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Biomedical subjects

A H Milam

Publications and source records attributed to A H Milam.

At least 55 records · Page 3Linked to original sources
PubMed1996

Clinicopathologic effects of the Q64ter rhodopsin mutation in retinitis pigmentosa.

PURPOSE: To correlate retinal histopathology with functional changes caused by the rhodopsin Q64ter mutation. METHODS: A 50-year-old female heterozygote was evaluated clinically and with psychophysical and electroretinographic measurements of rod and cone function. The retinas obtained after death were examined microscopically, including immunolabeling with antibodies against the C- and N-termini of rhodopsin. RESULTS: On clinical examination 4 months before death, patient's acuity was 20/60, and she had midperipheral scotomas with retained function centrally and in the far periphery. The rod electroretinogram (ERG) was undetectable, and the cone ERG was reduced in amplitude with abnormal receptoral and postreceptoral responses. A previous study of the phenotype of mildly affected family members of the donor suggested that the rod outer segments (ROS) were shortened and that only wild-type rhodopsin was functional. The retinas contained only scattered cones in the midperiphery; the maculas and far peripheral regions contained reduced numbers of rods and cones with short to absent outer segments. The ROS appeared to contain wild-type, but not mutant, rhodopsin, and many peripheral rods had sprouted long rhodopsin-positive neurites that projected into the inner retina. Many cone synapses were abnormal, and the axons of some peripheral cones reached the inner plexiform layer. CONCLUSIONS: Microscopic changes in the donor retinas correlated well with the abnormalities in visual function in the patient donor and other family members. Postreceptoral ERG defects may relate to the abnormal photoreceptor processes found in the inner retina.

Aged↗
PubMed1996

Light-induced acceleration of photoreceptor degeneration in transgenic mice expressing mutant rhodopsin.

PURPOSE: Mutations at various loci on the rhodopsin gene have been shown to cause autosomal dominant retinitis pigmentosa (ADRP). One of the most common is a point mutation (P23H) near the N-terminus of the protein. The authors have studied the effects of light deprivation on the rate of degeneration in pigmented transgenic mice expressing the P23H mutation as well as two additional mutations near the N-terminus of opsin (V20G, P27L). METHODS: Transgenic and normal littermates were reared in darkness or in cyclic light (approximately 7 foot-candle) for periods of 2, 4, or 6 months. Retinal structure and function were evaluated by electroretinography, retinal densitometry, light microscopy, and TUNEL labeling. RESULTS: Retinas of normal animals, whether reared in darkness or in cyclic light, had no structural or functional abnormalities. The rate of photoreceptor degeneration in dark-reared transgenic mice was significantly slower than in transgenic mice raised under cyclic light conditions. Differences between the two groups of animals were evident in the retinal histology, the electroretinographically determined sensitivity to photic stimulation, and the rhodopsin levels in the retina. TUNEL labeling of retinal wholemounts showed that cyclic light-reared animals had a threefold higher incidence of photoreceptor cell death than their dark-reared counterparts; the density of apoptotic cells was greatest in the inferior retina, the region most severely affected in patients with the P23H mutation. In comparison, photoreceptor cell death was more uniformly distributed across the retina in dark-reared transgenic mice. CONCLUSIONS: These findings suggest that light activation of rhodopsin contributes to the severity of the degenerative disease resulting from the P23H opsin mutation, and they raise the possibility that minimizing exposure to light may help to prolong useful vision of patients with this form of retinitis pigmentosa.

Animals↗
PubMed1996

Sub-retinal pigment epithelial deposits in a dominant late-onset retinal degeneration.

PURPOSE: To determine the pathogenesis of an autosomal dominant late-onset retinal degeneration by studies of the retinal histopathology, phenotype of family members, and candidate genes for the disease. METHODS: The retina from an 80-year-old patient donor was prepared for light and electron microscopy, including special stains and immunocytochemistry. Family members were examined clinically and with retinal function tests. Rhodopsin, peripherin/RDS, and TIMP3 genes were screened for mutations, and linkage analysis was performed with short tandem repeat polymorphisms flanking these genes. RESULTS: Affected family members had nyctalopia in the sixth decade of life and severe visual loss developed by the eighth decade. The donor retina showed marked loss of photoreceptors except in the inferior periphery. A thick layer of extracellular deposits was present between the RPE and Bruch's membrane in all retinal regions. A 70-year-old affected family member had a retinopathy resembling retinitis pigmentosa. Her 42-year-old daughter had a patch of punctate yellow-white lesions in one fundus and abnormal dark adaptation. The 50-year-old son of the donor had normal fundi but abnormal dark adaptation and electroretinography. No mutations were detected in the coding sequence of the rhodopsin, peripherin/RDS, and TIMP3 genes. Rhodopsin and TIMP3 were further excluded with linkage analysis. CONCLUSIONS: This novel retinal degeneration shares histopathologic and clinical features with both Sorsby fundus dystrophy and retinitis pigmentosa. The sub-RPE deposits may disrupt the exchange of nutrients and metabolites between the retina and the choriocapillaris, leading to photoreceptor dysfunction and degeneration.

