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Sample records for human lens epithelium

  1. Derivation of Multiple Cranial Tissues and Isolation of Lens Epithelium-Like Cells From Human Embryonic Stem Cells

    PubMed Central

    2013-01-01

    Human embryonic stem cells (hESCs) provide a powerful tool to investigate early events occurring during human embryonic development. In the present study, we induced differentiation of hESCs in conditions that allowed formation of neural and non-neural ectoderm and to a lesser extent mesoderm. These tissues are required for correct specification of the neural plate border, an early embryonic transient structure from which neural crest cells (NCs) and cranial placodes (CPs) originate. Although isolation of CP derivatives from hESCs has not been previously reported, isolation of hESC-derived NC-like cells has been already described. We performed a more detailed analysis of fluorescence-activated cell sorting (FACS)-purified cell populations using the surface antigens previously used to select hESC-derived NC-like cells, p75 and HNK-1, and uncovered their heterogeneous nature. In addition to the NC component, we identified a neural component within these populations using known surface markers, such as CD15 and FORSE1. We have further exploited this information to facilitate the isolation and purification by FACS of a CP derivative, the lens, from differentiating hESCs. Two surface markers expressed on lens cells, c-Met/HGFR and CD44, were used for positive selection of multiple populations with a simultaneous subtraction of the neural/NC component mediated by p75, HNK-1, and CD15. In particular, the c-Met/HGFR allowed early isolation of proliferative lens epithelium-like cells capable of forming lentoid bodies. Isolation of hESC-derived lens cells represents an important step toward the understanding of human lens development and regeneration and the devising of future therapeutic applications. PMID:23341438

  2. Derivation of multiple cranial tissues and isolation of lens epithelium-like cells from human embryonic stem cells.

    PubMed

    Mengarelli, Isabella; Barberi, Tiziano

    2013-02-01

    Human embryonic stem cells (hESCs) provide a powerful tool to investigate early events occurring during human embryonic development. In the present study, we induced differentiation of hESCs in conditions that allowed formation of neural and non-neural ectoderm and to a lesser extent mesoderm. These tissues are required for correct specification of the neural plate border, an early embryonic transient structure from which neural crest cells (NCs) and cranial placodes (CPs) originate. Although isolation of CP derivatives from hESCs has not been previously reported, isolation of hESC-derived NC-like cells has been already described. We performed a more detailed analysis of fluorescence-activated cell sorting (FACS)-purified cell populations using the surface antigens previously used to select hESC-derived NC-like cells, p75 and HNK-1, and uncovered their heterogeneous nature. In addition to the NC component, we identified a neural component within these populations using known surface markers, such as CD15 and FORSE1. We have further exploited this information to facilitate the isolation and purification by FACS of a CP derivative, the lens, from differentiating hESCs. Two surface markers expressed on lens cells, c-Met/HGFR and CD44, were used for positive selection of multiple populations with a simultaneous subtraction of the neural/NC component mediated by p75, HNK-1, and CD15. In particular, the c-Met/HGFR allowed early isolation of proliferative lens epithelium-like cells capable of forming lentoid bodies. Isolation of hESC-derived lens cells represents an important step toward the understanding of human lens development and regeneration and the devising of future therapeutic applications. PMID:23341438

  3. Virtual-screening targeting Human Immunodeficiency Virus type 1 integrase-lens epithelium-derived growth factor/p75 interaction for drug development.

    PubMed

    Gu, Wan-Gang; Liu, Bai-Nan; Yuan, Jun-Fa

    2015-02-01

    Three integrase (IN) inhibitors have been approved by FDA for clinical treatment of Human Immunodeficiency Virus (HIV) infection. This stimulates more researchers to focus their studies on this target for anti-HIV drug development. Three steps regarding of IN activity have been validated for inhibitor discovery: strand transfer, 3'-terminal processing, and IN-lens epithelium-derived growth factor (LEDGF)/p75 interaction. Among them, IN-LEDGF/p75 interaction is a new target validated in recent years. Emergence of drug-resistant virus strains makes this target appealing to pharmacologists. Compared with the traditional screening methods such as AlphaScreen and cell-based screening developed for IN inhibitor discovery, virtual screening is a powerful technique in modern drug discovery. Here we summarized the recent advances of virtual-screening targeting IN-LEDFG/p75 interaction. The combined application of virtual screening and experiments in drug discovery against IN-LEDFG/p75 interaction sheds light on anti-HIV research and drug discovery. PMID:25230778

  4. The Effects of Silicone Hydrogel Lens Wear on the Corneal Epithelium and Risk for Microbial Keratitis

    PubMed Central

    Robertson, Danielle M.

    2012-01-01

    Previous studies using animal models and human clinical trials have demonstrated that the use of low oxygen transmissible contact lens materials produce corneal epithelial surface damage resulting in increased Pseudomonas aeruginosa (PA) adhesion and raft-mediated internalization into surface corneal epithelial cells. These findings led to the testable clinical predictions that: (1) microbial keratitis (MK) risk is expected to be greatest during the first 6 months of wear; (2) there is no difference between 6 and 30 night extended wear; and (3) that wear of hyper-oxygen transmissible lenses would reduce the reported incidence of infection. Subsequent epidemiological studies have confirmed the first two predictions; however, increased oxygen transmissibility with silicone hydrogel (SiHy) lens wear has not altered the overall incidence of MK. In this review, more recent clinical and basic studies that investigate epithelial alterations and bacterial adhesion to corneal epithelial cells following wear of SiHy lenses with and without concomitant exposure to chemically preserved multipurpose solutions (MPS) will be examined. The collective results of these studies demonstrate that even in the absence of lens-related hypoxia, MPS induce ocular surface changes during SiHy lens wear which are associated with a pathophysiological increase in PA adherence and internalization in the corneal epithelium, and therefore, predict an increased risk for PA-MK. In addition, new data supporting an interactive role for inflammation in facilitating PA adherence and internalization in the corneal epithelium will also be discussed. PMID:23266590

  5. Identification of an evolutionarily conserved domain in human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) that binds HIV-1 integrase.

    PubMed

    Cherepanov, Peter; Devroe, Eric; Silver, Pamela A; Engelman, Alan

    2004-11-19

    Human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) protein was recently identified as a binding partner for HIV-1 integrase (IN) in human cells. In this work, we used biochemical and bioinformatic approaches to define the domain organization of LEDGF/p75. Using limited proteolysis and deletion mutagenesis we show that the protein contains a pair of evolutionarily conserved domains, assuming about 35% of its sequence. Whereas the N-terminal PWWP domain had been recognized previously, the second domain is novel. It is comprised of approximately 80 amino acid residues and is both necessary and sufficient for binding to HIV-1 IN. Strikingly, the integrase binding domain (IBD) is not unique to LEDGF/p75, as a second human protein, hepatoma-derived growth factor-related protein 2 (HRP2), contains a homologous sequence. LEDGF/p75 and HRP2 IBDs avidly bound HIV-1 IN in an in vitro GST pull-down assay and each full-length protein potently stimulated HIV-1 IN activity in vitro. LEDGF/p75 and HRP2 are predicted to share a similar domain organization and have an evident evolutionary and likely functional relationship. PMID:15371438

  6. Influence of amphotericin B on the sodium pump of porcine lens epithelium.

    PubMed

    Delamere, N A; Dean, W L; Stidam, J M; Moseley, A E

    1996-02-01

    Active transport by Na(+)-K(+)-ATPase in the monolayer of lens epithelium is vital for the regulation of sodium and potassium levels within the mass of fiber cells that make up the bulk of the lens. In this study, experiments were conducted using porcine lenses to test whether Na(+)-K(+)-ATPase activity in the epithelium is altered when the permeability of lens cell plasma membranes is increased by the ionophore amphotericin B. After 24 h, sodium was significantly (P < 0.01) elevated in lenses exposed to 5 or 10 microM amphotericin B. Amphotericin B stimulated 86Rb uptake, probably through an increase of cytoplasmic sodium concentration due to increased inward sodium leak; the rate of ouabain-sensitive potassium (86Rb) uptake by intact lenses was significantly increased by amphotericin B at 5 microM (P < 0.05) and 10 microM (P < 0.01). After 24 h, the epithelium from lenses exposed to amphotericin B had an Na(+)-K(+)-ATPase activity that was more than twofold higher (P < 0.01) than the Na(+)-K(+)-ATPase activity in control lenses. By immunoblot, there was an increase in Na(+)-K(+)-ATPase catalytic (alpha) subunit immunoreactive polypeptide in the epithelium of lenses exposed to amphotericin B. The increase stemmed from a marked increase of Na(+)-K(+)-ATPase alpha 2-immunoreactive polypeptide but little change in the amount of alpha 1-immunoreactive protein. As judged by immunoblot experiments, the amount of Na(+)-K(+)-ATPase beta 1-immunoreactive polypeptide also appeared to be higher in the epithelium of amphotericin B-treated lenses compared with control lenses. In summary, these results suggest that in response to a permeability challenge with amphotericin B, the porcine lens epithelium is able to increase the activity of Na(+)-K(+)-ATPase. The same permeability challenge also appears to stimulate the biosynthesis of Na(+)-K(+)-ATPase catalytic subunit as well as glycoprotein subunit polypeptides. PMID:8779908

  7. Epithelium

    MedlinePlus

    The term "epithelium" refers to layers of cells that line hollow organs and glands. It is also those cells that make ... Epithelium. In: Kierszenbaum AL, Tres LL. Histology and Cell Biology - An Introduction to Pathology , 3rd ed. Philadelphia, ...

  8. Structural Properties of HIV Integrase. Lens Epithelium-derived Growth Factor Oligomers

    SciTech Connect

    Gupta, K.; Diamond, T; Hwang, Y; Bushman, F; Van Duyne, G

    2010-01-01

    Integrase (IN) is the catalytic component of the preintegration complex, a large nucleoprotein assembly critical for the integration of the retroviral genome into a host chromosome. Although partial crystal structures of human immunodeficiency virus IN alone and its complex with the integrase binding domain of the host factor PSIP1/lens epithelium-derived growth factor (LEDGF)/p75 are available, many questions remain regarding the properties and structures of LEDGF-bound IN oligomers. Using analytical ultracentrifugation, multiangle light scattering, and small angle x-ray scattering, we have established the oligomeric state, stoichiometry, and molecular shapes of IN {center_dot} LEDGF complexes in solution. Analyses of intact IN tetramers bound to two different LEDGF truncations allow for placement of the integrase binding domain by difference analysis. Modeling of the small angle x-ray scattering envelopes using existing structural data suggests domain arrangements in the IN oligomers that support and extend existing biochemical data for IN {center_dot} LEDGF complexes and lend new insights into the quaternary structure of LEDGF-bound IN tetramers. These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication.

  9. Histatin-1 Expression in Human Lacrimal Epithelium

    PubMed Central

    Pasha, Zeeshan; Jaboori, Assraa Jassim; Jassim, Sarmad H.; Jain, Sandeep; Aakalu, Vinay K.

    2016-01-01

    Background Study of human lacrimal cell biology is limited by poor access to tissue samples, heterogeneous cell composition of tissue and a lack of established lacrimal epithelial markers. In order to further our understanding of lacrimal cell biology, we sought to find a better marker for human lacrimal epithelial cells, compared to what has been reported in the literature. Methods We utilized human Muller’s muscle conjunctival resection (MMCR) specimens containing accessory lacrimal gland (ALG) and cadaveric main lacrimal gland (MLG) as sources of lacrimal tissue. Candidate markers were sought using human ALG tissue from MMCR specimens, isolated by laser capture microdissection (LCM). Affymetrix® analysis was performed on total RNA isolated from FFPE samples to profile transcription in ALG. MMCR tissue sections were assessed by immunofluorescence using antibodies for histatin-1, lactoferrin, E-cadherin (E-cad) and alpha-smooth muscle actin (ASMA). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed to analyze the expression of histatin-1, E-cad and lactoferrin from cadaveric MLG. Results Histatin-1 is expressed in ALG and MLG, localizes to lacrimal epithelium, and to a greater degree than do other putative lacrimal epithelial markers. Conclusions Histatin-1 is a good marker for human lacrimal epithelium in ALG and MLG and can be used to identify lacrimal cells in future studies. PMID:26824896

  10. Human vomeronasal epithelium development: An immunohistochemical overview.

    PubMed

    Dénes, Lóránd; Pap, Zsuzsanna; Szántó, Annamária; Gergely, István; Pop, Tudor Sorin

    2015-06-01

    The vomeronasal organ (VNO) is the receptor structure of the vomeronasal system (VNS) in vertebrates. It is found bilaterally in the submucosa of the inferior part of the nasal septum. There are ongoing controversies regarding the functionality of this organ in humans. In this study we propose the immunohistochemical evaluation of changes in components of the human vomeronasal epithelium during foetal development. We used 45 foetuses of different age, which were included in three age groups. After VNO identification immunohistochemical reactions were performed using primary antibodies against the following: neuron specific enolase, calretinin, neurofilament, chromogranin, synaptophysin, cytokeratin 7, pan-cytokeratin and S100 protein. Digital slides were obtained and following colorimetric segmentation, surface area measurements were performed. The VNO was found in less than half of the studied specimens (42.2%). Neuron specific enolase and calretinin immunoexpression showed a decreasing trend with foetal age, while the other neural/neuroendocrine markers were negative in all specimens. Cytokeratin 7 expression increased with age, while Pan-Ctk had no significant variations. S100 protein immunoexpression also decreased around the VNO. The results of the present work uphold the theory of regression of the neuroepithelium that is present during initial stages of foetal development. PMID:26132837

  11. A central role for vimentin in regulating repair function during healing of the lens epithelium

    PubMed Central

    Menko, A. S.; Bleaken, B. M.; Libowitz, A. A.; Zhang, L.; Stepp, M. A.; Walker, J. L.

    2014-01-01

    Mock cataract surgery provides a unique ex vivo model for studying wound repair in a clinically relevant setting. Here wound healing involves a classical collective migration of the lens epithelium, directed at the leading edge by an innate mesenchymal subpopulation of vimentin-rich repair cells. We report that vimentin is essential to the function of repair cells as the directors of the wound-healing process. Vimentin and not actin filaments are the predominant cytoskeletal elements in the lamellipodial extensions of the repair cells at the wound edge. These vimentin filaments link to paxillin-containing focal adhesions at the lamellipodial tips. Microtubules are involved in the extension of vimentin filaments in repair cells, the elaboration of vimentin-rich protrusions, and wound closure. The requirement for vimentin in repair cell function is revealed by both small interfering RNA vimentin knockdown and exposure to the vimentin-targeted drug withaferin A. Perturbation of vimentin impairs repair cell function and wound closure. Coimmunoprecipitation analysis reveals for the first time that myosin IIB is associated with vimentin, linking vimentin function in cell migration to myosin II motor proteins. These studies reveal a critical role for vimentin in repair cell function in regulating the collective movement of the epithelium in response to wounding. PMID:24478454

  12. Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium

    PubMed Central

    Sun, Ning; Shibata, Brad; Hess, John F.

    2016-01-01

    Purpose The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age. Methods Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6. Results CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 μm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely

  13. Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

    PubMed

    Barandika, Olatz; Ezquerra-Inchausti, Maitane; Anasagasti, Ander; Vallejo-Illarramendi, Ainara; Llarena, Irantzu; Bascaran, Lucia; Alberdi, Txomin; De Benedetti, Giacomo; Mendicute, Javier; Ruiz-Ederra, Javier

    2016-10-01

    In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate. PMID:27497833

  14. Characterization of side population cells from human airway epithelium.

    PubMed

    Hackett, Tillie-Louise; Shaheen, Furquan; Johnson, Andrew; Wadsworth, Samuel; Pechkovsky, Dmitri V; Jacoby, David B; Kicic, Anthony; Stick, Stephen M; Knight, Darryl A

    2008-10-01

    The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil-sensitive efflux of the DNA-binding dye Hoechst 33342. Using flow cytometry, CD45(-) SP, CD45(+) SP, and non-SP cells were isolated and sorted. CD45(-) SP cells made up 0.12% +/- 0.01% of the total epithelial cell population in normal airway but 4.1% +/- 0.06% of the epithelium in asthmatic airways. All CD45(-) SP cells showed positive staining for epithelial-specific markers cytokeratin-5, E-cadherin, ZO-1, and p63. CD45(-) SP cells exhibited stable telomere length and increased colony-forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non-SP cells, fewer than 100 CD45(-) SP cells were able to generate a multilayered and differentiated epithelium in air-liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short- and long-term proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium. Disclosure of potential conflicts of interest is found at the end of this article. PMID:18653771

  15. Analysis of human crystalline lens accommodation.

    PubMed

    Chien, Chang-Hai M; Huang, Tseng; Schachar, Ronald A

    2006-01-01

    The behavior of the human crystalline lens during accommodation is analytically studied. The lens is modeled as a closed axisymmetrical membrane shell of varying thickness enclosing an incompressible liquid. To simulate zonular tension associated with lenticular accommodation, an axisymmetrical radial force or displacement is imposed around the shell equator. Two second-order, simultaneous, nonlinear governing differential equations are derived. Numerical results, obtained from the investigation of human lens profiles of three independently published MRI images and a drawing of a microphotograph, demonstrate that when zonular traction within the physiological force range of the ciliary muscle is exerted, both central lens thickness and central optical power increase. Qualitatively, these increases are independent of lens shape. However, the magnitude of these changes is dependent on the initial profile of the lens and is enhanced by the "natural" variation in capsular thickness. Only when a pulling force significantly exceeds the force capacity of the ciliary muscle does the lens flatten and its central thickness and optical power decrease. PMID:16023655

  16. The elastic constants of the human lens

    PubMed Central

    Fisher, R. F.

    1971-01-01

    1. When the lens is spun around its antero-posterior polar axis in an apparatus designed for the purpose, high speed photography can be used to record its changing profile. By this method a variable radial centrifugal force can be applied to the lens which mimics the pull of the zonule. 2. If the lens is not stressed at its centre beyond 100 Nm-2 it behaves as a truly elastic body. When stressed beyond this limit visco-elastic strain is produced at its poles. 3. The human lens has isotropic elastic properties at the extremes of life, but at the other times Young's Modulus of Elasticity varies with the direction in which it is measured. 4. Young's Modulus of Elasticity of the lens varies with age, polar elasticity and equatorial elasticity, at birth being 0·75 × 103 and 0·85 × 103 Nm-2 respectively, while at 63 years of age both are equal to 3 × 103 Nm-2. 5. A comparison of Young's Modulus of the young human lens with that of the rabbit and cat shows that the polar elasticity of the lenses of these animals was 5 times greater in the young rabbit, and 21 times greater in the adult cat. Equatorial elasticities of the rabbit and human lens were equal, while in the cat the equatorial elasticity was four times greater. 6. A mathematical model showing the lens substance possessing a nucleus of lower isotropic elasticity than that of the isotropic elastic cortex surrounding it, accounts for the difference between polar and equatorial elasticity of the intact adult lens. 7. The implications of these findings are discussed in relation to: (i) accommodation and the rheological properties of the lens; (ii) possible differences in the physical state of the lenticular proteins in the cortex and nucleus which may account for the senile variations in Young's Modulus of Elasticity in these regions of the lens; (iii) the loss of accommodation due solely to an increase in Young's Modulus of Elasticity of the lens between the ages of 15 and 60. This would amount to 44% of the

  17. Microbes on the human vaginal epithelium

    PubMed Central

    Hyman, Richard W.; Fukushima, Marilyn; Diamond, Lisa; Kumm, Jochen; Giudice, Linda C.; Davis, Ronald W.

    2005-01-01

    Using solely a gene-based procedure, PCR amplification of the 16S ribosomal RNA gene coupled with very deep sequencing of the amplified products, the microbes on 20 human vaginal epithelia of healthy women have been identified and quantitated. The Lactobacillus content on these 20 healthy vaginal epithelia was highly variable, ranging from 0% to 100%. For four subjects, Lactobacillus was (virtually) the only bacterium detected. However, that Lactobacillus was far from clonal and was a mixture of species and strains. Eight subjects presented complex mixtures of Lactobacillus and other microbes. The remaining eight subjects had no Lactobacillus. Instead, Bifidobacterium, Gardnerella, Prevotella, Pseudomonas, or Streptococcus predominated. PMID:15911771

  18. Cell proliferation in the human gallbladder epithelium: effect of distension.

    PubMed Central

    Putz, P; Willems, G

    1979-01-01

    DNA synthesis activity in the epithelium of the human gallbladder was studied through in vitro labelling of mucosal specimens with 3H-thymidine and autoradiography. The specimens were taken at the time of a surgical operation. Eight 'normal' gallbladders and six distended gallbladders from patients with carcinomatous obstruction of the common bile duct were examined. Proliferative activity was very low in the normal and significantly higher in the distended gallbladders. Images Figure PMID:437558

  19. Zinc uptake in vitro by human retinal pigment epithelium

    SciTech Connect

    Newsome, D.A.; Rothman, R.J.

    1987-11-01

    Zinc, an essential trace element, is present in unusually high concentrations in the chorioretinal complex relative to most other tissues. Because little has been known about the interactions between the retinal pigment epithelium and free or protein-associated zinc, we studied /sup 65/Zn uptake by human retinal pigment epithelium in vitro. When monolayers were exposed to differing concentrations from 0 to 30 microM /sup 65/Zn in Dulbecco's modified Eagle's medium with 5.4 gm/l glucose at 37 degrees C and 4 degrees C, we observed a temperature-dependent saturable accumulation of the radiolabel. With 15 microM /sup 65/Zn, we saw a biphasic pattern of uptake with a rapid first phase and a slower second phase over 120 min. Uptake of /sup 65/Zn was inhibited by iodacetate and cold, and reduced approximately 50% by the addition of 2% albumin to the labelling medium. Neither ouabain nor 2-deoxyglucose inhibited uptake. Cells previously exposed to /sup 65/Zn retained approximately 70% of accumulated /sup 65/Zn 60 min after being changed to radiolabel-free medium. Following removal of cells from the extracellular matrix adherent to the dish bottom, a variable amount of nonspecific binding of /sup 65/Zn to the residual matrix was demonstrated. These observations are consistent with a facilitated type of transport and demonstrate the ability of human retinal pigment epithelium in vitro to accumulate and retain zinc.

  20. Transactivation of involucrin, a marker of differentiation in keratinocytes, by lens epithelium-derived growth factor (LEDGF).

    PubMed

    Kubo, E; Fatma, N; Sharma, P; Shinohara, T; Chylack, L T; Akagi, Y; Singh, D P

    2002-07-26

    Human involucrin (hINV), first appears in the cytosol of keratinocytes and ultimately cross-linked to membrane proteins via transglutaminase and forms a protective barrier as an insoluble envelope beneath the plasma membrane. Although the function and evolution of involucrin is known, the regulation of its gene expression is not well understood. An analysis of the hINV gene sequence, upstream of the transcription start site (-534 to +1 nt) revealed the presence of potential sites for binding of lens epithelium-derived growth factor (LEDGF); stress response element (STRE; A/TGGGGA/T) and heat shock element (HSE; nGAAn). We reported earlier that LEDGF activates stress-associated genes by binding to these elements and elevates cellular resistance to various stresses. Here, gel-shift and super-shift assays confirm the binding of LEDGF to the DNA fragments containing HSEs and STREs that are present in the involucrin gene promoter. Furthermore, hINV promoter linked to CAT reporter gene, cotransfected in human corneal simian virus 40-transformed keratinocytes (HCK), was transactivated by LEDGF significantly. In contrast, the activity of hINV promoter bearing mutations at the WT1 (containing HSE and STRE), WT2 (containing STRE) and WT3 (containing STRE) binding sites was diminished. In addition, in HCK cell over-expressing LEDGF, the levels of hINV mRNA and hINV protein are increased by four to five-fold. LEDGF is inducible to oxidants. Cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate production of H(2)O(2), showed higher levels of LEDGF mRNA. Furthermore, our immunohistochemical studies revealed that hINV protein is found in the cytoplasm of HCK cells over-expressing LEDGF, but not detectable in the normal HCK cells or HCK cells transfected with vector. This regulation appears to be physiologically important, as over-expression of HCK with LEDGF increases the expression of the endogenous hINV gene and may provide new insight to understand

  1. Expression of stanniocalcin in the epithelium of human choroid plexus.

    PubMed

    Franzén, A M; Zhang, K Z; Westberg, J A; Zhang, W M; Arola, J; Olsen, H S; Andersson, L C

    2000-12-29

    Stanniocalcin (STC) is a 28 kD glycoprotein hormone originally found in bony fish in which it regulates calcium/phosphate homeostasis and protects against hypercalcemia. The recently characterized mammalian STC shows about 70% homology with fish STC. The epithelial cells of proximal tubuli in human and rat kidney and brain neurons have been found to express STC. Here we show that the epithelium of the choroid plexus, already at 16 weeks of fetal age, and of plexus papillomas, synthesize and express STC. Our findings suggest that STC may be of importance for the distribution of calcium and phosphate between the cerebrospinal fluid and blood. PMID:11134638

  2. In vitro reconstruction of human junctional and sulcular epithelium

    PubMed Central

    Dabija-Wolter, G; Bakken, V; Cimpan, M R; Johannessen, A C; Costea, D E

    2013-01-01

    BACKGROUND The aim of this study was to develop and characterize standardized in vitro three-dimensional organotypic models of human junctional epithelium (JE) and sulcular epithelium (SE). METHODS Organotypic models were constructed by growing human normal gingival keratinocytes on top of collagen matrices populated with gingival fibroblasts (GF) or periodontal ligament fibroblasts (PLF). Tissues obtained were harvested at different time points and assessed for epithelial morphology, proliferation (Ki67), expression of JE-specific markers (ODAM and FDC-SP), cytokeratins (CK), transglutaminase, filaggrin, and basement membrane proteins (collagen IV and laminin1). RESULTS The epithelial component in 3- and 5-day organotypics showed limited differentiation and expressed Ki-67, ODAM, FDC-SP, CK 8, 13, 16, 19, and transglutaminase in a similar fashion to control JE samples. PLF supported better than GF expression of CK19 and suprabasal proliferation, although statistically significant only at day 5. Basement membrane proteins started to be deposited only from day 5. The rate of proliferating cells as well as the percentage of CK19-expressing cells decreased significantly in 7- and 9-day cultures. Day 7 organotypics presented higher number of epithelial cell layers, proliferating cells in suprabasal layers, and CK expression pattern similar to SE. CONCLUSION Both time in culture and fibroblast type had impact on epithelial phenotype. Five-day cultures with PLF are suggested as JE models, 7-day cultures with PLF or GF as SE models, while 9-day cultures with GF as gingival epithelium (GE) models. Such standard, reproducible models represent useful tools to study periodontal bacteria–host interactions in vitro. PMID:22947066

  3. Lens epithelium-derived growth factor (LEDGF) delays photoreceptor degeneration in explants of rd/rd mouse retina.

    PubMed

    Ahuja, P; Caffé, A R; Holmqvist, I; Söderpalm, A K; Singh, D P; Shinohara, T; van Veen, T

    2001-09-17

    Lens epithelium derived growth factor (LEDGF) has been shown to rescue embryonic chick photoreceptor cells from serum starvation and heat stress, light damaged photoreceptor cells in Lewis rats, and photoreceptor cells in RCS rats. The aim of our study is to study the rescue effect of LEDGF on photoreceptor cells in the rd/rd mouse using our long-term serum free organ culture. At the end of this culture period of 21-26 days LEDGF treated rd mouse retina showed an increased photoreceptor survival compared to the untreated controls. LEDGF has no effect on expression and localization of opsin and arrestin in the rod photoreceptor cells when RPE is present. The protective potency of LEDGF on the retinal photoreceptor cells is similar to that of BDNF. LEDGF is known to activate heat shock proteins (Hsps) and the elevated Hsps are also reported to suppress apoptosis. PMID:11588609

  4. Human lens modeling and biometric measurement technique

    NASA Astrophysics Data System (ADS)

    Huang, Yanqiao

    This dissertation conducts theoretical and instrumental aspects of research aiming at extending knowledge and understanding of the optical design of the human eye. The first part of the thesis describes a newly constructed dynamic eye model that includes a gradient index (GRIN) lens to simulate eye accommodation. The GRIN profile of the crystalline lens is defined by a single continuous GRIN equation with optical power variability. In describing the lens accommodation process, different expansion coefficients are given to the lens nucleus and cortex to mimic lens dynamics. A relaxed state eye, a 4-D accommodated eye and a 10-D accommodated eye are simulated on a computer for studying and analyzing the first order and third order properties. This eye model can be further improved if giving accurate biometric measurement data on accommodating eyes. The second part of the thesis proposes an original interferometric technique that has potential for non-invasive ocular biometric measurements. This technique, termed spatial coherence interferometry, utilizes spatially incoherent monochromatic light as the illumination source, and employs the principle of low coherence interferometry to perform optical sectioning. Generalized coherence function for a multi-layer sample is derived and the theoretical axial longitudinal resolution is formulated. A spatial coherence interferometer with tunable coherence length is built, and detailed instrumental design and specifications are illustrated. Factors affecting system longitudinal resolution are examined. The instrument is first tested on plane mirrors for characterizing the longitudinal resolution. Various experiments are conducted including target searching, curved surface profiling and multi-layer sample sectioning. Finally en face surface profiling is performed on a pair of life size model eyes, and full field interferograms from various ocular surfaces are generated sequentially due to optical sectioning. In future research

  5. An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

    NASA Astrophysics Data System (ADS)

    Welsh, Michael J.

    1986-06-01

    The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

  6. Porphyromonas gingivalis invades human pocket epithelium in vitro.

    PubMed

    Sandros, J; Papapanou, P N; Nannmark, U; Dahlén, G

    1994-01-01

    The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro. Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis. PMID:8113953

  7. Human milk hyaluronan enhances innate defense of the intestinal epithelium.

    PubMed

    Hill, David R; Rho, Hyunjin K; Kessler, Sean P; Amin, Ripal; Homer, Craig R; McDonald, Christine; Cowman, Mary K; de la Motte, Carol A

    2013-10-01

    Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn. PMID:23950179

  8. Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium*

    PubMed Central

    Hill, David R.; Rho, Hyunjin K.; Kessler, Sean P.; Amin, Ripal; Homer, Craig R.; McDonald, Christine; Cowman, Mary K.; de la Motte, Carol A.

    2013-01-01

    Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn. PMID:23950179

  9. Glyoxalase I activity and immunoreactivity in the aging human lens

    PubMed Central

    Mailankot, Maneesh; Padmanabha, Smitha; Pasupuleti, NagaRekha; Major, Denice; Howell, Scott

    2013-01-01

    Glyoxalase I (GLOI) is the first enzyme of the glyoxalase system that catalyzes the metabolism of reactive dicarbonyls, such as methylglyoxal (MGO). During aging and cataract development, human lens proteins are chemically modified by MGO, which is likely due to inadequate metabolism of MGO by the glyoxalase system. In this study, we have determined the effect of aging on GLOI activity and the immunoreactivity and morphological distribution of GLOI in the human lens. A monoclonal antibody was developed against human GLOI. GLOI immunoreactivity was strongest in the anterior epithelial cells and weaker in rest of the lens. Cultured human lens epithelial cells showed immunostaining throughout the cytoplasm. In the human lens, GLOI activity and immunoreactivity both decreased with age. We believe that this would lead to promotion of MGO-modification in aging lens proteins. PMID:19238574

  10. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed Central

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-01-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS. PMID:160851

  11. Activation of human lymphocytes by supernatants from human thymic epithelium.

    PubMed

    Goust, J M; Vesole, D H; Fudenberg, H H

    1979-11-01

    Supernatants from human thymic epithelial cells (TS) were found to have a mitogenic effect on cultured human peripheral blood mononuclear cells and to potentiate their responses to lectins. This was not observed with culture supernatants from the human cell lines AV-3 and HeLa or from the murine cell line L-929. The maximum potentiating effects were observed with pokeweed mitogen (PWM) and phytohaemagglutinin (PHA), whereas the response to concanavalin A (Con A) was only slightly enhanced. TS also potentiated the mixed lymphocyte culture (MLC) response of normal T cells and thymocytes cultured with mitomycin C-treated B lymphoid cell lines. The mitogenic effect of TS was time-dependent and paralleled the appearance of lymphoid colonies in semi-solid agar. Chromatographical separation of concentrated serum-free TS on Sephadex G-100 yielded an active fraction of molecular weight 15,000--25,000 which had all the activities of unseparated TS. PMID:160851

  12. Transcriptome analysis and molecular signature of human retinal pigment epithelium

    PubMed Central

    Strunnikova, N.V.; Maminishkis, A.; Barb, J.J.; Wang, F.; Zhi, C.; Sergeev, Y.; Chen, W.; Edwards, A.O.; Stambolian, D.; Abecasis, G.; Swaroop, A.; Munson, P.J.; Miller, S.S.

    2010-01-01

    Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed ‘signature’ genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases. PMID:20360305

  13. Evaluation of equations for describing the human crystalline lens

    NASA Astrophysics Data System (ADS)

    Giovanzana, Stefano; Schachar, Ronald A.; Talu, Stefan; Kirby, Roger D.; Yan, Eric; Pierscionek, Barbara K.

