Gut memories do not fade: epigenetic regulation of lasting gut homing receptor expression in CD4+ memory T cells.

The concept of a "topographical memory" in lymphocytes implies a stable expression of homing receptors mediating trafficking of lymphocytes back to the tissue of initial activation. However, a significant plasticity of the gut-homing receptor α4ß7 was found in CD8+ T cells, questioning the concept. We now demonstrate that α4ß7 expression in murine CD4+ memory T cells is, in contrast, imprinted and remains stable in the absence of the inducing factor retinoic acid (RA) or other stimuli from mucosal environments. Repetitive rounds of RA treatment enhanced the stability of de novo induced α4ß7. A novel enhancer element in the murine Itga4 locus was identified that showed, correlating to stability, selective DNA demethylation in mucosa-seeking memory cells and methylation-dependent transcriptional activity in a reporter gene assay. This implies that epigenetic mechanisms contribute to the stabilization of α4ß7 expression. Analogous DNA methylation patterns could be observed in the human ITGA4 locus, suggesting that its epigenetic regulation is conserved between mice and men. These data prove that mucosa-specific homing mediated by α4ß7 is imprinted in CD4+ memory T cells, reinstating the validity of the concept of "topographical memory" for mucosal tissues, and imply a critical role of epigenetic mechanisms.

Signed, sealed and delivered: "big tobacco" in Hollywood, 1927-1951.

OBJECTIVE: Smoking in movies is associated with adolescent and young adult smoking initiation. Public health efforts to eliminate smoking from films accessible to youth have been countered by defenders of the status quo, who associate tobacco imagery in "classic" movies with artistry and nostalgia. The present work explores the mutually beneficial commercial collaborations between the tobacco companies and major motion picture studios from the late 1920s through the 1940s. METHODS: Cigarette endorsement contracts with Hollywood stars and movie studios were obtained from internal tobacco industry documents at the University of California, San Francisco (UCSF) Legacy Tobacco Documents Library and the Jackler advertising collection at Stanford. RESULTS: Cigarette advertising campaigns that included Hollywood endorsements appeared from 1927 to 1951, with major activity in 1931-2 and 1937-8 for American Tobacco Company's Lucky Strike, and in the late 1940s for Liggett & Myers' Chesterfield. Endorsement contracts and communication between American Tobacco and movie stars and studios explicitly reveal the cross-promotional value of the campaigns. American Tobacco paid movie stars who endorsed Lucky Strike cigarettes US$218,750 in 1937-8 (equivalent to US$3.2 million in 2008) for their testimonials. CONCLUSIONS: Hollywood endorsements in cigarette advertising afforded motion picture studios nationwide publicity supported by the tobacco industry's multimillion US dollar advertising budgets. Cross-promotion was the incentive that led to a synergistic relationship between the US tobacco and motion picture industries, whose artefacts, including "classic" films with smoking and glamorous publicity images with cigarettes, continue to perpetuate public tolerance of onscreen smoking. Market-based disincentives within the film industry may be a solution to decouple the historical association between Hollywood films and cigarettes.

[To be or not to be a Treg: epigenetic regulation of Foxp3 expression in regulatory T cells]. / To be or not to be a Treg: Epigenetische Regulation der Foxp3-Expression in regulatorischen T-Zellen.

Regulatory T cells (Treg) harbor great therapeutic potential for the treatment of autoimmune diseases due to their potent suppressive capacity. The majority of these cells express the transcription factor Foxp3, which is critical for both development and function of Treg. We discuss here our recent data indicating a contribution of epigenetic regulation for the permanent expression of Foxp3 in stable Treg a finding that is of significant importance if Treg are devised for clinical applications.

Low dose latrunculin-A inhibits dexamethasone-induced changes in the actin cytoskeleton and alters extracellular matrix protein expression in cultured human trabecular meshwork cells.

