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PubMed1988

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion.

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.

Amnion↗
PubMed1995

The human amnion is a site of MHC class Ib expression: evidence for the expression of HLA-E and HLA-G.

The expression of HLA class I Ag by term human amnion epithelial cells was investigated. In immunostaining and FACS analysis, mAb to monomorphic class I Ag reacted extensively with amnion cells, whereas polymorphic mAb reactivity was more limited and variable. Further studies were conducted on amnion cell preparations containing negligible contaminants. Northern analysis with use of locus-specific probes demonstrated that amnion expresses two class Ib genes, HLA-E and HLA-G. Radio-immunoprecipitation with use of monomorphic mAb identified two fully glycosylated cell surface class I H chains of 44 and 41 kDa; polymorphic mAbs failed to immunoprecipitate the 41-kDa product, although 44-kDa products, typical of class Ia Ag, were identified in some preparations. Class I H chains were isolated from amnion by affinity chromatography. Microsequencing revealed that the first nine residues of the N-terminus of the 41-kDa product aligned perfectly only with HLA-E. Overall, amnion at term appears to express class Ib Ag with limited class Ia Ag. HLA-G is therefore expressed in two extrafetal epithelia: amnion and trophoblast. Identification of the class Ib protein HLA-E in amnion epithelium may have implications for preterm labor that can be associated with infection of the placental membranes.

Amnion↗
PubMed1996

Microscopic and biochemical analysis of the viability and permeability of guinea pig amnion and chorion leave in vitro.

Tissue viability and permeability of guinea pig amnion and chorion leave were analyzed microscopically and biochemically. The vital dyes T1111 and fluorescein diacetate were used to locate and determine the integrity of cell plasma membranes in early and late tissue in vitro using confocal laser scanning microscopy and scanning electron microscopy. Early amnion and chorion laeve were each found to contain a single epithelial cell layer, composed of membrane-intact cells. In contrast, plasma membrane lesions were present throughout the epithelium of late amnion. Late chorion laeve contained both regions of intact and damaged epithelial cells on its maternal side. There was also a layer of membrane-intact squamous cells on the fetal side of late chorion laeve. ATP measurements confirmed that early fetal membranes were viable after incubation in isotonic salt solutions at physiological pH. Late amnion was depleted of ATP stores while late chorion laeve retained its capacity for generating energy. These viability markers indicate that late guinea pig amnion is not a viable tissue in vitro, while late chorion laeve is a viable but probably degenerating tissue. Confocal X-Z scans were used to trace the movement of T1111 through the tissue as an indication of permeability to free solutes. Whereas dye will permeate across the main thickness of early amnion and chorion leave, it did not pass between cells, but was blocked, presumably by a line of tight junctions. Late amnion was characterized by the complete permeability to T1111. Late chorion leave contained regions where solute migration was blocked, but overall was a permeable tissue. These results provide an important context for the interpretation of molecular movement across fetal membranes.

Adenosine Triphosphate↗
PubMed1990

Evidence of a role for protein kinase C in epidermal growth factor-induced prostaglandin E2 synthesis in amnion cells.

Human amnion cells synthesize and release prostaglandin E2 in response to epidermal growth factor. The protein kinase C activator, phorbol 12-myristate, 13-acetate also stimulates amnion cell prostaglandin E2 synthesis. We used a human amnion cell line (WISH) to conduct in vitro experiments to investigate a potential role of protein kinase C in the signal transduction pathway leading to epidermal growth factor-induced prostaglandin E2 production. Pretreatment of cultured amnion cells with a low, nonstimulating dose of phorbol 12-myristate, 13-acetate potentiated the action of epidermal growth factor in causing prostaglandin E2 production as measured by radioimmunoassay. The protein kinase C-selective inhibitor staurosporine inhibited epidermal growth factor-induced prostaglandin E2 production, further suggesting a role for protein kinase C in epidermal growth factor action. Experiments were conducted in which amnion cells were rendered protein kinase C-deficient by chronic exposure to phorbol ester, which has been shown to down-regulate the enzyme. In these cells, epidermal growth factor caused prostaglandin E2 synthesis at levels comparable to native (non-protein kinase C-deficient) cells. We conclude that protein kinase C plays a more modulatory than direct role in the epidermal growth factor signal transduction cascade that leads to prostaglandin E2 production by amnion cells. We propose a bifurcating transduction scheme in which, under conditions of protein kinase C inactivation, epidermal growth factor alone causes prostaglandin E2 synthesis. When protein kinase C is activated by as yet unknown endogenous substances, the epidermal growth factor responsiveness of the amnion cells is greatly enhanced. This pathway could have important implications in a feed-forward mechanism regulating the level of prostaglandin E2 production during the onset of labor.