    2013-03-01

    Accurate mathematical descriptions of the human crystalline lens surface shape are required to properly understand the nature of functional adaptations that occur when the lens shape alters to changes in refractive power. Using least squares method, the total mean normal distance, smoothness, rate of change of the transverse and sagittal radii of curvatures and continuity at the lens equator between eight mathematical functions: conic, figuring conicoid, generalized conic, Hermans conic patch, Urs polynomial, Urs 10th order Fourier series, Chien, and Giovanzana, and 17 human crystalline lenses were evaluated. The mean differences of the fits of all the equations to the whole lens and to the central 8 mm of the lens surfaces were >24 μm with comparable standard deviations. When considering fit smoothness and continuity at the equator, the Giovanzana and Chien functions are most representative of the lens surface.

  14. Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

    PubMed

    Zhang, Pingbo; Kirby, David; Dufresne, Craig; Chen, Yan; Turner, Randi; Ferri, Sara; Edward, Deepak P; Van Eyk, Jennifer E; Semba, Richard D

    2016-04-01

    The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194. PMID:26834087

  15. MORPHOMETRIC ASPECTS OF CILIARY DISTRIBUTION AND CILIOGENESIS IN HUMAN NASAL EPITHELIUM

    EPA Science Inventory

    Observations of freeze-fracture preparations of human nasal epithelium have provided a unique perspective of the spatial distribution of epithelial cell cilia unattainable by more conventional ultrastructural techniques. The initial stages of ciliogenesis were characterized ultra...

  16. Increased expression of osteonectin/SPARC mRNA and protein in age-related human cataracts and spatial expression in the normal human lens

    PubMed Central

    Kantorow, Marc; Huang, Quingling; Yang, Xian-jie; Sage, E. Helene; Magabo, Kristine S.; Miller, Kevin M.; Horwitz, Joseph

    2010-01-01

    Purpose We have previously reported increased levels of Osteonectin/SPARC transcript in age-related cataractous compared to normal human lenses. The purpose of the present study was to evaluate the corresponding levels of osteonectin/SPARC protein in age-related cataractous relative to normal lenses and to evaluate the levels of osteonectin/SPARC transcript in specific types of age-related human cataracts. The spatial expression of osteonectin/SPARC was also evaluated in normal human lenses. Methods Specific types of age-related cataracts were collected and graded. Normal human lenses were microdissected into epithelia and fibers. Osteonectin/SPARC protein levels were monitored by Western immunoblotting, and transcript levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Osteonectin/SPARC expression patterns were examined by RT-PCR and by immunostaining. Results Higher levels of osteonectin/SPARC protein were detected in age-related cataractous relative to normal human lenses. Increased levels of osteonectin/SPARC transcript were also detected in posterior-subcapsular and nuclear cataractous lenses relative to normal lenses. Osteonectin/SPARC transcripts were detected in the lens epithelium but not fibers. Osteonectin/SPARC protein levels were highest in the peripheral lens epithelium. Conclusions Consistent with our previous studies on osteonectin/SPARC mRNA levels, osteonectin/SPARC protein levels were also elevated in cataractous compared to normal human lenses. Increased levels of osteonectin/SPARC mRNA were also found in nuclear and posterior-subcapsular cataracts relative to normal lenses. Osteonectin/SPARC expression is confined to the lens epithelium, and osteonectin/SPARC levels are highest in the peripheral lens epithelium. PMID:10756178

  17. The Structure-Specific Recognition Protein 1 Associates with Lens Epithelium-Derived Growth Factor Proteins and Modulates HIV-1 Replication.

    PubMed

    Lopez, Angelica P; Kugelman, Jeffrey R; Garcia-Rivera, Jose; Urias, Eduardo; Salinas, Sandra A; Fernandez-Zapico, Martin E; Llano, Manuel

    2016-07-17

    The lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin-bound protein essential for efficient lentiviral integration. Genome-wide studies have located LEDGF/p75 inside actively transcribed genes where it mediates lentiviral integration. Although its role in HIV-1 integration is clearly established, the role of LEDGF/p75-associated proteins in HIV-1 infection remains unexplored. Using protein-protein interaction assays, we demonstrated that LEDGF/p75 complexes with a chromatin-remodeling complex facilitates chromatin transcription (FACT), a heterodimer of the structure-specific recognition protein 1 (SSRP1) and the human homolog of suppressor of Ty 16 (hSpt16). Detailed analysis of the interaction of LEDGF/p75 with the FACT complex indicates that LEDGF/p75 interacts with SSRP1 in an hSpt16-independent manner that requires the PWWP domain of LEDGF proteins and the HMG domain of SSRP1. Functional characterizations demonstrate a LEDGF/p75-independent role of SSRP1 in the regulation of HIV-1 replication. shRNA-mediated partial knockdown of SSRP1 reduces HIV-1 infection, but not Murine Leukemia Virus, in human CD4(+) T cells. Similarly, SSRP1 knockdown affects infection by HIV-1-derived viruses that express genes from the viral LTR but not from an internal immediate-early CMV promoter, suggesting a role of SSRP1 in LTR-driven gene expression but not in viral DNA integration. Together, our data demonstrate for the first time the association of LEDGF proteins with the FACT complex and give further support to a role of SSRP1 in HIV-1 infection. PMID:27216501

  18. Determination of human lens capsule permeability and its feasibility as a replacement for Bruch's membrane.

    PubMed

    Lee, Christina J; Vroom, Jonathan A; Fishman, Harvey A; Bent, Stacey F

    2006-03-01

    We have investigated human anterior lens capsule as a potential replacement for Bruch's membrane as a treatment for age-related macular degeneration. Any substrate to replace Bruch's membrane should possess certain characteristics to maintain proper function of the overlying retina. One of the important properties of Bruch's membrane is allowing the flow of nutrients and waste between the retinal pigment epithelium and the choriocapillaris. Here, we measured the permeability of the lens capsule by studying the diffusion of various molecular weight FITC-dextran molecules. Expressions for extraction of diffusion coefficients from concentration vs. time data from a blind-well chamber apparatus were derived for both a single and double membrane experiments. The diffusion coefficients in the lens capsule were found to be in the range of 10(-6) to 10(-10)cm2/s. We demonstrated a power law relationship, with the diffusion coefficient possessing a -0.6 order dependence on molecular weight. The molecular weight exclusion limit was determined to be 150+/-40 kDa. We have compared this value with reported values of Bruch's membrane molecular weight exclusion limit and find that the lens capsule has the potential to act as a substitute Bruch's membrane. PMID:16199085

  19. Biosynthesis, Characterization, and Efficacy in Retinal Degenerative Diseases of Lens Epithelium-derived Growth Factor Fragment (LEDGF1–326), a Novel Therapeutic Protein*

    PubMed Central

    Baid, Rinku; Upadhyay, Arun K.; Shinohara, Toshimichi; Kompella, Uday B.

    2013-01-01

    For vision-threatening retinitis pigmentosa and dry age-related macular degeneration, there are no United States Food and Drug Administration (FDA)-approved treatments. We identified, biosynthesized, purified, and characterized lens epithelium-derived growth factor fragment (LEDGF1–326) as a novel protein therapeutic. LEDGF1–326 was produced at about 20 mg/liter of culture when expressed in the Escherichia coli system, with about 95% purity and aggregate-free homogeneous population with a mean hydrodynamic diameter of 9 ± 1 nm. The free energy of unfolding of LEDGF1–326 was 3.3 ± 0.5 kcal mol−1, and melting temperature was 44.8 ± 0.2 °C. LEDGF1–326 increased human retinal pigment epithelial cell viability from 48.3 ± 5.6 to 119.3 ± 21.1% in the presence of P23H mutant rhodopsin-mediated aggregation stress. LEDGF1–326 also increased retinal pigment epithelial cell FluoSphere uptake to 140 ± 10%. Eight weeks after single intravitreal injection in Royal College of Surgeons (RCS) rats, LEDGF1–326 increased the b-wave amplitude significantly from 9.4 ± 4.6 to 57.6 ± 8.8 μV for scotopic electroretinogram and from 10.9 ± 5.6 to 45.8 ± 15.2 μV for photopic electroretinogram. LEDGF1–326 significantly increased the retinal outer nuclear layer thickness from 6.34 ± 1.6 to 11.7 ± 0.7 μm. LEDGF1–326 is a potential new therapeutic agent for treating retinal degenerative diseases. PMID:23640891

  20. The concentration of light in the human lens.

    PubMed Central

    Merriam, J C

    1996-01-01

    PURPOSE: This thesis explores the idea that light energy, especially ultraviolet light, contributes to the unequal distribution of cataract around the world and to the development of cortical opacities. METHODS: In the first section, the thesis reviews historical concepts of the function of the lens and the nature of cataract, epidemiologic data on the global distribution of cataract, and clinical observations of the predominant location of cortical opacification. Second, computer ray tracings and geometric optics demonstrate the passage of light of varying angle of incidence within the lens. Third, two models of the human eye are used to study the refraction of light by the cornea and lens and illustrate the concentration of energy at the equatorial plane of the lens. RESULTS: Cataract prevalence increases with proximity to the earth's equator, and cortical cataract is most common in the inferior and inferonasal lens. Theoretical studies and the eye models both demonstrate that the concentration of light within the lens increases with angle of incidence, and the eye models suggest that the inferior and inferonasal lens receives significantly more energy than other sections of the lens. CONCLUSION: The prevalence of cataract and exposure to ultraviolet energy both increase with decreasing latitude. The most common location of cortical cataract in the inferonasal lens is consistent with the greater dose of light energy received by this portion of the lens. These studies suggest that the global distribution of cataract and the development of cortical cataract are at least in part dependent on the dose of ultraviolet light received by the lens. Images FIGURE 1 FIGURE 2 FIGURE 27 FIGURE 28 FIGURE 29 FIGURE 31 FIGURE 32 FIGURE 33 FIGURE 34 FIGURE 36 FIGURE 37 FIGURE 38 FIGURE 50 FIGURE 51 FIGURE 52 FIGURE 53 FIGURE 54 FIGURE 56 FIGURE 60 FIGURE 61 FIGURE 63 FIGURE 64 FIGURE 65 FIGURE 68 FIGURE 69 FIGURE 70 FIGURE 71 PMID:8981716

  1. Osteogenic differentiation of human lens epithelial cells might contribute to lens calcification.

    PubMed

    Balogh, Enikő; Tóth, Andrea; Tolnai, Emese; Bodó, Tímea; Bányai, Emese; Szabó, Dóra Júlia; Petrovski, Goran; Jeney, Viktória

    2016-09-01

    Calcification of the human lens has been described in senile cataracts and in young patients with congenital cataract or chronic uveitis. Lens calcification is also a major complication of cataract surgery and plays a role in the opacification of intraocular lenses. A cell-mediated process has been suggested in the background of lens calcification, but so far the exact mechanism remained unexplored. Lens calcification shares remarkable similarities with vascular calcification; in both pathological processes hydroxyapatite accumulates in the soft tissue. Vascular calcification is a regulated, cell-mediated process in which vascular cells undergo osteogenic differentiation. Our objective was to investigate whether human lens epithelial cells (HuLECs) can undergo osteogenic transition in vitro, and whether this process contributes to lens calcification. We used inorganic phosphate (Pi) and Ca to stimulate osteogenic differentiation of HuLECs. Osteogenic stimuli (2.5mmol/L Pi and 1.2mmol/L Ca) induced extracellular matrix mineralization and Ca deposition in HuLECs with the critical involvement of active Pi uptake. Osteogenic stimuli almost doubled mRNA expressions of osteo-/chondrogenic transcription factors Runx2 and Sox9, which was accompanied by a 1.9-fold increase in Runx2 and a 5.5-fold increase in Sox9 protein expressions. Osteogenic stimuli induced mRNA and protein expressions of alkaline phosphatase and osteocalcin in HuLEC. Ca content was higher in human cataractous lenses, compared to non-cataractous controls (n=10). Osteocalcin, an osteoblast-specific protein, was expressed in 2 out of 10 cataractous lenses. We conclude that osteogenic stimuli induce osteogenic differentiation of HuLECs and propose that this mechanism might play a role in lens calcification. PMID:27318027

  2. Dynamic optometer. [for electronic recording of human lens anterior surface

    NASA Technical Reports Server (NTRS)

    Wilson, D. C.

    1974-01-01

    A dynamic optometer that electronically records the position of the anterior surface of the human lens is described. The geometrical optics of the eye and optometer, and the scattering of light from the lens, are closely examined to determine the optimum conditions for adjustment of the instrument. The light detector and associated electronics are also considered, and the operating conditions for obtaining the best signal-to-noise ratio are determined.

  3. Identification of Distinct Layers Within the Stratified Squamous Epithelium of the Adult Human True Vocal Fold

    PubMed Central

    Dowdall, Jayme R.; Sadow, Peter M.; Hartnick, Christopher; Vinarsky, Vladimir; Mou, Hongmei; Zhao, Rui; Song, Phillip C.; Franco, Ramon A.; Rajagopal, Jayaraj

    2016-01-01

    Objectives/Hypothesis A precise molecular schema for classifying the different cell types of the normal human vocal fold epithelium is lacking. We hypothesize that the true vocal fold epithelium has a cellular architecture and organization similar to that of other stratified squamous epithelia including the skin, cornea, oral mucosa, and esophagus. In analogy to disorders of the skin and gastrointestinal tract, a molecular definition of the normal cell types within the human vocal fold epithelium and a description of their geometric relationships should serve as a foundation for characterizing cellular changes associated with metaplasia, dysplasia, and cancer. Study Design Qualitative study with adult human larynges. Methods Histologic sections of normal human laryngeal tissue were analyzed for morphology (hematoxylin and eosin) and immunohistochemical protein expression profile, including cytokeratins (CK13 and CK14), cornified envelope proteins (involucrin), basal cells (NGFR/p75), and proliferation markers (Ki67). Results We demonstrated that three distinct cell strata with unique marker profiles are present within the stratified squamous epithelium of the true vocal fold. We used these definitions to establish that cell proliferation is restricted to certain cell types and layers within the epithelium. These distinct cell types are reproducible across five normal adult larynges. Conclusion We have established that three layers of cells are present within the normal adult stratified squamous epithelium of the true vocal fold. Furthermore, replicating cell populations are largely restricted to the parabasal strata within the epithelium. This delineation of distinct cell populations will facilitate future studies of vocal fold regeneration and cancer. Level of Evidence N/A. PMID:25988619

  4. Determination of Dideoxyosone Precursors of AGEs in Human Lens Proteins

    PubMed Central

    Linetsky, Mikhail; Johar, Kaid; Meltretter, Jasmin; Padmanabha, Smitha; Parmar, Trilok; Vasavada, Abhay R.; Pischetsrieder, Monika; Nagaraj, Ram H.

    2011-01-01

    Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation end products (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-{[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl) benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 AU/mg protein vs. 31.7±19.5 AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates. PMID:21820400

  5. Determination of dideoxyosone precursors of AGEs in human lens proteins.

    PubMed

    Linetsky, Mikhail; Kaid Johar, S R; Meltretter, Jasmin; Padmanabha, Smitha; Parmar, Trilok; Vasavada, Abhay R; Pischetsrieder, Monika; Nagaraj, Ram H

    2011-10-01

    Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates. PMID:21820400

  6. Spatial distribution of metabolites in the human lens.

    PubMed

    Tamara, Semen O; Yanshole, Lyudmila V; Yanshole, Vadim V; Fursova, Anjella Zh; Stepakov, Denis A; Novoselov, Vladimir P; Tsentalovich, Yuri P

    2016-02-01

    Spatial distribution of 34 metabolites along the optical and equatorial axes of the human lens has been determined. For the majority of metabolites, the homogeneous distribution has been observed. That suggests that the rate of the metabolite transformation in the lens is low due to the general metabolic passivity of the lens fiber cells. However, the redox processes are active in the lens; as a result, some metabolites, including antioxidants, demonstrate the "nucleus-depleted" type of distribution, whereas secondary UV filters show the "nucleus-enriched" type. The metabolite concentrations at the lens poles and equator are similar for all metabolites under study. The concentric pattern of the "nucleus-depleted" and "nucleus-enriched" distributions testifies that the metabolite distribution inside the lens is mostly governed by a passive diffusion, relatively free along the fiber cells and retarded in the radial direction across the cells. No significant difference in the metabolite distribution between the normal and cataractous human lenses was found. PMID:26500196

  7. Muscarinic cholinergic and alpha 2-adrenergic receptors in the epithelium and muscularis of the human ileum

    SciTech Connect

    Lepor, H.; Rigaud, G.; Shapiro, E.; Baumann, M.; Kodner, I.J.; Fleshman, J.W. )

    1990-04-01

    The aim of this study was to characterize the binding and functional properties of muscarinic cholinergic (MCh) and alpha 2-adrenergic receptors in the human ileum to provide insight into pharmacologic strategies for managing urinary and fecal incontinence after bladder and rectal replacement with intestinal segments. MCh and alpha 2-adrenergic binding sites were characterized in the epithelium and muscularis of eight human ileal segments with 3H-N-methylscopolamine and 3H-rauwolscine, respectively. The dissociation constant for 3H-N-methylscopolamine in the epithelium and muscularis was 0.32 +/- 0.07 nmol/L and 0.45 +/- 0.10 nmol/L, respectively (p = 0.32). The MCh receptor content was approximately eightfold greater in the muscularis compared with the epithelium (p = 0.008). The dissociation constant for 3H-rauwolscine in the muscularis and epithelium was 2.55 +/- 0.42 nmol/L and 2.03 +/- 0.19 nmol/L, respectively (p = 0.29). The alpha 2-adrenoceptor density was twofold greater in the epithelium compared with the muscularis (p = 0.05). Noncumulative concentration-response experiments were performed with carbachol, an MCh agonist, and UK-14304, a selective alpha 2-adrenergic agonist. The epithelium did not contract in the presence of high concentrations of carbachol and UK-14304. The muscularis preparations were responsive only to carbachol. The muscularis contains primarily MCh receptors mediating smooth muscle contraction. The alpha 2-adrenoceptors are localized primarily to the epithelium and may regulate water secretion in the intestine. The distribution and functional properties of ileal MCh and alpha 2-adrenergic receptors provide a theoretic basis for the treatment of incontinence after bladder and rectal replacement with intestinal segments.

  8. Mathematical models for the shape analysis of human crystalline lens

    NASA Astrophysics Data System (ADS)

    Giovanzana, Stefano; Talu, Stefan

    2012-01-01

    The objective of this paper is to present an analysis of mathematical models of the human crystalline lens. Seven existing models presented in the literature were investigated: conic, figuring conicoid, generalized conic, Hermans conic patch, Kasprzak hyperbolic cosine, Urs 10th-order Fourier series and Giovanzana parametric models. The analyzed models describe the shape for a data set of human crystalline lenses with ages from 6 to 82 years. The results highlight the difficulty and complexity of the task of choosing the most appropriate model for the crystalline lens shape.

  9. Method for quantifying optical properties of the human lens

    DOEpatents

    Loree, T.R.; Bigio, I.J.; Zuclich, J.A.; Shimada, Tsutomu; Strobl, K.

    1999-04-13

    A method is disclosed for quantifying optical properties of the human lens. The present invention includes the application of fiberoptic, OMA-based instrumentation as an in vivo diagnostic tool for the human ocular lens. Rapid, noninvasive and comprehensive assessment of the optical characteristics of a lens using very modest levels of exciting light are described. Typically, the backscatter and fluorescence spectra (from about 300- to 900-nm) elicited by each of several exciting wavelengths (from about 300- to 600-nm) are collected within a few seconds. The resulting optical signature of individual lenses is then used to assess the overall optical quality of the lens by comparing the results with a database of similar measurements obtained from a reference set of normal human lenses having various ages. Several metrics have been identified which gauge the optical quality of a given lens relative to the norm for the subject`s chronological age. These metrics may also serve to document accelerated optical aging and/or as early indicators of cataract or other disease processes. 8 figs.

  10. Method for quantifying optical properties of the human lens

    DOEpatents

    Loree, deceased, Thomas R.; Bigio, Irving J.; Zuclich, Joseph A.; Shimada, Tsutomu; Strobl, Karlheinz

    1999-01-01

    Method for quantifying optical properties of the human lens. The present invention includes the application of fiberoptic, OMA-based instrumentation as an in vivo diagnostic tool for the human ocular lens. Rapid, noninvasive and comprehensive assessment of the optical characteristics of a lens using very modest levels of exciting light are described. Typically, the backscatter and fluorescence spectra (from about 300- to 900-nm) elicited by each of several exciting wavelengths (from about 300- to 600-nm) are collected within a few seconds. The resulting optical signature of individual lenses is then used to assess the overall optical quality of the lens by comparing the results with a database of similar measurements obtained from a reference set of normal human lenses having various ages. Several metrics have been identified which gauge the optical quality of a given lens relative to the norm for the subject's chronological age. These metrics may also serve to document accelerated optical aging and/or as early indicators of cataract or other disease processes.

  11. Robust bioengineered 3D functional human intestinal epithelium

    PubMed Central

    Chen, Ying; Lin, Yinan; Davis, Kimberly M.; Wang, Qianrui; Rnjak-Kovacina, Jelena; Li, Chunmei; Isberg, Ralph R.; Kumamoto, Carol A.; Mecsas, Joan; Kaplan, David L.

    2015-01-01

    Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments. PMID:26374193

  12. FEM simulation of the human lens compared to ex vivo porcine lens cutting pattern: a possible treatment of presbyopia

    NASA Astrophysics Data System (ADS)

    Ripken, T.; Breitenfeld, P.; Fromm, M.; Oberheide, U.; Gerten, G.; Lubatschowski, H.

    2006-02-01

    The most probable reason for presbyopia is an age related loss of elasticity of the lens. It progresses typically during the whole life and at the age of about 45 it leads to a considerable loss of the ability to accommodate within the next decade. However, both, the ciliary muscle and the lens capsule stay active and elastic, respectively. With respect to this, one concept is to regain the deformability of the lens without changing the capsule or zonular apparatus. Since the investigations of Ripken et al. proofed that the flexibility of the presbyopic lens tissue can be increased through the creation of fs-laser induced microcuts inside the lens, this is one possible approach to treat presbyopia. On this account a finite-element-method model with ANSYS of the human lens during accommodation will be presented. The analysis premises all lens materials to be linear elastic and allow large displacements. A first analysis of this method for the treatment of presbyopia is accomplished. Therefore the mechanical analysis of untreated and treated lens are compared. In addition ex-vivo elasticity measurements of untreated and treated lenses will be presented. As a result an improvement of the flexibility of the lens tissue is found and as its consequence a change of the lens radii of curvature is established. After suitable processing of the output data the change in optical power between untreated and treated lenses are calculated. The finite element simulation shows similar behaviour compared to the treated porcine lenses.

  13. Nanoceria have no genotoxic effect on human lens epithelial cells

    NASA Astrophysics Data System (ADS)

    Pierscionek, Barbara K.; Li, Yuebin; Yasseen, Akeel A.; Colhoun, Liza M.; Schachar, Ronald A.; Chen, Wei

    2010-01-01

    There are no treatments for reversing or halting cataract, a disease of the structural proteins in the eye lens, that has associations with other age-related degenerative conditions such as Alzheimer's disease. The incidence of cataract and associated conditions is increasing as the average age of the population rises. Protein folding diseases are difficult to assess in vivo as proteins and their age-related changes are assessed after extraction. Nanotechnology can be used to investigate protein changes in the intact lens as well as for a potential means of drug delivery. Nanoparticles, such as cerium oxide (CeO2) which have antioxidant properties, may even be used as a means of treating cataract directly. Prior to use in treatments, nanoparticle genotoxicity must be tested to assess the extent of any DNA or chromosomal damage. Sister chromatid exchanges were measured and DNA damage investigated using the alkaline COMET assay on cultured human lens epithelial cells, exposed to 5 and 10 µg ml-1 of CeO2 nanoparticles (nanoceria). Nanoceria at these dosages did not cause any DNA damage or significant increases in the number of sister chromatid exchanges. The absence of genotoxic effects on lens cells suggests that nanoceria, in the doses and exposures tested in this study, are not deleterious to the eye lens and have the potential for use in studying structural alterations, in developing non-surgical cataract treatments and in investigating other protein folding diseases.

  14. Thioredoxin reductase activity may be more important than GSH level in protecting human lens epithelial cells against UVA light.

    PubMed

    Padgaonkar, Vanita A; Leverenz, Victor R; Bhat, Aparna V; Pelliccia, Sara E; Giblin, Frank J

    2015-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2 , 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm(-2) of UVA radiation (338-400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. PMID:25495870

  15. Thioredoxin Reductase Activity may be More Important than GSH Level in Protecting Human Lens Epithelial Cells Against UVA Light

    PubMed Central

    Padgaonkar, Vanita A.; Leverenz, Victor R.; Bhat, Aparna V.; Pelliccia, Sara E.; Giblin, Frank J.

    2014-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2, 3% and 20%, were employed during a 1 hr exposure of the cells to 25 J/cm2 of UVA radiation (338-400nm wavelength, peak at 365nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well-tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. PMID:25495870

  16. LENS: web-based lens for enrichment and network studies of human proteins

    PubMed Central

    2015-01-01

    Background Network analysis is a common approach for the study of genetic view of diseases and biological pathways. Typically, when a set of genes are identified to be of interest in relation to a disease, say through a genome wide association study (GWAS) or a different gene expression study, these genes are typically analyzed in the context of their protein-protein interaction (PPI) networks. Further analysis is carried out to compute the enrichment of known pathways and disease-associations in the network. Having tools for such analysis at the fingertips of biologists without the requirement for computer programming or curation of data would accelerate the characterization of genes of interest. Currently available tools do not integrate network and enrichment analysis and their visualizations, and most of them present results in formats not most conducive to human cognition. Results We developed the tool Lens for Enrichment and Network Studies of human proteins (LENS) that performs network and pathway and diseases enrichment analyses on genes of interest to users. The tool creates a visualization of the network, provides easy to read statistics on network connectivity, and displays Venn diagrams with statistical significance values of the network's association with drugs, diseases, pathways, and GWASs. We used the tool to analyze gene sets related to craniofacial development, autism, and schizophrenia. Conclusion LENS is a web-based tool that does not require and download or plugins to use. The tool is free and does not require login for use, and is available at http://severus.dbmi.pitt.edu/LENS. PMID:26680011

  17. [Human amniotic epithelium (HAE) as a possible source of stem cells (SC)].

    PubMed

    García-López, Guadalupe; García-Castro, Irma Lydia; Avila-González, Daniela; Molina-Hernández, Anayansi; Flores-Herrera, Héctor; Merchant-Larios, Horacio; Díaz-Martínez, Fabián

    2015-01-01

    There have been major recent advances in the field of developmental biology due to the investigation on stem cells (SC). Stem cells are characterized by their capacity of auto-renewal and differentiation to different cellular phenotypes. Based on the developmental stage, they can be classified into two different types: embryonic SCs and adult SCs. It has been widely reported that several problems need to be resolved before their possible clinical applications. As a result, fetal membranes have been suggested as an alternative source of SCs. In the human amniotic epithelium, the presence of markers of pluripotent SC´s has been reported, and its capacity as a feeder layer for expansion of different SC types. Also, fetal membranes are a discarded product after delivery, and thus there are not any ethical issues related to its use. In conclusion, the human amniotic epithelium can be a strong candidate for regenerative medicine. PMID:25739486

  18. HNF1 regulates critical processes in the human epididymis epithelium.

    PubMed

    Browne, James A; Yang, Rui; Eggener, Scott E; Leir, Shih-Hsing; Harris, Ann

    2016-04-15

    The luminal environment of the epididymis participates in sperm maturation and impacts male fertility. It is dependent on the coordinated expression of many genes encoding proteins with a role in epithelial transport. We identified cis-regulatory elements for critical genes in epididymis function, by mapping open chromatin genome-wide in human epididymis epithelial (HEE) cells. Bioinformatic predictions of transcription factors binding to the regulatory elements suggested an important role for hepatocyte nuclear factor 1 (HNF1) in the transcriptional program of these cells. Chromatin immunoprecipitation and deep sequencing (ChIP-seq) revealed HNF1 target genes in HEE cells. In parallel, the contribution of HNF1 to the transcriptome of HEE cells was determined by RNA-seq, following siRNA-mediated depletion of both HNF1α and HNF1β transcription factors. Repression of these factors caused differential expression of 1892 transcripts (902 were downregulated and 990 upregulated) in comparison to non-targeting siRNAs. Differentially expressed genes with HNF1 ChIP-seq peaks within 20 kb were subject to gene ontology process enrichment analysis. Among the most significant processes associated with down-regulated genes were epithelial transport of water, phosphate and bicarbonate, all critical processes in epididymis epithelial function. Measurements of intracellular pH (pHi) confirmed a role for HNF1 in regulating the epididymis luminal environment. PMID:26808453

  19. Human Reconstituted Nasal Epithelium, a promising in vitro model to assess impacts of environmental complex mixtures.

    PubMed

    Bardet, Gaëlle; Mignon, Virginie; Momas, Isabelle; Achard, Sophie; Seta, Nathalie

    2016-04-01

    Considering the impact of respiratory diseases around the world, appropriate experimental tools to help understand the mechanisms involved in such diseases are becoming essential. Our aim was to investigate the cellular and morphological reactivity of a human Reconstituted Nasal Epithelium (hRNE) to evaluate the impact of environmental complex mixture (ECM), with tobacco smoke as a model, after three weeks of repeated exposures. Staining of hRNE showed a multilayered ciliated epithelium, with a regular cilia beats, and a mucus production. When hRNE was exposed to ECM for 5 min once or twice a week, during 3 weeks, significant changes occurred: IL-8 production significantly increased 24h after the first exposure compared with Air-exposure and only during the first week, without any loss of tissue integrity. Immunostaining of F-actin cytoskeleton showed a modification in cellular morphology (number and diameter). Taken together our results indicate that hRNE is well suited to study the cellular and morphological effects of repeated exposures to an environmental complex mixture. Human reconstituted epithelium models are currently the best in vitro representation of human respiratory tract physiology, and also the most robust for performing repeated exposures to atmospheric pollutants. PMID:26631767

  20. In vivo antioxidant gene expression in human airway epithelium of normal individuals exposed to 100% O2.

    PubMed

    Erzurum, S C; Danel, C; Gillissen, A; Chu, C S; Trapnell, B C; Crystal, R G

    1993-09-01

    Human bronchial epithelium is exquisitely sensitive to high O2 levels, with tracheobronchitis usually developing after 12 h of exposure to 100% O2. To evaluate whether this vulnerability results from inability of the bronchial epithelium to provide adequate antioxidant protection, we quantified antioxidant gene expression in bronchial epithelium of normal volunteers at baseline and after exposure to 100% O2 in vivo. After 14.8 +/- 0.2 h of 100% O2, 24 of 33 individuals had evidence of tracheobronchitis. Baseline gene expression of CuZn superoxide dismutase (SOD), MnSOD, and catalase in bronchial epithelium was very low (CuZnSOD 4.1 +/- 0.8 transcripts/cell, MnSOD 5.1 +/- 0.9, catalase 1.3 +/- 0.2), with control gamma-actin expression relatively abundant (50 +/- 6 transcripts/cell). Importantly, despite 100% O2 exposure sufficient to cause tracheobronchitis in most individuals, antioxidant mRNA transcripts/cell in bronchial epithelium did not increase (P > 0.5). Catalase activity in bronchial epithelium did not change after exposure to hyperoxia (P > 0.05). Total SOD activity increased mildly (P < 0.01) but not sufficiently to protect the epithelium. Together, the very low levels of expression of intracellular antioxidant enzymes and the inability to upregulate expression at the mRNA level with oxidant stress likely have a role in human airway epithelium susceptibility to hyperoxia. PMID:8226538

  1. Design and analysis of an adaptive lens that mimics the performance of the crystalline lens in the human eye

    NASA Astrophysics Data System (ADS)

    Santiago-Alvarado, Agustin; Cruz-Félix, Angel S.; Iturbide-Jiménez, F.; Martínez-López, M.; Ramírez-Como, M.; Armengol-Cruz, V.; Vásquez-Báez, I.

    2014-09-01

    Tunable lenses are optical systems that have attracted much attention due to their potential applications in such areas like ophthalmology, machine vision, microscopy and laser processing. In recent years we have been working in the analysis and performance of a liquid-filled variable focal length lens, this is a lens that can modify its focal length by changing the amount of water within it. Nowadays we extend our study to a particular adaptive lens known as solid elastic lens (SEL) that it is formed by an elastic main body made of Polydimethylsiloxane (PDMS Sylgard 184). In this work, we present the design, simulation and analysis of an adaptive solid elastic lens that in principle imitates the accommodation process of the crystalline lens in the human eye. For this work, we have adopted the parameters of the schematic eye model developed in 1985 by Navarro et al.; this model represents the anatomy of the eye as close as possible to reality by predicting an acceptable and accurate quantity of spherical and chromatic aberrations without any shape fitting. An opto-mechanical analysis of the accommodation process of the adaptive lens is presented, by simulating a certain amount of radial force applied onto the SEL using the finite element method with the commercial software SolidWorks®. We also present ray-trace diagrams of the simulated compression process of the adaptive lens using the commercial software OSLO®.

  2. Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium.

