Minjuan, Wu; Jun, Xiong; Shiyun, Shao; Sha, Xu; Haitao, Ni
Early repair of skin injury and maximal restoration of the function and appearance have become important targets of clinical treatment. In the present study, we observed the healing process of skin defects in nude mice and structural characteristics of the new skin after transplantation of isolated and cultured adipose derived mesenchymal stem cells (ADMSCs) onto the human acellular amniotic membrane (AAM). The result showed that ADMSCs were closely attached to the surface of AAM and grew well 24 h after seeding. Comparison of the wound healing rate at days 7, 14, and 28 after transplantation showed that ADMSCs seeded on AAM facilitated the healing of full-thickness skin wounds more effectively as compared with either hAM or AAM alone, indicating that ADMSCs participated in skin regeneration. More importantly, we noticed a phenomenon of hair follicle development during the process of skin repair. Composite ADMSCs and AAM not only promoted the healing of the mouse full-thickness defects but also facilitated generation of the appendages of the affected skin, thus promoting restoration of the skin function. Our results provide a new possible therapy idea for the treatment of skin wounds with respect to both anatomical regeneration and functional restoration. PMID:27597871
Sánchez, Pedro L; Fernández-Santos, M Eugenia; Espinosa, M Angeles; González-Nicolas, M Angeles; Acebes, Judith R; Costanza, Salvatore; Moscoso, Isabel; Rodríguez, Hugo; García, Julio; Romero, Jesús; Kren, Stefan M; Bermejo, Javier; Yotti, Raquel; Del Villar, Candelas Pérez; Sanz-Ruiz, Ricardo; Elizaga, Jaime; Taylor, Doris A; Fernández-Avilés, Francisco
Perfusion decellularization of cadaveric hearts removes cells and generates a cell-free extracellular matrix scaffold containing acellular vascular conduits, which are theoretically sufficient to perfuse and support tissue-engineered heart constructs. This article contains additional data of our experience decellularizing and testing structural integrity and composition of a large series of human hearts, "Acellular human heart matrix: a critical step toward whole heat grafts" (Sanchez et al., 2015) . Here we provide the information about the heart decellularization technique, the valve competence evaluation of the decellularized scaffolds, the integrity evaluation of epicardial and myocardial coronary circulation, the pressure volume measurements, the primers used to assess cardiac muscle gene expression and, the characteristics of donors, donor hearts, scaffolds and perfusion decellularization process.
amniotic Fluid Derived Stem Cells (AFS). PRINCIPAL INVESTIGATOR: Thomas L. Smith, PhD CONTRACTING ORGANIZATION: Wake Forest University Health Sciences...Acellular Nerve Allografts plus amniotic Fluid Derived Stem Cells (AFS). 5a. CONTRACT NUMBER W81XWH-13-1-0309 5b. GRANT NUMBER OR120157 5c...year include successful seeding of AFS into ANA. This accomplishment also documented that these cells remained viable up to 72 hours after seeding. The
Sherris, David A; Oriel, Brad S
Rhinoplasty often relies on graft material for structural support in the form of cartilage, bone grafts, or fascia. In addition, pliable grafts are often helpful for contouring and can function as a barrier. Unfortunately, grafts carry the disadvantage of requiring an additional donor site, with associated complications. Human acellular dermal matrix (ADM) biological implants offer an exciting alternative for structural support and nonstructural implantation in rhinoplasty procedures. To examine the efficacy of ADM placement in rhinoplasty and septoplasty, the authors report the results from a series of 51 patients. In this series, there were no cases of infection, skin discoloration, seroma formation, septal perforation, significant resorption, extrusion, or other complications related to ADM placement. Therefore, the authors believe that ADM offers a safe and effective alternative to traditional grafting methods for functional and aesthetic rhinoplasty.