Adult↗
PubMed1995

Rhodopsin phosphorylation and dephosphorylation in vivo.

Rhodopsin is an important member of the superfamily of G protein-coupled receptors. In vitro studies have suggested that multiphosphorylation of rhodopsin is a pivotal step in phototransduction. Because the in vitro biochemical experiments were conducted under nonphysiological conditions, we investigated the phosphorylation of mouse rhodopsin in vivo and determined the sites of phosphorylation and the time course of dephosphorylation. We found that a single phosphate group is incorporated into the rhodopsin molecule in a light-dependent manner, primarily at Ser338 after flashes and at Ser334 after continuous illumination. Dephosphorylation of these sites had different kinetics and spatial distribution in rod outer segments. Dephosphorylation of Ser338 was complete within 30 min, while Ser334 was dephosphorylated much slower (requiring up to 60 min), correlating with the regeneration of rhodopsin. These results suggest that phosphorylation of Ser338 and Ser334 plays different roles in phototransduction.

Amino Acid Sequence↗
PubMed1995

Melanoma-associated retinopathy.

PURPOSE/METHODS: A 64-year-old man with a history of maxillary antrum melanoma had abrupt-onset night blindness and photopsia. RESULTS/CONCLUSIONS: Ophthalmologic examination and electroretinogram suggested melanoma-associated retinopathy. Serum produced a weak but specific labeling of human bipolar cells by indirect immunofluorescence. Complete excision of melanoma may have contributed to low levels of circulating autoantibodies. The identity of the retinal bipolar antigen recognized by melanoma-associated retinopathy autoantibodies is needed for more accurate diagnosis.

Electroretinography↗
PubMed1995

Pattern of retinal dysfunction in acute zonal occult outer retinopathy.

PURPOSE: To elucidate the pathophysiology of the diseases recently grouped under the name of acute zonal occult outer retinopathy (AZOOR). METHODS: Twenty-four patients with the diagnosis of multiple evanescent white dot syndrome, acute idiopathic blind-spot enlargement syndrome, acute macular neuroretinopathy, multifocal choroiditis, or pseudo-presumed ocular histoplasmosis syndrome, or a combination of these diagnoses, were evaluated on one or more visits with full-field electroretinograms (ERGs) and kinetic and static perimetry. Sera and IgG fractions were tested using immunofluorescence on cryostat sections of fixed and unfixed human and rat retina. RESULTS: Patients with AZOOR as a group showed abnormal results for all eight ERGs and two visual field parameters measured. Interocular asymmetry was a prominent feature of the patients' test results. Logistic regression indicated that interocular differences in ERG parameters significantly increased the efficacy of identifying patients with AZOOR beyond that of the parameter values alone. Visual field area correlated well with ERG a-wave amplitude. Serial visual function tests in a subset of patients showed that there could be short-term partial recovery or progressive loss of function. Autoantibodies to a specific retinal cell type were not detected. CONCLUSIONS: All patients with AZOOR showed a pattern of visual dysfunction that was photoreceptor in origin, patchy in its distribution across the retina, and asymmetric in the two eyes. There was considerable variability in the severity and the natural history of retinal dysfunction in the patients studied. The full-field ERG was found to be a valuable adjunct in diagnosing AZOOR, especially when the expected interocular variation in measured parameters was known for control subjects. No evidence was obtained for autoantibodies to any retinal cell type in the patients with AZOOR who were tested.

Acute Disease↗
PubMed1995

Histopathology of bone spicule pigmentation in retinitis pigmentosa.

