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MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.

Purpose: To demonstrate the presence of natriuretic peptide receptors (NPRs) in primary human corneal epithelial cells (p-CEPI), SV40-immortalized CEPI cells (CEPI-17-CL4) and in human corneal epithelium, and to define the pharmacology of natriuretic peptide (NP)-induced cGMP accumulation., Methods: NPR presence was shown by RT-PCR, western blot analysis, and indirect immunofluoresence. cGMP accumulation was determined using an enzyme immunoassay., Results: p-CEPI and CEPI-17-CL4 cells expressed mRNAs for NPR-A and NPR-B. Proteins for both NPRs were present in these cells and in human corneal epithelium. C-type NP (CNP), atrial NP (ANP) and brain NP (BNP) stimulated the accumulation of cGMP in a concentration-dependent manner in p-CEPI cells (potency; EC(50s)): CNP (1-53 amino acids) EC(50)=24+/-5 nM; CNP fragment (32-53 amino acids) EC(50)=51+/-8 nM; ANP (1-28 amino acids) EC(50)=>10 microM; BNP (32 amino acids) EC(50)>10 microM (all n=3-4). While the NPs were generally more potent in the CEPI-17-CL4 cells than in p-CEPI cells (n=4-9; p<0.01), the rank order of potency of the peptides was essentially the same in both cell types. Effects of CNP fragment in p-CEPI and CEPI-17-CL4 cells were potently blocked by HS-142-1, an NPR-B receptor subtype-selective antagonist (K(i)=0.25+/-0.05 microM in CEPI-CL4-17; K(i)=0.44+/-0.09 microM in p-CEPIs; n=6-7) but less so by an NPR-A receptor antagonist, isatin (K(i)=5.3-7.8 microM, n=3-7)., Conclusions: Our studies showed the presence of NPR-A and NPR-B (mRNAs and protein) in p-CEPI and CEPI-17-CL4 cells and in human corneal epithelial tissue. However, detailed pharmacological studies revealed NPR-B to be the predominant functionally active receptor in both cell-types whose activation leads to the generation of cGMP. While the physiologic role(s) of the NP system in corneal function remains to be delineated, our multidisciplinary findings pave the way for such future investigations.

Purpose: The family of natriuretic peptides (NPs); atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) as well as three associated receptors (NPRs); natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B), and natriuretic peptide receptor C (NPR-C) has never been documented in human lens epithelial cells. The study described herein was designed to demonstrate both expression and functionality of components of the natriuretic peptides and natriuretic peptide receptors in the human lens epithelial cell line, HLE-B3 and in normal human lens epithelial cell cultures (nHLE)., Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) along with confirmation by DNA sequencing and real-time quantitative RT-PCR was used to identify and demonstrate expression of mRNA for the natriuretic peptide family. Authentication of protein expression of the natriuretic peptide receptors was determined by using formaldehyde-fixed, Saponin-permeabilized cells (HLE-B3) or methanol:acetone-fixed and permeabilized cells (nHLE) using conventional immunofluorescence techniques. Enzyme-linked immunosorbent assay was used to determine cyclic GMP (cGMP) activity as stimulated by exogenous addition of natriuretic peptides., Results: Using RT-PCR with confirmation by DNA sequencing and real-time quantitative RT-PCR, HLE-B3 cells were shown to express mRNA for ANP, BNP, and CNP along with their associated receptors. Conventional immunofluorescence on the permeabilized cells confirmed positive diffuse staining indicating the presence of the three natriuretic peptide receptors in both HLE-B3 and nHLE cells. All three natriuretic peptides educe a cGMP response in the rank order CNP>>ANP approximately BNP indicating that the natriuretic peptide family is functional in HLE-B3 cells., Conclusions: The data indicates that ANP, BNP, and CNP and natriuretic peptide receptor transcripts are expressed and are functional in human lens epithelial cells. The cellular expression of NPs and NPRs, as well as the demonstration that all three NPs activate guanylyl cyclase suggests a potential role in maintaining lens epithelial cell homeostasis.

