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Biomedical subjects

A H Milam

Publications and source records attributed to A H Milam.

At least 37 records · Page 2Linked to original sources
PubMed1998

Guanylate-cyclase-inhibitory protein is a frog retinal Ca2+-binding protein related to mammalian guanylate-cyclase-activating proteins.

Two guanylate-cyclase-activating proteins (GCAP) encoded by a tail-to-tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca2+-binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca2+], but inhibits GC in high [Ca2+], and is therefore termed guanylate-cyclase-inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca2+-binding motif of GCIP is disabled for Ca2+ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca2+-binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca2+], competing with GCAP1 and GCAP2 for GC regulatory sites.

Amino Acid Sequence↗
PubMed1998

Recoverin-associated retinopathy: a clinically and immunologically distinctive disease.

PURPOSE: To compare the immune response to retinal antigens in a patient with a clinical condition resembling cancer-associated retinopathy with the immune responses of patients with other retinal degenerations or uveitis. METHODS: Cellular and humoral immune responses to retinal S-antigen and recoverin were determined in one patient with disease resembling cancer-associated retinopathy, three patients with other retinal degenerations, and eight patients with uveitis. RESULTS: A cellular immune response against recoverin was found only in the patient with the condition resembling cancer-associated retinopathy. Elevated levels of antibody against recoverin were found in this patient and in one of the three patients with a retinal degeneration, but in none of the eight patients with uveitis. In contrast, moderate lymphocyte responses to retinal S-antigen were found in most of the patients studied, and this response did not distinguish among the patient groups. Levels of serum antibodies against retinal S-antigen were also similar in all patients tested. Serum from the patient with disease resembling cancer-associated retinopathy produced strong immunostaining of the rods, cones, outer plexiform layer, and some cone bipolar cells, but serum from the patients with uveitis or other retinal degenerations did not show specific reactivity with the retina. CONCLUSIONS: We propose that this immunologically and clinically distinctive condition be termed recoverin-associated retinopathy, and we suggest that a cellular immune response against recoverin may be a distinguishing feature of the disorder.

Adenolymphoma↗
PubMed1998

Abnormal cone synapses in human cone-rod dystrophy.

OBJECTIVE: Little is known of the cytopathology of photoreceptors in human inherited retinal dystrophies that initially affect the central retina, including the macula. The current study sought to determine the cytologic features of dysfunctional cone and rod photoreceptors, as well as the pattern of degeneration of the cells in representative cases of central retinal dystrophy. STUDY DESIGN: Comparative human tissue study. MATERIALS: Four human donor eyes with the following forms of central retinal dystrophy: cone-rod dystrophy (CRD), central areolar choroidal dystrophy, Bardet-Biedl syndrome, and cone dystrophy-cerebellar ataxia. The cytologic features of retinal photoreceptors in these eyes were compared with those in an eye with retinitis pigmentosa and six normal human eyes. METHODS AND OUTCOME MEASURES: Immunocytochemistry and electron microscopy were used to evaluate the retinal histopathology in the donor eyes. RESULTS: Cone numbers were decreased in the case of CRD, particularly in the central and far peripheral retina, and both cone and rod outer segments were slightly shortened. Occasional degenerate cones had dense cytoplasm and pyknotic nuclei dislocated sclerad to the external-limiting membrane. The most prominent alteration in this retina was marked enlargement and distortion of the cone photoreceptor pedicles, which contained reduced numbers of synaptic vesicles. The retina with central areolar choroidal dystrophy contained a few cones with similarly abnormal synapses. However, comparable cone synapse abnormalities were not observed in the cases of Bardet-Biedl syndrome, cone dystrophy-cerebellar ataxia, retinitis pigmentosa, or in the normal retinas. CONCLUSIONS: The functional consequences of the cone synapse abnormalities in CRD are not known but may correlate with the electroretinographic abnormalities documented in some cases of CRD. To our knowledge, comparable synapse changes have not been noted in either rods or cones in other forms of retinal dystrophy, including retinitis pigmentosa, suggesting that different cytopathologic mechanisms may be involved.

Adult↗
PubMed1998

Histopathology of the human retina in retinitis pigmentosa.

The term, 'retinitis pigmentosa', refers to a heterogeneous group of inherited diseases that cause degeneration of rod and cone photoreceptors in the human retina. Loss of photoreceptor cells is usually followed by alterations in the retinal pigment epithelium and retinal glia. Ultimately, degenerative changes occur in the inner retinal neurons, blood vessels, and optic nerve head. This chapter provides background information on the genetics of retinitis pigmentosa and a summary of the histopathologic alterations in human retinas caused by this disease. Detailed information is provided on the effects of the primary disease process on the rod photoreceptors and changes in the other retinal components, all of which are important considerations for understanding and developing therapies for retinitis pigmentosa.

