Peripheral Lamina Research Articles

Lamina Cribrosa Microstructure in Nonhuman Primates With Naturally Occurring Peripapillary Retinal Nerve Fiber Layer Thinning.

The lamina cribrosa (LC) is hypothesized to be the site of initial axonal damage in glaucoma with the circumpapillary retinal nerve fiber layer thickness (RNFL-T) widely used as a standard metric for quantifying the glaucomatous damage. The purpose of this study was to determine in vivo, 3-dimensional (3D) differences in the microstructure of the LC in eyes of nonhuman primates (NHPs) with naturally occurring glaucoma. Spectral-domain optical coherence tomography (OCT) scans (Leica, Chicago, IL, USA) of the optic nerve head were acquired from a colony of 50 adult rhesus monkeys suspected of having high prevalence of glaucoma. The RNFL-T was analyzed globally and in quadrants using a semi-automated segmentation software. From a set of 100 eyes, 18 eyes with the thinnest global RNFL-T were selected as the study group and 18 eyes with RNFL-T values around the 50th percentile were used as controls. A previously described automated segmentation algorithm was used for LC microstructure analysis. Parameters included beam thickness, pore diameter and their ratio (beam-to-pore ratio [BPR]), pore area and shape parameters, beam and pore volume, and connective tissue volume fraction (CTVF; beam volume/total volume). The LC microstructure was analyzed globally and in the following volumetric sectors: quadrants, central and peripheral lamina, and three depth slabs (anterior, middle, and posterior). Although no significant difference was detected between groups for age, weight, or disc size, the study group had significantly thinner RNFL than the control group (P < 0.01). The study group had significantly smaller global and sectoral pore diameter and larger BPR compared with the control group. Across eyes, the global RNFL-T was associated positively with pore diameter globally. BPR and CTVF were significantly and negatively associated with the corresponding RNFL-T in the superior quadrant. Global and sectoral microstructural differences were detected when comparing thin and normal RNFL-T eyes. Whether these LC differences are the cause of RNFL damage or the result of remodeling of the LC requires further investigation. Our findings indicate structural alterations in the LC of NHP exhibiting natural thinning of the RNFL, a common characteristic of glaucomatous damage.

Interleukin-24 regulates mucosal remodeling in inflammatory bowel diseases

BackgroundRecently, increased interleukin (IL)-24 expression has been demonstrated in the colon biopsies of adult patients with inflammatory bowel disease (IBD). However, the role of IL-24 in the pathomechanism of IBD is still largely unknown.MethodsPresence of IL-24 was determined in the samples of children with IBD and in the colon of dextran sodium sulfate (DSS) treated mice. Effect of inflammatory factors on IL24 expression was determined in peripheral blood (PBMCs) and lamina propria mononuclear cells (LPMCs). Also, the impact of IL-24 was investigated on HT-29 epithelial cells and CCD-18Co colon fibroblasts. Expression of tissue remodeling related genes was investigated in the colon of wild type (WT) mice locally treated with IL-24 and in the colon of DSS treated WT and Il20rb knock out (KO) mice.Results Increased amount of IL-24 was demonstrated in the serum and colon samples of children with IBD and DSS treated mice compared to that of controls. IL-1β, LPS or H2O2 treatment increased the expression of IL24 in PBMCs and LPMCs. IL-24 treatment resulted in increased amount of TGF-β and PDGF-B in HT-29 cells and enhanced the expression of extracellular matrix (ECM)-related genes and the motility of CCD-18Co cells. Similarly, local IL-24 treatment increased the colonic Tgfb1 and Pdgfb expression of WT mice. Moreover, expression of pro-fibrotic Tgfb1 and Pdgfb were lower in the colon of DSS treated Il20rb KO compared to that of WT mice. The disease activity index of colitis was less severe in DSS treated Il20rb KO compared to WT mice.ConclusionOur study suggest that IL-24 may play a significant role in the mucosal remodeling of patients with IBD by promoting pro-fibrotic processes.

Lamins in the nuclear interior - life outside the lamina.