We determined the effects of a low dose of the actin-disrupting agent latrunculin (LAT)-A on dexamethasone (DEX)-induced changes in actin organization, focal adhesions, and production of extracellular matrix proteins in cultured human trabecular meshwork (HTM) cells. HTM cells were cultured to a highly confluent stage with stable endothelium-like morphology and incubated with 0.1 or 0.2 microM DEX and/or 0.1 microM LAT-A. Changes in the actin cytoskeleton and vinculin-containing focal contacts were evaluated by immunofluorescence microscopy. Expression of thrombospondin-1 (TSP1) and fibronectin (FN) in HTM cells was evaluated by Western blot analysis. The results showed that DEX induced morphological changes and actin reorganization in HTM cells. The cells partly recovered after DEX withdrawal, but the addition of low dose LAT-A hastened the recovery. In addition, DEX failed to induce changes when co-incubated with LAT-A for at least 4 weeks, and for at least 2 weeks when cells were pre-treated with LAT-A for 2 weeks. HTM cells treated with 0.1 microM LAT-A only for 5 days showed mild disorganization of the actin cytoskeleton and focal adhesions, which persisted during the 4 weeks of treatment. DEX stimulated production of FN in HTM cells independent of LAT-A treatment. LAT-A and, to a lesser extent, DEX inhibited production of TSP1 by HTM cells. Although LAT-A is not a DEX receptor antagonist, it is able to prevent the effects of DEX on the actin cytoskeleton in cultured HTM cells at a dose subthreshold for increasing outflow facility in monkeys. This suggests that LAT-A at low doses may be useful in treating steroid and other glaucomas. TSP1 may be an important target of LAT-A in HTM cells and modulation of TSP may influence the actin cytoskeleton of the trabecular meshwork (TM), and consequently, intraocular pressure.

Association of the myocilin mt.1 promoter variant with the worsening of glaucomatous disease over time.

A major variant of myocilin (MYOC) [TIGR/MYOC mt.1 (-1000 C/G)], present in the gene's promoter, is found to be associated with more rapid progression of the glaucoma disease state. Time-to-event analyses using the Cox proportional hazards model produced substantial statistical evidence that this TIGR/MYOC mt.1(+) variant accelerates worsening for both optic disc and visual field measures of disease progression. These analyses were based on evaluations of 147 patients with primary open-angle glaucoma (POAG) over 35 years of age with an average follow-up of approximately 15 years. Our analyses showed that there are independent effects of the variant on disease progression, taking into account other relevant disease-related baseline risk factors, including age, family history, initial drug treatment, initial surgical treatment, diabetes, gender, myopia, and initial disease severity. The finding that a TIGR/MYOC mt.1(+) determination provided a strong marker for glaucoma progression, above and beyond the other baseline risk factors, suggests a clinical utility in testing for this promoter genotype.

Association of a single nucleotide polymorphism in the TIGR/MYOCILIN gene promoter with the severity of primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is a highly prevalent optic neuropathy and a major cause of irreversible blindness, with elevation of intraocular pressure (IOP) being a primary risk factor. The trabecular meshwork-inducible glucocorticoid response (TIGR)/MYOCILIN (MYOC) gene coding region is mutated in 3-4% of POAG patients. Here, in a retrospective study of 142 POAG patients, we evaluated the influence on glaucoma phenotype of a novel biallelic polymorphism (-1000C/G) located in the upstream region of the MYOC gene. Allele frequencies were similar among patients and controls. However, the G allele (frequency 17.6%), also designated as MYOC.mt1, was associated with an increased IOP (+4.9 mmHg, p=0.0004) and a more damaged visual field (p=0.02). Both effects were predominant in females. Moreover, whereas IOP in MYOC.mt1 noncarriers decreased very markedly to the normal range between diagnosis and inclusion in the study (p=3 x 10(-5) in both males and females), reflecting successful therapy, it decreased less noticeably in MYOC.mt1+ male patients (p=0.005) and not at all in MYOC.mt1+ female patients. MYOC.mt1 appears therefore to be an indicator of poor IOP control and greater visual field damage in diagnosed POAG patients, potentially due to a lack of response to therapeutic intervention. Its typing might help in the selection of treatment paradigms for the management of POAG patients.

Effect of H-7 on cultured human trabecular meshwork cells.