Alkaloids↗
PubMed1984

Characterization of prostaglandin formation by human amnion cells in monolayer culture.

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E2 is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E2 by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E2 than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E2; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E2 biosynthesis in this tissue.

Amnion↗
PubMed1984

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue.

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained in vitro were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E2. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E2 release on the concentration of isoproterenol were similar, each response was maximal at 10(-6) M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E2 from amnion discs. Although prostaglandin E2, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, may be regulators of prostaglandin production by the amnion.

Amnion↗
PubMed1992

The mechanisms of preterm labour; the interaction between amnion cells and leukocytes in the metabolism of arachidonic acid.

Chorioamnionitis is frequently associated with preterm labour. We have used a cell culture model system to examine the effects of leukocytes upon the metabolism of endogenous arachidonic acid from within amnion cells. We have demonstrated that activated leukocytes release substances which increase the overall release and metabolism of endogenous arachidonic acid within amnion cells causing an increase in prostaglandin E2 production as well as a smaller increase in non-cyclo-oxygenase metabolism. When amnion cells and leukocytes are cultured together, in addition to prostaglandin E2 production by amnion cells, arachidonic acid released by the amnion cells appears to be metabolised by leucocytes to prostaglandin F2 alpha, prostacyclin and thromboxane A2. Prostaglandins E2 and F2 alpha are the principal cyclo-oxygenase products of this interaction. We postulate that chorioamnionitis stimulates preterm labour not only by causing an increase in prostaglandin E2 synthesis by amnion cells but by metabolism of amnion derived arachidonic acid to the powerfully oxytocic prostaglandin F2 alpha by leukocytes.

Amnion↗
PubMed1990

Amnion cell prostaglandin E2 stimulatory activity in chorion laeve-conditioned medium.

In this study we demonstrate the presence of a stimulant(s) to amnion cell prostaglandin (PG) E2 production in chorion-conditioned medium (CCM). The CCM induced a dose-dependent increase in amnion cell PGE2 production. This stimulatory activity was eliminated by heat and protease treatment. Maximal stimulation of amnion PGE2 by CCM did not occur until after 2 h of incubation, and treatment with cycloheximide (1 microgram/ml) effectively eliminated the ability of the amnion cells to respond to CCM. Additionally, CCM and arachidonic acid (2-40 microM) were synergistic in their stimulatory actions on amnion PGE2 production. CCM-treated amnion cells recover more quickly from acetylsalicylic acid pretreatment as compared to control. It is concluded that CCM contains a heat-labile protein which stimulates amnion cell PGE2 production by induction of prostaglandin endoperoxide synthase activity.

Amnion↗
PubMed1993

Epidermal growth factor and transforming growth factor-alpha enhance the interleukin-1- and tumor necrosis factor-stimulated prostaglandin E2 production and the interleukin-1 specific binding on amnion cells.