The regenerative inadequacy of the injured myocardium leads to adverse remodeling, cardiac dysfunction, and heart disease. Stem cell-replacement of damaged myocardium faces major challenges such as inappropriate differentiation, cellular uncoupling, scar formation, and accelerated apoptosis of transplanted cells. These challenges can be met by engineering an in vitro system for delivering stem cells capable of cardiac differentiation, tissue integration, and resistance to oxidative stress. In this study, we describe the formation of three-dimensional (3D) cell aggregates ("cardiospheres") by putative stem cells isolated from adult dog myocardium using poly-L-ornithine. De novo formation of cardiospheres in growth factor-containing medium occurred over a period of 2-3 weeks, but accelerated to 2-3 days when seeded on poly-L-ornithine. Older cardiospheres developed foci of "beating" cells upon co-culture with rat neonatal cardiomyocytes. Cardiospheres contained cells that exhibited characteristics of undifferentiated cells; differentiating cardiomyocytes with organized contractile machinery; and vascular cells capable of forming "vessel-like" networks. They exhibited strong resistance to elevated concentrations of hydrogen peroxide in culture and survived subcutaneous injections without undergoing neoplastic transformation up to 3 weeks post-transplantation. These findings suggest that cardiospheres are potentially useful for delivering functional stem cells to the damaged heart., ((c) 2008 Wiley-Liss, Inc.)

The influence of sexual category as a modifier of cellular function is underinvestigated. Whether sex differences affect estrogen-mediated mitochondrial cytoprotection was determined using cell cultures of normal human lens epithelia (nHLE) from postmortem male and female donors. Experimental indicators assessed included differences in estrogen receptor-beta (ERbeta) isoform expression, receptor localization in mitochondria, and estrogen-mediated prevention of loss of mitochondrial membrane potential using the potentiometric fluorescent compound JC-1 after nHLE were exposed to peroxide. The impact of wild-type ERbeta (wtERbeta1) was also assessed using wtERbeta1 siRNA to suppress expression. A triple-primer PCR assay was employed to determine the proportional distribution of the receptor isoforms (wtERbeta1, -beta2, and -beta5) from the total ERbeta message pool in male and female cell cultures. Irrespective of sex, nHLE express wtERbeta1 and the ERbeta2 and ERbeta5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in peripheral mitochondrial arrays and perinuclear mitochondria as well as nuclear staining in both cell populations. The ERbeta2 and ERbeta5 isoforms were distributed primarily in the nucleus and cytosol, respectively; no association with the mitochondria was detected. Both male and female nHLE treated with E(2) (1 muM) displayed similar levels of protection against peroxide-induced oxidative stress. In conjunction with acute oxidative insult, RNA suppression of wtERbeta1 elicited the collapse of mitochondrial membrane potential and markedly diminished the otherwise protective effects of E(2). Thus, whereas the estrogen-mediated prevention of mitochondrial membrane permeability transition is sex independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtERbeta1 dependent.

Purpose: 14-3-3 is a highly conserved, ubiquitously expressed family of proteins. At least seven mammalian isoforms (beta, epsilon, gamma, eta, theta, sigma, and zeta) are known. These proteins associate with over 200 different target molecules and activate several downstream signaling cascades involved in the regulation of metabolism, cell cycle, apoptosis, protein trafficking, transcription, stress responses, and malignant transformations. We are interested in the role of these proteins in the mechanisms regulating homeostasis and the pathologies of the human ocular surface. Therefore, our purpose is to determine the expression of the 14-3-3 proteins in the human cornea, the conjunctiva, and the primary cells comprising these tissues., Methods: Using immunofluorescence, we determined the expression of 14-3-3 beta, epsilon, gamma, eta, theta, sigma, and zeta in paraffin sections of the human cornea and conjunctiva. Using indirect immunofluorescence and western blot analysis, we also determined the expression of these isoforms in primary corneal epithelial cells, keratocytes, endothelial cells, and primary conjunctival epithelial cells. The expressions of these isoforms in primary epithelial and endothelial cells were compared with the same expressions in several corneal cell lines. Western blot analysis was used to determine the presence of 14-3-3 isoforms in the culture medium from corneal epithelial cells, cell lines, and the tear fluid., Results: All the 14-3-3 isoforms were expressed in the corneal and conjunctival epithelia as well as primary epithelial cells and cell lines. Expression of 14-3-3 sigma was confined to epithelial cells and was secreted into the culture medium of primary cells and cell lines. We also report for the first time that two of the secreted isoforms, 14-3-3 gamma and zeta, are also present in the human tear fluid., Conclusions: We have determined that all the mammalian 14-3-3 isoforms are expressed in the human cornea, conjunctiva, and the component cells and that the 14-3-3 sigma isoform was found to be epithelial cell specific. We propose that the intracellular and extracellular presence of 14-3-3 sigma suggest its involvement in the epithelia specific signaling pathways.