    PubMed

    Walsham, Alistair D S; MacKenzie, Donald A; Cook, Vivienne; Wemyss-Holden, Simon; Hews, Claire L; Juge, Nathalie; Schüller, Stephanie

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains. PMID:26973622

  3. Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium

    PubMed Central

    Walsham, Alistair D. S.; MacKenzie, Donald A.; Cook, Vivienne; Wemyss-Holden, Simon; Hews, Claire L.; Juge, Nathalie; Schüller, Stephanie

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains. PMID:26973622

  4. Vitamin C Degradation Products and Pathways in the Human Lens*

    PubMed Central

    Nemet, Ina; Monnier, Vincent M.

    2011-01-01

    Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation are poorly understood. Here we have determined the levels of vitamin C oxidation and degradation products dehydroascorbic acid, 2,3-diketogulonic acid, 3-deoxythreosone, xylosone, and threosone in the human lens using o-phenylenediamine to trap both free and protein-bound adducts. In the protein-free fraction and water-soluble proteins (WSP), all five listed degradation products were identified. Dehydroascorbic acid, 2,3-diketogulonic acid, and 3-deoxythreosone were the major products in the protein-free fraction, whereas in the WSP, 3-deoxythreosone was the most abundant measured dicarbonyl. In addition, 3-deoxythreosone in WSP showed positive linear correlation with age (p < 0.05). In water-insoluble proteins, only 3-deoxythreosone and threosone were detected, whereby the level of 3-deoxythreosone was ∼20 times higher than the level of threosone. The identification of 3-deoxythreosone as the major degradation product bound to human lens proteins provides in vivo evidence for the non-oxidative pathway of dehydroascorbate degradation into erythrulose as a major pathway for vitamin C degradation in vivo. PMID:21885436

  5. No turnover in lens lipids for the entire human lifespan

    PubMed Central

    Hughes, Jessica R; Levchenko, Vladimir A; Blanksby, Stephen J; Mitchell, Todd W; Williams, Alan; Truscott, Roger JW

    2015-01-01

    Lipids are critical to cellular function and it is generally accepted that lipid turnover is rapid and dysregulation in turnover results in disease (Dawidowicz 1987; Phillips et al., 2009; Liu et al., 2013). In this study, we present an intriguing counter-example by demonstrating that in the center of the human ocular lens, there is no lipid turnover in fiber cells during the entire human lifespan. This discovery, combined with prior demonstration of pronounced changes in the lens lipid composition over a lifetime (Hughes et al., 2012), suggests that some lipid classes break down in the body over several decades, whereas others are stable. Such substantial changes in lens cell membranes may play a role in the genesis of age-related eye disorders. Whether long-lived lipids are present in other tissues is not yet known, but this may prove to be important in understanding the development of age-related diseases. DOI: http://dx.doi.org/10.7554/eLife.06003.001 PMID:25760082

  6. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

    SciTech Connect

    Roberts, Joan E. Wielgus, Albert R. Boyes, William K. Andley, Usha Chignell, Colin F.

    2008-04-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C{sub 60}(OH){sub 22-26}] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C{sub 60}(OH){sub 22-26} in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 {mu}M. Exposure to either UVA or visible light in the presence of > 5 {mu}M fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 {mu}M lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein {alpha}-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C{sub 60}(OH){sub 22-26} is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo.

  7. Purification and characterization of factors produced by Aspergillus fumigatus which affect human ciliated respiratory epithelium.

    PubMed Central

    Amitani, R; Taylor, G; Elezis, E N; Llewellyn-Jones, C; Mitchell, J; Kuze, F; Cole, P J; Wilson, R

    1995-01-01

    The mechanisms by which Aspergillus fumigatus colonizes the respiratory mucosa are unknown. Culture filtrates of eight of nine clinical isolates of A. fumigatus slowed ciliary beat frequency and damaged human respiratory epithelium in vitro. These changes appeared to occur concurrently. Culture filtrates of two clinical isolates of Candida albicans had no effect on ciliated epithelium. We have purified and characterized cilioinhibitory factors of a clinical isolate of A. fumigatus. The cilioinhibitory activity was heat labile, reduced by dialysis, and partially extractable into chloroform. The activity was associated with both high- and low-molecular-weight factors, as determined by gel filtration on Sephadex G-50. A low-molecular-weight cilioinhibitory factor was further purified by reverse-phase high-performance liquid chromatography and shown by mass spectrometry to be gliotoxin, a known metabolite of A. fumigatus. Gliotoxin significantly slowed ciliary beat frequency in association with epithelial damage at concentrations above 0.2 microgram/ml; other Aspergillus toxins, i.e., fumagillin and helvolic acid, were also cilioinhibitory but at much higher concentrations. High-molecular-weight (> or = 35,000 and 25,000) cilioinhibitory materials had neither elastolytic nor proteolytic activity and remain to be identified. Thus, A. fumigatus produces a number of biologically active substances which slow ciliary beating and damage epithelium and which may influence colonization of the airways. PMID:7543879

  8. Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.

    PubMed

    Hill, David R; Kessler, Sean P; Rho, Hyunjin K; Cowman, Mary K; de la Motte, Carol A

    2012-08-31

    Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human β-defensin 2 (HβD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HβD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HβD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HβD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HβD2 protein. PMID:22761444

  9. Th2-type cytokine-induced mucus metaplasia decreases susceptibility of human bronchial epithelium to rhinovirus infection.

    PubMed

    Jakiela, Bogdan; Gielicz, Anna; Plutecka, Hanna; Hubalewska-Mazgaj, Magdalena; Mastalerz, Lucyna; Bochenek, Grazyna; Soja, Jerzy; Januszek, Rafal; Aab, Alar; Musial, Jacek; Akdis, Mübeccel; Akdis, Cezmi A; Sanak, Marek

    2014-08-01

    Human rhinoviruses (RVs) are a major cause of exacerbations in asthma and other chronic airway diseases. A characteristic feature of asthmatic epithelium is goblet cell metaplasia and mucus hypersecretion. Bronchial epithelium is also an important source of lipid mediators, including pro- and antiinflammatory eicosanoids. By using air-liquid interface cultures of airway epithelium from patients with asthma and nonasthmatic control subjects, we compared RV16 replication-induced changes in mRNA expression of asthma candidate genes and eicosanoid production in the epithelium with or without IL-13-induced mucus metaplasia. Mucus metaplastic epithelium was characterized by a 20-fold less effective replication of RV16 and blunted changes in gene expression; this effect was seen to the same extent in patients with asthma and control subjects. We identified ciliary cells as the main target for RV16 by immunofluorescence imaging and demonstrated that the numbers of ciliary cells decreased in RV16-infected epithelium. RV16 infection of mucociliary epithelium resulted in overexpression of genes associated with bronchial remodeling (e.g., MUC5AC, FGF2, and HBEGF), induction of cyclooxygenase-2, and increased secretion of prostaglandins. These responses were similar in both studied groups. These data indicate that structural changes associated with mucus metaplasia renders airway epithelium less susceptible to RV infection. Thus, exacerbations of the lung disease caused by RV may result from severe impairment in mucociliary clearance or activation of immune defense rather than from preferential infection of mucus metaplastic epithelium. Repeated rhinoviral infections of compromised epithelium may contribute to the remodeling of the airways. PMID:24588727

  10. On the growth and internal structure of the human lens

    PubMed Central

    Augusteyn, Robert C

    2010-01-01

    Growth of the human lens and the development of its internal features are examined using in vivo and in vitro observations on dimensions, weights, cell sizes, protein gradients and other properties. In vitro studies have shown that human lens growth is biphasic, asymptotic until just after birth and linear for most of postnatal life. This generates two distinct compartments, the prenatal and the postnatal. The prenatal growth mode leads to the formation of an adult nuclear core of fixed dimensions and the postnatal, to an ever-expanding cortex. The nuclear core and the cortex have different properties and can readily be physically separated. Communication and adhesion between the compartments is poor in older lenses. In vivo slit lamp examination reveals several zones of optical discontinuity in the lens. Different nomenclatures have been used to describe these, with the most common recognizing the embryonic, foetal, juvenile and adult nuclei as well as the cortex and outer cortex. Implicit in this nomenclature is the idea that the nuclear zones were generated at defined periods of development and growth. This review examines the relationship between the two compartments observed in vitro and the internal structures revealed by slit lamp photography. Defining the relationship is not as simple as it might seem because of remodeling and cell compaction which take place, mostly in the first 20 years of postnatal life. In addition, different investigators use different nomenclatures when describing the same regions of the lens. From a consideration of the dimensions, the dry mass contents and the protein distributions in the lens and in the various zones, it can be concluded that the juvenile nucleus and the layers contained within it, as well as most of the adult nucleus, were actually produced during prenatal life and the adult nucleus was completed within 3 months after birth, in the final stages of the prenatal growth mode. Further postnatal growth takes place

  11. Electrogenic transport and K+ ion channel expression by the human endolymphatic sac epithelium

    PubMed Central

    Kim, Sung Huhn; Kim, Bo Gyung; Kim, Jin Young; Roh, Kyung Jin; Suh, Michelle J.; Jung, JinSei; Moon, In Seok; Moon, Sung K.; Choi, Jae Young

    2015-01-01

    The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K+ channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K+ channels in the electrogenic transport of human ES epithelium. The identified K+ channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K+ transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid. PMID:26655723

  12. Reconstituted Human Upper Airway Epithelium as 3-D In Vitro Model for Nasal Polyposis

    PubMed Central

    de Borja Callejas, Francisco; Martínez-Antón, Asunción; Alobid, Isam; Fuentes, Mireya; Cortijo, Julio; Picado, César

    2014-01-01

    Background Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described. Objectives 1st) To develop and characterize the mucociliar differentiation overtime of human epithelial cells of chronic rhinosinusitis with nasal polyps (CRSwNP) in ALI culture system; 2nd) To corroborate that 3D in vitro model of NP reconstituted epithelium maintains, compared to control nasal mucosa (NM), an inflammatory function. Methods Epithelial cells were obtained from 9 NP and 7 control NM, and differentiated in ALI culture for 28 days. Mucociliary differentiation was characterized at different times (0, 7, 14, 21, and 28 days) using ultrastructure analysis by electron microscopy; ΔNp63 (basal stem/progenitor cell), β-tubulin IV (cilia), and MUC5AC (goblet cell) expression by immunocytochemistry; and mucous (MUC5AC, MUC5B) and serous (Lactoferrin) secretion by ELISA. Inflammatory function of ALI cultures (at days 0, 14, and 28) through cytokine (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70) and chemokine (RANTES, MIG, MCP-1, IP-10, eotaxin-1, and GM-CSF) production was analysed by CBA (Cytometric Bead Array). Results In both NP and control NM ALI cultures, pseudostratified epithelium with ciliated, mucus-secreting, and basal cells were observed by electron microscopy at days 14 and 28. Displaying epithelial cell re-differentation, β-tubulin IV and MUC5AC positive cells increased, while ΔNp63 positive cells decreased overtime. No significant differences were found overtime in MUC5AC, MUC5B, and lactoferrin secretions between both ALI cultures. IL-8 and GM-CSF were significantly increased in NP compared to control NM regenerated epithelia. Conclusion Reconstituted epithelia from human NP epithelial cells cultured in ALI system provides a 3D in vitro model

  13. Epithelium integrity is crucial for the relaxant activity of brain natriuretic peptide in human isolated bronchi

    PubMed Central

    Matera, Maria G; Calzetta, Luigino; Passeri, Daniela; Facciolo, Francesco; Rendina, Erino A; Page, Clive; Cazzola, Mario; Orlandi, Augusto

    2011-01-01

    BACKGROUND AND PURPOSE Brain natriuretic peptide (BNP) plays an important role in several biological functions, including bronchial relaxation. Here, we have investigated the role of BNP and its cognate receptors in human bronchial tone. EXPERIMENTAL APPROACH Effects of BNP on responses to carbachol and histamine were evaluated in non-sensitized, passively sensitized, epithelium-intact or denuded isolated bronchi and in the presence of methoctramine, Nω-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine. Natriuretic peptide receptors (NPRs) were investigated by immunohistochemistry, RT-PCR and real-time PCR. Release of NO and acetylcholine from bronchial tissues and cultured BEAS-2B bronchial epithelial cells was also investigated. KEY RESULTS BNP reduced contractions mediated by carbachol and histamine, with decreased Emax (carbachol: 22.7 ± 4.7%; histamine: 59.3 ± 1.8%) and increased EC50 (carbachol: control 3.33 ± 0.88 µM, BNP 100 ± 52.9 µM; histamine: control 16.7 ± 1.7 µM, BNP 90 ± 30.6 µM); BNP was ineffective in epithelium-denuded bronchi. Among NPRs, only atrial NPR (NPR1) transcripts were detected in bronchial tissue. Bronchial NPR1 immunoreactivity was detected in epithelium and inflammatory cells but faint or absent in airway smooth muscle cells. NPR1 transcripts in bronchi increased after incubation with BNP, but not after sensitization. Methoctramine and quinine abolished BNP-induced relaxant activity. The latter was associated with increased bronchial mRNA for NO synthase and NO release, inhibited by L-NAME and aminoguanidine. In vitro, BNP increased acetylcholine release from bronchial epithelial cells, whereas NO release was unchanged. CONCLUSIONS AND IMPLICATIONS Epithelial cells mediate the BNP-induced relaxant activity in human isolated bronchi. PMID:21410689

  14. Demonstration of carboxylesterase in cytology samples of human nasal respiratory epithelium

    SciTech Connect

    Rodgers, D.A.; Nikula, K.J.; Avila, K.

    1995-12-01

    The epithelial lining of the nasal airways is a target for responses induced by a variety of toxicant exposures. The high metabolic capacity of this tissue has been suggested to play a role in both protection of the airways through detoxication of certain toxicants, as well as in activation of other compounds to more toxic metabolites. Specifically, nasal carboxylesterase (CE) has been shown to mediate the toxicity of inhaled esters and acrylates by converting them to more toxic acid and alcohol metabolites which can be cytotoxic and/or carcinogenic to the nasal mucosa. Due to difficulties in extrapolating rodent models to human, new paradigms using human cells and tissues are essential to understanding and evaluating the metabolic processes in human nasal epithelium.

  15. Cell proliferation in the human mammary epithelium. Differential contribution by epithelial and myoepithelial cells.

    PubMed Central

    Joshi, K.; Smith, J. A.; Perusinghe, N.; Monoghan, P.

    1986-01-01

    The ductal system of the human breast consists of epithelial, myoepithelial, and basal clear cells. By labeling ducts and alveoli dissected from reduction mammoplasty specimens with 3H-thymidine in vitro and labeling human breast organoids xenografted in nude mice in vivo, it was found that cellular proliferation in the human breast is virtually confined to epithelial and basal clear cells. A pulse label of 3H-thymidine in organ culture explants was followed over a period of time, and it was found that myoepithelial cells originate from a precursor cell population within the mammary epithelium after a number of cell divisions. Myoepithelial cells were not seen to divide when fully mature. Images Figure 1 Figure 2 PMID:3740213

  16. A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells

    PubMed Central

    Ogaki, Soichiro; Morooka, Mayu; Otera, Kaito; Kume, Shoen

    2015-01-01

    The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes. PMID:26616277

  17. A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium.

    PubMed

    Speroni, Lucia; Sweeney, Michael F; Sonnenschein, Carlos; Soto, Ana M

    2016-01-01

    The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression. PMID:26891095

  18. A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.

    PubMed

    Ogaki, Soichiro; Morooka, Mayu; Otera, Kaito; Kume, Shoen

    2015-01-01

    The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes. PMID:26616277

  19. A breakdown in communication? Understanding the effects of aging on the human small intestine epithelium

    PubMed Central

    2015-01-01

    In the intestine, a single layer of epithelial cells sealed together at their apical surfaces by tight junctions helps to prevent the luminal commensal and pathogenic micro-organisms and their toxins from entering host tissues. The intestinal epithelium also helps to maintain homoeostasis in the mucosal immune system by expressing anti-inflammatory cytokines in the steady state and inflammatory cytokines in response to pathogens. Although the function of the mucosal immune system is impaired in elderly humans, the molecular mechanisms which cause this dramatic functional decline are poorly understood. Our current understanding of the effects of aging on the physical and immunological properties of the intestinal epithelial barrier is also very limited. In this issue of Clinical Science, Man et al. provide further insight into the effects of aging on small intestinal barrier function in humans and the influence that gut luminal micro-organisms may have on it. Using human terminal ileal biopsy tissues they show that intestinal permeability to solutes, but not macromolecules, was significantly increased in the intestines of elderly humans. This was accompanied by elevated expression of the pro-inflammatory cytokine interleukin (IL)-6 which appeared to modulate claudin-2 expression and solute permeability in the epithelium. Conversely, IL-8 synthesis in response to flagellin stimulation was reduced in intestines of the elderly subjects, but was not associated with effects on Toll-like receptor 5 (TLR5) expression. These data provide an important advance in our understanding on the effects of aging on intestinal permeability and innate mucosal immune responsiveness in elderly humans. PMID:26186738

  20. MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

    PubMed Central

    Greene, Whitney A.; Muñiz, Alberto.; Plamper, Mark L.; Kaini, Ramesh R.; Wang, Heuy-Ching

    2014-01-01

    The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types. PMID:24999033

  1. Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

    PubMed Central

    Lau, Megan E.; Hunstad, David A.

    2013-01-01

    The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder. PMID:24300797

  2. A novel interleukin 33/ST2 signaling regulates inflammatory response in human corneal epithelium.

    PubMed

    Lin, Jing; Zhang, Lili; Zhao, Guiqiu; Su, Zhitao; Deng, Ruzhi; Pflugfelder, Stephen C; Li, De-Quan

    2013-01-01

    Interleukin (IL) 33, a member of IL-1 cytokine family, is well known to promote Th2 type immune responses by signaling through its receptor ST2. However, it is not clear whether ST2 is expressed by mucosal epithelium, and how it responds to IL-33 to induce inflammatory mediators. This study was to identify the presence and function of ST2 and explore the role of IL-33/ST2 signaling in regulating the inflammatory cytokine production in corneal epithelial cells. Human corneal tissues and cultured primary human corneal epithelial cells (HCECs) were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of inflammatory cytokine and chemokine. The mRNA expression was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 mRNA and protein were detected in donor corneal epithelium and cultured HCECs, and ST2 signal was enhanced by exposure to IL-33. IL-33 significantly stimulated the production of inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokine IL-8 by HCECs at both mRNA and protein levels. The stimulated production of inflammatory mediators by IL-33 was blocked by ST2 antibody or soluble ST2 protein. Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein phosphorylation and nuclear translocation, and also suppressed the production of these inflammatory cytokines and chemokine induced by IL-33. These findings demonstrate that ST2 is present in human corneal epithelial cells, and IL-33/ST2 signaling plays an important role in regulating IL-33 induced inflammatory responses in ocular surface. PMID:23585867

  3. A Novel Interleukin 33/ST2 Signaling Regulates Inflammatory Response in Human Corneal Epithelium

    PubMed Central

    Lin, Jing; Zhang, Lili; Zhao, Guiqiu; Su, Zhitao; Deng, Ruzhi; Pflugfelder, Stephen C.; Li, De-Quan

    2013-01-01

    Interleukin (IL) 33, a member of IL-1 cytokine family, is well known to promote Th2 type immune responses by signaling through its receptor ST2. However, it is not clear whether ST2 is expressed by mucosal epithelium, and how it responds to IL-33 to induce inflammatory mediators. This study was to identify the presence and function of ST2 and explore the role of IL-33/ST2 signaling in regulating the inflammatory cytokine production in corneal epithelial cells. Human corneal tissues and cultured primary human corneal epithelial cells (HCECs) were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of inflammatory cytokine and chemokine. The mRNA expression was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 mRNA and protein were detected in donor corneal epithelium and cultured HCECs, and ST2 signal was enhanced by exposure to IL-33. IL-33 significantly stimulated the production of inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokine IL-8 by HCECs at both mRNA and protein levels. The stimulated production of inflammatory mediators by IL-33 was blocked by ST2 antibody or soluble ST2 protein. Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein phosphorylation and nuclear translocation, and also suppressed the production of these inflammatory cytokines and chemokine induced by IL-33. These findings demonstrate that ST2 is present in human corneal epithelial cells, and IL-33/ST2 signaling plays an important role in regulating IL-33 induced inflammatory responses in ocular surface. PMID:23585867

  4. Gene Expression and Functional Annotation of the Human and Mouse Choroid Plexus Epithelium

    PubMed Central

    Janssen, Sarah F.; van der Spek, Sophie J. F.; ten Brink, Jacoline B.; Essing, Anke H. W.; Gorgels, Theo G. M. F.; van der Spek, Peter J.; Jansonius, Nomdo M.; Bergen, Arthur A. B.

    2013-01-01

    Background The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and

  5. Morphological appearances of human lens epithelial cells in culture.

    PubMed

    Power, W; Neylan, D; Collum, L

    1993-01-01

    A system for culturing human lens epithelial cells in the laboratory was developed. The morphological appearances of the cells was studied using phase contrast, scanning and transmission electron microscopy. Cell marker studies using monoclonal antibodies to cytokeratin, vimentin and epithelial membrane antigen were also performed. There was a marked increase in cell size as a function of time in culture. After 3 to 4 weeks cells showed early signs of ageing. By 6 to 8 weeks the majority of the cells had become very irregular in shape and demonstrated irregularities of the plasma membrane and intra-cytoplasmic vacuole formation. The cells stained strongly for vimentin and epithelial membrane antigen. Staining with cytokeratin was somewhat weaker. This culture technique provides us with a suitable model for studying the growth behavior of these cells. PMID:7512459

  6. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

    PubMed Central

    Groh, V; Bahram, S; Bauer, S; Herman, A; Beauchamp, M; Spies, T

    1996-01-01

    Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8901601

  7. Inherent tone of human bronchus: role of eicosanoids and the epithelium

    PubMed Central

    Watson, N; Magnussen, H; Rabe, K F

    1997-01-01

    Airway preparations of different species possess varying degrees of inherent tone which is the result of different metabolites of arachidonic acid in different species. In human bronchial smooth muscle in vitro we have investigated the effects of 5-lipoxygenase inhibition (zileuton, 10 μM), cyclo-oxygenase inhibition (indomethacin, 1 μM) and mechanical epithelium removal on inherent tone. The shunting of arachidonic acid by inhibition of one or other of these enzymes, as a possible explanation for the effects observed, has also been investigated. Zileuton caused a significant fall in tone either alone (−107±33 mg) or after cyclo-oxygenase inhibition (−203±48 mg) and this effect was not significantly altered by epithelial removal (−191±43 mg alone; −333±88 mg after indomethacin). Indomethacin increased tone when applied alone (160±94 mg), but this effect only reached statistical significance after 5-lipoxygenase inhibition, (210±81 mg; P<0.05). Epithelial removal did not alter the effect of indomethacin when applied alone (213±97 mg), but significantly reduced the effect of indomethacin after 5-lipoxygenase inhibition (34±23 mg; P<0.05). These data suggest that inherent tone in human bronchus is largely the result of contractile 5-lipoxygenase products. However, the involvement of cyclo-oxygenase products cannot entirely be discounted, since in the presence of 5-lipoxygenase inhibition contractile and relaxant eicosanoids originating from the bronchial epithelium appear to influence significantly inherent tone. PMID:9249244

  8. Association of Single Nucleotide Polymorphisms in the Lens Epithelium-Derived Growth Factor (LEDGF/p75) with HIV-1 Infection Outcomes in Brazilian HIV-1+ Individuals

    PubMed Central

    Caetano, Diogo Gama; Teixeira, Sylvia Lopes Maia; Guimarães, Monick Lindenmeyer; Campos, Dayse Pereira; Veloso, Valdilea Gonçalves; Babic, Dunja Z.; Stevenson, Mario; Moraes, Milton Ozório; Morgado, Mariza Gonçalves

    2014-01-01

    The lens epithelium-derived growth factor p75 (LEDGF/p75), coded by the PSIP1 gene, is an important host co-factor that interacts with HIV-1 integrase to target integration of viral cDNA into active genes. The aim of this study was to investigate the association of SNPs in the PSIP1 gene with disease outcome in HIV-1 infected patients. We performed a genetic association study in a cohort of 171 HIV-1 seropositive Brazilian individuals classified as rapid progressors (RP, n = 69), typical progressors (TP, n = 79) and long-term nonprogressors (LTNP, n = 23). The exonic SNP rs61744944 and 9 tag SNPs were genotyped. A group of 192 healthy subjects was analyzed to determine the frequency of SNPs and haplotypes in the general population. Linkage disequilibrium (LD) analyses indicated that the SNPs analyzed were not in high LD (r2<0.8). Logistic regression models suggested that patients carrying the T allele rs61744944 (472L) were more likely to develop a LTNP phenotype (OR = 4.98; p = 0.05) as compared to TP group. The same trend was observed when LTNPs were compared to the RP group (OR = 3.26). Results of haplotype analyses reinforced this association, since the OR values obtained for the haplotype carrying allele T at rs61744944 also reflected an association with LTNP status (OR = 6.05; p = 0.08 and OR = 3.44; p = 0.12 for comparisons to TP and RP, respectively). The rare missense variations Ile436Ser and Thr473Ile were not identified in the patients enrolled in this study. Gene expression analyses showed lower LEDGF/p75 mRNA levels in peripheral blood mononuclear cells obtained from HIV-1 infected individuals. However, these levels were not influenced by any of the SNPs investigated. In spite of the limited number of LTNPs, these data suggest that the PSIP1 gene could be associated with the outcome of HIV-1 infection. Further analyses of this gene may guide the identification of causative variants to help predict disease course

  9. Fluorescence diagnosis of the status of the human lens in vivo

    NASA Astrophysics Data System (ADS)

    Vladimirova, E. S.; Salmin, V. V.; Salmina, A. B.; Oskirko, S. A.; Lazarenko, V. I.; Provorov, A. S.

    2012-03-01

    We have studied fluorescence spectra of the human lens in vivo for healthy eyes and in different stages of senile cataract development. We propose a spectral criterion, the lens opacity index, allowing us to differentiate between stages of cataract development. We show a high correlation between the stage of cataract development and the opacity index. We propose an empirical expression for determining the stage of senile cataract development from the value of the lens opacity index. The technique has been clinically tested.

  10. Effect of putative pheromones on the electrical activity of the human vomeronasal organ and olfactory epithelium.

    PubMed

    Monti-Bloch, L; Grosser, B I

    1991-10-01

    The summated receptor potential was recorded from the vomeronasal organ (VNO) and olfactory epithelium (OE) of 49 human subjects of both sexes (18 to 55 years old) using surface non-polarizable silver-silver chloride electrodes. 15-25 pg of human putative pheromones, clove oil and a diluent were administered to the VNO or the OE in 0.3-1 s pulses from a 0.05 mm dia cannula connected to a multichannel delivery system. Local stimulation of the VNO produces negative potentials of 1.8-11.6 mV showing adaptation. Responses are not obtained when the recording electrode is placed in the nasal respiratory mucosa. Pheromone ER-830 significantly stimulates the male VNO (P less than 0.01; n = 20), while ER-670 produces a significant effect on female subjects (P less than 0.001; n = 20). The other pheromones tested do not show significantly different effects in both male and female (P greater than 0.1). Similar quantities of odorant or diluent produce an insignificant effect on the VNO. Stimulation of the OE with clove oil produces depolarization of 12.3 +/- 3.9 mV, while pheromones do not show a significant effect. Our results show that the VNO is a functional organ in adult humans having receptor sites for human putative pheromones. PMID:1892788

  11. A2E and lipofuscin distributions in macaque retinal pigment epithelium are similar to human.

    PubMed

    Pallitto, Patrick; Ablonczy, Zsolt; Jones, E Ellen; Drake, Richard R; Koutalos, Yiannis; Crouch, Rosalie K; Donello, John; Herrmann, Julia

    2015-10-01

    The accumulation of lipofuscin, an autofluorescent aging marker, in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD). Lipofuscin contains several visual cycle byproducts, most notably the bisretinoid N-retinylidene-N-retinylethanolamine (A2E). Previous studies with human donor eyes have shown a significant mismatch between lipofuscin autofluorescence (AF) and A2E distributions. The goal of the current project was to examine this relationship in a primate model with a retinal anatomy similar to that of humans. Ophthalmologically naive young (<10 years., N = 3) and old (>10 years., N = 4) Macaca fascicularis (macaque) eyes, were enucleated, dissected to yield RPE/choroid tissue, and flat-mounted on indium-tin-oxide-coated conductive slides. To compare the spatial distributions of lipofuscin and A2E, fluorescence and mass spectrometric imaging were carried out sequentially on the same samples. The distribution of lipofuscin fluorescence in the primate RPE reflected previously obtained human results, having the highest intensities in a perifoveal ring. Contrarily, A2E levels were consistently highest in the periphery, confirming a lack of correlation between the distributions of lipofuscin and A2E previously described in human donor eyes. We conclude that the mismatch between lipofuscin AF and A2E distributions is related to anatomical features specific to primates, such as the macula, and that this primate model has the potential to fill an important gap in current AMD research. PMID:26223373

  12. Finite element method-simulation of the human lens during accommodation

    NASA Astrophysics Data System (ADS)

    Breitenfeld, P.; Ripken, T.; Lubatschowski, H.

    2005-08-01

    A finite-element-method model with ANSYS 8.0 of a 29 year old human lens during accommodation will be presented. The required data, to draw and calculate a two dimensional, axis-symmetric model of the human lens is inherited from various sources. Furthermore the analysis premises all lens materials to be linear elastic and allows large displacements. A first analysis of a possible method for the treatment of presbyopia by fs-laser induced microcuts is accomplished. Therefore a mechanical analysis of an untreated and a treated lens are compared. As a result an improvement of the flexibility of the lens tissue is found and as its consequence a change of the lens' radii of curvature is established. After a suitable processing of the output data a linear Gaussian ray trace is performed and a minor change in the optical power between the untreated and treaded human lens is perceived. By calculation of the discrete optical power of the anterior and posterior surface on the one hand and the overall optical power on the other hand an interpretation of the effectiveness resulting from the treatment is offered. It is ascertained that the change in optical power of the anterior lens surface is increased while the optical power of the posterior lens surface is decreased, almost compensating each other. A possible explanation for this phenomenon is given and a suggestion of how to increase the effectiveness of the treatment is discussed.

  13. CONSTITUTIVE AND STIMULATED MCP-1, GROA, B, AND Y EXPRESSION IN HUMAN A AIRWAY EPITHELIUM AND BRONCHOALVEOLAR MACROPHAGES

    EPA Science Inventory

    Constitutive expression of mRNAs for GROa, GROB, GROY, and MCP-1, belonging to the chemokine family of 8-10 kD cytokines with chemotactic properties for granulocytes and monocytes, has been identified in freshly isolated human nasal and bronchial epithelium, and in bronchoalveola...

  14. Activation of the IL-6/JAK/STAT3 signaling pathway in human middle ear cholesteatoma epithelium

    PubMed Central

    Liu, Wei; Xie, Shumin; Chen, Xing; Rao, Xingwang; Ren, Hongmiao; Hu, Bing; Yin, Tuanfang; Xiang, Yuyan; Ren, Jihao

    2014-01-01

    Interleukin-6 (IL-6) is one of the most important cytokines which has been shown to play a critical role in the pathogenesis of cholesteatoma. In this study, we aimed to investigate the expression of interleukin-6 (IL-6) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in middle ear cholesteatoma epithelium in an effort to determine the role of IL-6/JAK/STAT3 signaling pathway in the pathogenesis of cholesteatoma. Immunohistochemistry was used to examine the expression of IL-6 and p-STAT3 in 25 human middle ear cholesteatoma samples and 15 normal external auditory canal (EAC) epithelium specimens. We also analyzed the relation of IL-6 and p-STAT3 expression levels to the degree of bone destruction in cholesteatoma. We found that the expression of IL-6 and p-STAT3 were significantly higher in cholesteatoma epithelium than in normal EAC epithelium (p<0.05). In cholesteatoma epithelium, a significant positive association was observed between IL-6 and p-STAT3 expression (p<0.05). However, no significant relationships were observed between the degree of bone destruction and the levels of IL-6 and p-STAT3 expression (p>0.05). To conclude, our results support the concept that IL-6/JAK/STAT3 signaling pathway is active and may play an important role in the mechanisms of epithelial hyper-proliferation responsible for cholesteatoma. PMID:24551293

  15. [Inhibition of adherence of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases from marine hydrobiontes].