Interleukin-1 (IL-1), tumor necrosis factor (TNF), epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha) stimulate prostaglandin E2 (PGE2) production by amnion cells whereas TGF-beta inhibits the PGE2 production. During labor occurring in the setting of infection, several of these cytokines may be simultaneously present in amniotic fluid. The aim of the present study was to examine whether these cytokines modify each others' effects on amnion cell PGE2 production. Amnion cells in monolayer culture were treated with IL-1, TNF, EGF, TGF-alpha, TGF-beta 1, their combination, or vehicle. The PGE2 production and the specific binding of radiolabeled IL-1 beta on the cells were measured. IL-1 or TNF in combination with EGF or TGF-alpha stimulated synergistically the production of PGE2 by amnion cells. TGF-beta 1 did not modify the PGE2-stimulatory effect of EGF/TGF-alpha. Untreated amnion cells expressed 1030 +/- 100 IL-1 beta receptors per cell with a binding affinity of 1.40 +/- 0.26 nM. Treatment with TGF-alpha increased the number of receptors to 3940 +/- 260 per cell with no change in binding affinity. The potentiation of the PGE2-stimulatory effect of IL-1 by TGF-alpha may be related to its ability to induce IL-1 receptors on amnion cells. The synergistic effects of cytokines on amnion cell PGE2 production may promote labor.

Amnion↗
PubMed2004

The novel antimicrobial peptide beta3-defensin is produced by the amnion: a possible role of the fetal membranes in innate immunity of the amniotic cavity.

BACKGROUND: Innate immunity evolved to eliminate microorganisms before, or after their entry into the tissues, but before enough antigen is available to activate an adaptive, immune response. Innate immunity is so successful that the majority of encountered microbes are neutralized. The beta-defensins are antimicrobial peptides produced by skin and mucosal surfaces and are an integral part of the innate immune system. The ability of the amnion cells, which are epithelial derivatives, to produce antimicrobial beta-defensins has not been explored. OBJECTIVE: This study was undertaken to test the hypothesis that amnion cells synthesize beta-defensins under either basal or stimulated conditions. METHODS: Amnion epithelial FL cells (ATCC CCL 62) were cultured in Ham's F12 and Dulbecco's modified Eagle medium plus 10% fetal calf serum until confluence, then replated into 24-well plates at 1.5 million cells per well. Cells from triplicate wells were harvested after 1, 3, 6, and 24 hours of exposure to microbial wall components (lipopolysaccharide [LPS]: 1 microg/mL or peptidoglycan [PG]: 10 microg/mL). Reverse transcription real-time polymerase chain reaction was performed with the use of human-specific primers for beta1, beta2, beta3, and beta4 defensins to compare basal messenger RNA (mRNA) levels of defensins and in response to treatment. beta-actin was used for standardization. Protein expression was investigated by immunofluorescence of the cells in culture, and by immunohistochemistry in paraffin sections of human fetal membranes from pregnancies with or without histologic chorioamnionitis. RESULTS: Amnion FL cells expressed mRNA for all known beta-defensins with beta3-defensin mRNA levels significantly higher compared with others ( P < .001, 1-way analysis of variance [ANOVA]). beta3 was the only beta-defensin whose mRNA was upregulated in response to the microbial mimics LPS (1-way ANOVA, P = .019) and PG (1-way ANOVA, P = .011). Immunofluorescence confirmed that beta3-defensin protein was present in cultured amnion cells, and upregulated in response to PG and LPS in distinct cells. Similarly, in tissue sections of human fetal membranes amnion epithelium was intensely positive for beta3-defensin protein by immunohistochemistry. Conspicuous beta3-defensin staining was also detected in the chorio-decidua. CONCLUSION: Amnion cells have the ability to produce beta-defensins. The beta3-defensin appears to be the predominant epithelial defensin expressed. Its induction by microbial mimics suggests that the amniotic epithelium may play a role in the innate immunity of the amniotic cavity.

Amnion↗
PubMed2006

Separation of amnion from choriodecidua is an integral event to the rupture of normal term fetal membranes and constitutes a significant component of the work required.

OBJECTIVE: This study was undertaken to determine the sequence of events that occur during fetal membrane (FM) rupture and to compare the biophysical properties of intact FM with its separated individual components (amnion and choriodecidua). STUDY DESIGN: FM physical properties were determined with computerized, specially adapted, industrial, strength testing equipment and the rupture sequence (in vitro) video documented. Separated individual FM component properties were compared with those of reapproximated components, and of intact FMs. RESULTS: The sequence of FM rupture was (1) FM components stretch together under load; (2) amnion separates from choriodecidua; (3) choriodecidua ruptures; (4) amnion distends further, nonelastically; and (5) amnion ruptures. In all FMs tested, amnion was stronger, stiffer, and more ductile than choriodecidua. The sum of work required to rupture separated FM components (amnion + choriodecidua), or reapproximated components, was significantly less than that of intact FMs. CONCLUSION: Separation of amnion from choriodecidua occurs as part of normal term FM rupture. FMs become significantly weaker as a result of this separation.