PURPOSE: To determine the effect of the serine-threonine kinase inhibitor H-7, which blocks actomyosin contractility and increases outflow facility in live monkeys, on morphology, cytoskeleton, and cellular adhesions of human trabecular meshwork (HTM) cells in culture. METHODS: Cultured HTM cells were videographically recorded and evaluated before and after exposure to H-7 at different concentrations. The subcellular distribution of the actin-based cytoskeleton and associated anchor proteins including vinculin, paxillin, and beta-catenin, as well as phosphotyrosine-containing proteins were evaluated by fluorescence immunocytochemistry and digital fluorescence microscopy. RESULTS: H-7 induced pronounced but reversible HTM cell thickening toward the cell center and deterioration of the actin cytoskeletal network. Cell-extracellular matrix (ECM) and cell-cell adhesions were also affected, but the beta-catenin-rich, vinculin-containing adherens junctions were clearly more resistant than focal contacts. Phosphotyrosine labeling in focal contacts was highly sensitive to H-7. CONCLUSIONS: H-7 induces alterations in cell shape, actin cytoskeleton, and associated focal adhesions in cultured HTM cells, which may be responsible for the effects of H-7 on outflow facility in live monkey eyes.

Reduced TIGR/myocilin protein in the monkey ciliary muscle after topical prostaglandin F(2alpha) treatment.

PURPOSE: Mutations in the trabecular meshwork inducible glucocorticoid response (TIGR) gene, also known as myocilin, have recently been linked to some forms of glaucoma. Recent studies have shown that TIGR protein also is expressed in the ciliary muscle. Because uveoscleral outflow, which traverses the ciliary muscle, is increased by prostaglandins (PGs), the present study assessed whether topical PGs alter the amount of TIGR protein within the ciliary muscle. METHODS: Vehicle was topically applied to one eye, and 2 microg PGF(2alpha)-isopropyl ester (PGF(2alpha)-IE) was applied to the other eye of cynomolgus monkeys twice daily for 5 days. Pressure reductions of 5 mm Hg in the PGF(2alpha)-IE-treated eyes were confirmed. The eyes were then fixed and paraffin sections were cut from each eye. The distribution of TIGR protein in the ciliary muscle was determined by confocal scanning laser microscopy. Additional sections were immunostained with a polyclonal antibody to recombinant TIGR protein or with a polyclonal antibody to a synthetic peptide corresponding to the leucine zipper region within the TIGR protein. Staining intensity in the ciliary muscle was assessed by measuring optical density (OD) along two line segments overlying the ciliary muscle, by using a high-resolution imaging densitometer. RESULTS: TIGR protein immunoreactivity was observed in ciliary muscle fibers throughout the ciliary muscle. Extracellular TIGR immunoreactivity colocalized with collagen type IV immunoreactivity. Intracellular staining also was present. Immunoreactivity was less intense in the sections from the PGF(2alpha)-IE-treated eyes compared with the vehicle-treated eyes. This was reflected in the reduction of mean OD scores in each monkey. Overall, the reduction of mean OD scores in the treated eyes was 42.1% +/- 9.9% (P < 0.005) with the anti-recombinant TIGR antibody and 27.3% +/- 10.4% with the anti-TIGR peptide antibody (P < 0.005). CONCLUSIONS: TIGR protein immunoreactivity was present both intracellularly and extracellularly in the ciliary muscle of the cynomolgus monkey. This suggests that extracellular TIGR protein is in contact with aqueous humor in the uveoscleral outflow pathway. Moreover, IOP-lowering topical PGF(2alpha)-IE treatment decreases the amount of TIGR protein in the ciliary muscle.

Growth and differentiation of human lens epithelial cells in vitro on matrix.

PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

Particle irradiation induces FGF2 expression in normal human lens cells.

Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells. Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2-3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.

Regulation of TIGR/MYOC gene expression in human trabecular meshwork cells.