    PubMed

    Zaporozhets, T S; Makarenkova, I D; Bakunina, I Iu; Burtseva, Iu V; Kusaĭkin, M I; Balabanova, L A; Zviagintseva, T N; Besednova, N N; Rasskazov, V A

    2010-01-01

    A possibility of adhesion inhibition of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases of marine hydrobiontes was investigated using alpha-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, total enzyme preparation and beta-1,3-glucanase from marine fungi Chaetomium, total enzyme preparation and beta-1,3-glucanase from marine mollusk Littorina kurila, and total enzyme preparation from crystalline style of marine mollusk Spisula sachalinensis were used. The enzymes were added to test-tubes containing buccal epithelial cells and/or the toxigenic bacterial strain C. diphtheriae No 1129, v. gravis. All the investigated enzymes were able to abort C. diphtheriae adherence, to human buccal epithelocytes. Inhibition of adhesion was more pronounced in the case of treatment of epithelocytes with highly purified enzymes of marine hydrobiontes in comparison with total enzyme preparations. The significant inhibition of C. diphtheriae adhesion was observed when the enzymes were added to the epithelocytes with the attached microorganisms. The results obtained show that glycoside hydrolases of marine hydrobiontes degrade any carbohydrates expressed on cell surface of bacterium or human buccal epithelocytes, impair unique lectin-carbohydrate interaction and prevent the adhesion. PMID:20695214

  16. Enterohemorrhagic Escherichia coli colonization of human colonic epithelium in vitro and ex vivo.

    PubMed

    Lewis, Steven B; Cook, Vivienne; Tighe, Richard; Schüller, Stephanie

    2015-03-01

    Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation. PMID:25534942

  17. Enterohemorrhagic Escherichia coli Colonization of Human Colonic Epithelium In Vitro and Ex Vivo

    PubMed Central

    Lewis, Steven B.; Cook, Vivienne; Tighe, Richard

    2014-01-01

    Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation. PMID:25534942

  18. Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors

    PubMed Central

    Teixeira, Vitor H; Nadarajan, Parthiban; Graham, Trevor A; Pipinikas, Christodoulos P; Brown, James M; Falzon, Mary; Nye, Emma; Poulsom, Richard; Lawrence, David; Wright, Nicholas A; McDonald, Stuart; Giangreco, Adam; Simons, Benjamin D; Janes, Sam M

    2013-01-01

    Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI: http://dx.doi.org/10.7554/eLife.00966.001 PMID:24151545

  19. Proton Irradiation Alters Expression of FGF-2 In Human Lens Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.

    1999-01-01

    We are investigating a role for proton radiation-induced changes in FGF-2 gene expression as part of the mechanism(s) underlying lens cell injury. Radiation injury to the human lens is associated with the induction of cataract following exposure to protons.

  20. Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

    SciTech Connect

    Greiner, J.W.; Malan-Shibley, L.B.; Janss, D.H.

    1980-01-28

    Studies were conducted to determine whether normal and/or neoplastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the in vitro addition of 10 ..mu..M DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 ..mu..M DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 ..mu..M BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, ..cap alpha..-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.

  1. AGEs in human lens capsule promote the TGFβ2-mediated EMT of lens epithelial cells: implications for age-associated fibrosis.

    PubMed

    Raghavan, Cibin T; Smuda, Mareen; Smith, Andrew J O; Howell, Scott; Smith, Dawn G; Singh, Annapurna; Gupta, Pankaj; Glomb, Marcus A; Wormstone, Ian Michael; Nagaraj, Ram H

    2016-06-01

    Proteins in basement membrane (BM) are long-lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial-to-mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age-dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC-MS/MS. The TGFβ2-mediated upregulation of the mRNA levels (by qPCR) of EMT-associated proteins was significantly enhanced in cells cultured on AGE-modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2-mediated α-smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2-mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes-associated fibrosis. PMID:26853893

  2. Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

    PubMed Central

    Beaulieu, Alexandre; Gravel, Émilie; Cloutier, Alexandre; Marois, Isabelle; Colombo, Éloïc; Désilets, Antoine; Verreault, Catherine; Leduc, Richard; Marsault, Éric

    2013-01-01

    Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place. PMID:23365447

  3. Differential expression of TYRP1 in adult human retinal pigment epithelium and uveal melanoma cells

    PubMed Central

    QIU, CHUN; LI, PENG; BI, JIANJUN; WU, QING; LU, LINNA; QIAN, GUANXIANG; JIA, RENBING; JIA, RONG

    2016-01-01

    Uveal melanoma (UM) is the most frequently occurring primary intraocular malignancy in adults. Tyrosinase (TYR) is a copper-containing enzyme and a type I membrane protein that is involved in the generation of melanin, the main pigment in vertebrates. TYR-related protein 1 (TYRP1) is regarded to have a crucial role in the immunotherapy of melanoma. As biomarkers, the TYR-related proteins, TYRP1 and TYRP2, exhibit specific expression in melanocytes, while also contributing to melanin synthesis within melanosomes. In the present study, the differential expression of TYRP1 was investigated at the mRNA, protein and morphological levels in four human UM cell lines (SP6.5, OM431, OCM1 and OCM290) and the human retinal pigment epithelium (RPE) cell line, using polymerase chain reaction, western blotting, immunocytochemistry and immunofluorescence staining. It was found that SP6.5 cells expressed the highest level of TYRP1, in comparison to SP6.5 OCM1 and OM431 cells, which produced less TYRP1, and OCM290 cells, which produced almost no TYRP1. No TYRP1 protein expression was identified in the RPE cell line. These findings indicate the potential use of TYRP1 in the development of therapy for UM. PMID:27073483

  4. A novel Bruch's membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells.

    PubMed

    Xiang, Ping; Wu, Kun-Chao; Zhu, Ying; Xiang, Lue; Li, Chong; Chen, Deng-Long; Chen, Feng; Xu, Guotong; Wang, Aijun; Li, Min; Jin, Zi-Bing

    2014-12-01

    Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3-5 μm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation. PMID:25220295

  5. Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.

    PubMed

    Milenkovic, Andrea; Brandl, Caroline; Milenkovic, Vladimir M; Jendryke, Thomas; Sirianant, Lalida; Wanitchakool, Potchanart; Zimmermann, Stephanie; Reiff, Charlotte M; Horling, Franziska; Schrewe, Heinrich; Schreiber, Rainer; Kunzelmann, Karl; Wetzel, Christian H; Weber, Bernhard H F

    2015-05-19

    In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies. PMID:25941382

  6. Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium

    SciTech Connect

    Koehler, C.; Ginzkey, C.; Friehs, G.; Hackenberg, S.; Froelich, K.; Scherzed, A.; Burghartz, M.; Kessler, M.; Kleinsasser, N.

    2010-06-01

    Cytotoxicity and genotoxicity of nitrogen dioxide (NO{sub 2}) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO{sub 2} in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract. Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO{sub 2}, 0.1 ppm NO{sub 2}, 1 ppm NO{sub 2}, 10 ppm NO{sub 2} and synthetic air for half an hour. After exposure, genotoxicity was evaluated by the alkaline single-cell microgel electophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively. The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO{sub 2} in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO{sub 2} in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.

  7. Ocimum sanctum extracts attenuate hydrogen peroxide induced cytotoxic ultrastructural changes in human lens epithelial cells.

    PubMed

    Halder, Nabanita; Joshi, Sujata; Nag, Tapas Chandra; Tandon, Radhika; Gupta, Suresh Kumar

    2009-12-01

    Hydrogen peroxide (H2O2) is the major oxidant involved in cataract formation. The present study investigated the effect of an aqueous leaf extract of Tulsi (Ocimum sanctum) against H2O2 induced cytotoxic changes in human lens epithelial cells (HLEC). Donor eyes of the age range 20-40 years were procured within 5-8 h of death. After several washings with gentamicin (50 mL/L) and betadine (10 mL/L), clear transparent lenses (n=6 in each group) were incubated in Dulbecco's modified Eagle's medium (DMEM) alone (normal) or in DMEM containing 100 microm of H2O2 (control) or in DMEM containing both H2O2 (100 microm) and 150 microg/mL of Ocimum sanctum extract (treated) for 30 min at 37 degrees C with 5% CO2 and 95% air. Following incubation, the semi-hardened epithelium of each lens was carefully removed, fixed and processed for electron microscopic studies. Thin sections (60-70 mm) were contrasted with uranyl acetate and lead citrate and viewed under a transmission electron microscope. Normal epithelial cells showed intact, euchromatic nucleus with few small vacuoles (diameter 0.58+/-0.6 microm) in well-demarcated cytoplasm. After treatment with H2O2, they showed pyknotic nuclei with clumping of chromatin and ill-defined edges. The cytoplasm was full of vacuoles (diameter 1.61+/-0.7 microm). The overall cellular morphology was typical of dying cells. Treatment of cells with Ocimum sanctum extract protected the epithelial cells from H2O2 insult and maintained their normal architecture. The mean diameter of the vacuoles was 0.66+/-0.2 microm. The results indicate that extracts of O. sanctum have an important protective role against H2O2 injury in HLEC by maintaining the normal cellular architecture. The protection could be due to its ability to reduce H2O2 through its antioxidant property and thus reinforcing the concept that the extracts can penetrate the HLEC membrane. PMID:19441070

  8. ABCD matrix of the human lens gradient-index profile: applicability of the calculation methods.

    PubMed

    Díaz, José Antonio

    2008-01-10

    The applicability of different approximate methods proposed to determine the paraxial properties of the gradient-index (GRIN) distribution resembling that of the human lens, by means of the system ABCD matrix, is tested. Thus, the parabolic-ray-path approximation has been extended to provide the ABCD matrix of a slab lens comprised of a rotationally GRIN medium. The results show that this method has good numerical stability, and it is also the easiest one in determining the Gaussian constants of the human lens GRIN profile. PMID:18188201

  9. RNA-Seq quantification of the human small airway epithelium transcriptome

    PubMed Central

    2012-01-01

    Background The small airway epithelium (SAE), the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq). Results The data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype. Conclusions These observations provide insights into the unique biology of human SAE by providing quantit-ative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking. PMID:22375630

  10. POU2AF1 Functions in the Human Airway Epithelium To Regulate Expression of Host Defense Genes.

    PubMed

    Zhou, Haixia; Brekman, Angelika; Zuo, Wu-Lin; Ou, Xuemei; Shaykhiev, Renat; Agosto-Perez, Francisco J; Wang, Rui; Walters, Matthew S; Salit, Jacqueline; Strulovici-Barel, Yael; Staudt, Michelle R; Kaner, Robert J; Mezey, Jason G; Crystal, Ronald G; Wang, Guoqing

    2016-04-01

    In the process of seeking novel lung host defense regulators by analyzing genome-wide RNA sequence data from normal human airway epithelium, we detected expression of POU domain class 2-associating factor 1 (POU2AF1), a known transcription cofactor previously thought to be expressed only in lymphocytes. Lymphocyte contamination of human airway epithelial samples obtained by bronchoscopy and brushing was excluded by immunohistochemistry staining, the observation of upregulation of POU2AF1 in purified airway basal stem/progenitor cells undergoing differentiation, and analysis of differentiating single basal cell clones. Lentivirus-mediated upregulation of POU2AF1 in airway basal cells induced upregulation of host defense genes, including MX1, IFIT3, IFITM, and known POU2AF1 downstream genes HLA-DRA, ID2, ID3, IL6, and BCL6. Interestingly, expression of these genes paralleled changes of POU2AF1 expression during airway epithelium differentiation in vitro, suggesting POU2AF1 helps to maintain a host defense tone even in pathogen-free condition. Cigarette smoke, a known risk factor for airway infection, suppressed POU2AF1 expression both in vivo in humans and in vitro in human airway epithelial cultures, accompanied by deregulation of POU2AF1 downstream genes. Finally, enhancing POU2AF1 expression in human airway epithelium attenuated the suppression of host defense genes by smoking. Together, these findings suggest a novel function of POU2AF1 as a potential regulator of host defense genes in the human airway epithelium. PMID:26927796

  11. Critical determinants of uptake and translocation of nanoparticles by the human pulmonary alveolar epithelium.

    PubMed

    Thorley, Andrew J; Ruenraroengsak, Pakatip; Potter, Thomas E; Tetley, Teresa D

    2014-11-25

    The ability to manipulate the size and surface properties of nanomaterials makes them a promising vector for improving drug delivery and efficacy. Inhalation is a desirable route of administration as nanomaterials preferentially deposit in the alveolar region, a large surface area for drug absorption. However, as yet, the mechanisms by which particles translocate across the alveolar epithelial layer are poorly understood. Here we show that human alveolar type I epithelial cells internalize nanoparticles, whereas alveolar type II epithelial cells do not, and that nanoparticles translocate across the epithelial monolayer but are unable to penetrate the tight junctions between cells, ruling out paracellular translocation. Furthermore, using siRNA, we demonstrate that 50 nm nanoparticles enter largely by passive diffusion and are found in the cytoplasm, whereas 100 nm nanoparticles enter primarily via clathrin- and also caveolin-mediated endocytosis and are found in endosomes. Functionalization of nanoparticles increases their uptake and enhances binding of surfactant which further promotes uptake. Thus, we demonstrate that uptake and translocation across the pulmonary epithelium is controlled by alveolar type I epithelial cells, and furthermore, we highlight a number of factors that should be considered when designing new nanomedicines in order to improve drug delivery to the lung. PMID:25360809

  12. In Vitro Spatial and Temporal Analysis of Mycoplasma pneumoniae Colonization of Human Airway Epithelium

    PubMed Central

    Prince, Oliver A.; Krunkosky, Thomas M.

    2014-01-01

    Mycoplasma pneumoniae is an important cause of respiratory disease, especially in school-age children and young adults. We employed normal human bronchial epithelial (NHBE) cells in air-liquid interface culture to study the interaction of M. pneumoniae with differentiated airway epithelium. These airway cells, when grown in air-liquid interface culture, polarize, form tight junctions, produce mucus, and develop ciliary function. We examined both qualitatively and quantitatively the role of mycoplasma gliding motility in the colonization pattern of developing airway cells, comparing wild-type M. pneumoniae and mutants thereof with moderate to severe defects in gliding motility. Adherence assays with radiolabeled mycoplasmas demonstrated a dramatic reduction in binding for all strains with airway cell polarization, independent of acquisition of mucociliary function. Adherence levels dropped further once NHBE cells achieved terminal differentiation, with mucociliary activity strongly selecting for full gliding competence. Analysis over time by confocal microscopy demonstrated a distinct colonization pattern that appeared to originate primarily with ciliated cells, but lateral spread from the base of the cilia was slower than expected. The data support a model in which the mucociliary apparatus impairs colonization yet cilia provide a conduit for mycoplasma access to the host cell surface and suggest acquisition of a barrier function, perhaps associated with tethered mucin levels, with NHBE cell polarization. PMID:24478073

  13. Frequent genomic alterations in epithelium measured by microsatellite instability following allogeneic hematopoietic cell transplantation in humans.

    PubMed

    Faber, Philipp; Fisch, Paul; Waterhouse, Miguel; Schmitt-Gräff, Annette; Bertz, Hartmut; Finke, Jürgen; Spyridonidis, Alexandros

    2006-04-15

    Although typically found in cancers, frameshift mutations in microsatellites have also been detected in chronically inflamed tissues. Allogeneic hematopoietic cell transplantation (HCT) may potentially produce chronic tissue stress through graft-versus-host reactions. We examined non-neoplastic epithelial tissues (colon, buccal) obtained 1 to 5061 days after human allogeneic HCT for the presence of genomic alterations at 3 tetranucleotide and 3 mononucleotide microsatellite loci. Novel bands indicative of microsatellite instability (MSI) at tetranucleotide repeats were detected in laser-microdissected colonic crypts and in buccal smears of 75% and 42% of patients who received an allograft, respectively. In contrast, no MSI was found in similar tissues from control subjects and from patients after intensive chemotherapy or in buccal cells from patients after autologous HCT. The MSI found in colon, which was often affected by graft-versus-host disease, was not due to loss of expression or nitrosylation of DNA repair proteins. MSI in clinically intact oral mucosa was more frequently found at later time points after HCT. MSI was also found in 3 posttransplant squamous cell cancers examined. Our data show that genomic alterations in epithelium regularly occur after allogeneic HCT and may be implicated in the evolution of posttransplantation diseases, including secondary cancer. PMID:16368884

  14. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells.

    PubMed

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-01-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases. PMID:27246808

  15. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    PubMed Central

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-01-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases. PMID:27246808

  16. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    NASA Astrophysics Data System (ADS)

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-06-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

  17. Critical Determinants of Uptake and Translocation of Nanoparticles by the Human Pulmonary Alveolar Epithelium

    PubMed Central

    2015-01-01

    The ability to manipulate the size and surface properties of nanomaterials makes them a promising vector for improving drug delivery and efficacy. Inhalation is a desirable route of administration as nanomaterials preferentially deposit in the alveolar region, a large surface area for drug absorption. However, as yet, the mechanisms by which particles translocate across the alveolar epithelial layer are poorly understood. Here we show that human alveolar type I epithelial cells internalize nanoparticles, whereas alveolar type II epithelial cells do not, and that nanoparticles translocate across the epithelial monolayer but are unable to penetrate the tight junctions between cells, ruling out paracellular translocation. Furthermore, using siRNA, we demonstrate that 50 nm nanoparticles enter largely by passive diffusion and are found in the cytoplasm, whereas 100 nm nanoparticles enter primarily via clathrin- and also caveolin-mediated endocytosis and are found in endosomes. Functionalization of nanoparticles increases their uptake and enhances binding of surfactant which further promotes uptake. Thus, we demonstrate that uptake and translocation across the pulmonary epithelium is controlled by alveolar type I epithelial cells, and furthermore, we highlight a number of factors that should be considered when designing new nanomedicines in order to improve drug delivery to the lung. PMID:25360809

  18. Basement membrane proteins promote progression of intraepithelial neoplasia in 3-dimensional models of human stratified epithelium.

    PubMed

    Andriani, Frank; Garfield, Jackie; Fusenig, Norbert E; Garlick, Jonathan A

    2004-01-20

    We have developed novel 3-dimensional in vitro and in vivo tissue models that mimic premalignant disease of human stratified epithelium in order to analyze the stromal contribution of extracellular matrix and basement membrane proteins to the progression of intraepithelial neoplasia. Three-dimensional, organotypic cultures were grown either on a de-epidermalized human dermis with pre-existing basement membrane components on its surface (AlloDerm), on a Type I collagen gel that lacked basement membrane proteins or on polycarbonate membranes coated with purified extracellular matrix proteins. When tumor cells (HaCaT-II4) were mixed with normal keratinocytes (4:1/normals:HaCaT-II4), tumor cells selectively attached, persisted and proliferated at the dermal-epidermal interface in vitro and generated dysplastic tissues when transplanted to nude mice only when grown in the presence of the AlloDerm substrate. This stromal interface was permissive for tumor cell attachment due to the rapid assembly of structured basement membrane. When tumor cells were mixed with normal keratinocytes and grown on polycarbonate membranes coated with individual extracellular matrix or basement membrane components, selective attachment and significant intraepithelial expansion occurred only on laminin 1 and Type IV collagen-coated membranes. This preferential adhesion of tumor cells restricted the synthesis of laminin 5 to basal cells where it was deposited in a polarized distribution. Western blot analysis revealed that tumor cell attachment was not due to differences in the synthesis or processing of laminin 5. Thus, intraepithelial progression towards premalignant disease is dependent on the selective adhesion of cells with malignant potential to basement membrane proteins that provide a permissive template for their persistence and expansion. PMID:14648700

  19. Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

    PubMed

    Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

    2015-07-15

    Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development. PMID:25758640

  20. Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

    PubMed Central

    Milenkovic, Andrea; Brandl, Caroline; Milenkovic, Vladimir M.; Jendryke, Thomas; Sirianant, Lalida; Wanitchakool, Potchanart; Zimmermann, Stephanie; Reiff, Charlotte M.; Horling, Franziska; Schrewe, Heinrich; Schreiber, Rainer; Kunzelmann, Karl; Wetzel, Christian H.; Weber, Bernhard H. F.

    2015-01-01

    In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies. PMID:25941382

  1. Content of trans fatty acids in human cheek epithelium: comparison with serum and adipose tissue.

    PubMed

    Abraham, Ransi A; Bahl, Vinay K; Parshad, Rajinder; Seenu, V; Roy, Ambuj; Golandaz, Smita; Dorairaj, Prabhakaran; Ramakrishnan, Lakshmy

    2013-01-01

    Studies pertaining to trans fatty acids (TFA), which have been implicated in development of chronic diseases, are more relevant in developing countries where nutrition transition is changing traditional habits and practices. Measuring TFA is an arduous task because of the need for fat biopsies. This study identifies a tissue, which can be easily accessed for analytical measurement of trans fatty acid. In this cross-sectional study, fatty acid in adipose tissue, cheek epithelium, and blood samples were assessed by gas chromatography. Spearman correlation coefficient was computed to study the correlation of fatty acid distribution among the three tissues. The correlation coefficient of total trans fatty acid between cheek epithelium and serum was 0.30 (P < 0.02) and between cheek epithelium and adipose tissue was 0.33 (P < 0.019). This study is the first to report trans fatty acid profile in cheek epithelium giving scope for utilizing the cheek epithelium as a tissue for objective assessment of trans fatty acid intake. PMID:24222900

  2. Human papillomavirus 16-associated cervical intraepithelial neoplasia in humans excludes CD8 T cells from dysplastic epithelium.

    PubMed

    Trimble, Cornelia L; Clark, Rachael A; Thoburn, Christopher; Hanson, Nicole C; Tassello, Jodie; Frosina, Denise; Kos, Ferdynand; Teague, Jessica; Jiang, Ying; Barat, Nicole C; Jungbluth, Achim A

    2010-12-01

    High-grade cervical dysplasia caused by human papillomavirus (HPV) type 16 is a lesion that should be susceptible to an HPV-specific immune response; disease initiation and persistence is predicated on expression of two viral Ags, E6 and E7. In immune-competent subjects, at least 25% of HPV16(+) high-grade cervical dysplasia lesions undergo complete regression. However, in the peripheral blood, naturally occurring IFN-γ T cell responses to HPV E6 and E7 are weak, requiring ex vivo sensitization to detect, and are not sufficiently sensitive to predict regression. In this study, we present immunologic data directly assessing cervical lymphocytes from this cohort. We found that nearly all cervical tissue T cells express the mucosal homing receptor, α(4)β(7) surface integrin. T cells isolated from dysplastic mucosa were skewed toward a central memory phenotype compared with normal mucosal resident T cells, and dysplastic lesions expressed transcripts for CCL19 and CCL21, raising the possibility that the tissue itself sustains a response that is not detectable in the blood. Moreover, lesion regression in the study window could retrospectively be predicted at study entry by the ability of CD8(+) T cells to gain access to lesional epithelium. Vascular endothelial expression of mucosal addressin cell adhesion molecule-1, the ligand that supports entry of α(4)β(7)(+) T cells into tissues, colocalized tightly with the distribution of CD8 T cells and was not expressed in persistent dysplastic epithelium. These findings suggest that dysregulated expression of vascular adhesion molecules plays a role in immune evasion very early in the course of HPV disease. PMID:21037100

  3. The Significance of the Discordant Occurrence of Lens Tumors in Humans versus Other Species

    PubMed Central

    Albert, Daniel M.; Phelps, Paul O.; Surapaneni, Krishna R.; Thuro, Bradley A.; Potter, Heather D.; Ikeda, Akihiro; Teixeira, Leandro B. C.; Dubielzig, Richard R.

    2015-01-01

    Purpose The purpose of this study was to determine in which species and under what conditions lens tumors occur. Design A review of data bases of available human and veterinary ocular pathological material and the previously reported literature. Participants Approximately 18,000 patients who had ocular surgical specimens submitted and studied at the University of Wisconsin School of Medicine and Public Health (UWSMPH) between 1920 and 2014 and 45,000 ocular veterinary cases from the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) between 1983 and 2014. Methods Material in two major archived collections at the University of Wisconsin medical and veterinary schools were studied for occurrence of lens tumors. Tumor was defined as “a new growth of tissue characterized by progressive, uncontrolled proliferation of cells.” In addition, cases presented at 3 major eye pathology societies (Verhoeff-Zimmerman Ophthalmic Pathology Society, Eastern Ophthalmic Pathology Society, and The Armed Forces Institute of Pathology Ophthalmic Alumni Society) from 1975 through 2014 were reviewed. Finally, a careful search of the literature was carried out. Approval from the IRB to carry out this study was obtained. Main Outcome Measures The presence of tumors of the lens. Results The database search and literature review failed to find an example of a lens tumor in humans. In contrast, examples of naturally occurring lens tumors were found in cats, dogs, rabbits, and birds. 4.5% of feline intraocular and adnexal neoplasms (234/5153) in the veterinary school database were designated as feline ocular post-traumatic sarcoma (FOPTS), a tumor previously demonstrated to be of lens epithelial origin. Similar tumors were seen in rabbit eyes, a bird, and in a dog. All four species with lens tumors had a history of either ocular trauma or protracted uveitis. The literature search also revealed cases where lens tumors were induced in zebrafish, rainbow trout, hamsters, and mice, by

  4. Coherent fiber optic sensor for early detection of cataractogenesis in a human eye lens

    NASA Technical Reports Server (NTRS)

    Dhadwal, Harbans S.; Ansari, Rafat R.; Dellavecchia, Michael A.

    1993-01-01

    A lensless backscatter fiber optic probe is used to measure the size distribution of protein molecules inside an excised, but intact, human eye lens. The fiber optic probe, about 5 mm in diameter, can be positioned arbitrarily close to the anterior surface of the eye; it is a trans-receiver, which delivers a Gaussian laser beam into a small region inside the lens and provides a coherent detection of the laser light scattered by the protein molecules in the backward direction. Protein sizes determined from the fast and slow diffusion coefficients show good correlation with the age of the lens and cataractogenesis.

  5. Imaging human retinal pigment epithelium cells using adaptive optics optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Liu, Zhuolin; Kocaoglu, Omer P.; Turner, Timothy L.; Miller, Donald T.

    2016-03-01

    Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, but are often compromised in ageing and major ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, and while biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. We present a novel method based on optical coherence tomography (OCT) equipped with adaptive optics (AO) that overcomes the associated technical obstacles. The method takes advantage of the 3D resolution of AO-OCT, but more critically sub-cellular segmentation and registration that permit organelle motility to be used as a novel contrast mechanism. With this method, we successfully visualized RPE cells and characterized their 3D reflectance profile in every subject and retinal location (3° and 7° temporal to the fovea) imaged to date. We have quantified RPE packing geometry in terms of cell density, cone-to-RPE ratio, and number of nearest neighbors using Voronoi and power spectra analyses. RPE cell density (cells/mm2) showed no significant difference between 3° (4,892+/-691) and 7° (4,780+/-354). In contrast, cone-to- RPE ratio was significantly higher at 3° (3.88+/-0.52:1) than 7° (2.31+/- 0.23:1). Voronoi analysis also showed most RPE cells have six nearest neighbors, which was significantly larger than the next two most prevalent associations: five and seven. Averaged across the five subjects, prevalence of cells with six neighbors was 51.4+/-3.58% at 3°, and 54.58+/-3.01% at 7°. These results are consistent with histology and in vivo studies using other imaging modalities.

  6. Prevalence of human papillomavirus in the cervical epithelium of Mexican women: meta-analysis

    PubMed Central

    2012-01-01

    Background Human Papillomavirus (HPV) in cervical epithelium has been identified as the main etiological factor in the developing of Cervical Cancer (CC), which has recently become a public health problem in Mexico. This finding has allowed for the development of vaccines that help prevent this infection. In the present study, we aimed to determine the prevalence and HPV type-distribution in Mexican women with CC, high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion (LSIL), and Normal cytology (N) to estimate the impact of the HPV vaccines. Methods The PubMed database was used to identify and review all articles that reported data on HPV prevalence in CC, precursor lesions, and normal cytology of Mexican women. Results A total of 8,706 samples of the tissues of Mexican women were stratified according to diagnosis as follows: 499 for CC; 364 for HSIL; 1,425 for LSIL, and 6,418 for N. According to the results, the most prevalent genotypes are the following: HPV16 (63.1%), -18 (8.6%), -58, and −31 (5%) for CC; HPV-16 (28.3%), 58 (12.6%), 18 (7.4%), and 33 (6.5%) for HSIL; HPV-16 (13.1%), 33 (7.4%), 18 (4.2%), and 58 (2.6%) for LSIL, and HPV-16 (3.4%), 33 (2.1%), 18, and 58 (1.2%) for N. Conclusions Taken together, genotypes 58 and 31 (10%) are more common than type 18 (8.6%) in CC. Therefore, the inclusion of these two genotypes in a second-generation vaccine would provide optimal prevention of CC in Mexico. PMID:23199368

  7. Quantitative Autofluorescence and Cell Density Maps of the Human Retinal Pigment Epithelium

    PubMed Central

    Ach, Thomas; Huisingh, Carrie; McGwin, Gerald; Messinger, Jeffrey D.; Zhang, Tianjiao; Bentley, Mark J.; Gutierrez, Danielle B.; Ablonczy, Zsolt; Smith, R. Theodore; Sloan, Kenneth R.; Curcio, Christine A.

    2014-01-01

    Purpose. Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. Methods. Retinal pigment epithelium–Bruch's membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. Results. Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. Conclusions. Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LF's role in AMD. PMID:25034602

  8. Changes in spectral properties and composition of lipofuscin fluorophores from human-retinal-pigment epithelium with age and pathology.

    PubMed

    Feldman, Tatiana B; Yakovleva, Marina A; Arbukhanova, Patimat M; Borzenok, Sergey A; Kononikhin, Alexey S; Popov, Igor A; Nikolaev, Evgeny N; Ostrovsky, Mikhail A

    2015-02-01

    Fundus autofluorescence mostly originates from bisretinoid fluorophores in lipofuscin granules, which accumulate in retinal-pigment-epithelium cells with age. The dynamics of accumulation, photo-oxidation, and photodegradation of bisretinoids during aging or in the presence of pathology have been insufficiently investigated. Changes in spectral properties and composition of human lipofuscin-granule fluorophores with age and pathology have now been investigated by a high-performance liquid chromatography method using spectrophotometric and fluorescent detectors connected in series. It was found that: (i) N-retinylidene-N-retinylethanolamine (A2E) fluorescence intensity is not predominant in the chloroform extract of human-cadaver-eye retinal pigment epithelium studied; bisretinoid photo-oxidation and photodegradation products have much higher fluorescent properties; (ii) the relative emission maximum in the fluorescence spectrum of suspended retinal-pigment-epithelium cells obtained from an individual human-cadaver eye without pathology is irrespective of donor age and falls within the range 575 ± 15 nm; in two cadaver eyes with signs of age-related macular degeneration, emission maxima were shifted by 23-36 nm towards the shortwave region; and (iii) the ratio of bisretinoid photo-oxidation and photodegradation products to unoxidized bisretinoids in the chloroform extract of cadaver-eye retinal pigment epithelium increases with donor age, from 0.69 ± 0.03 to 1.32 ± 0.04. The differences in fluorescence properties between chloroform extracts obtained from cadaver eyes with and without signs of age-related macular degeneration could be used to increase the potential of fundus autofluorescence imaging as a noninvasive diagnostic method. PMID:25471291

  9. Dose conversion coefficients for neutron exposure to the lens of the human eye

    SciTech Connect

    Manger, Ryan P; Bellamy, Michael B; Eckerman, Keith F

    2011-01-01

    Dose conversion coefficients for the lens of the human eye have been calculated for neutron exposure at energies from 1 x 10{sup -9} to 20 MeV and several standard orientations: anterior-to-posterior, rotational and right lateral. MCNPX version 2.6.0, a Monte Carlo-based particle transport package, was used to determine the energy deposited in the lens of the eye. The human eyeball model was updated by partitioning the lens into sensitive and insensitive volumes as the anterior portion (sensitive volume) of the lens being more radiosensitive and prone to cataract formation. The updated eye model was used with the adult UF-ORNL mathematical phantom in the MCNPX transport calculations.

  10. Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium

    PubMed Central

    Kinnick, Tyson R.; Stanton, J. Brett; Johnson, Adiv A.; Lynch, Ronald M.; Marmorstein, Lihua Y.

    2015-01-01

    Purpose Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. Methods Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. Results Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control

  11. Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers

    PubMed Central

    Poussin, Carine; Weisensee, Dirk; Gebel, Stephan; Hengstermann, Arnd; Sewer, Alain; Belcastro, Vincenzo; Xiang, Yang; Ansari, Sam; Wagner, Sandra; Hoeng, Julia; Peitsch, Manuel C.

    2013-01-01

    Organotypic culture of human primary bronchial epithelial cells is a useful in vitro system to study normal biological processes and lung disease mechanisms, to develop new therapies, and to assess the biological perturbations induced by environmental pollutants. Herein, we investigate whether the perturbations induced by cigarette smoke (CS) and observed in the epithelium of smokers' airways are reproducible in this in vitro system (AIR-100 tissue), which has been shown to recapitulate most of the characteristics of the human bronchial epithelium. Human AIR-100 tissues were exposed to mainstream CS for 7, 14, 21, or 28 min at the air-liquid interface, and we investigated various biological endpoints [e.g., gene expression and microRNA profiles, matrix metalloproteinase 1 (MMP-1) release] at multiple postexposure time points (0.5, 2, 4, 24, 48 h). By performing a Gene Set Enrichment Analysis, we observed a significant enrichment of human smokers' bronchial epithelium gene signatures derived from different public transcriptomics datasets in CS-exposed AIR-100 tissue. Comparison of in vitro microRNA profiles with microRNA data from healthy smokers highlighted various highly translatable microRNAs associated with inflammation or with cell cycle processes that are known to be perturbed by CS in lung tissue. We also found a dose-dependent increase of MMP-1 release by AIR-100 tissue 48 h after CS exposure in agreement with the known effect of CS on this collagenase expression in smokers' tissues. In conclusion, a similar biological perturbation than the one observed in vivo in smokers' airway epithelium could be induced after a single CS exposure of a human organotypic bronchial epithelium-like tissue culture. PMID:23355383

  12. Effect of holmium and erbium laser action on the human lens: an in-vitro study

    NASA Astrophysics Data System (ADS)

    Kasprzak, Jan; Kecik, Dariusz

    1997-10-01

    We investigated the holmium and erbium lasers operating at the medium IR range, used for cataract surgery. The main advantage of these lasers action on biological structures is total absorption of radiation by superficial layers. During the study of the lens emulsification process we found that the mechanical properties of the lens nucleus were of crucial importance for the rate of emulsification. The soft lenses were fragmented and emulsified after 200-700 pulses, while the hard lens required 5000 or more pulses while complete emulsification was not achieved. The results are promising and show that the holmium and erbium lasers can be used for human lens emulsification during ECCE. For clinical purposes, however, it is necessary to construct a suitable fiberoptic tip to be used in cataract removal. It seems that lasers whose beam is in the medium IR range could be used in many ophthalmic operations.

