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Biomedical subjects

A H Milam

Publications and source records attributed to A H Milam.

At least 19 recordsLinked to original sources
PubMed2001

Multiple phosphorylated isoforms of NRL are expressed in rod photoreceptors.

NRL, a bZIP transcription factor of the Maf subfamily, interacts with the homeodomain protein CRX and synergistically regulates rhodopsin expression. Here we report that six isoforms of NRL (29-35 kDa) are generated by phosphorylation and expressed specifically in the mammalian retina. The anti-NRL antibody also cross-reacts with a cytosolic 45-kDa protein, which is detected in neuronal tissues but is not encoded by the NRL gene. In both human retinal cell cultures and sections of fetal and adult human retina, NRL is present in the nuclei of developing and mature rods but not cones. We propose that NRL regulates rod photoreceptor-specific gene expression and is involved in rod differentiation.

Alkaline Phosphatase↗
PubMed2001

Localization of pigment epithelium derived factor (PEDF) in developing and adult human ocular tissues.

PURPOSE: To localize pigment epithelium-derived factor (PEDF) in developing and adult human ocular tissues. METHODS: PEDF was localized in fetal and adult eyes by immunofluorescence with a polyclonal antibody (pAb) against amino acids 327-343 of PEDF, or a monoclonal antibody (mAb) against the C-terminal 155 amino acids of PEDF. Specificity of the antibodies was documented by Western blotting. PEDF mRNA was localized in adult retina by in situ hybridization. RESULTS: In developing retinas (7.4 to 21.5 fetal weeks, Fwks), pAb anti-PEDF labeled retinal pigment epithelium (RPE) granules, developing cones, some neuroblasts and many cells in the ganglion cell layer (GCL). In adult retinas, pAb anti-PEDF labeled rod and cone cytoplasm and nuclei of rods but not cones. Cells in the INL and GCL, choroid, corneal epithelium and endothelium, and ciliary body were also pAb PEDF-positive. Preadsorption of pAb anti-PEDF with the immunizing peptide blocked specific labeling in retina and other tissues, except for photoreceptor outer segments. In agreement with the immunolocalization with pAb anti-PEDF, in situ hybridization revealed PEDF mRNA in the RPE, photoreceptors, inner nuclear layer cells and ganglion cells in adult retina. In developing retinas 18 Fwks and older, and in adult retinas, mAb anti-PEDF labeled the interphotoreceptor matrix (IPM). Western blots of retina, cornea, and ciliary body/iris with pAb anti-PEDF produced several bands at about 46 kDa. With mAb anti-PEDF, retina produced one band at about 46 kDa; cornea and ciliary body/iris had several bands at about 46 kDa. CONCLUSIONS: PEDF, originally reported as a product of RPE cells, is present in photoreceptors and inner retinal cell types in developing and adult human eyes. Photoreceptors and RPE may secrete PEDF into the IPM.

Aged↗
PubMed2001

Concentric retinitis pigmentosa: clinicopathologic correlations.

Progressive concentric (centripetal) loss of vision is one pattern of visual field loss in retinitis pigmentosa. This study provides the first clinicopathologic correlations for this form of retinitis pigmentosa. A family with autosomal dominant concentric retinitis pigmentosa was examined clinically and with visual function tests. A post-mortem eye of an affected 94 year old family member was processed for histopathology and immunocytochemistry with retinal cell specific antibodies. Unrelated simplex/multiplex patients with concentric retinitis pigmentosa were also examined. Affected family members of the eye donor and patients from the other families had prominent peripheral pigmentary retinopathy with more normal appearing central retina, good visual acuity, concentric field loss, normal or near normal rod and cone sensitivity within the preserved visual field, and reduced rod and cone electroretinograms. The eye donor, at age 90, had good acuity and function in a central island. Grossly, the central region of the donor retina appeared thinned but otherwise normal, while the far periphery contained heavy bone spicule pigment. Microscopically the central retina showed photoreceptor outer segment shortening and some photoreceptor cell loss. The mid periphery had a sharp line of demarcation where more central photoreceptors were near normal except for very short outer segments and peripheral photoreceptors were absent. Rods and cones showed abrupt loss of outer segments and cell death at this interface. It is concluded that concentric retinitis pigmentosa is a rare but recognizable phenotype with slowly progressive photoreceptor death from the far periphery toward the central retina. The disease is retina-wide but shows regional variation in severity of degeneration; photoreceptor death is severe in the peripheral retina with an abrupt edge between viable and degenerate photoreceptors. Peripheral to central gradients of unknown retinal molecule(s) may be defective or modify photoreceptor degeneration in concentric retinitis pigmentosa.