Amnion↗
PubMed2006

Premature rupture of the fetal membranes: is the amnion the major determinant?

OBJECTIVE: The objective of the study was to evaluate the relative contributions of amnion and chorion to the strength of fetal membranes and to correlate these findings with gestational age. STUDY DESIGN: Fetal membranes from 78 pregnancies were tested for biaxial puncture force using a blunt, instrumented probe with a low-force load cell connected through a load cell conditioner to an oscilloscope. The average of 2 to 4 tests performed on independent regions of the membrane was recorded. Means and SDs were calculated through the gestational age ranges of less than 32, 32 to 36, or 37 weeks or longer. Linear regression analysis was performed across gestational age after grouping data by labor and mode of delivery. RESULTS: There were trends toward decreasing puncture force with gestational age for both chorioamnion and amnion for both vaginal deliveries and cesarean sections. The trends were significant by linear regression for labored deliveries but not unlabored cesarean sections for both chorioamnion and amnion alone. There was no trend in chorion puncture force with either gestational age or delivery mode and the mean puncture force values were, on average, half those for the amnion. CONCLUSION: The amnion is significantly stronger than the chorion when subjected to biaxial strength testing. The amnion but not the chorion is significantly affected by the chemical and mechanical changes during gestation and the labor process. These data will help direct future studies on the effects of clinical and molecular modulators of inflammation on membrane rupture thresholds with special emphasis on the biochemical and structural changes in the amnion.

Adult↗
PubMedDate not supplied

Dexamethasone fails to inhibit the induction of cytosolic phospholipase A(2) expression by interleukin-1beta in cultured primary human amnion fibroblasts.

OBJECTIVE: To investigate the interaction of dexamethasone and interleukin-1beta (IL-1beta) on the expression of cytosolic phospholipase A(2) (cPLA(2)), the enzyme catalyzing the first reaction in the formation of prostaglandins, in cultured primary human amnion fibroblasts. DESIGN AND METHODS: Human amnion fibroblasts were prepared from fetal amnion collected at term and were treated with dexamethasone with or without interleukin-1beta for 24h. Prostaglandin E(2) (PGE(2)) output and cPLA(2) expression in cultured amnion fibroblasts were measured with radioimmunoassay, quantitative real-time polymerase chain reaction, Western blotting and cPLA(2) promoter-driven luciferase reporter gene activity. RESULTS: Both dexamethasone and IL-1beta caused a significant increase in prostaglandin E(2) output, cPLA(2) mRNA and protein expression in cultured human amnion fibroblasts. Both dexamethasone and IL-1beta stimulated cPLA(2) promoter-driven luciferase reporter gene activity. There was no obvious antagonistic or synergistic effect of combined treatment of dexamethasone and IL-1beta on PGE(2) output, cPLA(2) expression or cPLA(2) promoter-driven luciferase reporter gene activity in cultured human amnion fibroblasts. CONCLUSION: The above findings suggest that paradoxically dexamethasone is a stimulator for both prostaglandin synthesis and cPLA(2) expression in human amnion fibroblasts. The interaction between dexamethasone and IL-1beta on prostaglandin synthesis and cPLA(2) expression is neither synergistic nor conventionally antagonistic.

Amnion↗
PubMed1997

Regulation of metallothionein expression in human amnion epithelial and mesenchymal cells.