  13. LENS* 4: a program for calculation of geometric optics of the human eye

    SciTech Connect

    Doss, J.D.

    1982-10-01

    A computer program (LENS* 4) has been written that may be used for ray-trace calculations of human or animal eyes. The user enters all relevant eye specifications, and the program calculates ray segments from a (specified) external object. From the ray calculations, the program yields data on image location, image distance from the retina, retinal spot size, and effective pupil diameter. The program also allows entry of simple corrective lens systems that simulate a contact lens or ordinary spectacles. Graphics output illustrating the eye cross section, corrective lens, and rays is provided either on the CRT, thermal printer, or graphics plotter. Although the program is written in BASIC, many of the statements (particularly in the graphics section) are peculiar to the Hewlett-Packard 9845B desktop computer.

  14. Permeability of human HT-29/B6 colonic epithelium as a function of apoptosis

    PubMed Central

    Bojarski, C; Gitter, A H; Bendfeldt, K; Mankertz, J; Schmitz, H; Wagner, S; Fromm, M; Schulzke, J D

    2001-01-01

    The barrier function of colonic epithelia is challenged by apoptotic loss of enterocytes. In monolayers of human colonic HT-29/B6 cells, apoptosis induced by camptothecin was assessed by poly-(ADP-ribose)-polymerase (PARP) cleavage, histone ELISA and DNA-specific fluorochrome staining (with 4′,6′-diamidino-2′-phenylindoladihydrochloride (DAPI)). Epithelial barrier function was studied in Ussing chambers by measuring transepithelial conductivity and unidirectional tracer fluxes. The ion permeability associated with single cell apoptoses was investigated with the conductance scanning technique. The spontaneous rate of apoptotic cells was 3.5 ± 0.3 % with an overall epithelial conductivity of 3.2 ± 0.1 mS cm−2. Camptothecin induced a time- and dose-dependent increase of apoptosis and permeability. With 20 μg ml−1 of camptothecin for 48 h, apoptosis increased 4.1-fold to 14.3 ± 1.5 % and the conductivity doubled to 6.4 ± 1.0 mS cm−2. While 3H-mannitol flux increased 3.8-fold and 3H-lactulose flux increased 2.6-fold, the flux of 3H-polyethylene glycol 4000 remained unchanged. Hence, the higher permeability was limited to molecules < 4000 Da. The local epithelial conductivity was higher at the sites of apoptosis than in non-apoptotic areas. With camptothecin the leaks associated with apoptosis became more numerous and more conductive, while in non-apoptotic areas the conductivity remained at control level. Hence, the camptothecin-induced increase in epithelial conductivity reflected the opening of apoptotic leaks and thus the results described, for the first time, epithelial permeability as a function of apoptosis only. The conductivity of apoptotic leaks contributed 5.5 % to the epithelial conductivity of controls and 60 % to the conductivity of monolayers treated with 20 μg ml−1 of camptothecin. Thus apoptosis increased the contribution of paracellular pathways to the overall epithelial permeability. Under control conditions the paracellular

  15. Interferon-gamma increased epithelial barrier function via upregulating claudin-7 expression in human submandibular gland duct epithelium.

    PubMed

    Abe, Ayumi; Takano, Kenichi; Kojima, Takashi; Nomura, Kazuaki; Kakuki, Takuya; Kaneko, Yakuto; Yamamoto, Motohisa; Takahashi, Hiroki; Himi, Tetsuo

    2016-06-01

    Tight junctions (TJs) are necessary for salivary gland function and may serve as indicators of salivary gland epithelial dysfunction. IgG4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition which disrupts the TJ associated epithelial barrier. The salivary glands are one of the most frequently involved organs in IgG4-RD, however, changes of the TJ associated epithelial barrier in salivary gland duct epithelium is poorly understood. Here, we investigated the regulation and function of TJs in human submandibular gland ductal epithelial cells (HSDECs) in normal and IgG4-RD. We examined submandibular gland (SMG) tissue from eight control individuals and 22 patients with IgG4-RD and established an HSDEC culture system. Immunohistochemistry, immunocytochemistry, western blotting, and measurement of transepithelial electrical resistance (TER) were performed. Claudin-4, claudin-7, occludin, and JAM-A were expressed at the apical side of the duct epithelium in submandibular gland (SMG) tissue and at the cell borders in HSDECs of normal and IgG4-RD. The expression and distribution of TJs in SMG tissue were not different in control individuals and patients with IgG4-RD in vivo and in vitro. Although interferon-gamma (IFNγ) generally disrupts the integrity and function of TJs, as manifested by decreased epithelial barrier function, IFNγ markedly increased the epithelial barrier function of HSDECs via upregulation of claudin-7 expression in HSDECs from patients with IgG4-RD. This is the first report showing an IFNγ-dependent increase in epithelial barrier function in the salivary gland duct epithelium. Our results provide insights into the functional significance of TJs in salivary gland duct epithelium in physiological and pathological conditions, including IgG4-RD. PMID:26956365

  16. PARP-1 inhibition influences the oxidative stress response of the human lens.

    PubMed

    Smith, Andrew J O; Ball, Simon S R; Bowater, Richard P; Wormstone, I Michael

    2016-08-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects. PMID:26990173

  17. PARP-1 inhibition influences the oxidative stress response of the human lens

    PubMed Central

    Smith, Andrew J.O.; Ball, Simon S.R.; Bowater, Richard P.; Wormstone, I. Michael

    2016-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects. PMID:26990173

  18. Vulnerability of the human airway epithelium to hyperoxia. Constitutive expression of the catalase gene in human bronchial epithelial cells despite oxidant stress.

    PubMed

    Yoo, J H; Erzurum, S C; Hay, J G; Lemarchand, P; Crystal, R G

    1994-01-01

    Although catalase is a major intracellular antioxidant, the expression of the human catalase gene appears to be limited in the airway epithelium, making these cells vulnerable to oxidant stress. The basis for this limited gene expression was examined by evaluation of the expression of the endogenous gene in human bronchial epithelial cells in response to hyperoxia. Hyperoxia failed to upregulate endogenous catalase gene expression, in contrast to a marked increase in expression of the heat shock protein gene. Sequence analysis of 1.7 kb of the 5'-flanking region of the human catalase gene showed features of a "house-keeping" gene (no TATA box, high GC content, multiple CCAAT boxes, and transcription start sites). Transfection of human bronchial epithelial cells with fusion genes composed of various lengths of the catalase 5'-flanking region and luciferase as a reporter gene showed low level constitutive promoter activity that did not change after exposure to hyperoxia. Importantly, using a replication-deficient recombinant adenoviral vector containing the human catalase cDNA, levels of catalase were significantly increased in human airway epithelial cells and this was associated with increased survival of the cells when exposed to hyperoxia. These observations provide a basis for understanding the sensitivity of the human airway epithelium to oxidant stress and a strategy for protecting the epithelium from such injury. PMID:8282800

  19. A normal and biotransforming model of the human bronchial epithelium for the toxicity testing of aerosols and solubilised substances.

    PubMed

    Prytherch, Zoë C; BéruBé, Kelly A

    2014-12-01

    In this article, we provide an overview of the experimental workflow by the Lung and Particle Research Group at Cardiff University, that led to the development of the two in vitro lung models - the normal human bronchial epithelium (NHBE) model and the lung-liver model, Metabo-Lung™. This work was jointly awarded the 2013 Lush Science Prize. The NHBE model is a three-dimensional, in vitro, human tissue-based model of the normal human bronchial epithelium, and Metabo-Lung involves the co-culture of the NHBE model with primary human hepatocytes, thus permitting the biotransformation of inhaled toxicants in an in vivo-like manner. Both models can be used as alternative test systems that could replace the use of animals in research and development for safety and toxicity testing in a variety of industries (e.g. the pharmaceutical, environmental, cosmetics, and food industries). Metabo-Lung itself is a unique tool for the in vitro detection of toxins produced by reactive metabolites. This 21st century animal replacement model could yield representative in vitro predictions for in vivo toxicity. This advancement in in vitro toxicology relies on filter-well technology that will enable a wide-spectrum of researchers to create viable and economic alternatives for respiratory safety assessment and disease-focused research. PMID:25635646

  20. K2P—A Novel Cross-Link from Human Lens Protein

    PubMed Central

    CHENG, RONGZHU; FENG, QI; ARGIROV, OGNYAN K.; ORTWERTH, BERYL J.

    2006-01-01

    We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by LC-MS and NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure as being 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentyl-amino)-3-hydroxy-2, 3-dihydropyridinium, a cross-link between the _-amino groups of two lysine residues and a five-carbon atom ring. We assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests revealed a significant enhancement of K2P in the early stage of brunescent cataract lens proteins (type I/II, 613 ± 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 ± 51 pmol/mg of water-soluble [WS] protein) when compared with aged normal human lens proteins (261 ± 93 pmol/mg of WISS protein or 23 ± 15 pmol/mg of WS protein). Furthermore, a gradual decrease of K2P in the late stages of brunescent cataract lenses with the development of the browning color in the lens argues different coloration mechanisms during the processes of normal aging and cataract development. This new cross-link may serve as a quantitatively significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract. PMID:16037238

  1. Light scattering of normal human lens I. Application of random density and orientation fluctuation theory.

    PubMed Central

    Bettelheim, F A; Paunovic, M

    1979-01-01

    Light-scattering intensities in the I parallel and I+ mode were obtained on thin sections of three human lenses. Random density and orientation fluctuation theory, without cross correlation, was employed to evaluate light-scattering parameters. Both the density correlation distances, as well as the orientation correlation distances, were related to structural elements in the lens fiber cell that have been observed by other investigators with different techniques. The magnitude of these fluctuations were evaluated, and it was demonstrated that the density fluctuations are the main contributors to light scattering in normal human lenses. Changes in the light-scattering parameters were evaluated as a function of position within the lens. The changes observed agree with the biochemical data in the literature that reflects that an aging process occurs when one proceeds from the periphery of the lens toward the center. PMID:262413

  2. UBIQUITIN-CONJUGATING ENZYME 3 DELAYS HUMAN LENS EPITHELIAL CELLS IN METAPHASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ubc3/Cdc34 is a ubiquitin-conjugating enzyme (Ubc) with well established functions in the G1-to-S-phase transition. Expecting to find similar effects in human lens epithelial cells (HLECs), the authors explored roles for this ubiquitin-conjugating enzyme in regulation of the HLEC cycle. Catalytical...

  3. A Simple Model of the Accommodating Lens of the Human Eye

    ERIC Educational Resources Information Center

    Oommen, Vinay; Kanthakumar, Praghalathan

    2014-01-01

    The human eye is often discussed as optically equivalent to a photographic camera. The iris is compared with the shutter, the pupil to the aperture, and the retina to the film, and both have lens systems to focus rays of light. Although many similarities exist, a major difference between the two systems is the mechanism involved in focusing an…

  4. Particle radiation alters expression of matrix metalloproteases resulting in ECM remodeling in human lens cells.

    PubMed

    Chang, P Y; Bjornstad, K A; Rosen, C J; Lin, S; Blakely, E A

    2007-06-01

    Relatively low doses of space radiation have been correlated with an increased incidence and earlier appearance of cataracts in space travelers. The lens is a radiosensitive organ of the body with a very obvious late end point of radiation damage--cataract. However, many molecular changes occur in the lens soon after radiation exposure and long before the appearance of an opacification. The goal of our research is to elucidate early mechanisms associated with particle radiation-induced cataractogenesis, with the ultimate goal of developing countermeasures. Normal, cultured non-immortalized human lens cells were grown on matrix-coated plastic tissue culture vessels and irradiated with particle beams at Lawrence Berkeley National Lab (LBNL) or at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Lab. Samples were harvested at different times after radiation exposure. Using a focused genetic approach, total RNA and protein extracts from control and irradiated samples were processed and probed for the expression of genes associated with extracellular matrix (ECM) proteases. Matrix metalloproteinases (MMPs) have previously been studied in adult postmortem human lenses, in post-cataract intraocular lens (IOL) surgery capsular bags and with immortalized human lens cell cultures. Significant differences exist in the expression pattern with these various model systems. We have evidence for the cell stage-specific expression of MMP family of genes during lens fiber differentiation, and for radiation-induced alterations in the misregulation of MMP expression. Our data indicate that radiation exposure may lead to differences in the expression of radiation stress responses, which may impact selective ECM remodeling and cell differentiation. PMID:17256179

  5. Effects of wheat germ agglutinin on human gastrointestinal epithelium: insights from an experimental model of immune/epithelial cell interaction.

    PubMed

    Dalla Pellegrina, Chiara; Perbellini, Omar; Scupoli, Maria Teresa; Tomelleri, Carlo; Zanetti, Chiara; Zoccatelli, Gianni; Fusi, Marina; Peruffo, Angelo; Rizzi, Corrado; Chignola, Roberto

    2009-06-01

    Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only approximately 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods. PMID:19332085

  6. Effects of wheat germ agglutinin on human gastrointestinal epithelium: Insights from an experimental model of immune/epithelial cell interaction

    SciTech Connect

    Pellegrina, Chiara Dalla; Perbellini, Omar; Scupoli, Maria Teresa; Tomelleri, Carlo; Zanetti, Chiara; Zoccatelli, Gianni; Fusi, Marina; Peruffo, Angelo; Rizzi, Corrado; Chignola, Roberto

    2009-06-01

    Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only {approx} 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.

  7. Honeycomb porous films as permeable scaffold materials for human embryonic stem cell-derived retinal pigment epithelium.

    PubMed

    Calejo, Maria Teresa; Ilmarinen, Tanja; Jongprasitkul, Hatai; Skottman, Heli; Kellomäki, Minna

    2016-07-01

    Age-related macular degeneration (AMD) is a leading cause of blindness in developed countries, characterised by the degeneration of the retinal pigment epithelium (RPE), a pigmented cell monolayer that closely interacts with the photoreceptors. RPE transplantation is thus considered a very promising therapeutic option to treat this disease. In this work, porous honeycomb-like films are for the first time investigated as scaffold materials for human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). By changing the conditions during film preparation, it was possible to produce films with homogeneous pore distribution and adequate pore size (∼3-5 µm), that is large enough to ensure high permeability but small enough to enable cell adherence and spreading. A brief dip-coating procedure with collagen type IV enabled the homogeneous adsorption of the protein to the walls and bottom of pores, increasing the hydrophilicity of the surface. hESC-RPE adhered and proliferated on all the collagen-coated materials, regardless of small differences in pore size. The differentiation of hESC-RPE was confirmed by the detection of specific RPE protein markers. These results suggest that the porous honeycomb films can be promising candidates for hESC-RPE tissue engineering, importantly enabling the free flow of ions and molecules across the material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1646-1656, 2016. PMID:26914698

  8. Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium

    PubMed Central

    Zhu, Yu; Carido, Madalena; Meinhardt, Andrea; Kurth, Thomas; Karl, Mike O.; Ader, Marius; Tanaka, Elly M.

    2013-01-01

    A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia. PMID:23358448

  9. GP41-specific Antibody Blocks Cell-free HIV-1 Transcytosis through Human Rectal Mucosa and Model Colonic Epithelium#

    PubMed Central

    Shen, Ruizhong; Drelichman, Ernesto R.; Bimczok, Diane; Ochsenbauer, Christina; Kappes, John C.; Tudor, Daniela; Bomsel, Morgane; Smythies, Lesley E.; Smith, Phillip D.

    2013-01-01

    Monostratified epithelial cells translocate HIV-1 from the apical to the basolateral surface via vesicular transcytosis. Since acutely transmitted HIV-1 is almost exclusively CCR5-tropic and human intestinal epithelial cells preferentially transcytose CCR5-tropic virus, we established epithelial monolayers using polarized HT-29 cells transduced to express CCR5, and an explant system using normal human rectal mucosa, to characterize biological parameters of epithelial cell transcytosis of HIV-1 and assess antiviral antibody blockade of transcytosis. The amount of cell-free HIV-1 transcytosed through the epithelial monolayer increased linearly in relation to the amount of virus applied to the apical surface, indicating transcytosis efficiency was constant (r2 = 0.9846, P<0.0001). The efficiency of HIV-1 transcytosis ranged between 0.05% and 1.21%, depending on the virus strain, producer cell type and gp120 V1-V3 loop signature. Inoculation of HIV-1 neutralizing antibodies to the immunodominant region (7B2) or the conserved membrane proximal external region (2F5) of gp41 or to cardiolipin (IS4) onto the apical surface of epithelial monolayers prior to inoculation of virus significantly reduced HIV-1 transcytosis. 2F5 was the most potent of these IgG1 mAbs. Dimeric IgA (dIgA) and monomeric IgA (mIgA), but not polymeric IgM, 2F5 antibodies also blocked HIV-1 transcytosis across the epithelium and, importantly, across explanted normal human rectal mucosa, with mIgA substantially more potent than dIgA in effecting transcytosis blockade. These findings underscore the potential role of transcytosis blockade in the prevention of HIV-1 transmission across columnar epithelium such as that of the rectum. PMID:20208001

  10. Dose conversion coefficients for monoenergetic electrons incident on a realistic human eye model with different lens cell populations

    NASA Astrophysics Data System (ADS)

    Nogueira, P.; Zankl, M.; Schlattl, H.; Vaz, P.

    2011-11-01

    The radiation-induced posterior subcapsular cataract has long been generally accepted to be a deterministic effect that does not occur at doses below a threshold of at least 2 Gy. Recent epidemiological studies indicate that the threshold for cataract induction may be much lower or that there may be no threshold at all. A thorough study of this subject requires more accurate dose estimates for the eye lens than those available in ICRP Publication 74. Eye lens absorbed dose per unit fluence conversion coefficients for electron irradiation were calculated using a geometrical model of the eye that takes into account different cell populations of the lens epithelium, together with the MCNPX Monte Carlo radiation transport code package. For the cell population most sensitive to ionizing radiation—the germinative cells—absorbed dose per unit fluence conversion coefficients were determined that are up to a factor of 4.8 higher than the mean eye lens absorbed dose conversion coefficients for electron energies below 2 MeV. Comparison of the results with previously published values for a slightly different eye model showed generally good agreement for all electron energies. Finally, the influence of individual anatomical variability was quantified by positioning the lens at various depths below the cornea. A depth difference of 2 mm between the shallowest and the deepest location of the germinative zone can lead to a difference between the resulting absorbed doses of up to nearly a factor of 5000 for electron energy of 0.7 MeV.

  11. Studies on the solubilization of the water-insoluble fraction from human lens and cataract.

    PubMed

    Ortwerth, B J; Olesen, P R

    1992-12-01

    Studies were carried out comparing the ability of urea extraction and sonication to solubilize the water-insoluble (WI) protein fraction from human lens tissue. Sonication and urea extraction were able to solubilize greater than 80% of the insoluble protein whether whole lenses or lens nuclei were used. This was true for normal lens and +1 cataracts; however, only 60% solubilization was obtained with the WI fraction from more advanced cataracts. Equal aliquots of a WI fraction from both pooled normal and pooled cataract lens nuclei were solubilized with and without reducing agents. The addition of dithiothreitol (DTT) had no significant effect on solubilization of the normal lens WI fraction. DTT did increase the protein solubilized from the cataract WI fraction by 30% with urea extraction; however, no increase was seen with sonication. When sodium borohydride was used as the reducing agent, essentially the same results were obtained. The solubilized protein populations were identical by SDS-PAGE and amino acid analysis. The addition of reducing agents had no effect on the amino acid content of the solubilized proteins with the single exception of lysine. This amino acid was markedly decreased in the proteins extracted in the presence of 40 mM sodium borohydride, but not with DTT. These data suggest that the borohydride not only increased the amount of protein solubilized, but likely also stabilized glycated lysine residues during the acid hydrolysis. Therefore, sonication readily provides a soluble preparation of the WI proteins from normal and cataract lens nuclei without the need for denaturing agents, however, disulfide-linked and lysine modified crystallins were best solubilized with urea. PMID:1486936

  12. Dose conversion coefficients for photon exposure of the human eye lens.

    PubMed

    Behrens, R; Dietze, G

    2011-01-21

    In recent years, several papers dealing with the eye lens dose have been published, because epidemiological studies implied that the induction of cataracts occurs even at eye lens doses of less than 500 mGy. Different questions were addressed: Which personal dose equivalent quantity is appropriate for monitoring the dose to the eye lens? Is a new definition of the dose quantity H(p)(3) based on a cylinder phantom to represent the human head necessary? Are current conversion coefficients from fluence to equivalent dose to the lens sufficiently accurate? To investigate the latter question, a realistic model of the eye including the inner structure of the lens was developed. Using this eye model, conversion coefficients for electrons have already been presented. In this paper, the same eye model-with the addition of the whole body-was used to calculate conversion coefficients from fluence (and air kerma) to equivalent dose to the lens for photon radiation from 5 keV to 10 MeV. Compared to the values adopted in 1996 by the International Commission on Radiological Protection (ICRP), the new values are similar between 40 keV and 1 MeV and lower by up to a factor of 5 and 7 for photon energies at about 10 keV and 10 MeV, respectively. Above 1 MeV, the new values (calculated without kerma approximation) should be applied in pure photon radiation fields, while the values adopted by the ICRP in 1996 (calculated with kerma approximation) should be applied in case a significant contribution from secondary electrons originating outside the body is present. PMID:21178237

  13. Progenitor Epithelium

    PubMed Central

    Marty-Santos, Leilani

    2015-01-01

    Insulin-producing β cells within the vertebrate fetal pancreas acquire their fate in a step-wise manner. Whereas the intrinsic factors dictating the transcriptional or epigenetic status of pancreatic lineages have been intensely examined, less is known about cell–cell interactions that might constitute a niche for the developing β cell lineage. It is becoming increasingly clear that understanding and recapitulating these steps may instruct in vitro differentiation of embryonic stem cells and/or therapeutic regeneration. Indeed, directed differentiation techniques have improved since transitioning from 2D to 3D cultures, suggesting that the 3D microenvironment in which β cells are born is critical. However, to date, it remains unknown whether the changing architecture of the pancreatic epithelium impacts the fate of cells therein. An emerging challenge in the field is to elucidate how progenitors are allocated during key events, such as the stratification and subsequent resolution of the pre-pancreatic epithelium, as well as the formation of lumens and branches. Here, we assess the progenitor epithelium and examine how it might influence the emergence of pancreatic multipotent progenitors (MPCs), which give rise to β cells and other pancreatic lineages. PMID:26216134

  14. Protective effects of Semiaquilegia adoxoides n-butanol extract against hydrogen peroxide-induced oxidative stress in human lens epithelial cells.

    PubMed

    Liang, Bing; Wei, Wei; Wang, Jianta; Zhang, Mingming; Xu, Ran; Wu, Fei; Xiao, Haitao; Tang, Lei

    2016-09-01

    Context Hydrogen peroxide (H2O2)-induced damage in the lens epithelium leads to cell death and cataract. Semiaquilegia adoxoides (DC.) Makino (Ranunculaceae), a folk medicine of Hmong (an ethnic group of China), has been traditionally used to treat cataract; however, the underlying molecular mechanism is yet to be uncovered. Objective This study aimed to investigate whether the n-butanol extract of S. adoxoides (nSA) is effective against the H2O2-induced oxidative stress in human lens epithelial (HLE) cells. Materials and methods Human lens epithelial (SRA 01/04) cells were stimulated by H2O2 (250 μM) in the presence or absence of nSA. The antioxidant effects of nSA were determined in terms of cell viability (MTT assay), apoptosis (AnnexinV/PI staining), radical scavenging capability (various enzymatic assays), loss of mitochondrial membrane potential (Rhodamine 123 staining), expression of apoptotic markers including caspase-3 and caspase-9 and the change of Bcl-2/Bax ratio (western blot) in the HLE cells. Results The results showed that pretreatment of nSA (250, 500 and 1000 μg/mL) markedly reduced H2O2-induced cellular apoptosis and malondialdehyde accumulation, but elevated the activities of total superoxide dismutase, catalase, glutathione peroxidase. Thus, the total antioxidative capability was enhanced upon the nSA treatment meanwhile the loss of mitochondrial membrane potential was prevented. Moreover, nSA at concentrations of 250, 500 and 1000 μg/mL also significantly suppressed the activation of caspase-3 and -9, and increased the Bcl-2/Bax ratio in the HLE cells. Discussion and conclusion Our findings suggested that nSA is a potential prophylactic agent in the prevention of cataractogeneis. PMID:26974044

  15. Spatial and Spectral Characterization of Human Retinal Pigment Epithelium Fluorophore Families by Ex Vivo Hyperspectral Autofluorescence Imaging

    PubMed Central

    Ben Ami, Tal; Tong, Yuehong; Bhuiyan, Alauddin; Huisingh, Carrie; Ablonczy, Zsolt; Ach, Thomas; Curcio, Christine A.; Smith, R. Theodore

    2016-01-01

    Purpose Discovery of candidate spectra for abundant fluorophore families in human retinal pigment epithelium (RPE) by ex vivo hyperspectral imaging. Methods Hyperspectral autofluorescence emission images were captured between 420 and 720 nm (10-nm intervals), at two excitation bands (436–460, 480–510 nm), from three locations (fovea, perifovea, near-periphery) in 20 normal RPE/Bruch's membrane (BrM) flatmounts. Mathematical factorization extracted a BrM spectrum (S0) and abundant lipofuscin/melanolipofuscin (LF/ML) spectra of RPE origin (S1, S2, S3) from each tissue. Results Smooth spectra S1 to S3, with perinuclear localization consistent with LF/ML at all three retinal locations and both excitations in 14 eyes (84 datasets), were included in the analysis. The mean peak emissions of S0, S1, and S2 at λex 436 nm were, respectively, 495 ± 14, 535 ± 17, and 576 ± 20 nm. S3 was generally trimodal, with peaks at either 580, 620, or 650 nm (peak mode, 650 nm). At λex 480 nm, S0, S1, and S2 were red-shifted to 526 ± 9, 553 ± 10, and 588 ± 23 nm, and S3 was again trimodal (peak mode, 620 nm). S1 often split into two spectra, S1A and S1B. S3 strongly colocalized with melanin. There were no significant differences across age, sex, or retinal location. Conclusions There appear to be at least three families of abundant RPE fluorophores that are ubiquitous across age, retinal location, and sex in this sample of healthy eyes. Further molecular characterization by imaging mass spectrometry and localization via super-resolution microscopy should elucidate normal and abnormal RPE physiology involving fluorophores. Translational Relevance Our results help establish hyperspectral autofluorescence imaging of the human retinal pigment epithelium as a useful tool for investigating retinal health and disease. PMID:27226929

  16. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

    PubMed Central

    Bennis, Anna; Gorgels, Theo G. M. F.; ten Brink, Jacoline B.; van der Spek, Peter J.; Bossers, Koen; Heine, Vivi M.; Bergen, Arthur A.

    2015-01-01

    Background The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE. Methods We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE. Results We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier. Conclusion These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular

  17. Caveolin-1 as a Novel Indicator of Wound-Healing Capacity in Aged Human Corneal Epithelium

    PubMed Central

    Rhim, Ji Heon; Kim, Jae Hoon; Yeo, Eui-Ju; Kim, Jae Chan; Park, Sang Chul

    2010-01-01

    Excess caveolin-1 has been reported to play a role in age-dependent hyporesponsiveness to growth factors in vitro. Therefore, we hypothesized that caveolin-1–dependent hyporesponsiveness to growth factors in aged corneal epithelial cells might be responsible for delayed wound healing in vivo. To test this hypothesis, we evaluated corneal wound-healing time by vital staining using fluorescein after laser epithelial keratomileusis (LASEK). We compared wound-healing times in young, middle-aged and elderly patients. We also examined caveolin-1 levels and other aging markers, such as p53 and p21, in the corneal epithelium. Elderly patients generally had higher caveolin-1 levels in the corneal epithelia than young patients. There were, however, variations among individuals with increased caveolin-1 in some young patients and decreased levels in some elderly patients. Wound-healing time after LASEK correlated well with the corneal caveolin-1 status. Therefore, we suggest that caveolin-1 status might be responsible for delayed wound healing in elderly patients after LASEK. Caveolin-1 status might be a regulator for wound-healing capacity and a novel target for in vivo adjustment. PMID:20644900

  18. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    SciTech Connect

    Manceur, Aziza P.; Tseng, Michael; Holowacz, Tamara; Witterick, Ian; Weksberg, Rosanna; McCurdy, Richard D.; Warsh, Jerry J.; Audet, Julie

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  19. Generation of Human Lens Epithelial-Like Cells From Patient-Specific Induced Pluripotent Stem Cells.

    PubMed

    Li, Dan; Qiu, Xiaodi; Yang, Jin; Liu, Tianjin; Luo, Yi; Lu, Yi

    2016-12-01

    Cataractogenesis begins from the dynamic lens epithelial cells (LECs) and adjacent fiber cells. LECs derived from cell lines cannot maintain the crystalline expression as the primary LECs. The current study aimed to efficiently generate large numbers of human LECs from patient-specific induced pluripotent stem cells (iPSCs). Anterior lens capsules were collected from cataract surgery and were used to culture primary hLECs. iPSCs were induced from these primary hLECs by lentiviral transduction of Oct4, Sox2, Klf4, and c-Myc. Then, the generated iPSCs were re-differentiated into hLECs by the 3-step addition of defined factor combinations (Noggin, BMP4/7, bFGF, and EGF) modified from an established method. During the re-differentiation process, colonies of interest were isolated using a glass picking tool and cloning cylinders based on the colony morphology. After two steps of isolation, populations of LEC-like cells (LLCs) were generated and identified by the expression of lens marker genes by qPCR, western blot and immunofluorescence staining. The study introduced a modified protocol to isolate LLCs from iPSCs by defined factors in a short time frame. This technique could be useful for mechanistic studies of lens-related diseases. J. Cell. Physiol. 231: 2555-2562, 2016. © 2016 Wiley Periodicals, Inc. PMID:26991066

  20. Stretch-dependent changes in surface profiles of the human crystalline lens during accommodation: A finite element study

    PubMed Central

    Pour, Hooman Mohammad; Kanapathipillai, Sangarapillai; Zarrabi, Khosrow; Manns, Fabrice; Ho, Arthur

    2015-01-01

    Background A nonlinear isotropic finite element (FE) model of a 29 year old human crystalline lens was constructed to study the effects of various geometrical parameters on lens accommodation. Methods The model simulates dis-accommodation by stretching of the lens and predicts the change in the lens capsule, cortex and nucleus surface profiles at select states of stretching/accommodation. Multiple regression analysis (MRA) is used to develop a stretch-dependent mathematical model relating the lens sagittal height to the radial position of the lens surface as a function of dis-accommodative stretch. A load analysis is performed to compare the FE results to empirical results from lens stretcher studies. Using the predicted geometrical changes, the optical response of the whole eye during accommodation was analysed by ray-tracing. Results Aspects of lens shape change relative to stretch were evaluated including change in diameter (d), central thickness (T) and accommodation (A). Maximum accommodation achieved was 10.29 D. From the MRA, the stretch-dependent mathematical model of the lens shape related lens curvatures as a function of lens ciliary stretch well (maximum mean-square residual error 2.5×10−3 µm, p<0.001). The results are compared with those from in vitro studies. Conclusions The FE and ray-tracing predictions are consistent with EVAS studies in terms of load and power change versus change in thickness. The mathematical stretch-dependent model of accommodation presented may have utility in investigating lens behaviour at states other than the relaxed or fully-accommodated states. PMID:25727940

  1. Coordinate Control of Expression of Nrf2-Modulated Genes in the Human Small Airway Epithelium Is Highly Responsive to Cigarette Smoking

    PubMed Central

    Hübner, Ralf-Harto; Schwartz, Jamie D; De Bishnu, P; Ferris, Barbara; Omberg, Larsson; Mezey, Jason G; Hackett, Neil R; Crystal, Ronald G

    2009-01-01

    Nuclear factor erythroid 2–related factor 2 (Nrf2) is an oxidant-responsive transcription factor known to induce detoxifying and antioxidant genes. Cigarette smoke, with its large oxidant content, is a major stress on the cells of small airway epithelium, which are vulnerable to oxidant damage. We assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample the small airway epithelium in healthy-nonsmoker and healthy-smoker, and gene expression was assessed using microarrays. Relative to nonsmokers, Nrf2 protein in the small airway epithelium of smokers was activated and localized in the nucleus. The human homologs of 201 known murine Nrf2-modulated genes were identified, and 13 highly smoking-responsive Nrf2-modulated genes were identified. Construction of an Nrf2 index to assess the expression levels of these 13 genes in the airway epithelium of smokers showed coordinate control, an observation confirmed by quantitative PCR. This coordinate level of expression of the 13 Nrf2-modulated genes was independent of smoking history or demographic parameters. The Nrf2 index was used to identify two novel Nrf2-modulated, smoking-responsive genes, pirin (PIR) and UDP glucuronosyltransferase 1-family polypeptide A4 (UGT1A4). Both genes were demonstrated to contain functional antioxidant response elements in the promoter region. These observations suggest that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cells, and that there is variability within the human population in the Nrf2 responsiveness to oxidant burden. PMID:19593404

  2. Molecular mechanism of ocular surface damage: Application to an in vitro dry eye model on human corneal epithelium

    PubMed Central

    De Servi, Barbara; Marasco, Daniela; Del Prete, Salvatore

    2011-01-01

    Purpose The present study was concerned with the development of a new experimental model of dry eye using human reconstructed in vitro corneal epithelium (HCE). The model is based on the use of adapted culture conditions that induce relevant modifications at the cellular and molecular level thus mimicking dry eye. Methods The HCE model was maintained in a controlled environmental setting (relative humidity <40% and 40 °C temperature) for 24 h and up to 72 h to induce dry eye. The evolution of the dry eye condition was assessed by histology, immunohistochemistry staining, scanning electron microscopy, and gene expression by using TaqMan gene assay technology (mucin-4 [MUC4], matrix metallopeptidase-9 [MMP9], tumor necrosis factor-α [TNF-α], and defensin β-2 [DEFB2). The effects of different commercially available tear substitutes on the induced dry eye condition were tested. Results This in vitro dry eye HCE model, that was well established within 24 h, has the characteristic features of a dry eye epithelium and could be satisfactorily used for preliminary assessment of the protective activity of some artificial tears. The transcriptional study of selected biomarkers showed an increase in MUC4, MMP9, TNF-α, and hBD-2 (DEFB2) gene expression. Conclusions By using a dynamic approach, we were able to define a biomarker gene signature of dry eye-induced effects that could be predictive of corneal damage in vivo and to discriminate the efficacy among different commercial artificial tears. PMID:21245952

  3. The effects of human serum to the morphology, proliferation and gene expression level of the respiratory epithelium in vitro.