Nuclear lamins are components of the peripheral lamina that define the mechanical properties of nuclei and tether heterochromatin to the periphery. A-type lamins localize also to the nuclear interior, but the regulation and specific functions of this nucleoplasmic lamin pool are poorly understood. In this Commentary, we summarize known pathways that are potentially involved in the localization and dynamic behavior of intranuclear lamins, including their post-translational modifications and interactions with nucleoplasmic proteins, such as lamina-associated polypeptide 2α (LAP2α; encoded by TMPO). In addition, new data suggest that lamins in the nuclear interior have an important role in chromatin regulation and gene expression through dynamic binding to both hetero- and euchromatic genomic regions and promoter subdomains, thereby affecting epigenetic pathways and chromatin accessibility. Nucleoplasmic lamins also have a role in spatial chromatin organization and may be involved in mechanosignaling. In view of this newly emerging concept, we propose that the previously reported cellular phenotypes in lamin-linked diseases are, at least in part, rooted in an impaired regulation and/or function of the nucleoplasmic lamin A/C pool.

Peripheral lamina cribrosa depth in primary open-angle glaucoma: a swept-source optical coherence tomography study of lamina cribrosa.

To investigate peripheral lamina cribrosa depth (PLCD) and its vertical-horizontal difference in eyes with primary open-angle glaucoma (POAG). Patients with POAG (n=90 eyes) and age-matched healthy individuals (n=90 eyes) underwent swept-source optical coherence tomography (SS-OCT) scans centered at the optic discs. The PLCD was defined as the vertical distance between the most peripheral visible end of anterior lamina cribrosa (LC) surface and the reference plane connecting the Bruch's membrane openings. The PLCD in each quadrant region and the vertical-horizontal PLCD difference were compared between the POAG and healthy eyes. The clinical factors associated with increased PLCD were evaluated. The PLCD was significantly larger in the POAG eyes than the control eyes at the horizontal (P=0.034) and vertical (P=0.001) meridians. The vertical PLCD was significantly larger than the horizontal PLCD, both in the POAG eyes (P<0.001) and in the control eyes (P=0.003). However, the vertical-horizontal PLCD difference was significantly larger in the POAG eyes (47±60 μm) than in the control eyes (18±54 μm, P=0.001). Multivariate regression showed a significant association of male gender (P=0.005), increased baseline IOP (P=0.043), and decreased MD of VF (P=0.025) with increased PLCD. The peripheral LC was displaced more posteriorly in the POAG eyes compared with the age-matched healthy eyes. In the POAG eyes, the peripheral LC was displaced more posteriorly at the vertical meridian than at the horizontal meridian. The peripheral LC in the vertical meridian might have increased IOP-related strain (deformation) compared with horizontal meridian in glaucomatous eyes.

Sa1760 miR-155 Is Crucial for Th17 Cell Development and Plays a Major Role in the Pathogenesis of Intestinal Inflammation

BACKGROUND & AIMS: Interferon gamma (IFNγ) responses and distinct CD8+ T cell transcriptional signatures can be linked to clinical manifestations of inflammatory bowl disease (IBD). CD39 is an ectonucleotidase and is typically associated with CD4+ T regulatory memory cells, which have the capacity to generate immunosuppressive adenosine. However, immunomodulatory effects of CD39 expression on CD8+ T cells, as in IBD, remain unknown. METHODS:CD39+CD8+ T cells were purified from peripheral blood (PB) and lamina propria (LP) of patients with Crohn's disease. Phenotypic features and functions of CD39+CD8+ T cells were assessed by flow cytometry and immunoblotting. RESULTS: CD39 expressing CD8+ T cells of patients with Crohn's disease are IFNγ-producing cells, and exhibited type 1 CD8+ T cell (Tc1) properties. CD3 and CD28-mediated synergistic stimulation of CD8+ T cells augment CD39 expression, boost reactive oxygen species (ROS) generation, and increase IFNγ production. CD39+CD8+ T cells preferentially express CD28, and exhibit robust ROS-JNK/NFkB signals and IFNγ production. Decreases in ROSmediated by inhibitors of NADPH oxidases (NOX) or knockdown of gp91phox (NOX2) in CD8+ T cells abrogate effects of TCR ligation, and decrease both CD39 expression and IFNγ production. Curiously, CD39+CD8+ T cells inhibit IFNγ production by CD39-CD8+ T cells via generation of adenosine, which is operational via the paracrine expression of Adenosine 2A (ADORA2A) receptor. CONCLUSIONS: CD8+ Tc1 cells express CD39, which is further boosted by cell activation and ROS, which in turn limit IFNg responses by these cells. Strategies to regulate ROS signal cascades and adenosine-mediated effects might have therapeutic potential in the treatment of Crohn's disease.