Adolescent↗
PubMed2001

Evaluation of retinal photoreceptors and pigment epithelium in a female carrier of choroideremia.

PURPOSE: To clarify the pathogenesis of choroideremia. STUDY DESIGN: Human tissue study. TISSUES: Eyes of an 88-year-old symptomatic female carrier of choroideremia (CHM) and six normal, age-matched donors. METHODS: The eyes were processed for histopathologic examination, including immunocytochemistry with an antibody against the CHM gene product, REP-1, and retinal cell-specific markers. RESULTS: The CHM carrier retina showed patchy degeneration, but the photoreceptor and retinal pigment epithelium (RPE) loss appeared to be independent. The choriocapillaris was normal except where retinal areas were severely degenerate. The CHM gene product, REP-1, was localized to the cytoplasm of rods but not cones. CONCLUSIONS: It has generally been considered that photoreceptor degeneration in CHM is secondary to loss of the choriocapillaris or RPE. This study suggests that the rod photoreceptors are a primary site of disease in CHM.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Immunocytochemical characterization of macular hole opercula.

OBJECTIVES: To immunocytochemically characterize the neural and glial elements of idiopathic full-thickness macular hole (FTMH) opercula excised during vitrectomy, and to correlate them with the outcome of surgery. METHODS: Opercula were collected from eyes undergoing vitrectomy for stage 3 FTMH and processed for transmission electron microscopy, light epifluorescence, and laser scanning confocal microscopy. Glia were identified using anti-glial fibrillary acid protein (GFAP), antivimentin, and anti-cellular retinaldehyde binding protein antibodies. Anti-phosphodiesterase gamma and antirhodopsin were used for cone and rod photoreceptors, and anticytokeratin was used for retinal pigment epithelium. The findings were correlated with the clinical data before and after surgery. For statistical analysis, data were combined with those of a previous study by the authors of 18 opercula. RESULTS: Opercula from 12 consecutive eyes of 12 patients were studied. In all opercula, GFAP, vimentin, and cellular retinaldehyde binding protein-positive glia were present. Six (50%) of 12 opercula contained more than 5 photoreceptors with somata and internal photoreceptor fibres, but lacking outer segments, demonstrating strong immunoreactivity to anti-phosphodiesterase gamma without antirhodopsin reactivity consistent with cones. Further, 2 (17%) of 12 opercula showed few cones (1-5 cones), and 4 (33%) of 12 contained only glia. Clinicopathologic correlation of the 30 opercula from the 2 studies showed that eyes with opercula containing more than 5 photoreceptors were associated with a worse anatomical closure rate after initial surgery, compared with those with fewer than 5 photoreceptors (P =.004). Once closure had been achieved with reoperation, median postoperative vision was similar in both groups (20/40 and 20/60, respectively). CONCLUSIONS: A spectrum of opercula occur in FTMH ranging from those containing only glia to those containing numerous cones. The extent of foveal neuroretinal tissue loss may affect the outcome of surgery.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Loss of cone molecular markers in rhodopsin-mutant human retinas with retinitis pigmentosa.