Paterson, P.G.; Zlotkin, S.H.; Sarkar, B. )
Amniotic fluid is a potential source of both nutritionally essential and toxic metals for the fetus. As the binding pattern of these metals in amniotic fluid may be one of the determining factors in their availability to the fetus, the objective of this study was to investigate metal binding in vitro. The binding of six trace metals, Mn(II), Ni(II), Zn(II), Cu(II), Cd(II), and Fe(III), to components of human amniotic fluid was studied by Sephadex G-100 gel filtration at physiological pH, using radioisotopes as tracers and 50 mM TRIS/HCl as the elution buffer. The amniotic fluid was collected at 16-16.5 weeks gestation by amniocentesis and pooled for analysis. Extensive amounts of Fe, Cu, Zn, and Cd and small amounts of Mn and Ni were bound to high molecular weight proteins with elution patterns similar to those seen for the binding of these metals in serum. In addition, large amounts of Fe, Mn, Ni and Cd and small amounts of Zn and Cu were associated with low molecular weight component(s). The identity of these latter components is unknown, but they play an important biological role in amniotic fluid.
Kirsner, Robert S; Bohn, Greg; Driver, Vickie R; Mills, Joseph L; Nanney, Lillian B; Williams, Marie L; Wu, Stephanie C
A chronic wound fails to complete an orderly and timely reparative process and places patients at increased risk for wound complications that negatively impact quality of life and require greater health care expenditure. The role of extracellular matrix (ECM) is critical in normal and chronic wound repair. Not only is ECM the largest component of the dermal skin layer, but also ECM proteins provide structure and cell signalling that are necessary for successful tissue repair. Chronic wounds are characterised by their inflammatory and proteolytic environment, which degrades the ECM. Human acellular dermal matrices, which provide an ECM scaffold, therefore, are being used to treat chronic wounds. The ideal human acellular dermal wound matrix (HADWM) would support regenerative healing, providing a structure that could be repopulated by the body's cells. Experienced wound care investigators and clinicians discussed the function of ECM, the evidence related to a specific HADWM (Graftjacket(®) regenerative tissue matrix, Wright Medical Technology, Inc., licensed by KCI USA, Inc., San Antonio, TX), and their clinical experience with this scaffold. This article distills these discussions into an evidence-based and practical overview for treating chronic lower extremity wounds with this HADWM.
Wolbank, Susanne; van Griensven, Martijn; Grillari-Voglauer, Regina; Peterbauer-Scherb, Anja
Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.
Seo, Young-Kwon; Song, Kye-Yong; Kim, Young-Jin; Park, Jung-Keug
In this study, an acellular artificial dermis, composed of human collagen and glycosaminoglycan (GAG) secreted by cultured human fibroblasts on a bovine collagen sponge, was developed. Much of the newly secreted extracellular matrix (ECM) remained after the cell removal process. The main theme of this study focused on the matrix, rather than the viable cell components of the skin, as the major dermal deficit in the wound. Both the acellular artificial and bioartificial dermises, containing viable cells with ECM, were significantly less soluble than the collagen sponge, and the relative GAG content in the bioartificial and acellular artificial dermises was approximately 115-120% of the chondroitin-6-sulfate (CS) content found in the collagen sponge. In the group receiving the collagen sponge, the wound area gradually decreased to approximately 10% of its original area, while in the groups receiving the bioartificial and acellular artificial dermises, the wound area also gradually decreased to approximately 60 and 50%, respectively, of the original size over the 5 weeks after grafting. Both the bioartificial and acellular artificial dermises formed thicker, denser collagen fibers; more new blood vessel formation was observed in both cases. The basement membrane of the regenerated epidermal-dermal junction was thicker and more linear in the acellular artificial dermis graft than in the collagen sponge graft. In conclusion, the wound healing effects of acellular artificial dermis are no less than those of the bioartificial dermis, and much better than the collagen sponge graft with respect to wound contraction, angiogenesis, collagen formation, and basement membrane repair.
Everson, R.B.; Milne, K.L.; Warbuton, D.; McClamrock, H.D.; Buchanan, P.D.
Extracts of amniocentesis samples from 144 women were tested for the presence of mutagenic substances using tester strain TA1538 in the Ames Salmonella/mammalian-microsome mutagenicity test. Because the volume of amniotic fluid in these samples was limited (generally less than 10 ml), the authors investigated modifications of this mutagenesis assay that could increase its ability to detect effects from small quantities of test material. Using mutagenicity in samples of urine from smokers as a model, it appeared that improved ability to detect small amounts of mutagen could be obtained by reducing volumes of media and reagents while keeping the amount of test sample constant. Tests of amniotic fluid extracts by this modified procedure showed small increases in revertants, about 50% above dimethylsulfoxide solvent control values. The increases suggest the presence of small amounts of mutagenic material in many of the amniotic fluid samples. At the doses employed, mutagenic activity in these samples was not associated with maternal smoking.