Changes in expression of phenotypic markers characterizing the cells comprising intact neurospheres are difficult to determine easily and accurately. The problem is compounded by the diversity of cell populations and phenotypes involved, and consequent non-uniform protein expression across the neurosphere wall, or around the circumference. Therefore, interpreting the effects of phenotype modifying conditions has been a complex and demanding task. Here, we report a novel direct method for measuring protein expression in immunofluoerescently labeled intact neurospheres by densitometric image analysis of optical cross-sections, obtained by scanning laser confocal microscopy. To demonstrate our methodology, hollow human neurospheres were exposed to basic fibroblast growth factor (FGF2), which reportedly induces neuronal commitment in monolayer cultures of neuroprogenitor cells derived from neurospheres. We determined that this treatment downregulated nestin and vimentin, protein markers accepted to indicate an immature, uncommitted phenotype. Neuron specific enolase was only marginally affected. Our strategy allows quantitation of changes in expression of marker proteins that is comparable to Western blot analysis. In addition to discriminating heterogeneity in protein expression, suitable optics may allow the resolution down to single cell level. We propose that this novel strategy, with or without confocal microscopy, may be applied to other biological systems. Analysis of protein expression by the cells comprising tubular or cylindrical cellular structures, or approximately spherical cell aggregates, can be performed efficiently using a small sample size.

Purpose: The healing process of some corneal wounds involves closure by stromal contraction and the renewal of the stratified epithelium. In wound gape injury such stromal contraction occurs with epithelial stratification. In previous in vitro studies of noncontracted and contracted corneal fibroblast-seeded collagen gels (FSCGs) it was shown that initiation of wound contraction by the myofibroblast phenotype (present within the wounded stroma) was dependent on vitronectin and/or fibronectin. This study considers one aspect of the epithelial-stromal interaction that occurs during wounding. The stratification of corneal epithelial cells on noncontracted and contracted corneal FSCGs was compared., Methods: Dissociated bovine corneal epithelial cells were seeded on noncontracted and contracted corneal FSCGs, and these assemblies were cultured for 7 days. The epithelium that formed was evaluated using laser confocal microscopy and immunohistochemical markers directed against cytokeratin 3, desmoplakin I and II, integrin alpha-6 subunit, laminin, and collagen VII. The characteristics of the epithelium were compared with stromal carriers comprised of dissociated bovine corneal epithelial cells seeded on intact stroma and basement membrane (stromal carrier biopsies)., Results: The stratified epithelium that developed on contracted corneal fibroblast-seeded collagen gels was similar to that formed on stromal carriers, whereas nonstratified epithelium formed on noncontracted FSCGs., Conclusions: These studies showed that the contracted state of fibroblast-seeded gels enhanced the development of well-organized, stratified corneal epithelium.

Purpose: The wound healing process in the corneal stroma involves the activation of corneal keratocytes and the expression of associated phenotypes (fibroblasts and myofibroblasts). One of these phenotypes, the myofibroblasts, synthesizes alpha-smooth muscle actin in order to affect wound closure by contracting the surrounding matrix. Excessive contraction results in the formation of unresolvable scars that are undesirable in the corneal stroma. The authors tested the effect of vitronectin and fibronectin on the contraction process associated with corneal wound healing., Methods: Collagen gels were prepared and were exposed to different treatments of fetal calf serum (FCS). The FCS used was either depleted of fibronectin and vitronectin or contained a known concentration of fibronectin, vitronectin, or both at 50 microg/ml. Contraction was measured using image analysis and cross sections of contracted gels were examined for alpha-smooth muscle actin expression using laser confocal microscopy., Results: Fibroblasts seeded in collagen gels paralleled the morphologic characteristics and cell distribution of keratocytes in unwounded cornea. Matrix contraction was dependent on the presence of fibronectin and/or vitronectin where myofibroblasts were present. The cell-mediated contraction process was maximal at 0.5 x 10(5) fibroblasts/ml., Conclusions: These studies showed that vitronectin or fibronectin is required for the myofibroblast-associated contraction to occur in this in vitro model of stromal wound healing. This model system shows a distinct potential for further studies relating to the corneal wound healing process.