PURPOSE: To examine the effect of rhodopsin mutations on cone photoreceptors in human retinas with retinitis pigmentosa (RP). METHODS: Four RP retinas with rhodopsin mutations and four normal retinas were examined by immunofluorescence with a battery of cell-specific antibodies against cone and rod cytoplasmic and outer segment membrane proteins. Areas of the retinas were studied that showed maximal preservation of photoreceptor structure. RESULTS: All four RP retinas showed loss of rods, ranging from mild (T-17-M), to more severe (P-23-H), to advanced degeneration (Q-64-ter and G-106-R). The majority of cones in the T-17-M and P-23-H retinas were cytologically normal but showed loss of immunoreactivity for the cytoplasmic proteins 7G6, calbindin, and X-arrestin. The cone outer segments (OS) remained positive for cone opsins and peripherin-2 (rds/peripherin). All remaining cones in the Q-64-ter and G-106-R retinas were degenerate, with short to absent OS, but had strong reactivity for these cytoplasmic and OS membrane markers. Cones in the maculas of the RP retinas were degenerate, with short to absent OS, but retained strong labeling for the cytoplasmic and OS proteins. CONCLUSIONS: Even before cones show cytologic changes in response to rod cell degeneration, they lose immunoreactivity for certain cytoplasmic proteins. These cones later show shortening and loss of OS, although their OS membrane proteins remain well labeled. Cones may down regulate expression of both cytoplasmic and outer segment membrane proteins in response to mutant rod cell dysfunction and/or cell death in human RP retinas. Such cytologic and immunocytochemical changes in the cones may presage death of these critical cells in the later stages of RP.

Aged↗
PubMed2000

Correction of ornithine accumulation prevents retinal degeneration in a mouse model of gyrate atrophy of the choroid and retina.

Deficiency of ornithine-delta-aminotransferase (OAT) in humans results in gyrate atrophy of the choroid and retina (GA), an autosomal recessive disorder characterized by ornithine accumulation and a progressive chorioretinal degeneration of unknown pathogenesis. To determine whether chronic, systemic reduction of ornithine can prevent this form of retinal degeneration, we used an arginine-restricted diet to maintain long term reduction of ornithine in a mouse model of OAT-deficiency (Oat(-/-)) produced by gene targeting. We evaluated the mice over a 12-month period by measurement of plasma amino acids, electroretinograms, and retinal histologic and ultrastructural studies. We found that an arginine-restricted diet substantially reduces plasma ornithine levels and completely prevents retinal degeneration in Oat(-/-). This result indicates that ornithine accumulation is a necessary factor in the pathophysiology of the retinal degeneration in GA and that restoration of OAT activity in retina is not required for effective treatment of GA.

Amino Acids↗
PubMed2000

Abnormalities in rod photoreceptors, amacrine cells, and horizontal cells in human retinas with retinitis pigmentosa.

PURPOSE: To evaluate changes in the rods and amacrine cells and horizontal cells in human retinas with retinitis pigmentosa. METHODS: Seven retinas from patient donors with retinitis pigmentosa and 14 age- and postmortem-matched normal human retinas were processed for immunocytochemistry and confocal microscopy. The following cell-specific antibodies were used: anti-rhodopsin (rods), anti-gamma-aminobutyric acid (amacrine cells), anticalbindin (cones and horizontal cells), anti-glial fibrillary acidic protein (astrocytes and reactive Müller cells), and anti-synaptophysin and anti-SV2 (synaptic vesicles). RESULTS: In retinal regions with significant photoreceptor loss, the rods, gamma-aminobutyric acid-positive amacrine cells, and calbindin-positive horizontal cells had undergone neurite sprouting. The rod, amacrine and horizontal cell neurites were associated with the surfaces of glial fibrillary acidic protein-immunoreactive Müller cells. Most rod neurites that projected into the inner retina contacted the somata of gamma-aminobutyric acid-positive amacrine cells. CONCLUSIONS: Rods, amacrine and horizontal cells undergo neurite sprouting in human retinas with retinitis pigmentosa. These changes in the retinal neurons may contribute to the electroretinographic abnormalities and progressive decline in vision noted by patients with retinitis pigmentosa. These alterations may also complicate strategies for treatment of retinitis pigmentosa.

Adult↗
PubMed2000

Histopathologic and immunocytochemical analysis of the retina and ocular tissues in Batten disease.