Matsubara, S; Tamada, T
In order to elucidate and characterize the transport pathway of the substances in the amniotic fluid, the permeability of the term human amnion was studied ultracytochemically, with lanthanum or horse radish peroxidase (HRP) as a tracer. Pieces of the term human amnion were exposed to the solutions containing lanthanum or HRP, and processed for electronmicroscopy. Precipitates indicating lanthanum or HRP were observed in the lateral intercellular spaces of the amniotic epithelial cells through the entire depth of the spaces. Generally, pinocytosis of HPR was not observed. In rare cases, however, diffuse uptake of HRP was noticed in the cells of the electron-lucent cytoplasm. These facts indicated that the human amniotic epithelium is quite permeable and that this particular intercellular pathway is important in the mechanism of the transfer of substances between the mother and the fetus.
Kesting, Marco Rainer; Loeffelbein, Denys John; Steinstraesser, Lars; Muecke, Thomas; Demtroeder, Cedric; Sommerer, Florian; Hoelzle, Frank; Wolff, Klaus-Dietrich
Fresh amniotic membrane has been used in medicine since 1910. The reconstruction of immunologic privileged ocular surfaces with cryopreserved amniotic membrane was introduced in the 1990s. The aim of this study was to analyze the use of cryopreserved human amniotic membrane (HAM) as a surgical patch in immunologic unprivileged anatomic sites. In part I of the investigation, the abdominal wall muscle of 36 rats was covered with mono- and multilayered HAM. After 3, 14, and 28 days, respectively, these grafts were evaluated macro- and microscopically. Multilayer samples displayed slower degradation and less inflammation compared with monolayer coverage. In part II of the study, abdominal wall closure with multilayer HAM and with polypropylene mesh was conducted in 20 rats. All rats showed sufficient closure after 21 days, but significantly lower intraabdominal adhesion formation was observed in the HAM rats. The results of this study might pave the way for the use of cryopreserved HAM as graft material in reconstructive surgery.
Peyman, A.; Gabriel, C.; Benedickter, H. R.; Fröhlich, J.
The dielectric properties of freshly delivered human placenta, umbilical cord and amniotic fluid have been acquired at 37 °C and in the frequency range of 200 MHz-10 GHz. The experimental data were fitted to a Cole-Cole expression. The results show that dielectric properties of the umbilical cord are significantly higher than placenta due to the presence of high water content Wharton's jelly. The results also demonstrate large differences in the dielectric properties of amniotic and cerebrospinal fluids. The data presented can be used in numerical simulations of the exposure of pregnant women to electromagnetic fields.
Thornton, Catherine A; Holloway, Judith A; Shute, Janis K; Holloway, John W; Diaper, Norma D; Warner, John O
CD4-positive cells are detectable in the human fetal gastrointestinal tract from 11 weeks of gestation. Interleukin-16 (IL-16) is a chemoattractant for CD4(+) cells and, via fetal swallowing of amniotic fluid, could mediate the influx of CD4(+) cells into the fetal gut. We have shown that IL-16 was detectable in human amniotic fluid at 16-18 weeks of gestation (mid-pregnancy) but was not detectable at term (late pregnancy; > 37 weeks of gestation). Similarly, mid-pregnancy, but not late pregnancy, amniotic fluid contained chemotactic activity for CD4(+) T cells, this activity was reduced by 58% in the presence of a neutralizing anti-IL-16 antibody. The levels of IL-16 in fetal plasma at 16-24 weeks of gestation were very high, and decreased significantly by 25-36 weeks but at > 37 weeks remained significantly higher than adult levels. IL-16 transcripts were detectable in whole tissue extracts of fetal gut, skin and placenta but not in amniocytes, and IL-16 immunoreactivity was detectable in cells within the lamina propria of the fetal gut and within the skin, where it was associated with the basement membrane. Neither IL-16 levels nor chemotactic activity for CD4(+) T cells in mid-pregnancy amniotic fluid was related to atopic outcomes at 1 year of age. IL-16 might have an important role in the early development of the human immune system and/or in regulating fetal and maternal immunological responsiveness during pregnancy.