Sa1761 Anti-TNF Therapy Switches on CD39+ FoxP3 Tregs in Association With Symptomatic and Endoscopic Remission in IBD

BACKGROUND & AIMS: Interferon gamma (IFNγ) responses and distinct CD8+ T cell transcriptional signatures can be linked to clinical manifestations of inflammatory bowl disease (IBD). CD39 is an ectonucleotidase and is typically associated with CD4+ T regulatory memory cells, which have the capacity to generate immunosuppressive adenosine. However, immunomodulatory effects of CD39 expression on CD8+ T cells, as in IBD, remain unknown. METHODS:CD39+CD8+ T cells were purified from peripheral blood (PB) and lamina propria (LP) of patients with Crohn's disease. Phenotypic features and functions of CD39+CD8+ T cells were assessed by flow cytometry and immunoblotting. RESULTS: CD39 expressing CD8+ T cells of patients with Crohn's disease are IFNγ-producing cells, and exhibited type 1 CD8+ T cell (Tc1) properties. CD3 and CD28-mediated synergistic stimulation of CD8+ T cells augment CD39 expression, boost reactive oxygen species (ROS) generation, and increase IFNγ production. CD39+CD8+ T cells preferentially express CD28, and exhibit robust ROS-JNK/NFkB signals and IFNγ production. Decreases in ROSmediated by inhibitors of NADPH oxidases (NOX) or knockdown of gp91phox (NOX2) in CD8+ T cells abrogate effects of TCR ligation, and decrease both CD39 expression and IFNγ production. Curiously, CD39+CD8+ T cells inhibit IFNγ production by CD39-CD8+ T cells via generation of adenosine, which is operational via the paracrine expression of Adenosine 2A (ADORA2A) receptor. CONCLUSIONS: CD8+ Tc1 cells express CD39, which is further boosted by cell activation and ROS, which in turn limit IFNg responses by these cells. Strategies to regulate ROS signal cascades and adenosine-mediated effects might have therapeutic potential in the treatment of Crohn's disease.

Sa1759 CD39 Expression by CD8+ T Cells Modulates Interferon γ-Mediated Responses in Crohn's Disease

BACKGROUND & AIMS: Interferon gamma (IFNγ) responses and distinct CD8+ T cell transcriptional signatures can be linked to clinical manifestations of inflammatory bowl disease (IBD). CD39 is an ectonucleotidase and is typically associated with CD4+ T regulatory memory cells, which have the capacity to generate immunosuppressive adenosine. However, immunomodulatory effects of CD39 expression on CD8+ T cells, as in IBD, remain unknown. METHODS:CD39+CD8+ T cells were purified from peripheral blood (PB) and lamina propria (LP) of patients with Crohn's disease. Phenotypic features and functions of CD39+CD8+ T cells were assessed by flow cytometry and immunoblotting. RESULTS: CD39 expressing CD8+ T cells of patients with Crohn's disease are IFNγ-producing cells, and exhibited type 1 CD8+ T cell (Tc1) properties. CD3 and CD28-mediated synergistic stimulation of CD8+ T cells augment CD39 expression, boost reactive oxygen species (ROS) generation, and increase IFNγ production. CD39+CD8+ T cells preferentially express CD28, and exhibit robust ROS-JNK/NFkB signals and IFNγ production. Decreases in ROSmediated by inhibitors of NADPH oxidases (NOX) or knockdown of gp91phox (NOX2) in CD8+ T cells abrogate effects of TCR ligation, and decrease both CD39 expression and IFNγ production. Curiously, CD39+CD8+ T cells inhibit IFNγ production by CD39-CD8+ T cells via generation of adenosine, which is operational via the paracrine expression of Adenosine 2A (ADORA2A) receptor. CONCLUSIONS: CD8+ Tc1 cells express CD39, which is further boosted by cell activation and ROS, which in turn limit IFNg responses by these cells. Strategies to regulate ROS signal cascades and adenosine-mediated effects might have therapeutic potential in the treatment of Crohn's disease.