    PubMed

    Yunus, Mohd Heikal Mohd; Siang, Kan Chan; Hashim, Nurul Izzati; Zhi, Ng Pei; Zamani, Nur Fathurah; Sabri, Primuharsa Putra; Busra, Mohd Fauzi; Chowdhury, Shiplu Roy; Idrus, Ruszymah Binti Haji

    2014-08-01

    The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. The present study focused on investigating the effects of human serum (HS) on the qualitative and quantitative properties of the human respiratory epithelium compared to the fetal bovine serum (FBS), as a supplement in culture. Respiratory epithelial (RE) cells derived from human nasal turbinate were co-cultured with fibroblasts, subsequently separated at 80-90% confluency by differential trypsinization. RE cells were then sub-cultured into 2 different plates containing 5% allogenic HS and FBS supplemented media respectively up to passage 1 (P1). Cell morphology, growth rate, cell viability and population doubling time were assessed under light microscope, and levels of gene expression were measured via real time reverse transcriptase-polymerase chain reaction (qRT-PCR). RE cells appeared as polygonal shape and expanded when cultured in HS whereas RE cells in FBS were observed to be easily matured thus limit the RE cells expansion. Proliferation rate of RE cells in HS supplemented media (7673.18 ± 1207.15) was 3 times higher compared to RE in FBS supplemented media (2357.68 ± 186.85). Furthermore, RE cells cultured in HS-supplemented media required fewer days (9.15 ± 1.10) to double in numbers compared to cells cultured in FBS-supplemented media (13.66 ± 0.81). Both the differences were significant (p<0.05). However, there were no significant differences in the viability of RE cells in both groups (p=0.105). qRT-PCR showed comparable expressions of gene Cytokeratin-14 (CK-14), Cytokeratin-18 (CK-18) and Mucin-5 subtype B (MUC5B) in RE cells cultured in both groups (p>0.05). In conclusion, HS is a comparatively better choice of media supplement in accelerating growth kinetics of RE cells in vitro thus producing a better quality of respiratory

  4. Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium.

    PubMed

    Lidgerwood, Grace E; Lim, Shiang Y; Crombie, Duncan E; Ali, Ray; Gill, Katherine P; Hernández, Damián; Kie, Josh; Conquest, Alison; Waugh, Hayley S; Wong, Raymond C B; Liang, Helena H; Hewitt, Alex W; Davidson, Kathryn C; Pébay, Alice

    2016-04-01

    We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening. PMID:26589197

  5. Hypericin-mediated photooxidative damage of α-crystallin in human lens epithelial cells.

    PubMed

    Ehrenshaft, Marilyn; Roberts, Joan E; Mason, Ronald P

    2013-07-01

    St. John's wort (Hypericum perforatum), a perennial herb native to Europe, is widely used for and seems to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and the UVA range, is considered to be one of the bioactive ingredients of St. John's wort, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. α-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescence confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths <400nm provides incomplete protection against α-crystallin modification and NFK accumulation, suggesting that even by wearing UV-blocking sunglasses, routine users of St. John's wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage. PMID:23453985

  6. Hypericin-Mediated Photooxidative Damage of α-crystallin in Human Lens Epithelial Cells

    PubMed Central

    Ehrenshaft, Marilyn; Roberts, Joan E.; Mason, Ronald P.

    2013-01-01

    St. John's wort (Hypericum perforatum), a perennial herb native to Europe, is widely used and appears to be effective in treatment of mild to moderate depression. Hypericin, a singlet oxygen-generating photosensitizer that absorbs in both the visible and UVA range, is considered to be one of the bioactive ingredients, and commercial preparations are frequently calibrated to contain a standard concentration. Hypericin can accumulate in ocular tissues, including lenses, and can bind in vitro to α-crystallin, a major lens protein. Alpha-crystallin is required for lens transparency and also acts as a chaperone to ensure its own integrity and the integrity of all lens proteins. Because there is no crystallin turnover, damage to α-crystallin is cumulative over the lifetime of the lens, and can lead to cataracts, the principal cause of blindness worldwide. In this work we study hypericin photosensitization of α-crystallin and detect extensive polymerization of bovine α-crystallin exposed in vitro to hypericin and UVA. We use fluorescent confocal microscopy to visualize binding between hypericin and α-crystallin in a human lens epithelial (HLE) cell line. Further, we show that UVA irradiation of hypericin-treated HLE cells results in a dramatic decrease in α-crystallin detection concurrent with a dramatic accumulation of the tryptophan oxidation product N-formylkynurenine (NFK). Examination of actin in HLE cells indicates that this cytoskeleton protein accumulates NFK resulting from hypericin-mediated photosensitization. This work also shows that filtration of wavelengths <400 nm provides incomplete protection against α-crystallin modifications and NFK accumulation, suggesting that even by wearing UV blocking sunglasses, routine users of St. John's wort cannot adequately shield their lenses from hypericin-mediated photosensitized damage. PMID:23453985

  7. Finite element implementation of a multiscale model of the human lens capsule.

    PubMed

    Burd, H J; Regueiro, R A

    2015-11-01

    An axisymmetric finite element implementation of a previously described structural constitutive model for the human lens capsule (Burd in Biomech Model Mechanobiol 8(3):217-231, 2009) is presented. This constitutive model is based on a hyperelastic approach in which the network of collagen IV within the capsule is represented by an irregular hexagonal planar network of hyperelastic bars, embedded in a hyperelastic matrix. The paper gives a detailed specification of the model and the periodic boundary conditions adopted for the network component. Momentum balance equations for the network are derived in variational form. These balance equations are used to develop a nonlinear solution scheme to enable the equilibrium configuration of the network to be computed. The constitutive model is implemented within a macroscopic finite element framework to give a multiscale model of the lens capsule. The possibility of capsule wrinkling is included in the formulation. To achieve this implementation, values of the first and second derivatives of the strain energy density with respect to the in-plane stretch ratios need to be computed at the local, constitutive model, level. Procedures to determine these strain energy derivatives at equilibrium configurations of the network are described. The multiscale model is calibrated against previously published experimental data on isolated inflation and uniaxial stretching of ex vivo human capsule samples. Two independent example lens capsule inflation analyses are presented. PMID:25957261

  8. Path-dependent human identification using a pyroelectric infrared sensor and fresnel lens arrays.

    PubMed

    Fang, Jian-Shuen; Hao, Qi; Brady, David J; Shankar, Mohan; Guenther, Bob D; Pitsianis, Nikos P; Hsu, Ken Y

    2006-01-23

    This paper presents a design and development of a low power consumption, and low cost, human identification system using a pyroelectric infrared (PIR) sensor whose visibility is modulated by a Fresnel lens array. The optimal element number of the lens array for the identification system was investigated and the experimental results suggest that the lens array with more elements can yield a better performance in terms of identification and false alarm rates. The other parameters of the system configuration such as the height of sensor location and sensor-to-object distance were also studied to improve spectral distinctions among sensory data of human objects. The identification process consists of two parts: training and testing. For the data training, we employed a principal components regression (PCR) method to cluster data with respect to different registered objects at different speed levels. The feature data of different objects walking along the same path in training yet at random speeds are then tested against the pre-trained clusters to decide whether the target is registered, and which member of the registered group it is. PMID:19503378

  9. Particle irradiation induces FGF2 expression in normal human lens cells

    NASA Technical Reports Server (NTRS)

    Chang, P. Y.; Bjornstad K, A.; Chang, E.; McNamara, M.; Barcellos-Hoff, M. H.; Lin, S. P.; Aragon, G.; Polansky, J. R.; Lui, G. M.; Blakely, E. A.

    2000-01-01

    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.

  10. Multiwalled carbon nanotubes induce altered morphology and loss of barrier function in human bronchial epithelium at noncytotoxic doses

    PubMed Central

    Snyder, Ryan J; Hussain, Salik; Rice, Annette B; Garantziotis, Stavros

    2014-01-01

    Multiwalled carbon nanotubes (MWCNTs) have seen increasing application in consumer products over the past decade, resulting in an increasing risk of human exposure. While numerous toxicological studies have been performed using acute high doses of various carbonaceous nanomaterials, the effects of longer-term, low doses of MWCNTs remain relatively unexplored. This study examined bronchoscopy-derived healthy human bronchial epithelial cells exposed in submerged culture to noncytotoxic doses of MWCNTs over 7 days. Under these conditions, doses as low as 3 μg/mL caused altered cell morphology, superficially resembling fibroblasts. Electrical impedance of the epithelial monolayer was greatly reduced following MWCNT exposure. However, Western blot and polymerase chain reaction showed no elevated expression of the fibroblast markers, vimentin, α-smooth muscle actin, or fibronectin, indicating that a mechanism other than epithelial–mesenchymal transition may be responsible for the changes. Phalloidin and tubulin immunostaining showed disruption of the cytoskeleton, and confocal imaging showed a reduction of the tight junction proteins, zona occludens 1 and occludin. We propose that MWCNTs interfere with the cytoskeleton of the lung epithelium, which can result in a harmful reduction in barrier function over time, even at noncytotoxic doses. PMID:25187712

  11. Quantitative assessment of normal and potentially premalignant epithelium at different levels of human colorectal crypts.

    PubMed

    Tipoe, G L; White, F H

    1998-04-01

    The present study uses morphometric techniques to assess whether altered differentiation patterns exist in PPM which might reflect its premalignant status. Samples were obtained from resected malignant lesions of large bowels of 10 Chinese patients. Normal (N) samples were biopsied from the margins of each resected large bowel. Potentially premalignant (PPM) mucosae were obtained from within 2 cm of the margins of the malignant lesions. Tissues were processed for histological examination and using strict criteria, colorectal crypts were divided into basal (B), intermediate (I) and surface (S) segments. Interactive digitisation of sections from each group was used to generate the following morphometric parameters in each segment: nuclear profile circularity indices (NSF and NCI); nuclear numerical density (NA and NV); the degree of deviation of the major nuclear axis in relation to the epithelial-connective junction (AGDMAX); cell height (CH); the distance between nuclear apex to cell apex (DNACA); the distance between cell base to nuclear apex (DCBNA); stratification index (SI)--the ratio of DCBNA and CH; and the volume density of mucous vacuoles in the reference epithelium (VVMV,EP). In comparisons of different segments within groups, the nuclei at the S segment of N and PPM crypts were more irregular and less circular in shape than nuclei from other segments. There was a shift of nuclear profile shape (NSF and NCI) from circular to ellipsiodal between B and S segments. In comparisons of similar segments between groups, no significant nuclear shape changes were detected in nuclei of PPM crypts when compared with nuclei in similar segments of N crypts and the pattern of nuclear shape alterations resembled those of normal crypts. In comparisons of different segments within groups of N and PPM crypts, AGDMAX, DNACA, DCBNA, CH and SI parameters demonstrated that epithelial cells at the I segments have more centrally positioned nuclei with the tallest epithelial height

  12. Von Hippel-Lindau gene expression on the human fallopian tube epithelium during the menstrual cycle.

    PubMed

    Lu, Yan-Yan; Zhu, Wei-Jie; Xie, Bao-Guo

    2015-06-01

    The Von Hippel-Lindau gene (VHL) is a tumor suppressor gene, which is widely expressed in kidney, lung, breast, ovary, and cervix. VHL gene mutations can induce VHL disease and tumorigenesis. However, whether this gene is expressed in the human fallopian tube has not been evaluated. The objectives of this study were to investigate whether the VHL gene is expressed in human fallopian tube, and to investigate its expression changes during the menstrual cycle. Twenty‑seven patients undergoing abdominal hysterectomy with adnexectomy for benign uterine disease were enrolled in the study. Human fallopian tubes were divided into proliferative stage (n=14) and secretory stage (n=13) according to the stage of the menstrual cycle they were isolated from. The expression of the VHL gene and protein was studied by reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunohistochemistry, respectively. The results revealed positive expression of the VHL protein in the cytoplasm of ciliated cells of the human fallopian tube. The mRNA and protein expression of VHL in the fallopian tubes was higher in the proliferative compared to the secretory phase of the menstrual cycle, but this difference was not significant (P>0.05). Overall, this study presents data on the VHL mRNA and protein expression in the human fallopian tube, which may be relevant to the process of differentiation of ciliated and secretory cells. PMID:25625420

  13. Identification of global gene expression differences between human lens epithelial and cortical fiber cells reveals specific genes and their associated pathways important for specialized lens cell functions

    PubMed Central

    Hawse, John R.; DeAmicis-Tress, Candida; Cowell, Tracy L.; Kantorow, Marc

    2005-01-01

    Purpose: In order to identify specific genes that may play important roles in maintaining the specialized functions of lens epithelial and fiber cells, we have analyzed the global gene expression profiles of these two cell types in the human lens. This analysis will also reveal those genes that are exclusively expressed in the epithelial and cortical fiber cells and those genes that may play important roles in the differentiation of epithelial cells to mature fiber cells. Methods: Oligonucleotide microarray hybridization was used to analyze the expression profiles of 22,215 genes between adult (average age greater than 56 years) human lens epithelial and cortical fiber cells. The expression levels of selected genes were further compared by semi-quantitative RT-PCR and selected genes were functionally clustered into common categories using the EASE bioinformatics software package. Results: Analysis of three separate microarray hybridizations revealed 1,196 transcripts that exhibit increased expression and 1,278 transcripts that exhibit decreased expression at the 2 fold or greater level between lens epithelial cells and cortical fiber cells on all three of the arrays analyzed. Of these, 222 transcripts exhibited increased expression and 135 transcripts exhibited decreased expression by an average of 5 fold or greater levels on all three arrays. Semi-quantitative RT-PCR analysis of 21 randomly selected genes revealed identical expression patterns as those detected by microarray hybridization indicating that the microarray data are accurate. Functional clustering of the identified gene expression patterns using the EASE program revealed a wide variety of biological pathways that exhibited altered expression patterns between the two cell types including mRNA processing, cell adhesion, cell proliferation, translation, protein folding, oxidative phosphorylation, and apoptosis, among others. Conclusions: These data reveal novel and previously identified gene expression

  14. Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution

    PubMed Central

    Starnes, Austin C; Huisingh, Carrie; McGwin, Gerald; Sloan, Kenneth R; Ablonczy, Zsolt; Smith, R. Theodore; Curcio, Christine A; Ach, Thomas

    2016-01-01

    Background The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. Methods Nineteen human RPE-flatmounts (9≤51years, 10>80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. Results A total of 11403 RPE cells at 200 locations were analyzed: 94.66 % mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. Conclusion This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies. PMID:26923500

  15. Expression of Cell Competition Markers at the Interface between p53 Signature and Normal Epithelium in the Human Fallopian Tube

    PubMed Central

    Kito, Masahiko; Maeda, Daichi; Kudo-Asabe, Yukitsugu; Sato, Naoki; Shih, Ie-Ming; Wang, Tian-Li; Tanaka, Masamitsu; Terada, Yukihiro; Goto, Akiteru

    2016-01-01

    There is a growing body of evidence regarding cell competition between normal and mutant mammalian cells, which suggest that it may play a defensive role in the early phase of carcinogenesis. In vitro study in the past has shown that overexpression of vimentin in normal epithelial cells at the contact surface with transformed cells is essential for the cell competition involved in epithelial defense against cancer. In this study, we attempted to examine cell competition in human tissue in vivo by investigating surgically resected human fallopian tubes that contain p53 signatures and serous tubal intraepithelial lesions (STILs), a linear expansion of p53-immunopositive/TP53 mutant tubal epithelial cells that are considered as precursors of pelvic high grade serous carcinoma. Immunofluorescence double staining for p53 and the cell competition marker vimentin was performed in 21 sections of human fallopian tube tissue containing 17 p53 signatures and 4 STILs. The intensities of vimentin expression at the interface between p53-positive cells at the end of the p53 signature/STIL and adjacent p53-negative normal tubal epithelial cells were compared with the background tubal epithelium. As a result, the average vimentin intensity at the interfaces relative to the background intensity was 1.076 (95% CI, 0.9412 – 1.211 for p53 signature and 0.9790 (95% CI, 0.7206 – 1.237) for STIL. Thus, it can be concluded that overexpression of the cell competition marker vimentin are not observed in human tissue with TP53 alterations. PMID:27258067

  16. Reflectance confocal microscopy of oral epithelial tissue using an electrically tunable lens

    NASA Astrophysics Data System (ADS)

    Jabbour, Joey M.; Malik, Bilal H.; Cuenca, Rodrigo; Cheng, Shuna; Jo, Javier A.; Cheng, Yi-Shing L.; Wright, John M.; Maitland, Kristen C.

    2014-02-01

    We present the use of a commercially available electrically tunable lens to achieve axial scanning in a reflectance confocal microscope. Over a 255 μm axial scan range, the lateral and axial resolutions varied from 1-2 μm and 4-14 μm, respectively, dependent on the variable focal length of the tunable lens. Confocal imaging was performed on normal human biopsies from the oral cavity ex vivo. Sub-cellular morphologic features were seen throughout the depth of the epithelium while axially scanning using the focus tunable lens.

  17. Gene expression in the human mammary epithelium during lactation: the milk fat globule transcriptome.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular physiology underlying human milk production is largely unknown because of limitations in obtaining tissue samples. Determining gene expression in normal lactating women would be a potential step toward understanding why some women struggle with or fail at breastfeeding their infants. R...

  18. Regulation of gene expression in human mammary epithelium: effect of breast pumping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known of the molecular regulation of human milk production because of limitations in obtaining mammary tissue from lactating women. Our objectives were to evaluate whether RNA isolated from breast milk fat globules (MFGs) could be an alternative to mammary biopsies and to determine whether...

  19. Determination of the Geometrical and Optical Properties of the Human Crystalline Lens Leading to a Model of Human Accommodation

    NASA Astrophysics Data System (ADS)

    Cook, Christopher Andrew

    Of the methods that have been developed (e.g., phakometry, NMI, etc.) for non-invasive measurement of the geometry of the anterior segment of the human eye, at present Scheimpflug photography offers the best resolution, and the highest accuracy. The primary obstacle encountered with this or any other image based method has been in directly obtaining quantitative measurements from the images. Image enhancement (grey-scale gradient analysis) and pattern recognition methods (Hough transformation and recursive least squares algorithms) are developed so that parametric representations of lens surfaces and zone boundaries can be obtained directly from the images. Methods to correct for nonlinear Scheimpflug camera reproduction ratios, and provide error estimates for geometrical parameters are also developed. Combined, these techniques yield representations of lens geometry having sufficient accuracy, to which paraxial ray tracing can be applied to determine lens optical properties using well posed optical models with one unknown. Scheimpflug images taken of 100 emmetropic individuals have been processed using these methods, and the results are given. Positional data including anterior chamber depth, lens thickness, anterior segment length, nuclear thickness and anterior and posterior cortical thickness are given for the unaccommodated state, and as functions of accommodative amplitude. Curvature data for the lens surfaces, nuclear boundaries, and optical substructure (zones of discontinuity) are given as with the positional data. An explanation of the mechanical basis of presbyopia is developed from these results. Error estimates for all quantities are included. In combination with separate characterizations of each subjects globe using keratometry, A-scan ultrasonography, pachymetry, and Hartinger refractometry, these data form the basis for paraxial optical modeling. Three models of lens optical properties are developed and examined. The first is based on the simple

  20. Glucocorticoid Clearance and Metabolite Profiling in an In Vitro Human Airway Epithelium Lung Model.

    PubMed

    Rivera-Burgos, Dinelia; Sarkar, Ujjal; Lever, Amanda R; Avram, Michael J; Coppeta, Jonathan R; Wishnok, John S; Borenstein, Jeffrey T; Tannenbaum, Steven R

    2016-02-01

    The emergence of microphysiologic epithelial lung models using human cells in a physiologically relevant microenvironment has the potential to be a powerful tool for preclinical drug development and to improve predictive power regarding in vivo drug clearance. In this study, an in vitro model of the airway comprising human primary lung epithelial cells cultured in a microfluidic platform was used to establish a physiologic state and to observe metabolic changes as a function of glucocorticoid exposure. Evaluation of mucus production rate and barrier function, along with lung-specific markers, demonstrated that the lungs maintained a differentiated phenotype. Initial concentrations of 100 nM hydrocortisone (HC) and 30 nM cortisone (C) were used to evaluate drug clearance and metabolite production. Measurements made using ultra-high-performance liquid chromatography and high-mass-accuracy mass spectrometry indicated that HC metabolism resulted in the production of C and dihydrocortisone (diHC). When the airway model was exposed to C, diHC was identified; however, no conversion to HC was observed. Multicompartmental modeling was used to characterize the lung bioreactor data, and pharmacokinetic parameters, including elimination clearance and elimination half-life, were estimated. Polymerse chain reaction data confirmed overexpression of 11-β hydroxysteroid dehydrogenase 2 (11βHSD2) over 11βHSD1, which is biologically relevant to human lung. Faster metabolism was observed relative to a static model on elevated rates of C and diHC formation. Overall, our results demonstrate that this lung airway model has been successfully developed and could interact with other human tissues in vitro to better predict in vivo drug behavior. PMID:26586376

  1. Widespread expression of serum amyloid A in histologically normal human tissues. Predominant localization to the epithelium.

    PubMed

    Urieli-Shoval, S; Cohen, P; Eisenberg, S; Matzner, Y

    1998-12-01

    Serum amyloid A (SAA) is an acute-phase reactant whose level in the blood is elevated to 1000-fold as part of the body's responses to various injuries, including trauma, infection, inflammation, and neoplasia. As an acute-phase reactant, the liver has been considered to be the primary site of expression. However, limited extrahepatic SAA expression was described in mouse tissues and in cells of human atherosclerotic lesions. Here we describe nonradioactive in situ hybridization experiments revealing that the SAA mRNA is widely expressed in many histologically normal human tissues. Expression was localized predominantly to the epithelial components of a variety of tissues, including breast, stomach, small and large intestine, prostate, lung, pancreas, kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and brain neurons. Expression was also observed in lymphocytes, plasma cells, and endothelial cells. RT-PCR analysis of selected tissues revealed expression of the SAA1, SAA2, and SAA4 genes but not of SAA3, consistent with expression of these genes in the liver. Immunohistochemical staining revealed SAA protein expression that co-localized with SAA mRNA expression. These data indicate local production of the SAA proteins in histologically normal human extrahepatic tissues. PMID:9815279

  2. Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys

    PubMed Central

    Schaap-Nutt, Anne; Scull, Margaret A.; Schmidt, Alexander C.; Murphy, Brian R.; Pickles, Raymond J.

    2010-01-01

    Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32°C and exhibited little productive replication at 37°C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates. PMID:20139039

  3. Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.

    PubMed

    Kamao, Hiroyuki; Mandai, Michiko; Okamoto, Satoshi; Sakai, Noriko; Suga, Akiko; Sugita, Sunao; Kiryu, Junichi; Takahashi, Masayo

    2014-02-11

    Age-related macular degeneration (AMD) causes severe visual impairment due in part to age-dependent impairment of retinal pigment epithelium (RPE). It has been suggested that autologous human induced pluripotent stem cells (hiPSCs) may represent a useful cell source for the generation of graft RPE. We generated hiPSC-derived RPE (hiPSC-RPE) cell sheets optimized to meet clinical use requirements, including quality, quantity, consistency, and safety. These cell sheets are generated as a monolayer of cells without any artificial scaffolds, express typical RPE markers, form tight junctions that exhibit polarized secretion of growth factors, and show phagocytotic ability and gene-expression patterns similar to those of native RPE. Additionally, upon transplantation, autologous nonhuman primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. These results suggest that autologous hiPSC-RPE cell sheets may serve as a useful form of graft for use in tissue replacement therapy for AMD. PMID:24527394

  4. [Ox-LDL down-regulates expression of pigment epithelium-derived factor in human umbilical vein endothelial cells].

    PubMed

    Liu, Jie; Yao, Shu-Tong; Zhai, Lei; Feng, Yue-Long; Song, Guo-Hua; Yu, Yang; Zhu, Ping; Qin, Shu-Cun

    2014-08-25

    Pigment epithelium-derived factor (PEDF) is a multifunctional protein with anti-inflammatory, antioxidant and antithrombotic properties and plays a protective role against atherosclerosis (AS). The purpose of the present study is to explore the effects of oxidized low density lipoprotein (ox-LDL) on the expression of PEDF in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were cultured and incubated with ox-LDL at different concentrations (6.25, 12.5, 25, 50, 100 and 150 mg/L) for 24 h. Apoptosis of endothelial cells were assayed by morphological staining and flow cytometry. The intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Cell viability was assayed by MTT assay. PEDF protein and mRNA expressions in HUVECs were analyzed by Western blot and quantitative real-time PCR, respectively. The results showed that ox-LDL significantly induced apoptosis, reduced cell viability, increased intracellular ROS levels and decreased the PEDF expression in HUVECs in a concentration-dependent manner. Ox-LDL at 50 mg/L obviously decreased the PEDF protein expression compared with control group (P < 0.05), whereas 25 mg/L ox-LDL already markedly reduced the PEDF mRNA expression (P < 0.05). In conclusion, the results suggest that ox-LDL down-regulates the PEDF expression through an increased ox-LDL-induced intracellular production of ROS. PMID:25131792

  5. Dose conversion coefficients for monoenergetic electrons incident on a realistic human eye model with different lens cell populations.

    PubMed

    Nogueira, P; Zankl, M; Schlattl, H; Vaz, P

    2011-11-01

    The radiation-induced posterior subcapsular cataract has long been generally accepted to be a deterministic effect that does not occur at doses below a threshold of at least 2 Gy. Recent epidemiological studies indicate that the threshold for cataract induction may be much lower or that there may be no threshold at all. A thorough study of this subject requires more accurate dose estimates for the eye lens than those available in ICRP Publication 74. Eye lens absorbed dose per unit fluence conversion coefficients for electron irradiation were calculated using a geometrical model of the eye that takes into account different cell populations of the lens epithelium, together with the MCNPX Monte Carlo radiation transport code package. For the cell population most sensitive to ionizing radiation-the germinative cells-absorbed dose per unit fluence conversion coefficients were determined that are up to a factor of 4.8 higher than the mean eye lens absorbed dose conversion coefficients for electron energies below 2 MeV. Comparison of the results with previously published values for a slightly different eye model showed generally good agreement for all electron energies. Finally, the influence of individual anatomical variability was quantified by positioning the lens at various depths below the cornea. A depth difference of 2 mm between the shallowest and the deepest location of the germinative zone can lead to a difference between the resulting absorbed doses of up to nearly a factor of 5000 for electron energy of 0.7 MeV. PMID:21983644

  6. Cell-Deposited Matrix Improves Retinal Pigment Epithelium Survival on Aged Submacular Human Bruch's Membrane

    PubMed Central

    Sugino, Ilene K.; Gullapalli, Vamsi K.; Sun, Qian; Wang, Jianqiu; Nunes, Celia F.; Cheewatrakoolpong, Noounanong; Johnson, Adam C.; Degner, Benjamin C.; Hua, Jianyuan; Liu, Tong; Chen, Wei; Li, Hong

    2011-01-01

    Purpose. To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. Methods. Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. Results. The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. Conclusions. Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes. PMID:21398292

  7. Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

    PubMed

    Maruotti, Julien; Sripathi, Srinivas R; Bharti, Kapil; Fuller, John; Wahlin, Karl J; Ranganathan, Vinod; Sluch, Valentin M; Berlinicke, Cynthia A; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z; Bhutto, Imran; Lutty, Gerard A; Zack, Donald J

    2015-09-01

    Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. PMID:26269569

  8. Generation of Functional Thymic Epithelium from Human Embryonic Stem Cells that Supports Host T Cell Development

    PubMed Central

    Parent, Audrey V.; Russ, Holger A.; Khan, Imran S.; LaFlam, Taylor N.; Metzger, Todd C.; Anderson, Mark S.; Hebrok, Matthias

    2013-01-01

    SUMMARY Inducing immune tolerance to prevent rejection is a key step toward successful engraftment of stem-cell-derived tissue in a clinical setting. Using human pluripotent stem cells to generate thymic epithelial cells (TECs) capable of supporting T cell development represents a promising approach to reach this goal; however, progress toward generating functional TECs has been limited. Here, we describe a robust in vitro method to direct differentiation of human embryonic stem cells (hESCs) into thymic epithelial progenitors (TEPs) by precise regulation of TGFβ, BMP4, RA, Wnt, Shh, and FGF signaling. The hESC-derived TEPs further mature into functional TECs that support T cell development upon transplantation into thymus-deficient mice. Importantly, the engrafted TEPs produce T cells capable of in vitro proliferation as well as in vivo immune responses. Thus, hESC-derived TEP grafts may have broad applications for enhancing engraftment in cell-based therapies as well as restoring age-and stress-related thymic decline. PMID:23684540

  9. Neuroblast long-term cell cultures from human fetal olfactory epithelium respond to odors.

    PubMed

    Vannelli, G B; Ensoli, F; Zonefrati, R; Kubota, Y; Arcangeli, A; Becchetti, A; Camici, G; Barni, T; Thiele, C J; Balboni, G C

    1995-06-01

    Primary cell cultures from human fetal olfactory neuroepithelium have been isolated, cloned, and propagated in continuous in vitro culture for approximately 1 year. The two clones we report here synthesize both neuronal proteins and olfactory-specific markers as well as the putative olfactory neurotransmitter, carnosine. In addition, patchclamp experiments reveal that these cells are electrically excitable. Following exposure to a panel of aromatic chemicals one of the cell cultures shows a specific increase in intracellular cAMP, indicating that some degree of functional maturity is expressed in vitro. The results suggest that these cells originate from the "stem cell" compartment that gives rise to mature olfactory receptor neurons. These long-term cell cultures represent models that will be useful in studying the mechanism(s) of olfaction and the regulation of olfactory neurogenesis and differentiation. PMID:7790915

  10. Ambient Levels of Air Pollution Induce Goblet-Cell Hyperplasia in Human Conjunctival Epithelium

    PubMed Central

    Novaes, Priscila; do Nascimento Saldiva, Paulo Hilário; Kara-José, Newton; Macchione, Mariângela; Matsuda, Monique; Racca, Lourdes; Berra, Alejandro

    2007-01-01

    Background Ocular mucosa is exposed constantly to the external environment, and chronic exposure to air pollution may affect the ocular surface. Objective We assessed the effect of air pollution on the ocular surface by combining determinations of individual exposure and conjunctival impression cytology. Methods A panel study was conducted with 29 volunteers recruited in two locations with different pollution levels: São Paulo (n = 13) and Divinolândia (n = 16). We assessed mean individual levels of nitrogen dioxide (NO2) exposure for 7 days, using a passive sampler. Impression cytology samples were obtained from inferior tarsal conjunctiva. Comparisons between the two groups in terms of NO2 exposure and goblet-cell counts were performed using the Student t-test. Correlations between goblet-cells counts and corresponding individual NO2 exposure levels were determined using Spearman’s correlation. Results Individuals living in São Paulo received a significantly (p = 0.005) higher dose of NO2 (mean 32.47; SD 9.83) than those living in Divinolândia (mean 19.33; SD 5.24). There was a steady increase in goblet-cell counts, proportional to NO2 exposure (Spearman’s correlation = 0.566, p = 0.001), with a dose–response pattern. Conclusions A positive and significant association between exposure to air pollution and goblet-cell hyperplasia in human conjunctiva was detected. The combination of simple measurements of exposure and impression cytology was an effective and noninvasive approach for characterizing human response to ambient levels of air pollution. PMID:18087595

  11. SWCNT suppress inflammatory mediator responses in human lung epithelium in vitro

    SciTech Connect

    Herzog, Eva Byrne, Hugh J.; Casey, Alan; Davoren, Maria; Lenz, Anke-Gabriele; Maier, Konrad L.; Duschl, Albert; Oostingh, Gertie Janneke

    2009-02-01

    Single-walled carbon nanotubes have gained enormous popularity due to a variety of potential applications which will ultimately lead to increased human and environmental exposure to these nanoparticles. This study was carried out in order to evaluate the inflammatory response of immortalised and primary human lung epithelial cells (A549 and NHBE) to single-walled carbon nanotube samples (SWCNT). Special focus was placed on the mediating role of lung surfactant on particle toxicity. The toxicity of SWCNT dispersed in cell culture medium was compared to that of nanotubes dispersed in dipalmitoylphosphatidylcholine (DPPC, the main component of lung lining fluid). Exposure was carried out for 6 to 48 h with the latter time-point showing the most significant responses. Moreover, exposure was performed in the presence of the pro-inflammatory stimulus tumour necrosis factor-{alpha} (TNF-{alpha}) in order to mimic exposure of stimulated cells, as would occur during infection. Endpoints evaluated included cell viability, proliferation and the analysis of inflammatory mediators such as interleukin (IL)-8, IL-6, TNF-{alpha} and macrophage chemoattractant protein-1 (MCP-1). Crocidolite asbestos was included as a well characterised, toxic fibre control. The results of this study showed that HiPco SWCNT samples suppress inflammatory responses of A549 and NHBE cells. This was also true for TNF-{alpha} stimulated cells. The use of DPPC improved the degree of SWCNT dispersion in A549 medium and in turn, leads to increased particle toxicity, however, it was not shown to modify NHBE cell responses.