A critical stage of cutaneous wound healing is the development and maturation of the epidermis. In the aged, and in certain pathologies, this repair process is compromised due to a variety of deficiencies, one of which is tissue oxygenation. Several phases of wound healing are dependent on adequate tissue oxygen levels, and hyperbaric oxygenation has been shown to transiently elevate these levels. The use of human cell monolayers, dermal equivalents and human skin equivalents provide excellent opportunities for studying wound healing using in vivo relevant models. The goal of this study was to examine the effect of hyperbaric oxygen on cell proliferation, differentiation, and matrix biosynthesis in monolayer cultures and epidermopoiesis in the developing skin equivalent. Normal human dermal fibroblasts, keratinocytes and melanocytes, dermal equivalents and skin equivalents were exposed to hyperbaric oxygen at pressures up to three atmospheres, for up to 10 consecutive daily treatments lasting 90 minutes each. Increase in fibroblast proliferation (cf., 30% at 1 atmosphere after 10 days treatment), was observed without a significant effect on proliferation of normal human melanocytes and glycosaminoglycan synthesis. Stimulation of collagen synthesis after two days of treatment was only significant at 1 atmosphere (about 20% increase) but this differential was not observed after 5 days of treatment. Hyperbaric oxygenation above 2 atmospheres, inhibited proliferation of fibroblasts and keratinocytes in cell monolayer cultures (e.g., a 10 day treatment at 3 atmospheres appeared cytostatic to keratinocytes). In contrast, hyperbaric treatment up to 3 atmospheres dramatically enhanced keratinocyte differentiation, and epidermopoiesis in the complete human skin equivalent. These results support the importance of hyperbaric oxygen therapy in wound healing, and should provide an insight into oxygen utilization during repair of peripheral human tissue. The results also show the utility of the human skin equivalent as a model for evaluation of parameters involved in wound healing.

The production of cytokines by human conjunctival epithelial cells following stimulation was investigated. Primary cultures of human conjunctival epithelial cells were characterized by morphology and keratin expression. Cultured epithelial cells were treated with varying concentrations of lipopolysaccharide, interleukin (IL)-1 beta, calcium ionophore A23187, or phorbol myristate acetate, and cytokine secretion was determined over specified intervals. Culture supernatants and cell lysates were analyzed by ELISA for IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF). With the exception of IL-1ra, unstimulated conjunctival epithelial cells produced cytokines at relatively low or undetectable levels. IL-1ra was detected in both culture supernatants and cell lysates under basal conditions. In response to stimuli, conjunctival epithelial cells secreted the proinflammatory cytokines TNF-alpha, IL-6, IL-8, and GM-CSF in a dose- and time-dependent fashion. After stimulation, the intracellular levels of IL-1ra increased in these cells but the supernatant-associated levels remained unchanged. None of the other cytokines evaluated (IL-1 beta, IL-3, IL-4, IL-5, and IL-11) were detected in supernatants or lysates of resting or stimulated cells. These findings suggest that conjunctival epithelial cells may contribute to the pathogenesis of human ocular diseases by production of proinflammatory cytokines. Further evaluation of these cells as targets of therapy is warranted.

The stem cells of the corneal epithelial lineage are confined to the basal cell layer of the limbus, a vascularized outer corneal rim. These slow cycling cells of great proliferative potential maintain the corneal epithelial mass. Since cell-cell communication plays an important role in development and differentiation, we conducted a comparative examination of the expression of two corneal connexins, C x 43 and C x 50, and the tracer transfer capacity of the limbal and corneal epithelia using the scrape loading technique. C x 43 is abundantly expressed in the basal cell layer of the epithelium covering the cornea, but is essentially absent from the mouse, human, neonatal rabbit, and chicken limbal epithelium. In the adult rabbit the limbal epithelium displays an overall weak C x 43 immunoreactivity, but C x 43-free isolated basal cells can be distinguished. C x 50 is expressed throughout the corneal epithelium of the three mammalian corneas, but is not detectable in the limbus. Scrape loading experiments in the rabbit yielded results which were consistent with the immunohistological findings; limbal epithelium lacked tracer (lucifer yellow) transfer capacity, strongly suggesting the absence of functional gap junctions. Altogether, our results demonstrate the incompetence of stem cells for gap junction-mediated cell-to-cell communication. This property may reflect the need of these unique cells to maintain a distinct intracellular environment.