PURPOSE: To describe the pathophysiologic features of retinal degeneration in Batten disease (juvenile neuronal ceroid lipofuscinosis [JNCL]) caused by mutations in the CLN3 gene. STUDY DESIGN: Comparative human tissue study. MATERIALS: The retina and other ocular tissues of a 22-year-old man with JNCL were compared with the same tissues of a healthy 30-year-old man. DNA from whole blood and RNA from retina were used for genotype analysis. METHODS: The retinas, corneas, conjunctiva, and ciliary body were processed for histopathologic and immunofluorescence analysis. Genomic DNA was subjected to polymerase chain reaction (PCR) and nucleotide sequence analyses. Reverse transcriptase/PCR and sequence analysis were performed on retinal RNA. RESULTS: The JNCL donor was heterozygous for a approximately 1 kb deletion in CLN3, as found in most JNCL patients. The other allele had a single base pair deletion in exon 6 that resulted in a frame shift. Gross pathology of the JNCL retina resembled that in retinitis pigmentosa, including deposits of bone spicule pigment. Histopathologic studies revealed loss of neurons from all retinal layers. Immunofluorescence labeling with antibodies to rhodopsin, recoverin, and cone opsin demonstrated degenerate rods and cones with short outer segments in the far periphery. Autofluorescent lipopigment granules were prominent in ganglion cells and some cells of the inner nuclear layer, but not in the photoreceptors. The retinal pigment epithelium (RPE) had fewer lipofuscin granules than the control specimen. Increased numbers of lipofuscin granules were found in the epithelia of the ciliary body and conjunctiva, but not in the cornea of the JNCL eye. CONCLUSIONS: Immunofluorescence studies revealed degenerate rods and cones in the far periphery. Lipofuscin granules were decreased in the RPE, consistent with loss of photoreceptor outer segments. The novel finding that degenerate photoreceptors did not contain autofluorescent inclusions suggests that granule accumulation may not precede photoreceptor degeneration in JNCL. The presence of normal photoreceptor proteins in the degenerate rods and cones suggests that these cells may be capable of functional regeneration if a therapy for Batten disease is developed.

Adult↗
PubMed2000

Dominant late-onset retinal degeneration with regional variation of sub-retinal pigment epithelium deposits, retinal function, and photoreceptor degeneration.

PURPOSE: To clarify the pathogenesis of late-onset retinal degeneration (L-ORD), an autosomal dominant disorder characterized by thick deposits of lipid-rich material between the retinal pigment epithelium (RPE) and Bruch's membrane. STUDY DESIGN: Comparative clinicopathologic case report and case series. TISSUES: Eyes of an 82-year-old L-ORD eye donor and an age-matched control. SUBJECTS: Five descendants of the eye donor and his affected sister. METHODS: The eyes were processed for histopathologic examination, including electron microscopy and immunohistochemistry. Family members were examined clinically and with retinal function tests. RESULTS: The L-ORD eye had sub-RPE deposits that were positive for lipid, including esterified and unesterified cholesterol. The deposits were thinnest in the macula, which retained the highest percentage of photoreceptors. In the periphery, RPE thinning and photoreceptor loss correlated with thickness of the sub-RPE deposits. The eye donor was asymptomatic until his late 50s, when he developed problems with adapting to darkness. At age 68, the eye donor had normal acuity but a midperipheral scotoma and subnormal electroretinograms (ERGs); visual loss was progressive. The five descendants (at the time of examination ages 44-58) of the eye donor and his affected sister, who were at 50/50 risk of inheriting L-ORD, had normal ERGs, but four showed defects in dark adaptation. The dark adaptation abnormalities had a distribution similar to the thickness of the sub-RPE deposits in the eye donor, with slow kinetics in the midperiphery and normal kinetics centrally. CONCLUSIONS: The L-ORD donor eye differed from a previous case in the regional distribution of sub-RPE deposits and photoreceptors. In the next generation of this L-ORD family, the first expression of disease, abnormal dark adaptation, mirrored the regional distribution of the deposits in the donor eye. The fine structure and staining characteristics of the sub-RPE deposits in L-ORD resemble those in age-related macular degeneration and Sorsby fundus dystrophy.

Adult↗
PubMed2000

Human melanoma-associated retinopathy (MAR) antibodies alter the retinal ON-response of the monkey ERG in vivo.