Sandor, Maryellen; Singh, Devinder; Silverman, Ronald P.; Xu, Hui; De Deyne, Patrick G.
Objective: We examined the differences in capsule formation between 2 commercially available human acellular dermal matrices in a nonhuman primate model. Methods: Primates were implanted dorsally with a subcutaneously placed tissue expander and randomized into 3 groups, receiving skin coverage only, coverage with non-irradiated freeze-dried human acellular dermal matrix, or coverage with gamma-irradiated human acellular dermal matrix. After 9 weeks, soft tissue around the tissue expander was excised and evaluated qualitatively and quantitatively to assess extent of inflammation (CD68 antibodies and interleukin-6 levels), degradation and fibrosis (matrix metalloproteinase-1 and procollagen-1 staining), and mechanical (tensile) strength. Results: Histological evaluation of tissue around the tissue expander indicated differences in host response, suggesting capsule presence in the gamma-irradiated matrix group but not the freeze-dried matrix group. The extent of local inflammation was much higher in the gamma-irradiated matrix group which demonstrated mean (standard deviation) localized interleukin-6 concentration of 67.3 (53.6) vs 16.3 (6.7) pg/mg protein in the non-irradiated matrix group. There was robust degradation and fibrotic response in the gamma-irradiated matrix group versus the freeze-dried matrix group. Mechanical testing indicated mean (standard deviation) ultimate tensile strength of 12.0 (7.1) N in the gamma-irradiated matrix group versus 99.3 (48.8) N in the freeze-dried matrix group. Conclusions: Enclosure of a tissue expander with human acellular dermal matrix untreated by gamma irradiation led to minimal inflammation and minimal evidence of fibrosis/capsule around the tissue expander compared with robust capsule formation around the tissue expander that was covered by a gamma-irradiated human acellular dermal matrix. PMID:24570768
Chehelcheraghi, Farzaneh; Eimani, Hossein; Homayoonsadraie, Seyed; Torkaman, Giti; Amini, Abdollah; Alavi Majd, Hamid; Shemshadi, Hashem
Background The necrotic skin flap represents a great challenge in plastic and reconstructive surgery. In this study, we evaluated the effect of bioscaffolds, acellular amniotic membranes (AAMs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) on random skin flap (RSF) survival in rats by applying a cell-free extracellular matrix scaffold as a supportive component for the growth and proliferation of BM-MSCs on RSFs. AAM matrix scaffolds were created by incubating AMs in ethylenediaminetetraacetic acid 0.05% at 37°C, and cell scrapers were used. Objectives The aim of the present study was to assess the effect of AAM as a scaffold in TE, and combined with transplanted BM-MSCs, on the survival of RSFs and on the biomechanical parameters of the incision-wound flap margins 7 days after flap elevation. Materials and Methods BM-MSCs and AAMs were transplanted into subcutaneous tissue in the flap area. On the 7th postoperative day, the surviving flap areas were measured using digital imaging software, and the flap tissue was collected for evaluation. Forty rats were randomly divided into four groups of 10 each: group 1 received an AAM injection; group 2 underwent BM-MSC transplantation; group 3 received both AAM injection + BM-MSC transplantation; and group 4 was the control group, receiving only saline. Results The survival area in the AAM/BM-MSC group was significantly higher than in the control group (18.49 ± 1.58 versus 7.51 ± 2.42, P < 0.05). The biomechanical assessment showed no significant differences between the experimental groups and the control group (P > 0.05), and there was no correlation with flap survival. Conclusions Our findings showed that the treatment of flaps with BM-MSC and AAM transplantations significantly promoted flap survival compared to a control group. The viability of the flap was improved by combining BM-MSCs with AAM matrix scaffolds. PMID:27621924
Sewell, H F; Matthews, J B; Flack, V; Jefferis, R
An antiserum raised to a partially purified preparation of secretory IgA isolated from human colostrum was shown to contain antibodies directed against human IgD. The inferred presence of IgD in the human colostrum was confirmed and also its association with antibody activity, as demonstrated by the presence of anti-E. coli antibodies. IgD was also shown to be present in whole saliva, parotid saliva and amniotic fluid, but could not be detected in jejunal juice. Images FIG. 1 FIG. 2 PMID:111882