Astrocytes↗
PubMed1998

Accumulation of tissue inhibitor of metalloproteinases-3 in human eyes with Sorsby's fundus dystrophy or retinitis pigmentosa.

BACKGROUND/AIMS: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is normally synthesised by the retinal pigment epithelium (RPE) and deposited in Bruch's membrane. Mutations in the TIMP3 gene cause Sorsby's fundus dystrophy (SFD), which is characterised by thickening of Bruch's membrane, choroidal neovascularisation, and photoreceptor degeneration. To elucidate the role of TIMP-3 in human retinal degenerative diseases, we immunolocalised TIMP-3 in eyes with SFD caused by the Ser-181-Cys TIMP3 gene mutation or retinitis pigmentosa (RP; not caused by TIMP3 mutations). METHODS: Standard light microscopic immunocytochemistry, including antigen retrieval, was used to localise TIMP-3 in paraffin sections of human eyes: two with SFD, three with different genetic forms of RP, and two normal. RESULTS: In the SFD eyes, the thickened Bruch's membrane was strongly TIMP-3 positive except where RPE cells had degenerated. Similarly, in the RP eyes, Bruch's membrane was TIMP-3 positive except where RPE cells were lost, consistent with ongoing RPE mediated turnover of TIMP-3 in this region. In areas of total photoreceptor loss, migrated RPE cells formed cuffs around blood vessels in the RP retinas. Thick, TIMP-3 positive extracellular matrix (ECM) deposits associated with the migrated RPE cells occluded some vascular lumina, correlating with the observed loss of inner retinal neurons in RP. CONCLUSIONS: TIMP-3 is a component of the increased ECM sequestered in Bruch's membrane in SFD. Further information is needed on normal TIMP-3/ECM interactions in Bruch's membrane and the effect of mutant TIMP-3 on this process. The finding of TIMP-3 accumulations in retinas with RP not caused by TIMP-3 mutations emphasises the importance of ECM remodelling in normal and diseased human eyes.

Aged↗
PubMed1998

Effects of retinal laser photocoagulation on photoreceptor basic fibroblast growth factor and survival.

PURPOSE: In an unpublished study, the authors found that immunoreactivity for basic fibroblast growth factor (bFGF) is increased in rod photoreceptors adjacent to long-standing laser burns in human diabetic retinas. The goal of this study was to determine whether laser photocoagulation produces a similar increase in photoreceptor bFGF and promotes survival of these cells in dystrophic rodent retinas. METHODS: Threshold (whitening) and subthreshold (nonwhitening) laser burns were made in retinas of normal and Royal College of Surgeons (RCS) rats and normal and rds mice. The retinas were processed for immunocytochemical and morphometric analyses. RESULTS: In nonlasered normal rat and mouse retinas, bFGF immunoreactivity was prominent in the nuclei of Müller cells and astrocytes. Photoreceptors were bFGF negative except for a zone of bFGF-immunoreactive rods near the ora serrata. Some photoreceptors in nonlasered retinas of RCS rats and rds mice became bFGF immunoreactive. After laser treatment, bFGF immunoreactivity was markedly increased in all photoreceptors flanking the threshold burns and within the subthreshold burns in normal and mutant rats and mice. In RCS rat retinas, photoreceptor bFGF immunoreactivity remained elevated within subthreshold burns and flanking the threshold burns, and photoreceptor survival was prolonged. In rds mouse retinas, increased bFGF immunoreactivity in photoreceptors was not sustained and their degeneration was not retarded. CONCLUSIONS: Laser treatment of RCS rat retinas produced a sustained increase in bFGF immunoreactivity in photoreceptors and prolonged their survival, but laser treatment of rds mouse retinas did not have a long-term effect on photoreceptor bFGF immunoreactivity or survival. Although species differences in laser effects on photoreceptor bFGF and survival are apparent, the finding that rods flanking laser burns in human retinas have sustained increases in bFGF immunoreactivity suggests that laser treatment may be useful for prolonging survival of mutant photoreceptors in retinas of patients with retinitis pigmentosa.

Adult↗
PubMed1998

Rhodopsin transgenic pigs as a model for human retinitis pigmentosa.