OBJECTIVE: In this study the potential for metallothionein expression in amnion epithelial and mesenchymal cells in response to cadmium was evaluated. STUDY DESIGN: Levels of metallothionein messenger ribonucleic acid were evaluated in freshly separated amnion epithelial and mesenchymal cells and in amnion cells in culture. RESULTS: The levels of metallothionein messenger ribonucleic acid in human amnion mesenchymal cells freshly isolated after delivery of term pregnancies were greater than those in epithelial cells of the same tissue. The levels in mesenchymal cells in monolayer culture at confluence also were greater than those in confluent epithelial cells propagated from the same tissue. In response to treatment with cadmium (100 nmol/L to 50 micromol/L), which is inhaled in cigarette smoke, the levels of metallothionein messenger ribonucleic acid in both cell types increased markedly in a dose-dependent manner, but the level was greater in epithelial cells at all concentrations of cadmium chloride tested. With cadmium chloride (10 micromol/L), the level of metallothionein messenger ribonucleic acid increased by as much as 1000-fold in epithelial cells and 10-fold in mesenchymal cells compared with untreated (control) cells. Dexamethasone and tetradecanoyl phorbol acetate also acted to increase the levels in amnion epithelial and mesenchymal cells but not nearly to the levels effected by cadmium. CONCLUSION: These findings are indicative that metallothionein expression in amnion epithelial cells is exquisitely sensitive to cadmium in concentrations similar to those in amniotic fluid of pregnancies of women who smoke cigarettes. We hypothesize that increased levels of metallothionein in amniotic fluid and amnion epithelial cells will bind and thereby may limit the availability of copper to the Cu++-dependent enzyme lysyl oxidase in mesenchymal cells and thereby impair the cross-linking of interstitial collagens, which is effected by this enzyme.

Amnion↗
PubMedDate not supplied

Prostaglandin endoperoxide synthase kinetics in human amnion before and after labor at term and following preterm labor.

To determine whether the kinetics of prostaglandin endoperoxide synthase (PGHS, commonly known as cyclooxygenase) in human amnion change with labor onset or between preterm and term labor, a specific enzyme assay was developed and characterized. The assay was linear for time (0-8 min) and protein concentration (5-30 micrograms/250 microliters incubation volume). The optimum pH was 8.0-8.5, and the enzyme reaction reached saturation at 10-20 microM arachidonic acid. Flufenamic acid was more efficacious than ibuprofen in the presence of 1 mM tryptophan in inhibiting enzyme activity. The Km and Vmax of PGHS were determined in 10 amnions obtained at elective caesarean section before labor onset (CS) at 39.3 +/- 0.8 wk gestational age (mean +/- SD, range = 38.5-41 wk) and 9 amnions obtained following spontaneous labor and vaginal delivery (SL) at 39.6 +/- 0.8 wk (range = 38.5-41 wk). The Km values were 1.4 +/- 1.2 mumol/l (CS) and 2.2 +/- 1.5 mumol/l (SL) (not different). However, the Vmax increased significantly (p < 0.05) from 11 +/- 8 (CS) to 19 +/- 4 (SL) pg PGE2/micrograms protein/min. In eight preterm amnions obtained following spontaneous labor and delivery at 32.9 +/- 2.1 wk (range = 29-36 wk), the Km and Vmax were 2.0 +/- 1.2 mumol/l and 17 +/- 9 pg PGE2/micrograms protein/min, respectively. Neither of these values was different from those of CS or SL amnions. None of the preterm pregnancies displayed histological evidence of infection. These results suggest that an increase in the mean amnion PGHS maximum velocity occurs in association with the onset of labor at term. The mean Vmax of PGHS in amnions obtained from idiopathic preterm spontaneous deliveries is between the CS and SL term values, reflecting, perhaps, multiple etiologies for preterm delivery.

Amnion↗
PubMedDate not supplied

The presence of cortisol receptors in the human amnion.