Glucocorticoid (GC) treatment of human trabecular meshwork (HTM) cells produces delayed, progressive cellular and extracellular protein/glycoprotein inductions with characteristics matching those for intraocular pressure elevation with corticosteroid eyedrops. The cloning of the Trabecular Meshwork Inducible Glucocorticoid Response (TIGR) gene from this system has suggested possible environmental and genetic influences in relation to glaucoma mechanisms. As reported here, the major GC-induced increase of TIGR expression in HTM cells is reduced approximately 4-fold by basic fibroblast growth factor (bFGF, 100-1000 pM), with a somewhat smaller inhibition noted with the thyroid hormone triiodothyronine (T3, 100 nM). Such endogenous 'protective' factors could help balance stimulatory effects on TIGR gene expression from 'stress' and/or mechanical perturbations in the trabecular meshwork. TIGR coding region mutations affecting the gene's olfactomedin (OLF) homology domain may also perturb biosynthetic pathways and cellular homeostatic functions. Our recent studies have shown the OLF domain corresponds to a major translocational 'pause', an area where critical processes for normal TIGR biogenesis are expected to take place. Observations that Glu323Lys (and other mutations early in the OLF domain) altered the pattern of paused protein intermediates provide possible clues to previously unexplained pathogenetic mechanisms. HTM cell transfection studies using TIGR-green fluorescent protein (GFP) fusions showed increased and altered distribution of the expressed protein with constructs missing the OLF domain, an effect also found with the Pro370 Leu mutation for early-onset glaucoma. The data suggest an activation of stress/apoptotic pathways in HTM cells as a potential mechanism for environmental/genetic interactions in glaucoma pathogenesis.

Effect of latrunculin-A on morphology and actin-associated adhesions of cultured human trabecular meshwork cells.

PURPOSE: Determine the effects of the actin cytoskeleton disrupting compound latrunculin-A (LAT-A) on morphology, cytoskeleton, and cellular adhesions of cultured human trabecular meshwork (HTM) cells. METHODS: HTM cells were cultured to high confluence with endothelial-like morphology and treated with LAT-A at different doses and duration. Topography of living cells was evaluated by videomicroscopy. Distribution and organization of the actin-based cytoskeleton, vinculin- and paxillin-containing focal contacts, and beta-catenin-rich intercellular adhesions were determined by immunofluorescence and digital microscopy. RESULTS: LAT-A induced pronounced but highly reversible rounding of HTM cells, intercellular separation, and disruption of actin filaments. beta-catenin-rich intercellular adherens junctions were particularly sensitive to LAT-A. Vinculin- and paxillin-containing focal contacts were only partially affected and appeared to be more resistant to the drug than the intercellular interactions. CONCLUSIONS: The increase in outflow facility in the living primate eye induced by LAT-A may be due to the disorganization and disruption of the actin cytoskeleton and its associated cellular adhesions in the trabecular meshwork.

Genetic screening in a large family with juvenile onset primary open angle glaucoma.

AIMS: A number of genetic loci have been implicated in the pathogenesis of primary open angle glaucoma (POAG). The aim of this study was to identify the genetic cause of POAG in a large Scottish family and, if possible, offer genetic screening and advice to family members. METHODS: Family members were examined to determine their disease status. Base excision sequence scanning was carried out in order to test for the presence of a POAG causing mutation at known genetic loci. Direct DNA sequencing was performed in order to determine the mutation sequence. RESULTS: All family members of known affected disease status and two family members of unknown disease status were found to have a mutation in the TIGR gene. The mutation resulted in the substitution of a glycine residue with an arginine residue at codon 252 (Gly252Arg). No other sequence variations were present in any members of the family. CONCLUSION: The Gly252Arg mutation in the TIGR gene results in the development of POAG in this family. It was possible to identify younger, currently unaffected, members of the family who carry the mutation and who are therefore at a very high risk of developing POAG themselves. This is the first demonstration that Gly252Arg can be a disease causing mutation rather than a benign polymorphism. The possible pathogenic mechanisms and wider implications of the mutation are considered.

Gene delivery and expression in human retinal pigment epithelial cells: effects of synthetic carriers, serum, extracellular matrix and viral promoters.

Non-viral gene therapy is a potential treatment to many incurable retinal diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various +/- charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamidoamine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% of the cells expressed histochemically detectable amounts of the gene after transfection with cationic lipid DNA complexes. In RPE cells, Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratios, expression levels of luciferase were > 10(9) light units/mg protein after transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 10(7)-10(9) light units/mg protein or less. In general, dendrimers and large molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene products. Degraded dendrimers and high molecular weight PEI exhibited the best combination of high activity and low toxicity in RPE cell transfection.

Immunofluorescence method for quantifying the trabecular meshwork glucocorticoid response (TIGR) protein in trabecular meshwork and Schlemm's canal cells.