PURPOSE: To further characterize the retinas of Pro3471Leu rhodopsin transgenic pigs, a model for human retinitis pigmentosa. METHODS: Retinas from normal and transgenic pigs, newborn to 20 months old, were processed for light and electron microscopic immunocytochemical examination. RESULTS: At birth, rod numbers were normal in the transgenic retinas, but their outer segments were short and disorganized and their inner segments contained stacks of rhodopsin-positive membranes. The newborn rod synapses lacked synaptic vesicles and ribbons and had numerous rhodopsin-positive, filopodia-like processes that extended past the cone synapses into the outer plexiform layer. Rod cell death was apparent by 2 weeks and was pronounced in the mid periphery and central regions by 6 weeks. Far peripheral rods were initially better preserved, but by 9 months virtually all rods had degenerated. Cones degenerated more slowly than rods, but by 4 weeks the cone synapses were shrunken and some mid peripheral cones had lost their immunoreactivity for phosphodiesterase-gamma, arrestin, and recoverin. From 9 months to 20 months, the cone outer segments shortened progressively, and more cones lost immunoreactivity for these proteins. CONCLUSIONS: The rhodopsin transgenic pig retina shares many cytologic features with human retinas with retinitis pigmentosa and provides an opportunity to examine the earliest stages in photoreceptor degeneration, about which little is known in humans. The finding of abnormal rhodopsin localization in newborn rods is consistent with misrouting of mutant rhodopsin as an early process leading to rod cell death. Novel changes in the photoreceptor synapses may correlate with early electrophysiological abnormalities in these retinas.

Animals↗
PubMed1998

Relation of optical coherence tomography to microanatomy in normal and rd chickens.

PURPOSE: To elucidate the relation between optical coherence tomography (OCT) scans and retinal histology in normal and retinal degeneration (rd) chickens. METHODS: Retinas from adult normal and rd chickens were examined in vivo with OCT at 850 nm and compared quantitatively with stained cryosections of unfixed retinas from the same locations. RESULTS: The nerve fiber layer (NFL) and inner plexiform layer (IPL) show homogeneous backscatter throughout their thicknesses. NFL reflectivity is approximately 0.6 log units higher than that of the IPL. The inner nuclear layer shows a low reflectivity; the properties of reflections from ganglion cell and outer nuclear layers are indeterminate. The outer retina and choroid form a large reflective complex. Photoreceptor inner segments produce the highest of these reflections in normal chicken retinas, approximately 1.5 log units higher than that of the IPL. The retinal pigment epithelium also has a relatively large backscatter coefficient and is the dominant reflector in rd retinas that lack photoreceptors. Choroidal pigment produces an intermediate level of backscatter and is the largest attenuator of signal at 850 nm. CONCLUSIONS: Quantified OCT signals have a predictable relationship to histology and pathology in chicken retinas. The results from rd retinas represent a first step toward in vivo quantitation of retinal structure in retinal degenerative disease.

Animals↗
PubMed1997

Localization of guanylate cyclase-activating protein 2 in mammalian retinas.

Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca2+, a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.

Animals↗
PubMed1997

Preservation of the inner retina in retinitis pigmentosa. A morphometric analysis.

OBJECTIVE: To determine the extent of preservation in the inner retina in retinitis pigmentosa (RP). METHODS: We analyzed sectioned maculae of 21 postmortem eyes with RP and 19 age-matched, normal, postmortem eyes. Eyes were divided into 2 groups: severe and moderate RP. Cell nuclei were counted in the outer nuclear, inner nuclear, and ganglion cell layers within thirty 100-microns intervals from the foveola to 1500-microns eccentricity. RESULTS: Statistically significant (P < or = .05) loss of both the outer nuclear and ganglion cell layers was present in the groups with moderate and severe RP when compared with the control groups. However, even in the group with severe RP, 30% of the ganglion cells were histologically intact. Similarly, 78% and 88% of the inner nuclear layer cells were preserved in the groups with severe and moderate RP, respectively. Different inheritance modes showed no statistically significant differences in any of the retinal layers. CONCLUSIONS: Despite a statistically significant (P < or = .05) loss of cells found in all retinal layers, a large percentage of the inner retinal neurons remained histologically intact. Current experimental therapies, such as photoreceptor transplantation and implantation of a visual prosthesis, are based on the premise that some inner retinal neurons are preserved after death of photoreceptors in RP. Our observations support this assumption.

Aged↗
PubMed1997

Tissue inhibitor of metalloproteinases-3 is a component of Bruch's membrane of the eye.