PURPOSE: Melanoma-associated retinopathy (MAR) is a paraneoplastic condition that causes visual symptoms of night-blindness and photopsias. The electroretinogram (ERG) of MAR patients is characteristically abnormal in a way that implicates retinal depolarizing bipolar cell (DBC) dysfunction. Whether an injection of IgG from MAR patients into the vitreous of monkeys would alter the ERG acutely as a demonstration of a functional basis for patients' visual symptoms was explored. METHODS: MAR IgG was isolated from three visually symptomatic melanoma patients. Control IgG was from melanoma patients with no vision problems. The ERG was monitored after intravitreal injections into monkey eyes. One eye was injected with 2-amino-4-phosphonobutyric acid (APB), which is known to block DBC ON-pathway responses. Retinal immunocytochemistry was performed using fluorescein isothiocyanate-labeled goat anti-human IgG. RESULTS: Within 1 to 3 hours after MAR IgG injection, the ERG photopic b-wave was diminished, with far less effect on the a- and d-waves. These changes are characteristic of DBC dysfunction and were similar to the effects of APB. The scotopic ERG b-wave, which reflects activity of rod-driven DBCs, showed a loss of amplitude and threshold sensitivity after MAR IgG. Retinal immunocytochemistry with anti-IgG antibody showed IgG penetration throughout the retinal layers, but staining was not specific for a single type of retinal neuron. CONCLUSIONS: Intravitreal injection of human MAR IgG altered the monkey ERG acutely in ways that implicate functional disruption of retinal DBC signaling. These results support the hypothesis that MAR IgG circulating antibodies are responsible for the reported visual symptoms. Bipolar cells in the ON-pathway appear to be affected more than OFF-pathway bipolar cells of the cone pathway in this acute preparation.

Aminobutyrates↗
PubMed2000

Localization of tubby-like protein 1 in developing and adult human retinas.

PURPOSE: To localize tubby-like protein 1 (TULP1) in developing and adult human retinas. METHODS: TULP1 was localized by immunofluorescence microscopy in human retinas, aged 8.4 fetal weeks to adult. TULP1-positive cells were identified by double labeling with antibodies specific for cones, rods, and astrocytes. RESULTS: In adult retinas, anti-TULP1 labels cone and rod inner segments, somata, and synapses; outer segments are TULP1-negative. A few inner nuclear and ganglion cells are weakly TULP1-positive. In fetal retinas, cells at the outer retinal border are TULP1-positive at 8.4 weeks. At 11 weeks, the differentiating central cones are strongly TULP1-reactive and some are positive for blue cone opsin. At 15.4 weeks, all central cones are strongly positive for TULP1 and many are reactive for red/green cone opsin. At 17.4 weeks, central rods are weakly TULP-reactive. In peripheral retina at 15.4 weeks to 1 month after birth, displaced cones in the nerve fiber layer are positive for TULP1, recoverin, and blue cone opsin. Some ganglion cells are weakly reactive for TULP1 at 11 weeks and later, but astrocytes and the optic nerve are TULP1-negative at all ages examined. CONCLUSIONS: The finding of TULP1 labeling of cones before they are reactive for blue or red/green cone opsin suggests an important role for TULP1 in development. TULP1 expression in both developing and mature cones and rods is consistent with a primary photoreceptor defect in retinitis pigmentosa (RP) caused by TULP1 mutations. Weak TULP1-immunolabeling of some inner retinal neurons in developing and adult retinas suggests that optic disc changes in patients with RP who have TULP1 mutations may be primary as well as secondary to photoreceptor degeneration.

Adult↗
PubMed1999

Cancer-associated retinopathy.

Patients with systemic cancer may have a variety of ocular complaints. Most commonly these are metastases or adverse effects of therapy. Paraneoplastic syndromes, like cancer-associated retinopathy, rarely cause ophthalmic symptoms. We describe a patient with a malignant mixed mullerian tumor and cancer-associated retinopathy who had circulating serum antibodies to recoverin and cells positive for recoverin in the tumor. We discuss the typical clinical symptoms as well as the pathophysiology of this uncommon disorder.

Antigens, Neoplasm↗
PubMed1999

Clinical and immunocytochemical findings in a case of melanoma-associated retinopathy.