  12. The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium

    PubMed Central

    Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B.; Weinberger, Dov; Sredni, Benjamin

    2016-01-01

    Purpose Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch’s membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Methods Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Results Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Conclusions Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases. PMID:27293373

  13. Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity

    PubMed Central

    Keskin, Derin B.; Reinhold, Bruce B.; Zhang, Guang Lan; Ivanov, Alexander R.; Karger, Barry L.; Reinherz, Ellis L.

    2015-01-01

    Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8–10mers) with predicted IC50 < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M13–11 to a high of >500 for M158–66 with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M158–66, natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M158–66/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M158–66. By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against “stealth” epitopes, overriding viral chicanery. PMID:25646416

  14. Expression of Epstein–Barr virus-induced gene 3 and other interleukin-12-related molecules by human intestinal epithelium

    PubMed Central

    Maaser, Christian; Egan, Laurence J; Birkenbach, Mark P; Eckmann, Lars; Kagnoff, Martin F

    2004-01-01

    Antigen-presenting cells, including dendritic cells, monocytes and macrophages, produce members of the interleukin-12 (IL-12) family that are important in initiating and maintaining cell-mediated immune responses. These include IL-12p35 and p19 that dimerize with IL-12p40 to form IL-12 (also termed IL-12p75) and IL-23, respectively, and Epstein–Barr virus-induced gene 3 (EBI3) protein (a protein related to IL-12p40), that forms a dimer with p28, termed IL-27. Intestinal epithelial cells, which are the initial site of contact between the host and enteric pathogens, can act as antigen-presenting cells, and are known to express mediators important in inflammatory and immune responses. In the current studies, we hypothesized that intestinal epithelial cells express members of the IL-12 family, which can function as an early signalling system important in mucosal immunity. Using in vitro and in vivo model systems of human intestinal epithelium, we demonstrate the regulated expression of EBI3, IL-12p35 and p19 by human intestinal epithelial cells. However, intestinal epithelial cells do not coexpress IL-12p40 or p28 that are required to generate heterodimeric IL-12p75, IL-23 and IL-27. To the extent that IL-12p35, p19 and EBI3 cannot form IL-12p75, IL-23 or IL-27 heterodimers in intestinal epithelial cells, these data suggest that those cells may express other, currently unknown, molecules that can associate with EBI3, IL-12p35 and/or p19 or, alternatively, intestinal epithelial cells may release IL-12-related molecules that by themselves, or in combination with other molecules in the mucosal microenvironment, mediate biological activities. PMID:15196212

  15. Nitrite Modification of Extracellular Matrix Alters CD46 Expression and VEGF Release in Human Retinal Pigment Epithelium

    PubMed Central

    Fields, Mark A.; Cai, Hui; Bowrey, Hannah E.; Moreira, Ernesto F.; Beck Gooz, Monika; Kunchithapautham, Kannan; Gong, Jie; Vought, Emma; Del Priore, Lucian V.

    2015-01-01

    Purpose Loss of CD46 has recently been implicated in choroidal neovascularization in mice. Herein we investigated the effect of nitrite modification of the extracellular matrix (ECM) as an in vitro model of “aging” and its effect on CD46 expression and vascular endothelial growth factor (VEGF) release in cocultured human retinal pigment epithelium (RPE). Methods ARPE-19 cells were plated onto RPE-derived ECM conditions (untreated; nitrite modified; nitrite modified followed by washing with Triton X-100; or nitrite modified followed by washing with Triton X-100 and coated with extracellular matrix ligands). Cells were cultured for 7 days and CD46 expression was analyzed by immunohistochemistry and Western blot. Additionally, CD46 short interfering RNA (siRNA) was transfected into ARPE-19 cells, and VEGF levels were determined by ELISA. Finally, in the same ECM conditions, ARPE-19 cells were challenged with normal human serum and VEGF levels determined by ELISA. Results CD46 is expressed on the basolateral surface of ARPE-19 cells on RPE-derived ECM. Nitrite modification of ECM reduced the expression of CD46 on ARPE-19 cells by 0.5-fold (P = 0.003) and increased VEGF release in ARPE-19 cells by 1.7-fold (P < 0.001). CD46 knockdown also increased release of VEGF on the apical and basal sides of ARPE-19 cells in culture by 1.3- (P = 0.012) and 1.2-fold (P = 0.017), respectively. Conclusions Nitrite modification of the ECM decreased CD46 expression and increased the release of VEGF from ARPE-19 cells. Changes in CD46 expression may lead to changes in VEGF and play a pathologic role in the development of age-related macular degeneration. PMID:26161984

  16. Expansive Generation of Functional Airway Epithelium From Human Embryonic Stem Cells

    PubMed Central

    McIntyre, Brendan A.S.; Alev, Cantas; Mechael, Rami; Salci, Kyle R.; Lee, Jung Bok; Fiebig-Comyn, Aline; Guezguez, Borhane; Wu, Yuping; Sheng, Guojun

    2014-01-01

    Production of human embryonic stem cell (hESC)-derived lung progenitors has broad applicability for drug screening and cell therapy; however, this is complicated by limitations in demarcating phenotypic changes with functional validation of airway cell types. In this paper, we reveal the potential of hESCs to produce multipotent lung progenitors using a combined growth factor and physical culture approach, guided by the use of novel markers LIFRα and NRP1. Lung specification of hESCs was achieved by priming differentiation via matrix-specific support, followed by air-liquid interface to allow generation of lung progenitors capable of in vitro maturation into airway epithelial cell types, resulting in functional characteristics such as secretion of pulmonary surfactant, ciliation, polarization, and acquisition of innate immune activity. This approach provided a robust expansion of lung progenitors, allowing in vivo assessment, which demonstrated that only fully differentiated hESC-derived airway cells were retained in the distal airway, where they aided in physiological recovery in immunocompromised mice receiving airway injury. Our study provides a basis for translational applications of hESCs for lung diseases. PMID:24300555

  17. Goat's eye integrated with a human cataractous lens: A training model for phacoemulsification.

    PubMed

    Sengupta, Sabyasachi; Dhanapal, Praveen; Nath, Manas; Haripriya, Aravind; Venkatesh, Rengaraj

    2015-03-01

    A relatively simple and inexpensive technique to train surgeons in phacoemulsification using a goat's eye integrated with a human cataractous nucleus is described. The goat's eye is placed on a bed of cotton within the lumen of a cylindrical container. This is then mounted on a rectangular thermocol so that the limbus is presented at the surgical field. After making a clear corneal entry with a keratome, the trainer makes a 5-5.5 mm continuous curvilinear capsulorhexis in the anterior lens capsule, creates a crater of adequate depth in the cortex and inserts the human nucleus within this crater in the goat's capsular bag. The surgical wound is sutured, and the goat's eye is ready for training. Creating the capsulorhexis with precision and making the crater of adequate depth to snugly accommodate the human nucleus are the most important steps to prevent excessive wobbling of the nucleus while training. PMID:25971179

  18. Goat's eye integrated with a human cataractous lens: A training model for phacoemulsification

    PubMed Central

    Sengupta, Sabyasachi; Dhanapal, Praveen; Nath, Manas; Haripriya, Aravind; Venkatesh, Rengaraj

    2015-01-01

    A relatively simple and inexpensive technique to train surgeons in phacoemulsification using a goat's eye integrated with a human cataractous nucleus is described. The goat's eye is placed on a bed of cotton within the lumen of a cylindrical container. This is then mounted on a rectangular thermocol so that the limbus is presented at the surgical field. After making a clear corneal entry with a keratome, the trainer makes a 5–5.5 mm continuous curvilinear capsulorhexis in the anterior lens capsule, creates a crater of adequate depth in the cortex and inserts the human nucleus within this crater in the goat's capsular bag. The surgical wound is sutured, and the goat's eye is ready for training. Creating the capsulorhexis with precision and making the crater of adequate depth to snugly accommodate the human nucleus are the most important steps to prevent excessive wobbling of the nucleus while training. PMID:25971179

  19. Carbon turnover in the water-soluble protein of the adult human lens

    PubMed Central

    Stewart, Daniel N.; Lango, Jozsef; Nambiar, Krishnan P.; Falso, Miranda J. S.; FitzGerald, Paul G.; Rocke, David M.; Hammock, Bruce D.

    2013-01-01

    Purpose Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the 14C bomb pulse. Methods Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The 14C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the 14C bomb pulse. Results The water-insoluble fractions possessed 14C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction. Conclusions As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining

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

    NASA Technical Reports Server (NTRS)

    Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

    2000-01-01

    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.

  1. Age and Smoking Related Changes in Metal Ion Levels in Human Lens: Implications for Cataract Formation

    PubMed Central

    Langford-Smith, Alex; Tilakaratna, Viranga; Lythgoe, Paul R.; Clark, Simon J.; Bishop, Paul N.; Day, Anthony J.

    2016-01-01

    Age-related cataract formation is the primary cause of blindness worldwide and although treatable by surgical removal of the lens the majority of sufferers have neither the finances nor access to the medical facilities required. Therefore, a better understanding of the pathogenesis of cataract may identify new therapeutic targets to prevent or slow its progression. Cataract incidence is strongly correlated with age and cigarette smoking, factors that are often associated with accumulation of metal ions in other tissues. Therefore this study evaluated the age-related changes in 14 metal ions in 32 post mortem human lenses without known cataract from donors of 11 to 82 years of age by inductively coupled plasma mass spectrometry; smoking-related changes in 10 smokers verses 14 non-smokers were also analysed. A significant age-related increase in selenium and decrease in copper ions was observed for the first time in the lens tissue, where cadmium ion levels were also increased as has been seen previously. Aluminium and vanadium ions were found to be increased in smokers compared to non-smokers (an analysis that has only been carried out before in lenses with cataract). These changes in metal ions, i.e. that occur as a consequence of normal ageing and of smoking, could contribute to cataract formation via induction of oxidative stress pathways, modulation of extracellular matrix structure/function and cellular toxicity. Thus, this study has identified novel changes in metal ions in human lens that could potentially drive the pathology of cataract formation. PMID:26794210

  2. TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium

    PubMed Central

    Zhao, Peter Y.; Gan, Geliang; Peng, Shaomin; Wang, Shao-Bin; Chen, Bo; Adelman, Ron A.; Rizzolo, Lawrence J.

    2015-01-01

    Purpose. Calcium regulates many functions of the RPE. Its concentration in the subretinal space and RPE cytoplasm is closely regulated. Transient receptor potential (TRP) channels are a superfamily of ion channels that are moderately calcium-selective. This study investigates the subcellular localization and potential functions of TRP channels in a first-passage culture model of human fetal RPE (hfRPE). Methods. The RPE isolated from 15- to 16-week gestation fetuses were maintained in serum-free media. Cultures were treated with barium chloride (BaCl2) in the absence and presence of TRP channel inhibitors and monitored by the transepithelial electrical resistance (TER). The expression of TRP channels was determined using quantitative RT-PCR, immunoblotting, and immunofluorescence confocal microscopy. Results. Barium chloride substantially decreased TER and disrupted cell–cell contacts when added to the apical surface of RPE, but not when added to the basolateral surface. The effect could be partially blocked by the general TRP inhibitor, lanthanum chloride (LaCl3, ~75%), or an inhibitor of calpain (~25%). Family member-specific inhibitors, ML204 (TRPC4) and HC-067047 (TRPV4), had no effect on basal channel activity. Expression of TRPC4, TRPM1, TRPM3, TRPM7, and TRPV4 was detected by RT-PCR and immunoblotting. The TRPM3 localized to the base of the primary cilium, and TRPC4 and TRPM3 localized to apical tight junctions. The TRPV4 localized to apical microvilli in a small subset of cells. Conclusions. The TRP channels localized to subdomains of the apical membrane, and BaCl2 was only able to dissociate tight junctions when presented to the apical membrane. The data suggest a potential role for TRP channels as sensors of [Ca2+] in the subretinal space. PMID:25736794

  3. Estrogen increases the permeability of the cultured human cervical epithelium by modulating cell deformability.

    PubMed

    Gorodeski, G I

    1998-09-01

    Estrogens increase secretion of cervical mucus in females. The objective of this research was to study the mechanisms of estrogen action. The experimental models were human CaSki (endocervical) and hECE (ectocervical) epithelial cells cultured on filters. Incubation in steroid-free medium increased transepithelial electrical resistance (RTE) and decreased epithelial permeability to the cell-impermeant acid pyranine. Estrogen treatment reversed the effects, indicating estrogen decreases epithelial paracellular resistance. The estrogen effect was time and dose related (EC50 approximately 1 nM) and specific (estradiol = diethylstilbestrol > estrone, estriol; no effect by progesterone, testosterone, or cortisol) and was blocked by progesterone, tamoxifen, and ICI-182780 (an estrogen receptor antagonist). Estrogen treatment did not modulate dilution potential or changes in RTE in response to diC8 or to low extracellular Ca2+ (modulators of tight junctional resistance). In contrast, estrogen augmented decreases in RTE in response to hydrostatic and hypertonic gradients [modulators of resistance of lateral intercellular space (RLIS)], suggesting estrogen decreases RLIS. Estrogen decreased cervical cell size, shortened response time relative to changes in cell size after hypertonic challenge, and augmented the decrease in cell size in response to hypertonic and hydrostatic gradients. Lowering luminal NaCl had no significant effect on RTE, and the Cl- channel blocker diphenylamine-2-carboxylate attenuated the hypertonicity-induced decrease in cell size to the same degree in control and estrogen-treated cells, suggesting estrogen effects on permeability and cell size are not mediated by modulating Na+ or Cl- transport. In contrast, estrogen increased cellular G-actin levels, suggesting estrogens shift actin steady-state toward G-actin and the cervical cell cytoskeleton toward a more flexible structure. We suggest that the mechanism by which estrogens decrease RLIS and

  4. Proteomics of the human endometrial glandular epithelium and stroma from the proliferative and secretory phases of the menstrual cycle.

    PubMed

    Hood, Brian L; Liu, Baoquan; Alkhas, Addie; Shoji, Yutaka; Challa, Rusheeswar; Wang, Guisong; Ferguson, Susan; Oliver, Julie; Mitchell, Dave; Bateman, Nicholas W; Zahn, Christopher M; Hamilton, Chad A; Payson, Mark; Lessey, Bruce; Fazleabas, Asgerally T; Maxwell, G Larry; Conrads, Thomas P; Risinger, John I

    2015-04-01

    Despite its importance in reproductive biology and women's health, a detailed molecular-level understanding of the human endometrium is lacking. Indeed, no comprehensive studies have been undertaken to elucidate the important protein expression differences between the endometrial glandular epithelium and surrounding stroma during the proliferative and midsecretory phases of the menstrual cycle. We utilized laser microdissection to harvest epithelial cells and stromal compartments from proliferative and secretory premenopausal endometrial tissue and performed a global, quantitative mass spectrometry-based proteomics analysis. This analysis identified 1224 total proteins from epithelial cells, among which 318 were differentially abundant between the proliferative and secretory phases (q < 0.05), and 1005 proteins from the stromal compartments, 19 of which were differentially abundant between the phases (q < 0.05). Several proteins were chosen for validation by immunohistochemistry in an independent set of uterine tissues, including carboxypeptidase M, tenascin C, neprilysin, and ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3). ENPP3, which was elevated in epithelial glandular cells in the secretory phase, was confirmed to be elevated in midsecretory-phase baboon uterine lavage samples and also observed to have an N-linked glycosylated form that was not observed in the proliferative phase. This study provides a detailed view into the global proteomic alterations of the epithelial cells and stromal compartments of the cycling premenopausal endometrium. These proteomic alterations during endometrial remodeling provide a basis for numerous follow-up investigations on the function of these differentially regulated proteins and their role in reproductive biology and endometrial pathologies. PMID:25695723

  5. Stimulation of Aquaporin-Mediated Fluid Transport by Cyclic GMP in Human Retinal Pigment Epithelium In Vitro

    PubMed Central

    Baetz, Nicholas W.; Stamer, W. Daniel; Yool, Andrea J.

    2012-01-01

    Purpose The retinal pigment epithelium (RPE) expresses aquaporin-1 (AQP1) and components of the natriuretic peptide signaling pathway. We hypothesized that stimulation of the natriuretic signaling pathway in RPE with atrial natriuretic peptide (ANP) and with membrane-permeable analogs of cGMP would induce a net apical-to-basal transport of fluid. Methods The hypothesis was tested using human RPE cultures that retain properties seen in vivo. Confluent monolayers were treated with ANP or membrane-permeable cGMP analogs in the presence of anantin, H-8, and an AQP1 inhibitor, AqB013. Fluid movement from the apical to basal chambers was measured by weight and used to calculate net fluid transport. Results Our results demonstrated a 40% increase in net apical-to-basal fluid transport by ANP (5 μM) that was inhibited completely by the ANP receptor antagonist anantin and a 60% increase in net apical-to-basal fluid transport in response to the extracellularly applied membrane-permeable cGMP analog pCPT-cGMP (50 μM), which was not affected by the protein kinase G inhibitor H-8. The aquaporin antagonist AqB013 (20 μM) inhibited the cGMP-stimulated RPE fluid flux. Conclusions The effect of cGMP is consistent with an enhancement of the net fluid flux in RPE mediated by AQP1 channels. Pharmacologic activation of cGMP signaling and concomitant stimulation of fluid uptake from the subretinal space could offer insights into a new approach to treating or reducing the risk of retinal detachment. PMID:22427546

  6. Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept.

    PubMed

    Boecker, Werner; Buerger, Horst

    2003-10-01

    Although experimental data clearly confirm the existence of self-renewing mammary stem cells, the characteristics of such progenitor cells have never been satisfactorily defined. Using a double immunofluorescence technique for simultaneous detection of the basal cytokeratin 5, the glandular cytokeratins 8/18 and the myoepithelial differentiation marker smooth muscle actin (SMA), we were able to demonstrate the presence of CK5+ cells in human adult breast epithelium. These cells have the potential to differentiate to either glandular (CK8/18+) or myoepithelial cells (SMA+) through intermediary cells (CK5+ and CK8/18+ or SMA+). We therefore proceeded on the assumption that the CK5+ cells are phenotypically and behaviourally progenitor (committed adult stem) cells of human breast epithelium. Furthermore, we furnish evidence that most of these progenitor cells are located in the luminal epithelium of the ductal lobular tree. Based on data obtained in extensive analyses of proliferative breast disease lesions, we have come to regard usual ductal hyperplasia as a progenitor cell-derived lesion, whereas most breast cancers seem to evolve from differentiated glandular cells. Double immunofluorescence experiments provide a new tool to characterize phenotypically progenitor (adult stem) cells and their progenies. This model has been shown to be of great value for a better understanding not only of normal tissue regeneration but also of proliferative breast disease. Furthermore, this model provides a new tool for unravelling further the regulatory mechanisms that govern normal and pathological cell growth. PMID:14521517

  7. EGCG prevents tryptophan oxidation of cataractous ocular lens human γ-crystallin in presence of H2O2.

    PubMed

    Chaudhury, Susmitnarayan; Ghosh, Ishita; Saha, Gautam; Dasgupta, Swagata

    2015-01-01

    Disruption of the short range order of proteins present in the ocular lens leads to cataract resulting in a loss of transparency. Human γ-crystallin (HGC), a water soluble protein present in the lens is known to aggregate with aging. A modified form of HGC (HGC(c)) was isolated from cataractous human ocular lens extract and the number of Trp residues that undergo oxidation was determined. The extent of oxidized Trp (N-formyl kynurenine) in HGC due to cataract formation was determined, primarily using fluorescence spectroscopy. The ability of (-)-epigallocatechin gallate (EGCG) to retain its antioxidant effect even in the presence of H2O2 was investigated. This was monitored by its ability to prevent the modification of intact Trp residues in HGC(c) isolated from cataractous human eye lens. Significant Trp fluorescence quenching occurs on interaction of the green tea component, EGCG with HGC(c) accompanied by a red shift. Docking studies were employed to substantiate the experimental results. As eye lens proteins are prone to oxidative stress it is essential that a clear understanding of the effects of the components generated in vivo vis-à-vis the antioxidant effects of natural polyphenols be obtained. PMID:25841365

  8. A Critical Meta-Analysis of Lens Model Studies in Human Judgment and Decision-Making

    PubMed Central

    Kaufmann, Esther; Reips, Ulf-Dietrich; Wittmann, Werner W.

    2013-01-01

    increase in values of the lens model components, b) reduced heterogeneity between studies, and c) increases the success of bootstrapping. We argue that psychometric meta-analysis is useful for accurately evaluating human judgment and show the success of bootstrapping. PMID:24391781

  9. Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways

    PubMed Central

    Deng, Xuefeng; Yan, Ziying; Cheng, Fang; Engelhardt, John F.; Qiu, Jianming

    2016-01-01

    Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family, and is an emerging human pathogenic respiratory virus. In vitro, HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells, we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably, HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase–related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and, more importantly, we identified that two Y-family DNA polymerases, Pol η and Pol κ, are involved in HBoV1 genome amplification. Overall, we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells, which is dependent on the cellular DNA damage and repair pathways. PMID:26765330

  10. A stochastic model of eye lens growth.

    PubMed

    Šikić, Hrvoje; Shi, Yanrong; Lubura, Snježana; Bassnett, Steven

    2015-07-01

    The size and shape of the ocular lens must be controlled with precision if light is to be focused sharply on the retina. The lifelong growth of the lens depends on the production of cells in the anterior epithelium. At the lens equator, epithelial cells differentiate into fiber cells, which are added to the surface of the existing fiber cell mass, increasing its volume and area. We developed a stochastic model relating the rates of cell proliferation and death in various regions of the lens epithelium to deposition of fiber cells and radial lens growth. Epithelial population dynamics were modeled as a branching process with emigration and immigration between proliferative zones. Numerical simulations were in agreement with empirical measurements and demonstrated that, operating within the strict confines of lens geometry, a stochastic growth engine can produce the smooth and precise growth necessary for lens function. PMID:25816743

  11. Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model

    PubMed Central

    Fan, Xingjun; Reneker, Lixing W.; Obrenovich, Mark E.; Strauch, Christopher; Cheng, Rongzhu; Jarvis, Simon M.; Ortwerth, Beryl J.; Monnier, Vincent M.

    2006-01-01

    Senile cataracts are associated with progressive oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. We hypothesized that the Maillard reaction, which leads browning and aroma development during the baking of foods, would occur between the lens proteins and the highly reactive oxidation products of vitamin C. To test this hypothesis, we engineered a mouse that selectively overexpresses the human vitamin C transporter SVCT2 in the lens. Consequently, lenticular levels of vitamin C and its oxidation products were 5- to 15-fold elevated, resulting in a highly compressed aging process and accelerated formation of several protein-bound advanced Maillard reaction products identical with those of aging human lens proteins. These data strongly implicate vitamin C in lens crystallin aging and may serve as a model for protein aging in other tissues particularly rich in vitamin C, such as the hippocampal neurons and the adrenal gland. The hSVCT2 mouse is expected to facilitate the search for drugs that inhibit damage by vitamin C oxidation products. PMID:17075057

  12. Infection of human urethral epithelium with Neisseria gonorrhoeae elicits an upregulation of host anti-apoptotic factors and protects cells from staurosporine-induced apoptosis.

    PubMed

    Binnicker, Matthew J; Williams, Richard D; Apicella, Michael A

    2003-08-01

    In order to better understand the host response to an infection with Neisseria gonorrhoeae, microarray technology was used to analyse the gene expression profile between uninfected and infected human urethral epithelium. The anti-apoptotic genes bfl-1, cox-2 and c-IAP-2 were identified to be upregulated approximately eight-, four- or twofold, respectively, following infection. Subsequent assays including RT-PCR, real time RT-PCR and RNase protection confirmed the increased expression of these apoptotic regulators, and identified that a fourth anti-apoptotic factor, mcl-1, is also upregulated. RT-PCR and RNase protection also showed that key pro-apoptotic factors including bax, bad and bak do not change in expression. Furthermore, our studies demonstrated that infection with the gonococcus partially protects urethral epithelium from apoptosis induced by the protein kinase inhibitor, staurosporine (STS). This work shows that following infection with Neisseria gonorrhoeae, several host anti-apoptotic factors are upregulated. In addition, a gonococcal infection protects host cells from subsequent STS-induced death. The regulation of host cell death by the gonococcus may represent a mechanism employed by this pathogen to survive and proliferate in host epithelium. PMID:12864814

  13. Progesterone-Based Intrauterine Device Use Is Associated with a Thinner Apical Layer of the Human Ectocervical Epithelium and a Lower ZO-1 mRNA Expression1

    PubMed Central

    Tjernlund, Annelie; Carias, Ann M.; Andersson, Sonia; Gustafsson-Sanchez, Susanna; Röhl, Maria; Petersson, Pernilla; Introini, Andrea; Hope, Thomas J.; Broliden, Kristina

    2015-01-01

    ABSTRACT Currently, whether hormonal contraceptives affect male to female human immunodeficiency virus (HIV) transmission is being debated. In this study, we investigated whether the use of progesterone-based intrauterine devices (pIUDs) is associated with a thinning effect on the ectocervical squamous epithelium, down-regulation of epithelial junction proteins, and/or alteration of HIV target cell distribution in the human ectocervix. Ectocervical tissue biopsies from healthy premenopausal volunteers using pIUDs were collected and compared to biopsies obtained from two control groups, namely women using combined oral contraceptives (COCs) or who do not use hormonal contraceptives. In situ staining and image analysis were used to measure epithelial thickness and the presence of HIV receptors in tissue biopsies. Messenger RNA levels of epithelial junction markers were measured by quantitative PCR. The epithelial thickness displayed by women in the pIUD group was similar to those in the COC group, but significantly thinner as compared to women in the no hormonal contraceptive group. The thinner epithelial layer of the pIUD group was specific to the apical layer of the ectocervix. Furthermore, the pIUD group expressed significantly lower levels of the tight junction marker ZO-1 within the epithelium as compared to the COC group. Similar expression levels of HIV receptors and coreceptors CD4, CCR5, DC-SIGN, and Langerin were observed in the three study groups. Thus, women using pIUD displayed a thinner apical layer of the ectocervical epithelium and reduced ZO-1 expression as compared to control groups. These data suggest that pIUD use may weaken the ectocervical epithelial barrier against invading pathogens, including HIV. PMID:25588510

  14. Progesterone-based intrauterine device use is associated with a thinner apical layer of the human ectocervical epithelium and a lower ZO-1 mRNA expression.

    PubMed

    Tjernlund, Annelie; Carias, Ann M; Andersson, Sonia; Gustafsson-Sanchez, Susanna; Röhl, Maria; Petersson, Pernilla; Introini, Andrea; Hope, Thomas J; Broliden, Kristina

    2015-03-01

    Currently, whether hormonal contraceptives affect male to female human immunodeficiency virus (HIV) transmission is being debated. In this study, we investigated whether the use of progesterone-based intrauterine devices (pIUDs) is associated with a thinning effect on the ectocervical squamous epithelium, down-regulation of epithelial junction proteins, and/or alteration of HIV target cell distribution in the human ectocervix. Ectocervical tissue biopsies from healthy premenopausal volunteers using pIUDs were collected and compared to biopsies obtained from two control groups, namely women using combined oral contraceptives (COCs) or who do not use hormonal contraceptives. In situ staining and image analysis were used to measure epithelial thickness and the presence of HIV receptors in tissue biopsies. Messenger RNA levels of epithelial junction markers were measured by quantitative PCR. The epithelial thickness displayed by women in the pIUD group was similar to those in the COC group, but significantly thinner as compared to women in the no hormonal contraceptive group. The thinner epithelial layer of the pIUD group was specific to the apical layer of the ectocervix. Furthermore, the pIUD group expressed significantly lower levels of the tight junction marker ZO-1 within the epithelium as compared to the COC group. Similar expression levels of HIV receptors and coreceptors CD4, CCR5, DC-SIGN, and Langerin were observed in the three study groups. Thus, women using pIUD displayed a thinner apical layer of the ectocervical epithelium and reduced ZO-1 expression as compared to control groups. These data suggest that pIUD use may weaken the ectocervical epithelial barrier against invading pathogens, including HIV. PMID:25588510

  15. Engineering Airway Epithelium

    PubMed Central

    Soleas, John P.; Paz, Ana; Marcus, Paula; McGuigan, Alison; Waddell, Thomas K.

    2012-01-01

    Airway epithelium is constantly presented with injurious signals, yet under healthy circumstances, the epithelium maintains its innate immune barrier and mucociliary elevator function. This suggests that airway epithelium has regenerative potential (I. R. Telford and C. F. Bridgman, 1990). In practice, however, airway regeneration is problematic because of slow turnover and dedifferentiation of epithelium thereby hindering regeneration and increasing time necessary for full maturation and function. Based on the anatomy and biology of the airway epithelium, a variety of tissue engineering tools available could be utilized to overcome the barriers currently seen in airway epithelial generation. This paper describes the structure, function, and repair mechanisms in native epithelium and highlights specific and manipulatable tissue engineering signals that could be of great use in the creation of artificial airway epithelium. PMID:22523471

  16. Spatial analysis of human lens aquaporin-0 post-translational modifications by MALDI mass spectrometry tissue profiling.

    PubMed

    Gutierrez, Danielle B; Garland, Donita; Schey, Kevin L

    2011-12-01

    Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 μm. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation. PMID:22036630

  17. AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

    PubMed

    Raghavan, Cibin T; Nagaraj, Ram H

    2016-08-01

    Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis. PMID:27263094

  18. Development, validation and implementation of an in vitro model for the study of metabolic and immune function in normal and inflamed human colonic epithelium.

    PubMed

    Pedersen, Gitte

    2015-01-01

    Ulcerative colitis (UC) and Crohn's disease (CD), collectively referred to as inflammatory bowel disease (IBD), are chronic immune disorders affecting the gastrointestinal tract. The aetiology of IBD remains an enigma, but increasing evidence suggests that the development of IBD may be triggered by a disturbance in the balance between gut commensal bacteria and host response in the intestinal mucosa. It is now known that epithelial cells have the capacity to secrete and respond to a range of immunological mediators and this suggests that these cells play a prominent role in the pathogenesis of IBD. Current knowledge about the intestinal epithelium has mainly been obtained using models based on animal cells, transformed human intestinal cell lines and isolated cells from resected colonic bowel segments. Species difference, malignant origin and confounders related to surgery, obviously make these cell models however less applicable for patophysiological studies. Consequently, there was a clear need for models of representative intestinal epithelial cells that would allow functional and dynamic studies of the differentiated human colonic epithelium in vitro. The primary purpose of this thesis was to explore and validate the optimal conditions for establishing a model based on short-term cultures of human colonic epithelial cells obtained from endoscopical biopsies. The cell cultures were accordingly used to describe the interplay between proinflammatory cytokines and colonic epithelium, with focus on alterations in viability, butyrate metabolism and secretion of a chemokine and metalloproteinases (MMP). Finally, the model was used to characterize expression and activation of receptors like toll like receptor (TLR)9 and peroxisome activated proliferators (PPAR)- known to be important players in regulation of innate and adaptive immune responses in human colonic epithelium. The results showed that it is possible to establish short-term cultures of representative, viable

  19. Studies on the Thomsen-Friedenreich antigen in human colon with the lectin Amaranthin. Normal and neoplastic epithelium express only cryptic T antigen.