Mutations in tissue inhibitor of metalloproteinases (TIMP)-3 are found in some patients with Sorsby's fundus dystrophy, a retinal degeneration characterized by abnormal deposits in Bruch's membrane and choroidal neovascularization. The purpose of this study was to localize TIMP-3 in the retina/choroid of normal human and animal eyes. Immunolabeling was performed on unfixed and fixed sections of human eyes aged 24 to 85 years and unfixed sections of baboon, chicken, cow, pig, and rat eyes using a monoclonal antibody against a human TIMP-3 synthetic peptide. The antibody produced strong immunolabeling of Bruch's membrane and drusen and weak labeling of retina blood vessels in unfixed human and baboon eyes. Unfixed chicken, cow, pig, and rat tissues showed no reactivity. After antigen retrieval, all fixed human eyes showed specific labeling of Bruch's membrane and drusen, which was strongest in eyes from elderly donors. The results indicate that TIMP-3 is an extracellular matrix component of Bruch's membrane. Thus, abnormal local function of TIMP-3 may lead to the characteristic Bruch's membrane deposits and choroidal neovascularization found in Sorsby's fundus dystrophy. Specific labeling of drusen raises the possibility that altered TIMP-3-mediated matrix remodeling may contribute to age-related degenerative changes in Bruch's membrane.

Adult↗
PubMed1997

Disease expression in X-linked retinitis pigmentosa caused by a putative null mutation in the RPGR gene.

PURPOSE: To determine the disease expression in X-linked retinitis pigmentosa (XLRP) caused by a putative null mutation in the RPGR (retinitis pigmentosa GTPase regulator) gene. METHODS: In a family with XLRP, haplotype analysis was performed with polymorphic microsatellite markers from the Xp chromosomal region, and genomic polymerase chain reaction sequencing was used to identify sequence variations in the RPGR gene. Hemizygotes and heterozygotes were evaluated clinically and with visual function tests. Optical coherence tomography (OCT) was performed on heterozygotes. Postmortem donor retinas from a heterozygote were examined by microscopy and immunocytochemistry. RESULTS: X-linked inheritance was confirmed by haplotype analysis using Xp markers. Sequence analysis of the RPGR gene identified a single base pair change, a G-->T transversion, that converts codon 52 GGA (Gly) to TGA (stop codon); the mutation segregates with the disease. A hemizygote in the third decade of life had barely measurable rod function and severely impaired cone function that diminished further over a 7-year interval. Heterozygotes varied in degree of disease expression from mild to severe. Perimetry showed loci with normal rod and cone sensitivity interspersed with loci having either equal rod and cone dysfunction or rod > cone dysfunction. Electroretinographic photoreceptor responses had equal reductions in rod and cone maximal amplitude. OCT cross sectional reflectance images of retinal regions with severe dysfunction showed reduced thickness of the retina and retinal pigment epithelium-choriocapillaris (RPE-CC) complex and increased reflections posteriorly. Regions with mild dysfunction showed similar OCT findings but with preserved retinal thickness. Retinal histopathology in a heterozygote revealed loss of photoreceptors throughout, with retention of only a few islands of cones with tiny or absent outer segments and rods lacking outer segments. CONCLUSIONS: This RPGR gene mutation, in its mildest expression in heterozygotes, causes a relatively equal disturbance of rod and cone photoreceptor function. Detectable structural change by OCT at the level of the RPE-CC can be present in patches of retina with minimal functional disturbance. More advanced disease stages in heterozygotes show greater rod than cone dysfunction, and the end stage in hemizygotes and heterozygotes is that of typical RP, with only barely detectable cone function from residual cones in a thinned retina with abnormal RPE and choriocapillaris.

Adult↗
PubMed1997

Cellular retinaldehyde-binding protein is expressed by oligodendrocytes in optic nerve and brain.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium and Müller glial cells of the retina, where it forms complexes with endogenous 11-cis-retinoids. We examined the distribution of CRALBP in extraretinal tissues using polyclonal antibodies (pAb) and monoclonal antibodies (mAb). A protein was detected by immunoblot analysis in extracts of bovine and rat brain and optic nerve but not in several other tissues. This protein had electrophoretic, chromatographic, and retinoid-binding properties identical to those of CRALBP from bovine retina. Comparison of the masses of tryptic peptides and of partial amino acid sequences derived from brain and retinal CRALBP indicated that the two proteins are probably identical. Immunoperoxidase cytochemistry and double labeling immunofluorescence revealed CRALBP(+) cells in brain that resembled oligondendrocytes and not astrocytes, microglial cells, or pinealocytes. In 11-day-old rat brain, approximately 11% of the CRALBP(+) cells were labeled with the Rip antibody, a marker for oligodendroglia. In developing rat optic nerve, the temporal appearance of CRALBP(+) cells corresponded to that of oligodendrocytes and not that of astrocytes. In adult rat and mouse optic nerves, the CRALBP(+) somata showed the same distribution as oligodendrocytes. No endogenous retinoids were associated with CRALBP isolated from dark-dissected adult bovine brain. The results suggest that CRALBP has functions in addition to retinoid metabolism and visual pigment regeneration.