Recent Structural Alteration of the Peripheral Lamina Cribrosa Near the Location of Disc Hemorrhage in Glaucoma

We investigated whether disc hemorrhage (DH) is associated with the recent structural alteration of the peripheral lamina cribrosa (LC) as assessed by enhanced depth imaging (EDI) spectral-domain optical coherence tomography (SD-OCT). Serial horizontal B-scan images were obtained by EDI SD-OCT from primary open-angle glaucoma (POAG) patients before and after the detection of DH (DH group, n = 45), and those who had no DH during the 1-year scan interval (non-DH group, n = 36). The images were processed using compensation and contrast enhancement. Then, 11 radial OCT images centered on the optic disc were generated from the 3-dimensionally reconstructed volume image. A recent structural alteration of the LC was defined when either the outward deformation of the anterior LC surface or radial disruption of the LC was identified in the temporal periphery. A recent structural alteration of the LC was found in 40 (88.9%) eyes in the DH group versus 4 (11.1%) eyes in the non-DH group. The amount of maximum outward deformation (55.82 ± 34.60 vs. 20.15 ± 4.28 μm) and radial disruption (69.87 ± 46.74 vs. 18.31 ± 1.17 μm) was larger in the DH group than in the non-DH group. The maximum LC alteration was observed within 1 clock-hour distance from the location of DH in all 40 eyes. The peripheral LC exhibited a recent alteration in eyes with DH. The alteration was correlated spatially with the location of the DH. These findings suggest that DH may result from microvascular damage incurred from alteration of the LC near its insertion.

Celiac Disease Resolution After Allogeneic Bone Marrow Transplantation is Associated with Absence of Gliadin-Specific Memory Response by Donor-Derived Intestinal T-cells

To elucidate the relative role of the immune system and intestinal epithelium in the ethiopatogenesis of Celiac disease (CD). A patient with childhood CD who underwent allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukemia was followed for 5years after resumption of gluten containing diet. Immunological memory to gliadin epitopes was assessed in the index patient and in 5 newly diagnosed CD patients by standard serology testing and by CFSE-based proliferation assays of peripheral blood CD4+ cells and of intestinal LPL towards gliadin-TTG antigens. Intestinal lymphocytes' origin was determined by combined immuno-histochemistry and fluorescent in-situ hybridiazation (FISH). Over 5years of follow-up after receiving BMT from a HLA-matched woman and cessation of gluten-free diet, the patient has remained well, with negative periodic antibodies assays and unremarkable serial duodenal biopsies. In vitro proliferation assays showed lack of a memory response of the patient's peripheral blood and lamina propria CD4+ T-cells towards TTG, gliadin or TTG-treated gliadin, whereas memory responses were evident in the newly diagnosed CD patients. Immuno-FISH of post-BMT duodenal mucosa showed that the chromosomal phenotype of all the epithelial cells was XY. In contrast, CD45+ lymphocytic lineage cells were all donor-derived XX cells, presumably originating in the transplanted bone marrow and re-populating the intestinal wall. CD resolution following allogeneic BMT is associated with absent gliadin-specific memory response, and with a dichotomous lymphocyte-epithelial chimeric intestine. These observations suggest that the pathogenesis of CD is critically dependent upon the immune system rather than the epithelial compartment.

Lamin-like analogues in plants: the characterization of NMCP1 in Allium cepa

The nucleoskeleton of plants contains a peripheral lamina (also called plamina) and, even though lamins are absent in plants, their roles are still fulfilled in plant nuclei. One of the most intriguing topics in plant biology concerns the identity of lamin protein analogues in plants. Good candidates to play lamin functions in plants are the members of the NMCP (nuclear matrix constituent protein) family, which exhibit the typical tripartite structure of lamins. This paper describes a bioinformatics analysis and classification of the NMCP family based on phylogenetic relationships, sequence similarity and the distribution of conserved regions in 76 homologues. In addition, NMCP1 in the monocot Allium cepa characterized by its sequence and structure, biochemical properties, and subnuclear distribution and alterations in its expression throughout the root were identified. The results demonstrate that these proteins exhibit many similarities to lamins (structural organization, conserved regions, subnuclear distribution, and solubility) and that they may fulfil the functions of lamins in plants. These findings significantly advance understanding of the structural proteins of the plant lamina and nucleoskeleton and provide a basis for further investigation of the protein networks forming these structures.