PURPOSE: To evaluate bone spicule pigmentation, a fundus feature in retinitis pigmentosa (RP) formed by migration of pigment-containing cells to perivascular sites in the inner retina. METHODS: The authors performed light and electron microscopy, including immunocytochemistry, on the retinas from ten patients with RP and five normal donors. RESULTS: The pigment-containing cells in regions of bone spicule pigmentation were derived from the retinal pigment epithelium (RPE). The translocated cells were remarkably polarized with a number of specializations characteristic of RPE cells in situ, but they did not contain lipofuscin granules and were not immunoreactive for cellular retinaldehyde-binding protein. The cells were linked by junctional complexes and formed epithelial layers around retinal vessels and next to the inner limiting membrane. Adjacent Müller cell processes contained glial fibrillary acidic protein-positive filaments and formed microvilli and intermediate junctions, resembling those in the external limiting membrane. Vascular endothelial cells adjacent to the translocated RPE cells were thin and fenestrated, resembling the choriocapillaris, and were separated from the pigmented cells by a layer of extracellular matrix similar in organization to Bruch membrane. Thickening of the matrix layer caused narrowing and occlusion of the vascular lumina. CONCLUSIONS: The lack of lipofuscin granules and cellular retinaldehyde-binding protein immunoreactivity in the translocated RPE cells is probably related to the loss of photoreceptors. The development of fenestrations in the endothelial cells correlates with the leakiness of retinal vessels to fluorescein observed in some cases of RP. Narrowing and occlusion of vascular lumina by thickening of the surrounding layer of extracellular matrix may contribute to the loss of inner retinal neurons found in RP. These changes in the RPE, blood vessels, glia, and inner neurons warrant consideration in designing therapies to restore vision to degenerate retinas.

Adult↗
PubMed1995

Mice lacking ornithine-delta-aminotransferase have paradoxical neonatal hypoornithinaemia and retinal degeneration.

Deficiency of ornithine-delta-aminotransferase (OAT) in humans causes hyperornithinaemia and gyrate atrophy (GA), a blinding chorioretinal degeneration. Surprisingly, OAT-deficient mice produced by gene targeting exhibit neonatal hypoornithinaemia and lethality, rescuable by short-term arginine supplementation. Post-weaning, these mice develop hyperornithinaemia similar to human GA patients. Subsequent studies in one human GA infant also showed transient hypoornithinaemia. Thus, the OAT reaction plays opposite roles in neonatal and adult mammals. Over several months, OAT-deficient mice develop a retinal degeneration with involvement of photoreceptors and pigment epithelium. OAT-deficient mice appear to be an excellent model of human GA.

Adult↗
PubMed1995

Rod photoreceptor neurite sprouting in retinitis pigmentosa.

In animal models for retinitis pigmentosa (RP), rod photoreceptors show abnormal distribution of rhodopsin prior to undergoing cell death. To elucidate the steps in degeneration of human photoreceptors, immunocytochemistry was performed on donor retinas from 15 RP patients and five normal subjects. Rhodopsin immunolabeling in the normal retinas was restricted to the rod outer segments. In the RP retinas, rhodopsin was present in shortened rod outer segments and in the surface membranes of the rod inner segments and somata. In regions of photoreceptor death, the surviving rods had sprouted rhodopsin-positive neurites that were closely associated with gliotic Müller cell processes and extended to the inner limiting membrane. Rods and cones in the RP maculas did not form neurites, but the axons of peripheral cones were abnormally elongated and branched. Double immunofluorescence labeling showed that the rod neurites bypassed the horizontal and rod bipolar cells that are normally postsynaptic to rod axons. To our knowledge, this is the first report of rod neurite sprouting in vivo. We were unable to find neurites on degenerate rods in old rds mice, an animal model for RP. The rod neurites in the human RP retinas resemble the long, branched processes formed by rods cultured on Müller cells or purified N-CAM. Neurite growth by surviving rods in the RP retinas may be a response to neurotrophic factor upregulation, loss of inhibitory factors, or changes in molecules associated with reactive Müller cells. Such changes in the retinal microenvironment may impede functional integration of transplanted photoreceptors. The contributions of the rhodopsin-positive rod neurites and abnormal cone axons to the functional abnormalities observed in RP are unknown.

Adult↗
PubMed1994

A splice variant of arrestin. Molecular cloning and localization in bovine retina.

Inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of the 48-kDa regulatory protein arrestin. We recently isolated a novel form of arrestin, termed p44, that is truncated at the COOH terminus (Palczewski, K., Buczylko, J., Ohguro, H., Annan, R. S., Carr, S. A., Crabb, J. W., Kaplan, M. W., Johnson, R. S., and Walsh, K. A. (1994) Protein Sci. 3, 319-329) and strongly inhibits Gt activation by non-phosphorylated rhodopsin. p44 is identical to arrestin except at the COOH terminus, where the 35 amino acids of arrestin are replaced by a single alanine residue. p44 is identified as a splice variant of arrestin based on the identical cDNA sequence of p44 with arrestin (except the 3' non-coding regions), the presence of an exon/intron junction at the Ser369 codon, and identical Southern hybridization patterns generated by the 3' non-coding portion of arrestin and p44. Immunocytochemistry reveals that p44 is localized in the photoreceptor outer segment, whereas arrestin is present throughout the cell. This specificity of localization to the outer segment is consistent with a role of p44 in the phototransduction cascade.

Alternative Splicing↗
PubMed1994

Autosomal dominant retinitis pigmentosa caused by the threonine-17-methionine rhodopsin mutation: retinal histopathology and immunocytochemistry.

The retinas from a 68-year-old man with autosomal dominant retinitis pigmentosa caused by the threonine-17-methionine rhodopsin mutation were studied. Patients with this mutation have a characteristic clinical phenotype that shows intra- and interfamilial consistency. The retinas were examined by light and electron microscopy, including immunocytochemistry with markers for rods, cones, the retinal pigment epithelium, the interphotoreceptor matrix and Müller cells, and the results were correlated with those from visual function tests performed fourteen months before death. Grossly, both retinas had heavy deposits of bone spicule-like pigmentation inferiorly, but the superior retinas showed much less pigmentation. The maculas contained no rods and an incomplete monolayer of cone somata, consistent with the patient's severely reduced central vision. The inferior retinas contained no rods and rare cone somata, correlating with an absolute scotoma in the superior field. The superior retinas had near-normal-appearing rods and cones in the far periphery and a gradient from the midperipheral to central retina of progressively shortened outer segments and loss of photoreceptors, consistent with the patient's reduced rod and cone electroretinograms and retained rod and cone sensitivities in the inferior peripheral field by perimetry. Immunocytochemistry with rod-specific markers was qualitatively normal in the superior mid to far peripheral retina. Electron microscopic immunogold labeling with anti-rhodopsin revealed similar densities of gold particles over rod outer segments of the RP and a normal donor retina. Degenerate photoreceptors in the superior equatorial region of the RP retina had short outer segments, and some rods had surface labeling with anti-rhodopsin of the inner segments, somata and synapses. The results indicate regional retinal differences of disease severity in this genotype and that rods in the superior peripheral retina have normal morphology despite the presence of the mutant allele. To the authors' knowledge, this is the first detailed study of rod photoreceptors in the retina of an RP patient with a rhodopsin mutation.

Aged↗
PubMed1994

Histopathology and immunocytochemistry of the neurosensory retina in fundus flavimaculatus.

BACKGROUND: Fundus flavimaculatus (Stargardt disease) is a group of inherited macular dystrophies in which central vision usually decreases in the first two decades of life. Previous histopathologic studies used light, scanning, and transmission electron microscopy to characterize the retinal pigment epithelium (RPE) in fundus flavimaculatus. The authors describe in detail the pathologic changes in the neurosensory retina, including use of specific immunocytochemical markers. METHODS: The eyes of a patient with fundus flavimaculatus were processed using Medcast and JB-4 plastic for light and electron microscopy, and cryomicrotomy and LR-white resin for immunocytochemistry. RESULTS: Changes in the RPE occurred in a peripheral/central gradient and included increased lipofuscin content and cell loss toward the macula. The changes in the retina paralleled those in the RPE, including accumulation of lipofuscin in photoreceptor inner segments, loss of photoreceptors, and reactive Müller cell hypertrophy. Immunocytochemistry using rod- and cone-specific markers showed abnormal photoreceptor morphology but qualitatively normal immunoreactivity, and there was strong reactivity for glial fibrillary acid protein in reactive Müller cells. Labeling for cellular retinaldehyde-binding protein was qualitatively normal in Müller cells, but was reduced in RPE cells that were engorged with lipofuscin. CONCLUSIONS: The histopathologic changes in the retina correlate with clinical progression of the disease process. Although abnormal lipofuscin metabolism has been implicated in the loss of vision in fundus flavimaculatus and other macular diseases, the mechanism is not understood. Based on the authors' observations and a review of recent literature on lipofuscin, the authors propose that all-trans-retinol dehydrogenase, a photoreceptor outer segment enzyme, may be defective in fundus flavimaculatus.

