutation of aspartate 3 to alanine produced a protein with 9% of wild-type activity, whereas mutation of asparagine 6 to alanine produced a protein with 52.9% of wild-type activity. Mutation of amino acids within the first intercysteine loop yielded variable results. Changing tyrosine 28 to aspartate or arginine 30 to leucine each produced proteins with essentially no monocyte chemoattractant activity. The side chains of these amino acids are predicted to point into a putative receptor binding cleft, and these loss-of-function mutations are consistent with this model. Also consistent is the retention of 60% of wild-type activity after mutation of serine 27 to glutamine, since the side chain of serine 27 is predicted to point away from the binding cleft. However, mutation of arginine 24, which lies outside of this area, to phenylalanine produced a protein with only 5% of wild-type activity, suggesting more complex interactions. Truncations of the carboxyl terminus, as well as mutation of aspartate 68 to leucine, generated proteins with 10-20% of wild-type activity. (Another carboxyl-terminal insertional mutation demonstrated that O-linked carbohydrate in MCP-1 alpha may be added to a threonine in the carboxyl-terminal region.) These findings are consistent with a structural model of dimeric MCP-1 which is similar to interleukin-8, in which amino acids that point into a cleft between the two carboxyl-terminal alpha-helices of the subunits are important for receptor binding. In addition, however, amino acids at the amino terminus and others outside of the interhelical cleft are also essential for activity. The carboxyl-terminal alpha-helix is not required for signaling per se but is required for maximal specific activity. Finally, four mutant proteins partially inhibited the ability of wild-type MCP-1 to attract monocytes in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine-beta (or C-C) family of cytokines. Murine MCP-1, first identified as the JE gene, differs from human MCP-1 in molecular size and extent of glycosylation. We have used Chinese hamster ovary cells to express recombinant murine MCP-1 and find that its predominant form is a microheterogeneous protein of M(r) approximately 25,000. Most of MCP-1's microheterogeneity is due to variable amounts of sialic acid that are terminally attached to a constant number of O-linked oligosaccharide chains per molecule. This carbohydrate, along with a small amount of N-linked carbohydrate, accounts for 50% of the apparent molecular size of murine MCP-1 and is not required for in vitro monocyte chemoattractant activity. Mutational analysis shows that most of the carbohydrate is added to a 49-amino acid C-terminal domain that is not present in human MCP-1 and is not required for in vitro biologic activity, suggesting that murine MCP-1 consists of an N-terminal domain containing monocyte chemoattractant activity and a heavily glycosylated C-terminal domain of as yet unknown function. MCP-1 produced in COS cells contains a small amount of sulfate, but Chinese hamster ovary-produced MCP-1 does not. The absence of sulfate does not alter MCP-1's in vitro chemoattractant properties. In vitro, highly purified murine MCP-1 attracts monocytes, but not neutrophils, with a specific activity similar to human MCP-1 (EC50 approximately 0.5 nM). Equilibrium binding experiments with human monocytes reveal the presence of approximately 3000 binding sites per cell with a Kd of 0.77 nM. In vivo, injection of up to 1 micrograms murine MCP-1 in a variety of murine strains induces the appearance of a sparse mixed inflammatory infiltrate. The disparity between MCP-1's in vitro and in vivo effects suggests that other factors may be required to elicit a full-blown monocyte chemotactic response to MCP-1 in vivo.

Target imbalance: disparity of Borrelia burgdorferi genetic material in synovial fluid from Lyme arthritis patients.

Lyme arthritis is a late manifestation of Lyme disease that results in episodic synovial inflammation and swelling. Although this process is thought to be driven directly by the spirochetal etiologic agent, Borrelia burgdorferi, the organism itself has been recovered by culture only twice. In contrast, polymerase chain reaction (PCR) studies are usually positive. This apparent discrepancy in 19 culture-negative synovial fluid specimens from 18 patients with Lyme arthritis was investigated. In all 19, DNA sequences characteristic of plasmid-encoded genes OspA and OspB were easily detected. However, despite equivalent or even superior analytic sensitivity for detection of cultured organisms, the reactivity of two genomic DNA targets was often weak or absent altogether in the clinical specimens. This apparent overrepresentation of B. burgdorferi plasmid sequences was found exclusively in clinical specimens and not in cultured organisms. The physiologic imbalance of genomic and plasmid DNA reactivity in B. burgdorferi infection may signal an underlying pathogenetic mechanism.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis.

BACKGROUND: Borrelia burgdorferi is difficult to detect in synovial fluid, which limits our understanding of the pathogenesis of Lyme arthritis, particularly when arthritis persists despite antibiotic therapy. METHODS: Using the polymerase chain reaction (PCR), we attempted to detect B. burgdorferi DNA in joint-fluid samples obtained over a 17-year period. The samples were tested in two separate laboratories with four sets of primers and probes, three of which target plasmid DNA that encodes outer-surface protein A (OspA). RESULTS: B. burgdorferi DNA was detected in 75 of 88 patients with Lyme arthritis (85 percent) and in none of 64 control patients. Each of the three OspA primer-probe sets was sensitive, and the results were moderately concordant in the two laboratories (kappa = 0.54 to 0.73). Of 73 patients with Lyme arthritis that was untreated or treated with only short courses of oral antibiotics, 70 (96 percent) had positive PCR results. In contrast, of 19 patients who received either parenteral antibiotics or long courses of oral antibiotics (> or = 1 month), only 7 (37 percent) had positive tests (P < 0.001). None of these seven patients had received more than two months of oral antibiotic treatment or more than three weeks of intravenous antibiotic treatment. Of 10 patients with chronic arthritis (continuous joint inflammation for one year or more) despite multiple courses of antibiotics, 7 had consistently negative tests in samples obtained three months to two years after treatment. CONCLUSIONS: PCR testing can detect B. burgdorferi DNA in synovial fluid. This test may be able to show whether Lyme arthritis that persists after antibiotic treatment is due to persistence of the spirochete.

Sub-pigment epithelial membranes after photocoagulation for diabetic macular edema.

OBJECTIVE: The chronic histopathologic effects of focal and grid argon laser photocoagulation were examined in eyes obtained at autopsy that had previously been treated for diabetic macular edema. The focus was on further characterizing fibrous sub-pigment epithelial membranes that previously had been shown to extend beyond burn edges. DESIGN: A total of 131 argon laser burns were evaluated in five eyes. Tissue was embedded in paraffin or glycol methacrylate, serially sectioned, and examined by light microscopy. MAIN OUTCOME MEASURE: Outer and inner nuclear layer defects were measured, and the frequency and extent of sub-pigment epithelial membranes was estimated. The presence of Müller cell processes among membranes was evaluated by immunostaining for glial fibrillary acidic protein and enzyme histochemical staining for carbonic anhydrase. RESULTS: Burns consistently produced defects in the outer nuclear layer that were larger than the spot size of the laser beam. Inner nuclear layer defects were present in only seven of 131 burns. Glycol methacrylate--embedded tissue sections from 73 burns showed sub-pigment epithelial membranes in all five eyes. In one eye, membranes were confluent between burns. In the remaining four eyes, 37 individual membranes were found among 53 burns, and 47% of membranes contained Müller cell processes. The membranes in paraffin-embedded tissue could not be adequately evaluated. CONCLUSIONS: After focal laser treatment for diabetic macular edema, the inner retina was usually spared. Fibrous sub-pigment epithelial membranes were frequent among burns in all five eyes, and they showed a conspicuous contribution by Müller cell processes. We speculate that by impairing the overlying pigment epithelium, these membranes may contribute to a progressive enlargement of laser scars.