Ultrastructural composition of nonhistone chromosomal skeleton at different stages of mitosis in situ

In interphase cells of the SPEV culture treated with Triton X-100, 2 M NaCl, and DNAse, in the presence of 2 mM CuCl2, we clearly revealed a stabilized nuclear protein material (NPM) composed of a peripheral lamina, residual nucleolus, and internal fibrillar network. This network is formed by thin fibrils 10–20 nm in diameter, which are also revealed in the nonhistone matrix of mitotic chromosomes at all stages of mitosis. In mitotic chromosomes, NPM is represented as a network of the 10–20-nm-thick fibrils without any features of the central-axial structures. Beginning from the middle prophase, it is possible to see approached sister chromatids in contact with each other in certain sites, similar to centromeres. At these sites, the thickness of fibrils increases up to 40–50 nm, whereas the fibrils themselves are disposed more tightly; this structure can be seen in the chromosome until telophase. At the end of telophase, the decondensation of chromosomes and formation of two new nuclei whose NPM is analogous to NPM of usual interphase nucleus are observed. Thus, the NPM elements can perform the role of a skeleton in both the interphase nucleus and mitotic chromosomes.

Nuclear protein matrix of giant nuclei of polythene chromosome organization of midge Chironomus plumosus determinates

Giant nuclei from salivary glands of the midge Chironomus plumosus were treated in situ with 2M NaCl detergent and nucleases to reveal residual nuclear matrix proteins (NMP). It was shown that, after the prestabilization of nonhistone proteins with 2 mM CuCl2, the polythene chromosome body preserved its morphologic integrity and banding pattern, even after the extraction of all histones and DNA. The stabilized NPM of polythene chromosomes can be observed in both light and electron microscopy; no interchromatin fibrillary-granular structures are revealed in the nucleus except for peripheral lamina. Using the immunocytochemical method, in polythene chromosomes, we managed to detect major nonhistone proteins (topoisomerase IIα and SMC 1) and some RNA-components. Besides, in giant nuclei of larvae of early stages there is observed BrDU incorporation visualizing sites of DNA synthesis, which also are connected with NPM of polythene chromosomes. Thus, it can be concluded that structure of NPM of giant nuclei of Chironomus plumosus has all properties of NPMs of usual interphase nuclei; furthermore, this NPM determines specific structure of the polythene chromosome.

Central corneal thickness, lamina cribrosa and peripapillary scleral histomorphometry in non-glaucomatous chinese eyes

To examine a potential correlation between a thin cornea, a thin lamina cribrosa and a thin peripapillary sclera in normal eyes of Chinese, as surrogate for a presumed association between a thin cornea and an increased glaucoma susceptibility. The histomorphometric study included 55 non-glaucomatous globes (55 patients; mean age: 41.6 ± 13.6 years) enucleated due to a malignant choroidal melanoma without involvement of the optic nerve or trabecular meshwork. The axial length ranged between 21 and 27 mm. Histological sections through the pupil and the optic disc were morphometrically evaluated. We measured the thickness of the central cornea, central and peripheral lamina cribrosa, and peripapillary sclera. The mean central corneal thickness (500 ± 68 μm) was statistically not significantly associated with the thickness of the central lamina cribrosa (207 ± 60 μm) (P = 0.62), peripheral lamina cribrosa (P = 0.61), peripapillary sclera close to the optic nerve canal (P = 0.18), peripapillary sclera just outside of the optic nerve meninges (P = 0.11), nor with the shortest distance between the prelaminar space and cerebrospinal fluid space (P = 0.09). In Chinese, central corneal thickness is not significantly correlated with the thickness of the central and peripheral lamina cribrosa and the thickness of the peripapillary sclera nor with the shortest distance between the intraocular space and the cerebrospinal fluid space. These histomorphometric data do not support a relationship between central corneal thickness and thickness of structures of the optic nerve head, and thus do not support an association between a thin cornea and increased structural glaucoma susceptibility.