Amino Acid Sequence↗
PubMed1996

A mouse model of gyrate atrophy of the choroid and retina. Early retinal pigment epithelium damage and progressive retinal degeneration.

Gyrate atrophy (GA) of the choroid and retina is a blinding chorioretinal degeneration caused by deficiency of ornithine delta-aminotransferase (OAT). The phenotype of GA is characterized by progressive concentric reduction of the visual fields and ornithine accumulation. To understand better the pathogenesis of GA and to develop a model to test therapeutic strategies, we produced an OAT-deficient mouse by gene targeting. Like human GA patients, adult OAT-deficient mice exhibit chronic hyperornithinemia to levels 10-15-fold above normal and massive ornithinuria. Slowly progressive retinal degeneration is reflected by a gradual decline in electroretinogram amplitudes over the first 12 mo of life. At 2 mo, the retinal pigment epithelium is histologically normal, but electron microscopy reveals sporadic degeneration of scattered pigment epithelial cells. By 6 mo there are more diffuse abnormalities of the pigment epithelium with accumulation of large phagosomes and crystalloid inclusions. Although morphologically normal at 2 mo, the photo-receptor outer segments become highly disorganized and shortened to 60% of control length by 10 mo. Additionally, there is cumulative loss of the photoreceptor cells, which reaches 33% by 10 mo and is most pronounced in the central region of the retina. Our results indicate that retinal pigment epithelial cells are the initial site of insult in GA and that the OAT-deficient mouse is an excellent animal model of GA in human patients.

Animals↗
PubMed1996

Alterations in anterior segment dimensions in eyes with retinitis pigmentosa.

BACKGROUND: In visually deprived primate eyes there is a correlation between alterations in growth parameters and changes in retinal neurotransmitters. Progressive retinal degeneration also leads to alterations in retinal neurotransmitters. In this study we wanted to investigate whether alterations in the dimensions of the anterior eye segment are also present in human donor eyes of patients with hereditary retinal degeneration. METHODS: Sagittal sections through anterior segments of 47 eyes from donors with different types of retinitis pigmentosa (RP) and of 59 eyes from normal donors were investigated morphometrically using a PC-based morphometric system. RESULTS: In the RP eyes there was a significant shortening of the distance between the peripheral end of Descemet's membrane and the posterior end of Schlemm's canal and of the length of the pars plana of the ciliary body. These changes varied markedly among the different types of RP. The most pronounced shortenings were found in eyes of men with X-linked recessive RP, which had the most severe retinal changes. RP eyes showed, in contrast to normal eyes, significant age-related elongation of the ciliary body and the chamber angle. A significant increase of the axial diameter with age was also found in RP eyes. CONCLUSION: Our study reveals for the first time that there are changes in the anterior segment dimensions in human eyes with hereditary retinal degeneration. Measurements of anterior eye segment parameters might provide additional indices for the severity of RP and for the diagnosis of RP subtypes.

Aged↗
PubMed1996

Acquired unilateral night blindness associated with a negative electroretinogram waveform.

PURPOSE: The authors performed clinical, electrophysiologic, psychophysical, and immunologic studies in a patient who presented with an acquired night blindness in one eye to better define the clinical and functional changes in this rare disorder. METHODS: In addition to an ophthalmologic examination, the patient underwent the measurement of electroretinogram responses, dark-adapted thresholds using a Tübingen perimeter (Oculus, Tubingen, Germany), color vision assessment, kinetic visual-field testing using a Goldman perimeter, and immunologic testing to determine if the serum contained autoantibodies to retinal bipolar cells. RESULTS: Fundus examination showed no clinically apparent abnormality in either eye. The patient showed a selective reduction in the b-wave amplitude of the rod electroretinogram and an abnormality of the cone electroretinogram ON response in the affected left eye, whereas the rod and cone electroretinograms of the right eye were normal. Rod thresholds in the affected eye were elevated markedly, whereas rod thresholds in the right eye were normal centrally and slightly elevated in the far periphery. Immunologic testing did not show circulating autoantibodies to retinal cells. CONCLUSIONS: The patient examined in this study showed phenotypic similarities to patients with congenital stationary night blindness and to patients with an acquired form of night blindness associated with cutaneous melanoma (MAR syndrome). The electroretinogram findings from the patient are consistent with an acquired defect in signal transmission from photoreceptors to ON-type bipolar cells. However, the etiology of this unique form of unilateral night blindness remains obscure.

Adult↗