PURPOSE: Decreased flow of aqueous humor through the trabecular meshwork (TM) and into Schlemm's canal (SC) is believed to be a predominant factor in the development of the elevated intraocular pressure found in primary open-angle and steroid glaucoma. Recent biochemical and genetic evidence has suggested that alterations in the expression of the TM glucocorticoid response (TIGR) protein within the chamber angle may play a role in the development of these glaucomas. To understand the process of TIGR induction in outflow pathway cells we developed an assay for TIGR expression that could distinguish both individual cell responses and also provide a semi-quantitative comparison between cell cultures. The present study demonstrates this approach, using digital image capture of immunofluorescently stained human TM and SC cells after treatment with dexamethasone (Dex). METHODS: Confluent cultures of human TM and SC cells were treated with 1 M Dex or vehicle control for 10 days. The cells were then fixed, permeabilized, and stained using polyclonal antibodies produced against recombinant TIGR. Digital images of fluorescently stained cells, using the same exposure time within an experiment, were evaluated by tabulating the staining intensity of all cells on each image. Between 10 and 40 cells were evaluated per image, 8-10 frames/ sample, 2-3 samples per treatment. Each cell was ranked as either 0 (background), 1+ (minor), 2+ (moderate) or 3+ (very bright staining). RESULTS: TM cells showed a significant basal level of TIGR staining. About 20% of control cells showed appreciable levels of TIGR staining, with intensity levels evenly distributed between 1, 2 and 3+. Dex treatment increased the number of TM cells expressing TIGR to 60-80%, with the majority of cells showing 2+ to 3+ staining throughout the cytoplasm. SC cells showed no basal TIGR staining, but Dex-treated cells exhibited TIGR staining in 6-15% of cells. SC cell TIGR staining varied between 1+ to 2+ in intensity, and showed a distribution different from TM cells. Staining in SC cells was localized to a ribbon-like compartment adjacent to the nucleus. Such perinuclear localization was rarely seen in TM cells. CONCLUSIONS: The low standard errors of the mean TIGR responses within each experiment, and the reproducibility between experiments for each cell type, suggests good reliability for this method. At the same time, the consistent, marked contrast between TM and SC cells in their response to glucocorticoids demonstrates that the assay can distinguish significant differences between cell types. The data support the view that Dex has a cell type specific effect on TIGR induction. The different extent, pattern and localization of TIGR staining between cell types suggests that TIGR expression in SC cells could play a functional role in the outflow pathway, but one that may be distinct from that played in TM cells.

A trabecular meshwork glucocorticoid response (TIGR) gene mutation affects translocational processing.

PURPOSE: To examine possible effects of the E323K mutation in the trabecular meshwork glucocorticoid response (TIGR) gene (also known as myocilin [MYOC]), using assays of translocational processing through the endoplasmic reticulum (ER). The E323K mutation was of particular interest, since the mutation shows a strong association with early onset open-angle glaucoma, but has a minimal predicted effect on protein structure. METHODS: Normal and mutant TIGR cDNA constructs were used to generate protein products in the presence of endoplasmic reticulum (ER) membranes, using an assay previously developed to detect alterations in the ER translocation function. "Paused" regions for potential protein modifications were defined by proteinase K (PK) sensitivity in the presence of ER membranes, with the ability to restart translocation when treated with EDTA. The effects of the E323K mutation were evaluated, as well as mutations located on either side of E323K (G246R, G364V, P370L) as the other mutations had substantial predicted structural changes in addition to clear disease associations. RESULTS: The native TIGR molecule was observed to have a paused region that corresponds to the region of highest olfactomedin (OLF) homology. The E323K mutation, located near the beginning of this region, dramatically altered the normal pattern of nascent proteins observed in the translocational pausing assay. A prominent band appeared with the E323K mutation, which could represent a new product or a marked enhancement of a faint band normally seen, approximately 3 kDa higher than the major paused band. The other TIGR mutants examined did not show this effect. CONCLUSIONS: The major translocational pause that starts near the beginning of the region of high OLF homology may help to explain the high frequency of glaucoma-associated mutations in this area. The observed effect of the E323K mutation on the products of translocational processing suggests a delay in the normal pausing process of TIGR biogenesis. This delay points to a potentially distinct pathogenic mechanism for E323K as compared with the other TIGR mutations so far evaluated.