Formation and rearrangement of spermatodesms in males of some Orthoptera Tettigoniidae

In the male genital tract of Tettigoniidae, the spermatodesms are composed of a limited number of spermatozoa whose nuclei and acrosomes are covered by a mucous cap. The formation of the cap begins in the testicular cyst during the lengthening of the apical prolongations of the spermatids and the spermatids’ simultaneous division into small bundles or spermatodesms. The cap material is formed from a loosely arranged material in the lumen of the cyst, probably produced by the secretory activity of the delimiting cells. Another characteristic aspect of the Tettigoniidae is the rearrangement of the cap inside the spermiduct that seems to start when material from the lumen of the organ enters from the basal part of the cap. Except for the fibrils of the peripheral lamina, the fibrils of the cap undergo a marked degradation process. The final organization of the spermatodesm cap is reached inside the seminal vesicles; it consists of the fibrillar peripheral lamina delimiting an interior made up of loosely arranged fibrils immersed in a finely granular matrix.

Increased expression of interleukin-23 induces proinflammatory cytokine secretion in inflammatory bowel disease

Objective To analyze expression of interleukin (IL)-23p19 and IL-23 receptor (IL-23R) in inflammatory bowel disease (IBD),and the role in the induction of peripheral blood T cell activation and proinflammatory cytokine secretion.Methods Peripheral blood (PB) and intestinal mueosal biopsies were collected from 12 patients with Crohn's disease (CD),25 patients with ulcerative colitis (UC) and 20 healthy controls.Expression of IL-23p19 was determined by immunohistochemistry and RT-PCR.IL-23R expression in CD4+,CD8+ T and NK cells from peripheral blood and lamina propria was analyzed by flow eytometry.Peripheral blood mononuclear ceils (PBMC) were isolated and cultured under stimulation with IL-23 and anti-CD3,and the levels of tumor necrosis factor α (TNFα) interferon (IFN)γ and IL-2 were determined by enzyme-linked immunosorbent assay (ELISA).Results The expression of II.-23p19 was significantly increased in inflamed mucosa of CD at both the transcriptional and translational levels compared with that in UC and healthy controls.IL-23R was mainly expressed in PB- and lamina propria-CD4+,CD8+T cells and NK cells from IBD patients,and markedly increased compared with controls (P＜0.05).IL-23 strongly triggered PBMC from IBD patients to produce significantly higher levels of IFN-γ,TNF-α and IL-2(P＜0.05).Conclusions IL-23p19 and IL-23R are highly expressed in IBD,particularly in CD,and may play an important role in the induction of T cell activation and proinflammatory cytokine secretion,suggesting that targeted therapy directed against IL-23 may have a therapeutic role in IBD. Key words: Inflammatory bowel disease; Interleukin-23; Receptor,cytokines

Thematic Review Series: Sphingolipids. Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.

Optic Nerve Hydropic Axonal Degeneration and Blocked Retrograde Axoplasmic Transport

To describe histopathologic features of anterior optic nerves of 12 eyes enucleated for sustained high ocular pressure from iris-ciliary body melanomas in 10 and choroidal melanomas with chronic angle closure in 2. In this retrospective study, we analyzed cases indexed in 2 eye pathology laboratories and reviewed the pertinent literature. Cases were identified from diagnostic indexes; microscopic study of slides stained with hematoxylin-eosin and Verhoeff-van Gieson, Mallory trichrome, periodic acid-Schiff, alcian blue, or colloidal iron for acid mucopolysaccharide; review of available clinical documentation; and analysis of features and photography. The main outcome measures were description of optic nerve heads, prelaminar atrophy, laminar posterior bowing, locations and density of hydropic axonal degeneration, blocked retrograde axoplasmic transport, posterior atrophy, and optic nerve disorganization with glial proliferation. Hydropic axonal degeneration was present in front of, within, and posterior to the lamina cribrosa in all 12 eyes. This degeneration extended diffusely and posteriorly from the peripheral lamina and was most dense centrally in 10 eyes. Retrolaminar changes compatible with blockage of retrograde axoplasmic transport were seen in 9 eyes. Posterior atrophy with disorganization and glial proliferation was seen in 10 eyes. No eye had classic glaucomatous atrophic cupping. Diffuse and centrally intense hydropic axonal degeneration and central blocked retrograde axoplasmic transport explain loss of central acuity, generalized contraction of visual field, and generalized optic atrophy without glaucomatous cupping in eyes with prolonged high-pressure secondary glaucoma.