Stratified cultures of human corneal epithelial cells were used as an in vitro model for the evaluation of chemical damage to the ocular surface. Plasmid-transfected human corneal epithelial cells (HCE-T cells; 10.014 pRSV-T), cultured on a collagen membrane at the air-liquid interface, form a stratified epithelium (the HCE-T model). Results showed the HCE-T cell line to be comparable to primary human corneal epithelial (HCE) cells in morphology, keratin expression, and calcium-mediated modulation of morphology. Intercellular junctions and other ultrastructural features common to human corneal epithelium were identified in stratified HCE-T cultures. Chemical effects on morphology and cell viability indicated that the HCE-T model was more resistant to chemical toxicity than HCE-T monolayer cultures. Barrier function established by the HCE-T model was determined by measuring transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (TER). Previous results demonstrated similar baseline TEP and TER values for HCE and HCE-T cultures. Stratified HCE-T cultures retained 96.4 +/- 2.2% of the fluorescein applied to the apical surface for 30 min, and attained a TER of 468 +/- 89 ohms x cm(2); these baseline values were maintained over a 20-day culture period. Chemically induced alterations were determined by measuring TEP and TER after 5-min exposures to sodium dodecyl sulfate, benzalkonium chloride, ethanol or isopropanol. These exposures resulted in dose-dependent increases in TEP, and reductions in TER and cell viability (MTT assay). Transmission electron microscopy revealed dose-dependent mechanisms of toxicity. Two days after toxicant treatments, some cultures recovered barrier properties related to TEP, but most had not repaired tight junctions (TER). Cell viability either did not recover, or continued to decline. The results indicate that TEP, TER and the MTT assay measure different properties of the cultures, and are useful endpoints for the evaluation of chemically-induced damage in the HCE-T model. (c) 1997 Elsevier Science Ltd.

Amiprilose hydrochloride has been shown to inhibit the proliferation of a number of hyperproliferative cell types including psoriatic skin cells. In the present study, the effects of amiprilose hydrochloride on human tissue equivalents were examined by incubating a) dermal equivalents, b) skin equivalents in the process of epidermalization, and c) mature skin equivalents, with varying concentrations of the drug. In all three models amiprilose hydrochloride concentrations of 0.1% (wt/vol) and lower were not toxic to fibroblasts and keratinocytes and did not interfere with the differentiation of the skin equivalent and the developing skin equivalent. When tested in dermal equivalents, concentrations of amiprilose hydrochloride between 0.1 and 0.5% resulted in changes in fibroblast morphology with development of large intracellular vacuoles, and concentrations greater than 5% were toxic. In mature skin equivalents, in addition to changes in fibroblast morphology, amiprilose hydrochloride in concentrations of 1 to 10% affected the epidermis. When 0.5% amiprilose hydrochloride was present in the developing skin equivalent during differentiation, the epidermal keratinocytes were also affected. Thus the morphology of basal keratinocytes was modified, the differentiation was incomplete, and the dermal-epidermal attachment was compromised. These studies suggest the possibility of an extracellular mechanism of action of amiprilose hydrochloride and delineate acceptable dosage ranges for the potential drug.

The accumulation of modified proteins in aging is well documented in many aging models. For example, the deamidated isoforms of triosephosphate isomerase accumulate in: (a) old erythrocytes, (b) fibroblasts from old donors, (c) fibroblasts aged in vitro, (d) premature-aging syndromes and (e) old cells in the eye lens. However, a fundamental remaining question is: 'Do such modified proteins interfere with cellular function?' It has been difficult to assess this question at the molecular level using whole-organism models and equally frustrating to evaluate the physiological significance of such changes using classical cellular models. Tissue equivalent systems (TES) provide an opportunity for examining the molecular basis and physiological consequences of modified proteins during aging. TES are composed of differentiating and proliferating heterogeneous cell types with symbiotic cell-cell and cell-matrix interactions. They closely resemble, both morphologically and functionally, the tissues from which they were derived. Aging studies utilizing TES can provide information on modifications of protein structures, isozyme patterns, enzymes of the cellular environmental protection system and metabolic parameters which may regulate protein synthesis and degradation.