Cytosol extracts of human amnion tissue contained high affinity binding of cortisol (Ka=2.48+/-1.06 x 10(9) M(-1); n = 30) and low capacity binding of cortisol (Nmax=279+/-15.5 fmol mg(-1) protein). Kinetic studies of cortisol binding resulted in a similar value of Ka to that obtained by Scatchard analysis. Nuclear extracts of amnion tissue contained high affinity binding of cortisol (Ka=5.8+/-1.91 x 10(7) M[-1]) and low binding capacity (Nmax=91.4+/-21.4 fmol mg(-1) protein). Ka values were an order of magnitude higher in cytosol than in blood serum when amnion and blood were obtained from the same individuals. Differences in competitive ligand binding, especially dexamethasone, were observed between the amnion receptor and transcortin in serum. Gel permeation chromatography gave only one peak at 320 kDa for amnion receptor and only one peak at 48 kDa for transcortin from serum. When amnion tissue was incubated with or without cortisol, cytosol receptor activity was significantly lower in cortisol treated tissue than in control. The nuclear extracted receptor activity was significantly higher in cortisol treated tissue than control. The Ka values from cortisol treated tissue were significantly lower from control. Together the data support the presence of a specific cortisol receptor in the human amnion that is different from transcortin.

Amnion↗
PubMed2004

Application of sterilised human amnion for reconstruction of the ocular surface.

BACKGROUND: Despite improved surgical techniques and the use of new medication, healing of corneal and conjunctival defects cannot always be achieved. In this connection the clinical use of human amnion, produced by different techniques, has represented a successful alternative for the past ten years. The purpose of the present investigation was the development of a clinically secure, therapeutically efficient, and easy-to-handle (transport, storage, application) allogenic amnion transplant. PATIENTS AND METHODS: A new method for an amnion preparation, which contains a sterilisation process in peracetic acid and a drying process in a laminar flow cabinet, was developed as an alternative to previous techniques described in the literature. Amnion transplantation was used to treat 41 patients, 36 of them with corneal ulcer. Further indications for amnion transplantation were symblepharon, descemetocele, as well as dehiscence of conjunctiva after cerclage. RESULTS: Seventy-three percent of cases showed postoperative improvement evidenced by constant vision, while 15 percent showed decreased vision. CONCLUSIONS: The study confirms the observations of previous investigators who consider amnion transplantation an efficient therapeutic method for a multitude of eye diseases. The new method described in this report, guarantees patients' safety by using a validated new sterilisation process against infections that can be transmitted by human tissue. At present this method constitutes the only process available in Germany, and is approved by the Federal Institute for Drugs and Medical Products (BfArM) for the manufacture of human amnion transplants as a finished medical product.

Adult↗
PubMed2003

Human placental amnion is a novel substrate for detecting autoantibodies in autoimmune bullous diseases by immunoblotting.

BACKGROUND: Identification of antigens by immunoblotting techniques, using epidermal and dermal extracts, is regarded as essential for making a definitive diagnosis in autoimmune bullous diseases (AIBDs). These procedures involve epidermal-dermal separation for subsequent protein extraction, which may result in partial loss of some antigenic polypeptides and changes in the conformational epitopes targeted by autoantibodies in AIBDs. It may therefore be necessary to use different substrates for consistent results. Objectives To evaluate the usefulness of human placental amnion extract as a substrate for immunoblotting in the diagnosis of AIBDs. METHODS: We checked the structural components of the desmosomes and basement membrane zone (BMZ) of amnion by electron microscopy. Using immunofluorescence and immunoblotting techniques, we tested the amnion immunoreactivity with antibodies to desmosomal and BMZ proteins, and with sera from 76 patients with AIBDs including pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid (BP), pemphigoid gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, paraneoplastic pemphigus and mucous membrane pemphigoid. RESULTS: The desmosomes and BMZ of the amnion tissue were ultrastructurally similar to those in skin. Antigen mapping confirmed that amnion contains all the proteins that were recognized by a panel of monoclonal and polyclonal antibodies. Immunoblotting showed that the antibodies clearly detected bands corresponding to desmogleins 1 and 3, desmocollins 1 and 2, desmoplakins 1 and 2, three subunits (alpha3, beta3 and gamma2) of laminin 5, BP antigens 1 and 2, the 97-kDa LAD antigen and type VII collagen. In addition, most of the patient sera (82%) reacted exclusively with their respective antigens. CONCLUSIONS: Harvesting proteins from amnion does not require epidermal-dermal separation, and a sufficient yield of desmosomal and hemidesmosomal proteins can be obtained. Therefore, amnion may be a more reliable source of substrate than skin samples for immunoblot analysis of AIBDs.

Amnion↗