Nuclear localization of flavivirus RNA synthesis in infected cells.

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.

262 TISSUE-SPECIFIC ANALYSIS OF PRION EXPRESSION IN EARLY BOVINE FETUSES

The prion protein (PrP) is best known for its mis-folded, pathogenic isoform, which is widely regarded as the infectious agent in transmissable spongiform encephalopathies. However, the role of normal, cellular PrP, a host-encoded 29-kD glycoprotein tethered to the cell membrane by a phosphatidyl-inositol glycane (GPI) anchor, is poorly understood. PrP binds copper with high affinity, has antioxidant activity and may play a role in cell adhesion and/or signaling. PrP is expressed in mouse embryos on 6.5 days post-coitum in extra-embryonic tissue and at 13.5 days in the central and peripheral nervous system, intestine, and dental lamina. Our previous data revealed PrP gene expression in bovine embryos throughout pre-implantation embryo development. As part of a larger effort to map the ontogeny of cellular PrP expression in cattle, we sought here to analyze in early bovine fetuses (1) total PrP gene expression by real-time quantitative PCR (QPCR), and (2) tissue-specific PrP expression by immunohistochemistry. Fetuses were obtained from donor cattle bred by artificial insemination (AI; Day 0) and subjected to mid-ventral laparotomy on Days 32 (n = 2) and 39 (n = 2). Immediately upon recovery, one fetus from each stage was placed in RNAlater for RNA isolation and the other fixed in 10% formalin for immunohistochemistry. RNA was isolated using an RNeasy� mini kit (Qiagen, Valencia, CA, USA). cDNA was generated by reverse transcription with random hexamer priming and used the ΔΔcT method for estimation of PrP expression by QPCR. Tissue-specific expression was determined by immunohistochemistry. Formalin-fixed fetuses were embedded in paraffin, sagittally sectioned, dehydrated, and subjected to an unmasking protocol that employed Vectorlab unmasking solution and autoclaving. Tissues were then probed with a primary anti-PrP monoclonal antibody (SAF 32; Cayman Chemical Company, Ann Arbor, MI, USA). Bound primary antibody was detected with a biotinylated horse anti-mouse secondary antibody complexed to horseradish peroxidase using the ABC kit (Vector Laboratories, Burlingame, CA, USA). Probed sections were then counterstained with hematoxolin and eosin. Neighboring sections, processed identically but to which no primary antibody was added, served as controls. PrP gene expression was detected by QPCR at both stages examined and tended to be higher in Day 39 compared to Day 32 fetuses. PrP immunoreactivity was found throughout the central and peripheral nervous systems, ganglia, nerve trunks, and neural cell populations of sensory organs in both Day 32 and Day 39 fetuses. PrP immunolabeling was also observed in the mesonephric kidney, liver, and heart in the Day 39 fetus. At both stages, immunoreactivity was most intense in the nervous system. Thus, PrP is expressed in a tissue-specific pattern in early bovine fetuses. Tissue distribution of fetal PrP expression appears similar to that of adult PrP. Moreover, PrP appears to be expressed in a developmentally regulated fashion in some tissues.

Lamin A/C speckles mediate spatial organization of splicing factor compartments and RNA polymerase II transcription.

The A-type lamins have been observed to colocalize with RNA splicing factors in speckles within the nucleus, in addition to their typical distribution at the nuclear periphery. To understand the functions of lamin speckles, the effects of transcriptional inhibitors known to modify RNA splicing factor compartments (SFCs) were examined. Treatment of HeLa cells with α-amanitin or 5,6-dichlorobenzimidazole riboside (DRB) inhibited RNA polymerase II (pol II) transcription and led to the enlargement of lamin speckles as well as SFCs. Removal of the reversible inhibitor DRB resulted in the reactivation of transcription and a rapid, synchronous redistribution of lamins and splicing factors to normal-sized speckles, indicating a close association between lamin speckles and SFCs. Conversely, the expression of NH2-terminally modified lamin A or C in HeLa cells brought about a loss of lamin speckles, depletion of SFCs, and down-regulation of pol II transcription without affecting the peripheral lamina. Our results suggest a unique role for lamin speckles in the spatial organization of RNA splicing factors and pol II transcription in the nucleus.