Amiprilose hydrochloride, a 3-substituted glucose derivative, was found to inhibit the proliferation of human fibroblasts and keratinocytes originating from psoriatic lesions. Fibroblasts and keratinocytes were obtained from skin biopsies of normal donors, and from the biopsies of active/involved and uninvolved sites of psoriatic donors. The cells were cultured as monolayers or as components of tissue equivalent models. Keratinocytes and fibroblasts originating from biopsies of psoriatically involved areas were shown to proliferate at a significantly higher rate than those derived from uninvolved areas. The antiproliferative effect of amiprilose hydrochloride was not observed with normal keratinocytes or fibroblasts from the skin of healthy donors or from uninvolved areas of psoriatic donors. Amiprilose hydrochloride was not cytotoxic to any of these cells at levels below 0.1%. The combination of the low cytotoxicity and the selective antiproliferative effect indicates that this compound may be a useful antipsoriatic agent. The use of monolayer cultures and tissue equivalent models in this study illustrates the utility of such a progressive strategy in the evaluation of potential topical pharmaceuticals.

Oxidized, regenerated cellulose (ORC) was surgically implanted on the uterine horns of rabbits, and its biodegradation was studied in vivo. Samples of peritoneal lavages, serum, and urine were collected during the degradation process and analyzed for carbohydrate components utilizing high-performance liquid chromatography with pulsed amperometric detection (h.p.l.c.-p.a.d.). Degradation was rapid, and oligomeric products were evident primarily in the peritoneal fluid from the implantation site, with no apparent accumulation in either the serum or the urine. The size distribution and the amount of the oligomeric products decreased after day one, and by day four peritoneal lavages were essentially free of oligomers. The structure of the products formed was consistent with the lability of the polymer in solution, and the kinetics of degradation paralleled the results of the previously reported in vitro studies. Rabbit peritoneal macrophages, when incubated with ORC in vitro were observed to readily ingest and hydrolyze the polymeric material. A mechanism of degradation consisting of chemical depolymerization, followed by enzymatic hydrolysis mediated by glycosidases endogenous to peritoneal macrophages, is proposed.

The in vitro solubilization and degradation of regenerated cellulose was studied under conditions which approximate those found in vivo, when the material is used as an adhesion barrier to assist normal wound repair. Factors affecting solubilization which were examined included the effects of serum or plasma, and the presence of hydrolytic enzymes. Products of the solubilization and degradation processes were examined by high performance liquid chromatography coupled with pulsed amperometric detection. The oxidized polymer readily undergoes chain shortening to give oligomers which, in the presence of plasma or serum, are further hydrolyzed to smaller fragments, including glucuronic acid and glucose. Proposed mechanisms of degradation are discussed.

A critical step in tissue and wound repair is the removal of eschar--accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developed an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ([3H]thymidine, [14C]leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.

A simple method for drying virus on inanimate objects (cover slips) under vacuum in the cold is described. Following this procedure virus maintains high titers (10(6-7)) for periods of 1-3 wk at -70 degrees C depending on the virus. For virucidal assay of disinfectants, cover slips are exposed to medium simulating the disinfectant (virus control) or disinfectant in an upright position in an Ultra-Vu cuvette. Cover slips are readily removed and placed in tissue culture medium for dilution of virus and determination of virus titer. Cytotoxicity of disinfectant is determined by exposing cover slip without virus to disinfectant, then placing it in medium, diluting the medium and incubating with the indicator cells. The use of this technique results in high titers of virus on cover slips, which are inanimate objects requiring minimal manipulation. The titration of virus or cytotoxicity in microplates is cell, medium, serum, and labware economical.

We have developed general methods for the synthesis of 4'-fluoro- and 4'-methoxynucleosides by addition of iodinemonofluoride or iodine and methanol across the double bond of suitably protected 4',5'-unsaturated pyrimidine and purine nucleosides. The structures of these adducts have been determined by a combination of chemical, spectroscopic, and electrophoretic methods. The 4'-methoxy- and the uridine analogs of nucleocidin have been synthesized from the corresponding 4'-fluorouridine and 4'-methoxyadenosine derivatives.