    PubMed

    Sata, T; Roth, J; Zuber, C; Stamm, B; Rinderle, S J; Goldstein, I J; Heitz, P U

    1992-02-01

    The lectin Amaranthin has been shown to be highly specific for the galactose beta 1,3 N-acetylgalactosamine-alpha and sialic acid alpha 2,3 galactose beta 1,3 N-acetylgalactosamine-alpha sequence which represents the Thomsen-Friedenreich (T) antigen and its cryptic form, respectively. Previously, we demonstrated the usefulness of gold-labeled Amaranthin for the histochemical detection of the T antigen and its cryptic form. Application of the galactose oxidase (GO)-Schiff sequence abolished lectin binding to the T antigen but not its cryptic form, and therefore permitted their differentiation. In the present study we have analyzed by light and electron microscopy the distribution and subcellular localization of Amaranthin binding sites in normal, dysplastic and neoplastic colonic epithelium. Furthermore, a monoclonal antibody raised against synthetic galactose bera 1,3 N-acetylgalactosamine-alpha-bovine serum albumin was applied as a reagent for the T antigen. In normal colonic mucosa, two different Amaranthin staining patterns existed: (a) reactivity restricted to the lower portion of the crypts which was principally observed in the left colon, and (b) reactivity along the entire length of the crypts and in the surface epithelium with goblet cell staining in the upper portion of the crypts which was principally observed in the right colon. This Amaranthin staining was resistant to GO-Schiff treatment. No immunostaining with the monoclonal anti-T antigen was observed. Investigation of transitional mucosa, adenocarcinomas of different degrees of differentiation and mucinous carcinomas as well as adenomas with different degrees of dysplasia all revealed positive Amaranthin staining. The lectin staining was resistant to GO-Schiff treatment, and immunolabeling with the monoclonal antibody against the T antigen was absent. These results indicate that only the cryptic form of the T antigen is expressed in normal, dysplastic and neoplastic human colonic epithelium. PMID

  20. Effect of contact lens material on cytotoxicity potential of multipurpose solutions using human corneal epithelial cells

    PubMed Central

    Tanti, N.C.; Crockett, B.; Mansour, L.; Jones, L.

    2011-01-01

    Purpose Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses. Methods The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance. Results A significant reduction in viability (between 30 to 50%) was observed with cells exposed to lenses soaked in ReNu and OFX. A significant downregulation of α3 and β1 integrins, with integrin expression ranging from 60% to 75% of control (cells with no lens), was also observed with OFX and ReNu-soaked lenses. With the exception of BA, all other lenses soaked in OFX resulted in significant caspase activation, whereby over 18% of cells stained positive for caspases. Minimal caspase activation was observed in cells exposed to ReNu and Solo soaked lenses. For both OFX and ReNu, exposing cells to at least a 5% dilution had a significant effect on viability and integrin expression. While Complete and Solo did not lead to reduction in viability, cells exposed to a 10% dilution showed reduced integrin expression down to less than 70% of control value. Comparing cell response to diluted MPS solutions and various MPS-soaked lenses showed that it is not possible to reliably use cell response to MPS dilution alone to assess MPS

  1. Inhibition of Unfolding and Aggregation of Lens Protein Human Gamma D Crystallin by Sodium Citrate

    PubMed Central

    Goulet, Daniel R.; Knee, Kelly M.; King, Jonathan A.

    2012-01-01

    Cataract affects 1 in 6 Americans over the age of 40, and is considered a global health problem. Cataract is caused by the aggregation of unfolded or damaged proteins in the lens, which accumulate as an individual ages. Currently, surgery is the only available treatment for cataract, however, small molecules have been suggested as potential preventative therapies. In this work, we study the effect of sodium citrate on the stability of Human γD Crystallin (HγD-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S HγD-Crys and I90F HγD-Crys. In equilibrium unfolding-refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-td of WT HγD-Crys and L5S HγD-Crys by 0.3 M GuHCl, the C-td by 0.6M GuHCl, and the single transition of I90F HγD-Crys by 0.4M GuHCl. In kinetic unfolding reactions, sodium citrate demonstrates a measurable stabilization effect only for the mutant I90F HγD-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F HγD-Crys can be observed, which was not observed in the absence of citrate. Rate of aggregation was measured using solution turbidity, and sodium citrate demonstrates negligible effect on rate of aggregation of WT HγD-Crys, but considerably slows the rate of aggregation of both L5S HγD-Crys and I90F HγD-Crys. The presence of sodium citrate dramatically slows refolding of WT HγD-Crys and I90F HγD-Crys, but has a significantly smaller effect on the refolding of L5S HγD-Crys. The differential stabilizing effect of sodium citrate suggests that the ion is binding to a partially unfolded conformation of the C-td, but a solution-based Hofmeister effect cannot be eliminated as a possible explanation for the effects observed. These results suggest that sodium citrate may be a potential preventative agent for cataract. PMID:21600897

  2. MALDI Imaging Mass Spectrometry Spatially Maps Age-Related Deamidation and Truncation of Human Lens Aquaporin-0

    PubMed Central

    Wenke, Jamie L.; Rose, Kristie L.; Spraggins, Jeffrey M.; Schey, Kevin L.

    2015-01-01

    Purpose To spatially map human lens Aquaporin-0 (AQP0) protein modifications, including lipidation, truncation, and deamidation, from birth through middle age using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Methods Human lens sections were water-washed to facilitate detection of membrane protein AQP0. We acquired MALDI images from eight human lenses ranging in age from 2 months to 63 years. In situ tryptic digestion was used to generate peptides of AQP0 and peptide images were acquired on a 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Peptide extracts were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and database searched to identify peptides observed in MALDI imaging experiments. Results Unmodified, truncated, and fatty acid–acylated forms of AQP0 were detected in protein imaging experiments. Full-length AQP0 was fatty acid acylated in the core and cortex of young (2- and 4-month) lenses. Acylated and unmodified AQP0 were C-terminally truncated in older lens cores. Deamidated tryptic peptides (+0.9847 Da) were mass resolved from unmodified peptides by FTICR MS. Peptide images revealed differential localization of un-, singly-, and doubly-deamidated AQP0 C-terminal peptide (239–263). Deamidation was present at 4 months and increases with age. Liquid chromatography–MS/MS results indicated N246 undergoes deamidation more rapidly than N259. Conclusions Results indicated AQP0 fatty acid acylation and deamidation occur during early development. Progressive age-related AQP0 processing, including deamidation and truncation, was mapped in human lenses as a function of age. The localization of these modified AQP0 forms suggests where AQP0 functions may change throughout lens development and aging. PMID:26574799

  3. Mitochondrial "movement" and lens optics following oxidative stress from UV-B irradiation: cultured bovine lenses and human retinal pigment epithelial cells (ARPE-19) as examples.

    PubMed

    Bantseev, Vladimir; Youn, Hyun-Yi

    2006-12-01

    Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV-B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-19 cells were irradiated with broadband UV-B radiation at energy levels of 0.5 and 1.0 J/cm(2). Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium-neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45 degrees on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria-specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta

  4. Melatonin and Sleep-Wake Rhythms before and after Ocular Lens Replacement in Elderly Humans

    PubMed Central

    Giménez, Marina; Beersma, Domien; Daan, Serge; van der Pol, Bert; Kanis, Martijn; van Norren, Dick; Gordijn, Marijke

    2016-01-01

    Light of short wavelengths has been shown to play a key role in non-image forming responses. Due to aging, the ocular lens becomes more yellow reducing the transmission of short wavelengths in the elderly. In the present study, we make use of cataract surgery to investigate the effects of a relative increase of short wavelength transmission on melatonin- and sleep-wake rhythms (N = 14). We observed, on average, a delay of the sleep-wake and the nocturnal melatonin rhythms after cataract surgery. This delay is tentatively attributed to a relatively large increase of light transmittance in the evening hours more than an increase of the already relatively high light intensities found in the daytime. The later phase that we observed after cataract surgery (clear lens) as compared to the earlier phase observed before cataract (yellowish lens) is in agreement with the general later phase reported in the young (clear lens) population. PMID:26891336

  5. A human papillomavirus type 18 E6/E7 transgene sensitizes mouse lens cells to human wild-type p53-mediated apoptosis.

    PubMed

    Nakamura, T; Williams-Simons, L; Westphal, H

    1997-06-26

    We have studied the concerted action of factors that influence the balance between cell proliferation and cell death in the developing lens of transgenic mice. We show that a human papillomavirus type 18 (HPV18) E6/E7 transgene that predominantly expresses the viral E7 gene product triggers apoptosis in a dose dependent manner, and causes retardation of lens growth or microphakia. E7 is known to inactivate pRB, the product of the retinoblastoma gene, and to enhance the action of p53. Our earlier work had demonstrated that over-expression of p53 itself can cause apoptosis of lens cells, and that a mutant p53 allele can interfere with this process. In the present study, we examined lenses that simultaneously express different constellations of the HPV18 E6/E7, wild-type and mutant human p53, and wild-type human pRB transgenes. We observed that lens cells expressing the HPV18 transgene are more sensitive to wild-type human p53 action than normal lens cells. As a result, there is severe microphakia in lenses that express both the HPV18 and the wild-type p53 transgenes. By contrast, apoptosis was reduced in lenses that co-expressed the HPV18 and either the pRB or the mutant p53 transgene. We conclude that levels of wild-type p53 are critical, and that any excess of p53 or suppression of pRB can cause cell death. Our results encourage attempts to counteract the deleterious action of human papillomaviruses in cervical cancer by a combination of measures that decrease cell proliferation and enhance apoptosis. PMID:9223662

  6. Human lens coloration and aging. Evidence for crystallin modification by the major ultraviolet filter, 3-hydroxy-kynurenine O-beta-D-glucoside.

    PubMed

    Hood, B D; Garner, B; Truscott, R J

    1999-11-12

    The human lens becomes increasingly yellow with age and thereby reduces our perception of blue light. This coloration is associated with lens proteins (crystallins), but its molecular basis was unknown. Here we show that the coloration occurs because of the interaction of crystallins with a UV filter compound, 3-hydroxykynurenine glucoside (3-OHKG). Crystallin modification results from deamination of the 3-OHKG amino acid side chain, yielding an unsaturated ketone that is susceptible to nucleophilic attack by cysteine, histidine, and lysine residues. This novel protein modification contributes to age-related lens coloration and may play a role in human nuclear cataractogenesis. PMID:10551806

  7. The Secreted Aspartyl Proteinases Sap1 and Sap2 Cause Tissue Damage in an In Vitro Model of Vaginal Candidiasis Based on Reconstituted Human Vaginal Epithelium

    PubMed Central

    Schaller, Martin; Bein, Matthias; Korting, Hans C.; Baur, Stefan; Hamm, Gerald; Monod, Michel; Beinhauer, Sabine; Hube, Bernhard

    2003-01-01

    Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, and SAP10 was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of SAP1, SAP4, and SAP5. Additional transcripts of SAP6 and SAP7 were detected at a later stage of the artificial infection (24 h). Similar SAP expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by C. albicans was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by C. albicans cells. Furthermore, SAP null mutants lacking either SAP1 or SAP2 had a drastically reduced potential to cause tissue damage even though SAP3, SAP4, and SAP7 were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking SAP3 or SAP4 to SAP6 was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in C. albicans vaginal infections. PMID:12761103

  8. Human testicular peritubular cells secrete pigment epithelium-derived factor (PEDF), which may be responsible for the avascularity of the seminiferous tubules.

    PubMed

    Windschüttl, S; Kampfer, C; Mayer, C; Flenkenthaler, F; Fröhlich, T; Schwarzer, J U; Köhn, F M; Urbanski, H; Arnold, G J; Mayerhofer, A

    2015-01-01

    Male fertility depends on spermatogenesis, which takes place in the seminiferous tubules of the testis. This compartment is devoid of blood vessels, which are however found in the wall of the seminiferous tubules. Our proteomic study using cultured human testicular peritubular cells (HTPCs) i.e. the cells, which form this wall, revealed that they constitutively secrete pigment epithelium-derived factor, PEDF, which is known to exert anti-angiogenic actions. Immunohistochemistry supports its presence in vivo, in the human tubular wall. Co-culture studies and analysis of cell migration patterns showed that human endothelial cells (HUVECs) are repulsed by HTPCs. The factor involved is likely PEDF, as a PEDF-antiserum blocked the repulsing action. Thus testicular peritubular cells, via PEDF, may prevent vascularization of human seminiferous tubules. Dihydrotestosterone (DHT) increased PEDF (qPCR) in HTPCs, however PEDF expression in the testis of a non-human primate occurs before puberty. Thus PEDF could be involved in the establishment of the avascular nature of seminiferous tubules and after puberty androgens may further reinforce this feature. Testicular microvessels and blood flow are known to contribute to the spermatogonial stem cell niche. Hence HTPCs via control of testicular microvessels may contribute to the regulation of spermatogonial stem cells, as well. PMID:26333415

  9. Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures — a new donor for cell therapy

    PubMed Central

    Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

    2016-01-01

    Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases. PMID:27009841

  10. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    PubMed Central

    Youn, Hyun-Yi; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. Results The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. Conclusions The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the

  11. Sustained-release genistein from nanostructured lipid carrier suppresses human lens epithelial cell growth

    PubMed Central

    Liu, Jin-Lu; Zhang, Wen-Ji; Li, Xue-Dong; Yang, Na; Pan, Wei-San; Kong, Jun; Zhang, Jin-Song

    2016-01-01

    AIM To design and investigate the efficacy of a modified nanostructured lipid carrier loaded with genistein (Gen-NLC) to inhibit human lens epithelial cells (HLECs) proliferation. METHODS Gen-NLC was made by melt emulsification method. The morphology, particle size (PS), zeta potentials (ZP), encapsulation efficiency (EE) and in vitro release were characterized. The inhibition effect of nanostructured lipid carrier (NLC), genistein (Gen) and Gen-NLC on HLECs proliferation was evaluated by cell counting kit-8 (CCK-8) assay, gene and protein expression of the proliferation marker Ki67 were evaluated with real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence analyses. RESULTS The mean PS of Gen-NLC was 80.12±1.55 nm with a mean polydispersity index of 0.11±0.02. The mean ZP was -7.14±0.38 mV and the EE of Gen in the nanoparticles was 92.3%±0.73%. Transmission electron microscopy showed that Gen-NLC displayed spherical-shaped particles covered by an outer-layer structure. In vitro release experiments demonstrated a prolonged drug release for 72h. The CCK-8 assay results showed the NLC had no inhibitory effect on HLECs and Gen-NLC displayed a much more prominent inhibitory effect on cellular growth compared to Gen of the same concentration. The mRNA and protein expression of Ki67 in LECs decreased significantly in Gen-NLC group. CONCLUSION Sustained drug release by Gen-NLCs may impede HLEC growth. PMID:27275415

  12. Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells.

    PubMed

    Lauf, Peter K; Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C

    2008-03-01

    This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs. PMID:18184876

  13. Complement and Humoral Adaptive Immunity in the Human Choroid Plexus: Roles for Stromal Concretions, Basement Membranes, and Epithelium

    PubMed Central

    Laule, Cornelia; Leung, Esther; Pavlova, Vladimira; Morgan, B. Paul; Esiri, Margaret M.

    2016-01-01

    The choroid plexus (CP) provides a barrier to entry of toxic molecules from the blood into the brain and transports vital molecules into the cerebrospinal fluid. While a great deal is known about CP physiology, relatively little is known about its immunology. Here, we show immunohistochemical data that help define the role of the CP in innate and adaptive humoral immunity. The results show that complement, in the form of C1q, C3d, C9, or C9neo, is preferentially deposited in stromal concretions. In contrast, immunoglobulin (Ig) G (IgG) and IgA are more often found in CP epithelial cells, and IgM is found in either locale. C4d, IgD, and IgE are rarely, if ever, seen in the CP. In multiple sclerosis CP, basement membrane C9 or stromal IgA patterns were common but were not specific for the disease. These findings indicate that the CP may orchestrate the clearance of complement, particularly by deposition in its concretions, IgA and IgG preferentially via its epithelium, and IgM by either mechanism. PMID:26994633

  14. Pigment-epithelium-derived factor (PEDF) occurs at a physiologically relevant concentration in human blood: purification and characterization.

    PubMed Central

    Petersen, Steen V; Valnickova, Zuzana; Enghild, Jan J

    2003-01-01

    Pigment epithelium-derived factor (PEDF) inhibits the formation of blood vessels in the eye by inducing apotosis in actively dividing endothelial cells. The activity of PEDF equals or supersedes that of other anti-angiogenic factors, including angiostatin, endostatin and thrombospondin-1. In addition, PEDF has the potential to promote the survival of neurons and affect their differentiation. Here we show that PEDF is present in plasma at a concentration of approx. 100 nM (5 microg/ml) or twice the level required to inhibit aberrant blood-vessel growth in the eye. Thus the systemic delivery of PEDF has the potential to affect angiogenesis or neurotrophic processes throughout the body, significantly expanding the putative physiological role of the protein. A complete map of all post-translational modifications revealed that authentic plasma PEDF carries an N-terminal pyroglutamate blocking group and an N-linked glycan at position Asn266. The pyroglutamate residue may regulate the activity of PEDF analogously to the manner in which it regulates thyrotropin-releasing hormone. PMID:12737624

  15. Confocal fluorescence microendoscopy of bronchial epithelium

    NASA Astrophysics Data System (ADS)

    Lane, Pierre M.; Lam, Stephen; McWilliams, Annette; Leriche, Jean C.; Anderson, Marshall W.; Macaulay, Calum E.

    2009-03-01

    Confocal microendoscopy permits the acquisition of high-resolution real-time confocal images of bronchial mucosa via the instrument channel of an endoscope. We report here on the construction and validation of a confocal fluorescence microendoscope and its use to acquire images of bronchial epithelium in vivo. Our objective is to develop an imaging method that can distinguish preneoplastic lesions from normal epithelium to enable us to study the natural history of these lesions and the efficacy of chemopreventive agents without biopsy removal of the lesion that can introduce a spontaneous regression bias. The instrument employs a laser-scanning engine and bronchoscope-compatible confocal probe consisting of a fiber-optic image guide and a graded-index objective lens. We assessed the potential of topical application of physiological pH cresyl violet (CV) as a fluorescence contrast-enhancing agent for the visualization of tissue morphology. Images acquired ex vivo with the confocal microendoscope were first compared with a bench-top confocal fluorescence microscope and conventional histology. Confocal images from five sites topically stained with CV were then acquired in vivo from high-risk smokers and compared to hematoxylin and eosin stained sections of biopsies taken from the same site. Sufficient contrast in the confocal imagery was obtained to identify cells in the bronchial epithelium. However, further improvements in the miniature objective lens are required to provide sufficient axial resolution for accurate classification of preneoplastic lesions.

  16. Profiling the microRNA Expression in Human iPS and iPS-derived Retinal Pigment Epithelium

    PubMed Central

    Wang, Heuy-Ching; Greene, Whitney A; Kaini, Ramesh R; Shen-Gunther, Jane; Chen, Hung-I H; Cai, Hong; Wang, Yufeng

    2014-01-01

    The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore, we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs, RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129–5p may play a role in promoting differentiation, while down-regulated miRNAs such as miR-367, miR-18b, and miR-20b are implicated in cell proliferation. Subsequent miRNA–target and network analysis revealed that these miRNAs are involved in cellular development, cell cycle progression, cell death, and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis, eye differentiation and development. PMID:25392691

  17. Identification of the in vivo truncation sites at the C-terminal region of alpha-A crystallin from aged bovine and human lens

    NASA Technical Reports Server (NTRS)

    Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Total alpha-A crystallin was purified from young versus old lens, followed by digestion with cyanogen bromide. Laser desorption mass spectrometry of the C-terminal fragment demonstrated age-dependent loss of one and five amino acids from the C-terminus of alpha-A crystallin from both bovine and human lens. These results demonstrate specific peptide bonds of alpha-A crystallin are cleaved during the aging process of the normal lens. The C-terminal region is cleaved in two places between the two hydroxyl-containing amino acids present in the sequence -P-S(T)-S-.

  18. Measurements of elastic modulus for human anterior lens capsule with atomic force microscopy: the effect of loading force.

    PubMed

    Tsaousis, Konstantinos T; Karagiannidis, Panagiotis G; Kopsachilis, Nikolaos; Symeonidis, Chrysanthos; Tsinopoulos, Ioannis T; Karagkiozaki, Varvara; Lamprogiannis, Lampros P; Logothetidis, Stergios

    2014-06-01

    The purpose of the study was to appraise the effect of loading force magnitude on the determination of the elastic modulus of the anterior lens capsule through atomic force microscopy. Four human anterior lens capsules taken during phacoemulsification cataract surgery were studied, free of epithelial cells, with atomic force microscopy. For the experiment, five different indentation loading forces were applied to near areas of the specimen. Experimental data was exported and analyzed according to the Hertz model to obtain the Young's modulus with regards to the elastic behavior of the material. Force-distance curves were acquired by applying a load of 2, 5, 10, 20 and 30 nN. When examining the results it was evident that determination of Young's modulus of the anterior lens capsule is dependent on the loading force concerning the examined range. Loading forces of 10 and 20 nN led to results without significant difference (p > 0.05) and more reproducible (coefficients of variation 12.4 and 11.7 %, respectively). PMID:24037592

  19. Safety profiles of anti-VEGF drugs: bevacizumab, ranibizumab, aflibercept and ziv-aflibercept on human retinal pigment epithelium cells in culture

    PubMed Central

    Malik, Deepika; Tarek, Mohamed; Caceres del Carpio, Javier; Ramirez, Claudio; Boyer, David; Kenney, M Cristina; Kuppermann, Baruch D

    2014-01-01

    Purpose To compare the safety profiles of antivascular endothelial growth factor (VEGF) drugs ranibizumab, bevacizumab, aflibercept and ziv-aflibercept on retinal pigment epithelium cells in culture. Methods Human retinal pigment epithelium cells (ARPE-19) were exposed for 24 h to four anti-VEGF drugs at 1/2×, 1×, 2× and 10× clinical concentrations. Cell viability and mitochondrial membrane potential assay were performed to evaluate early apoptotic changes and rate of overall cell death. Results Cell viability decreased at 10× concentrations in bevacizumab (82.38%, p=0.0001), aflibercept (82.68%, p=0.0002) and ziv-aflibercept (77.25%, p<0.0001), but not at lower concentrations. However, no changes were seen in cell viability in ranibizumab-treated cells at all concentrations including 10×. Mitochondrial membrane potential was slightly decreased in 10× ranibizumab-treated cells (89.61%, p=0.0006) and 2× and 10× aflibercept-treated cells (88.76%, 81.46%; p<0.01, respectively). A larger reduction in mitochondrial membrane potential was seen at 1×, 2× and 10× concentrations of bevacizumab (86.53%, 74.38%, 66.67%; p<0.01) and ziv-aflibercept (73.50%, 64.83% and 49.65% p<0.01) suggestive of early apoptosis at lower doses, including the clinical doses. Conclusions At clinical doses, neither ranibizumab nor aflibercept produced evidence of mitochondrial toxicity or cell death. However, bevacizumab and ziv-aflibercept showed mild mitochondrial toxicity at clinically relevant doses. PMID:24836865

  20. Characterization of mesenchymal cells beneath cornification of the fetal epithelium and epidermis at the face: an immunohistochemical study using human fetal specimens

    PubMed Central

    Kim, Ji Hyun; Jin, Zhe Wu; Murakami, Gen

    2016-01-01

    Fetal development of the face involves a specific type of cornification in which keratinocytes provide a mass or plug to fill a cavity. The epithelial-mesenchymal interaction was likely to be different from that in the usual skin. We examined expression of intermediate filaments and other mesenchymal markers beneath cornification in the fetal face. Using sections from 5 mid-term human fetuses at 14–16 weeks, immunohistochemistry was conducted for cytokeratins (CK), vimentin, nestin, glial fibrilary acidic protein, desmin, CD34, CD68 and proliferating cell nuclear antigen (PCNA). Fetal zygomatic skin was composed of a thin stratum corneum and a stratum basale (CK5/6+, CK14+, and CK19+) and, as the intermediate layer, 2–3 layered large keratinocytes with nucleus. The basal layer was lined by mono-layered mesenchymal cells (CD34+ and nestin+). Some of basal cells were PCNA-positive. In the keratinocyte plug at the external ear and nose, most cell nuclei expressed PCNA, CK5/6, CK14, and CK19. Vimentin-positive mesenchymal cells migrated into the plug. The PCNA-positive nucleus as well as mesenchymal cell migration was not seen in the lip margin in spite of the thick keratinocyte layer. The lingual epithelium were characterized by the CK7-positive stratum corneum as well as the thick mesenchymal papilla. CD68-positive macrophages were absent in the epidermis/epithelium. Being different from usual cornification of the skin, loss of a mesenchymal monolayer as well as superficial migration of mesenchymal cells might connect with a specific differentiation of keratinocyte to provide a plug at the fetal nose and ear. PMID:27051567

  1. Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents.

    PubMed

    Müssig, Eva; Steinberg, Thorsten; Kohl, Annette; Chamulitrat, Walee; Komposch, Gerda; Tomakidi, Pascal

    2008-04-01

    Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2 weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits alpha6 and beta1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of beta1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin alpha6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and alpha6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation. PMID:18188601

  2. Characterization of mesenchymal cells beneath cornification of the fetal epithelium and epidermis at the face: an immunohistochemical study using human fetal specimens.

    PubMed

    Kim, Ji Hyun; Jin, Zhe Wu; Murakami, Gen; Cho, Baik Hwan

    2016-03-01

    Fetal development of the face involves a specific type of cornification in which keratinocytes provide a mass or plug to fill a cavity. The epithelial-mesenchymal interaction was likely to be different from that in the usual skin. We examined expression of intermediate filaments and other mesenchymal markers beneath cornification in the fetal face. Using sections from 5 mid-term human fetuses at 14-16 weeks, immunohistochemistry was conducted for cytokeratins (CK), vimentin, nestin, glial fibrilary acidic protein, desmin, CD34, CD68 and proliferating cell nuclear antigen (PCNA). Fetal zygomatic skin was composed of a thin stratum corneum and a stratum basale (CK5/6+, CK14+, and CK19+) and, as the intermediate layer, 2-3 layered large keratinocytes with nucleus. The basal layer was lined by mono-layered mesenchymal cells (CD34+ and nestin+). Some of basal cells were PCNA-positive. In the keratinocyte plug at the external ear and nose, most cell nuclei expressed PCNA, CK5/6, CK14, and CK19. Vimentin-positive mesenchymal cells migrated into the plug. The PCNA-positive nucleus as well as mesenchymal cell migration was not seen in the lip margin in spite of the thick keratinocyte layer. The lingual epithelium were characterized by the CK7-positive stratum corneum as well as the thick mesenchymal papilla. CD68-positive macrophages were absent in the epidermis/epithelium. Being different from usual cornification of the skin, loss of a mesenchymal monolayer as well as superficial migration of mesenchymal cells might connect with a specific differentiation of keratinocyte to provide a plug at the fetal nose and ear. PMID:27051567

  3. Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

    PubMed

    Poornejad, Nafiseh; Nielsen, Jeffery J; Morris, Ryan J; Gassman, Jason R; Reynolds, Paul R; Roeder, Beverly L; Cook, Alonzo D

    2016-03-01

    Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix. PMID:26589294

  4. Expression of K6W-ubiquitin inhibits proliferation of human lens epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway plays an important role in controlling the cell cycle. The purpose of this study was to examine if expression of a dominant negative form of ubiquitin can inhibit the proliferation of lens epithelial cells. Dominant negative K6W-ubiquitin was expressed in cultured hu...

  5. Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter–Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth

    PubMed Central

    Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W. K.; Hsieh, Chia-Ling

    2016-01-01

    Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor–promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter–driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers. PMID:27054343

  6. Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter-Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth.

    PubMed

    Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W K; Hsieh, Chia-Ling

    2016-01-01

    Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers. PMID:27054343

  7. Dose conversion coefficients for electron exposure of the human eye lens.

    PubMed

    Behrens, R; Dietze, G; Zankl, M

    2009-07-01

    Recent epidemiological studies suggest a rather low dose threshold (below 0.5 Gy) for the induction of a cataract of the eye lens. Some other studies even assume that there is no threshold at all. Therefore, protection measures have to be optimized and current dose limits for the eye lens may be reduced in the future. Two questions arise from this situation: first, which dose quantity is related to the risk of developing a cataract, and second, which personal dose equivalent quantity is appropriate for monitoring this dose quantity. While the dose equivalent quantity H(p)(0.07) has often been seen as being sufficiently accurate for monitoring the dose to the lens of the eye, this would be questionable in the case when the dose limits were reduced and, thus, it may be necessary to generally use the dose equivalent quantity H(p)(3) for this purpose. The basis for a decision, however, must be the knowledge of accurate conversion coefficients from fluence to equivalent dose to the lens. This is especially important for low-penetrating radiation, for example, electrons. Formerly published values of conversion coefficients are based on quite simple models of the eye. In this paper, quite a sophisticated model of the eye including the inner structure of the lens was used for the calculations and precise conversion coefficients for electrons with energies between 0.2 MeV and 12 MeV, and for angles of radiation incidence between 0 degrees and 45 degrees are presented. Compared to the values adopted in 1996 by the International Commission on Radiological Protection (ICRP), the new values are up to 1000 times smaller for electron energies below 1 MeV, nearly equal at 1 MeV and above 4 MeV, and by a factor of 1.5 larger at about 1.5 MeV electron energy. PMID:19502705

  8. Ionizing Irradiation Not Only Inactivates Clonogenic Potential in Primary Normal Human Diploid Lens Epithelial Cells but Also Stimulates Cell Proliferation in a Subset of This Population

    PubMed Central

    Fujimichi, Yuki; Hamada, Nobuyuki

    2014-01-01

    Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that

  9. Malignant transformation of human gastric epithelium cells via reactive oxygen species production and Wnt/β-catenin pathway activation following 40-week exposure to ochratoxin A.

    PubMed

    Jia, Xin; Cui, Jinfeng; Meng, Xinxing; Xing, Lingxiao; Shen, Haitao; Wang, Juan; Liu, Jing; Wang, Yuan; Lian, Weiguang; Zhang, Xianghong

    2016-03-01

    Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogenic to humans. We previously demonstrated that OTA treatment induced oxidative damage in human gastric epithelium cells (GES-1) in vitro. In this study, we found that long-term OTA treatment could result in increased proliferation, migration, and invasion abilities of GES-1 cells and induce anchorage-independent growth of cells in soft agar. Inoculation of OTA-treated GES-1 cells resulted in the formation of tumor xenografts in Balb/c nude mice in vivo, confirming that long-term OTA treatment can induce the malignant transformation of GES-1 cells. In addition, we found that long-term OTA treatment induced oxidative stress and activated the Wnt/β-catenin pathway, including the nuclear transition of β-catenin and the upregulation of the downstream molecules of the pathway. Finally, pretreatment with the antioxidant N-acetyl-L-cysteine (NAC) inhibited ROS formation and activation of the Wnt pathway in OTA-transformed GES-1 cells, which decreased the tumor formation abilities of these cells after inoculation in nude mice. These findings suggest that long-term OTA exposure induces the malignant transformation of GES-1 cells via intracellular ROS production and activation of the Wnt/β-catenin signaling pathway. PMID:26721203

  10. Cosmetics Europe multi-laboratory pre-validation of the SkinEthic™ reconstituted human corneal epithelium test method for the prediction of eye irritation.

    PubMed

    Alépée, N; Bessou-Touya, S; Cotovio, J; de Smedt, A; de Wever, B; Faller, C; Jones, P; Le Varlet, B; Marrec-Fairley, M; Pfannenbecker, U; Tailhardat, M; van Goethem, F; McNamee, P

    2013-08-01

    Cosmetics Europe, The Personal Care Association, known as Colipa before 2012, conducted a program of technology transfer and assessment of Within/Between Laboratory (WLV/BLV) reproducibility of the SkinEthic™ Reconstituted Human Corneal Epithelium (HCE) as one of two human reconstructed tissue eye irritation test methods. The SkinEthic™ HCE test method involves two exposure time treatment procedures - one for short time exposure (10 min - SE) and the other for long time exposure (60 min - LE) of tissues to test substance. This paper describes pre-validation studies of the SkinEthic™ HCE test method (SE and LE protocols) as well as the Eye Peptide Reactivity Assay (EPRA). In the SE WLV study, 30 substances were evaluated. A consistent outcome with respect to viability measurement across all runs was observed with all substances showing an SD of less than 18%. In the LE WLV study, 44 out of 45 substances were consistently classified. These data demonstrated a high level of reproducibility within laboratory for both the SE and LE treatment procedures. For the LE BLV, 19 out of 20 substances were consistently classified between the three laboratories, again demonstrating a high level of reproducibility between laboratories. The results for EPRA WLV and BLV studies demonstrated that all substances analysed were categorised similarly and that the method is reproducible. The SkinEthic™ HCE test method entered into the experimental phase of a formal ECVAM validation program in 2010. PMID:23524228