Female↗
PubMed1993

Strategies for rescue of retinal photoreceptor cells.

A variety of genetic and environmental factors cause degeneration of retinal photoreceptors. This review focuses on current strategies to rescue defective, but still viable, rods and cones, including transplantation of normal retinal pigment epithelium cells, corrective gene therapy, administration of survival/growth factors, protection from the damaging effects of light, and dietary supplementation of vitamin A.

Animals↗
PubMed1993

Autoantibodies against retinal bipolar cells in cutaneous melanoma-associated retinopathy.

PURPOSE: This study's goal was to determine the pathophysiology of the retinopathy that occurs in patients with metastatic cutaneous melanoma and sudden onset of night blindness, the so-called melanoma-associated retinopathy (MAR) syndrome. We tested the hypothesis that sera from two MAR patients contained autoantibodies that reacted with "on" bipolar cells of the human retina. METHODS: Immunofluorescence was performed on cryostat sections of unfixed normal human retinas. Sera and IgG fractions were tested from the two MAR patients and 38 control subjects (28 patients with metastatic melanoma, but no visual symptoms; two patients with non-MAR retinopathy; and eight normal subjects). RESULTS: The sera and IgG fractions from both MAR patients but from none of the control subjects produced heavy immunostaining of bipolar cells, which were identified as rod bipolars by a double labeling procedure using anti-protein kinase C. CONCLUSIONS: We hypothesize that MAR patients generate autoantibodies against a melanoma antigen that cross react with bipolar cells of the retina. These antibodies, by an unknown mechanism, may cause abnormalities of the rod and cone systems that are characteristic of MAR.

Adult↗
PubMed1992

Ultrastructure of connecting cilia in different forms of retinitis pigmentosa.

The connecting (sensory) cilium of rods and cones is the stalk that separates the outer segment, which contains visual pigment in stacks of membrane discs, from the inner segment, which contains cytoplasmic organelles involved in protein synthesis. There are conflicting reports on the occurrence of abnormal motile cilia in patients with retinitis pigmentosa (RP) and very few ultrastructural studies of photoreceptor connecting cilia in retinas from patients with RP. Defective connecting cilia could lead to the outer segment atrophy and degeneration that are characteristic of RP. The present study addresses the hypothesis that photoreceptor connecting cilia, as observed in cross section by electron microscopy, are defective in RP. We examined retinas from five patients with RP and four controls and found morphologic defects in the connecting cilia of one RP patient with type 2 Usher syndrome (86% abnormal, P less than .0001) but not in our sample of patients with X-linked (n = 2), simplex (n = 1), or autosomal dominant (n = 1) RP.

Adolescent↗
PubMed1992

Morphometric analysis of macular photoreceptors and ganglion cells in retinas with retinitis pigmentosa.

There have been a number of histopathologic studies of retinas that were taken post mortem from patients with retinitis pigmentosa (RP), but few have addressed the question of transneuronal degeneration of ganglion cells secondary to photoreceptor death. We studied sectioned maculae that were obtained from 41 patients with different genetic forms of RP: autosomal dominant (n = 11); X-linked (n = 9); and simplex (n = 21). We also studied sectioned maculae that were taken from 20 age-matched normal subjects. We counted cell bodies in the photoreceptor and ganglion cell layers at 100-microns (0.35 degrees) intervals from the foveola to 1500-microns eccentricity and compared the mean cell counts among each group with RP. Each RP type had significantly fewer (P < .05) photoreceptors than those of the control group at each 100-microns interval. At eccentricities of 700 to 1500 microns, the retinas with X-linked and autosomal dominant RP had significantly fewer (P < .05) ganglion cells than those of the control group; the simplex RP mean ganglion cell counts were significantly lower (P < .05) than those of the control group, from 1000 to 1500 microns. The mean photoreceptor and ganglion cell counts had a .43 correlation (P < .001) in the zone of 700 to 1500 microns, consistent with transneuronal ganglion cell degeneration. Current experimental attempts to restore vision in diseased retinas by simulating or replacing photoreceptors are based on the premise that ganglion cells are retained after photoreceptor death. Our findings support this assumption.

Adolescent↗