OBJECTIVE: To describe an unusual case of melanoma-associated retinopathy (MAR). DESIGN: Retrospective, observational case report and experimental study. PARTICIPANTS: A 61-year-old man with a history of cutaneous melanoma, acquired bilateral central scotomas, and night blindness. INTERVENTION: Serial full-field electroretinography (ERG) and Goldmann perimetry were performed. Serum was screened for cancer-associated retinopathy (CAR) antibodies by Western blotting. Sections of human and rat retina were examined by immunofluorescence microscopy to determine whether retinal cells were reactive with this patient's serum. A metastatic workup was performed. MAIN OUTCOME MEASURES: Electroretinography, Goldmann visual field testing, and immunocytochemistry were performed. RESULTS: The results were as follows: (1) The ERG showed a profound loss of the b-wave amplitude and a "negative" b-wave characteristic of congenital stationary night blindness; (2) a central scotoma and peripheral constriction were identified on Goldmann visual field tests; (3) as in other patients with MAR, bipolar cells in human and rat retinas were immunolabeled with this patient's serum; and (4) a previously unsuspected focus of metastatic melanoma was discovered. CONCLUSIONS: Recognition of this condition may help to identify an occult focus of metastatic melanoma.

Blotting, Western↗
PubMed1999

Retinal rod photoreceptor-specific gene mutation perturbs cone pathway development.

Rod-specific photoreceptor dystrophies are complicated by the delayed death of genetically normal neighboring cones. In transgenic (Tg) swine with a rod-specific (rhodopsin) gene mutation, cone photoreceptor physiology was normal for months but later declined, consistent with delayed cone cell death. Surprisingly, cone postreceptoral function was markedly abnormal when cone photoreceptor physiology was still normal. The defect was localized to hyperpolarizing cells postsynaptic to the middle wavelength-sensitive cones. Recordings throughout postnatal development indicated a failure of cone circuitry maturation, a novel mechanism of secondary cone abnormality in rod dystrophy. The results have implications for therapy for human retinal dystrophies and raise the possibility that rod afferent activity plays a role in the postnatal maturation of cone retinal circuitry.

Amino Acid Substitution↗
PubMed1999

Morphometric analysis of the extramacular retina from postmortem eyes with retinitis pigmentosa.

PURPOSE: To evaluate the degree of inner retinal preservation in the extramacular regions of postmortem retinitis pigmentosa (RP) eyes. METHODS: Eighteen RP retinas and 11 age-matched healthy retinas were sectioned for morphometric analysis by light microscopy. The 18 RP retinas were classified by disease severity and mode of inheritance. Cell nuclei in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) were counted in adjacent 125-microm segments from an area spanning the region between 4 mm and 10 mm from the fovea. RESULTS: A mixed-effects model showed a decrease in mean cell counts for each of the cell layers when the severity groups and inheritance types compared with those of control retinas. There was no statistically significant difference in the number of nuclei preserved in the INL and GCL in the moderate group compared with the severe group. Results from the INL counts for the different inheritance types of RP showed a higher overall mean percentage of cells was preserved for the autosomal dominant RP (ADRP) group when compared with the X-linked (XLRP) and simplex RP groups. Analysis of the GCL counts revealed significantly more counts only in the ADRP group compared with the XLRP group; the other group comparisons were not significant. CONCLUSIONS: Retinitis pigmentosa results in cell loss in all retinal layers, with the most profound loss in the ONL, followed by the GCL and then the INL. The preservation of the INL and GCL in the extramacular region is less than that previously reported for the macular region of the same retinas.

Aged↗
PubMed1998

Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man.

Mutations in the gene encoding rhodopsin, the visual pigment in rod photoreceptors, lead to retinal degeneration in species from Drosophila to man. The pathogenic sequence from rod cell-specific mutation to degeneration of rods and cones remains unclear. To understand the disease process in man, we studied heterozygotes with 18 different rhodopsin gene mutations by using noninvasive tests of rod and cone function and retinal histopathology. Two classes of disease expression were found, and there was allele-specificity. Class A mutants lead to severely abnormal rod function across the retina early in life; topography of residual cone function parallels cone cell density. Class B mutants are compatible with normal rods in adult life in some retinal regions or throughout the retina, and there is a slow stereotypical disease sequence. Disease manifests as a loss of rod photoreceptor outer segments, not singly but in microscopic patches that coalesce into larger irregular areas of degeneration. Cone outer segment function remains normal until >75% of rod outer segments are lost. The topography of cone loss coincides with that of rod loss. Most class B mutants show an inferior-nasal to superior-temporal retinal gradient of disease vulnerability associated with visual cycle abnormalities. Class A mutant alleles behave as if cytotoxic; class B mutants can be relatively innocuous and epigenetic factors may play a major role in the retinal degeneration.

Adult↗