Sandmann, G H; Eichhorn, S; Vogt, S; Adamczyk, C; Aryee, S; Hoberg, M; Milz, S; Imhoff, A B; Tischer, T
Meniscus tears are frequent indications for arthroscopic evaluation which can result in partial or total meniscectomy. Allografts or synthetic meniscus scaffolds have been used with varying success to prevent early degenerative joint disease in these cases. Problems related to reduced initial and long-term stability, as well as immunological reactions prevent widespread clinical use so far. Therefore, the aim of this study was to develop a new construct for tissue engineering of the human meniscus based on an acellular meniscus allograft. Human menisci (n = 16) were collected and acellularized using the detergent sodium dodecyl sulfate as the main ingredient or left untreated as control group. These acellularized menisci were characterized biomechanically using a repetitive ball indentation test (Stiffness N/mm, residual force N, relative compression force N) and by histological (hematoxylin-eosin, phase-contrast) as well as immunohistochemical (collagen I, II, VI) investigation. The processed menisci histologically appeared cell-free and had biomechanical properties similar to the intact meniscus samples (p > 0.05). The collagen fiber arrangement was not altered, according to phase-contrast microscopy and immunohistochemical labeling. The removal of the immunogenic cell components combined with the preservation of the mechanically relevant parts of the extracellular matrix could make these scaffolds ideal implants for future tissue engineering of the meniscus.
Ranzoni, Anna M.; Corcelli, Michelangelo; Hau, Kwan-Leong; Kerns, Jemma G.; Vanleene, Maximilien; Shefelbine, Sandra; Jones, Gemma N.; Moschidou, Dafni; Dala-Ali, Benan; Goodship, Allen E.; De Coppi, Paolo; Arnett, Timothy R.; Guillot, Pascale V.
The impaired maturation of bone-forming osteoblasts results in reduced bone formation and subsequent bone weakening, which leads to a number of conditions such as osteogenesis imperfecta (OI). Transplantation of human fetal mesenchymal stem cells has been proposed as skeletal anabolic therapy to enhance bone formation, but the mechanisms underlying the contribution of the donor cells to bone health are poorly understood and require further elucidation. Here, we show that intraperitoneal injection of human amniotic mesenchymal stem cells (AFSCs) into a mouse model of OI (oim mice) reduced fracture susceptibility, increased bone strength, improved bone quality and micro-architecture, normalised bone remodelling and reduced TNFα and TGFβ sigalling. Donor cells engrafted into bones and differentiated into osteoblasts but importantly, also promoted endogenous osteogenesis and the maturation of resident osteoblasts. Together, these findings identify AFSC transplantation as a countermeasure to bone fragility. These data have wider implications for bone health and fracture reduction. PMID:27995994
Drewek, M J; Bruner, J P; Whetsell, W O; Tulipan, N
It has been proposed that the myelodysplastic components of a myelomeningocele are secondarily damaged as the result of exposure to amniotic fluid, the so-called 'two-hit' hypothesis. The critical time at which this secondary insult might occur has not been clearly defined. The present study addresses this issue by quantitatively assessing the toxic effects of human amniotic fluid of various gestational ages upon organotypic cultures of rat spinal cord. Using an assay for lactate dehydrogenase efflux to evaluate toxicity in such spinal cord cultures, we found that the amniotic fluid became toxic at approximately 34 weeks' gestation. This toxic effect of amniotic fluid appears to emerge rather suddenly. Accordingly, it seems reasonable to suggest that prevention of exposure of vulnerable spinal cord tissue to this toxicity by surgical closure of a myelomeningocele defect prior to the emergence of toxicity in amniotic fluid may prevent injury to vulnerable myelodysplastic spinal cord tissue.
Sitkiewicz, Izabela; Green, Nicole M.; Guo, Nina; Bongiovanni, Ann M.; Witkin, Steven S.; Musser, James M.
Background For more than 100 years, group A Streptococcus has been identified as a cause of severe and, in many cases, fatal infections of the female urogenital tract. Due to advances in hospital hygiene and the advent of antibiotics, this type of infection has been virtually eradicated. However, within the last three decades there has been an increase in severe intra- and post-partum infections attributed to GAS. Methodology We hypothesized that GAS alters its transcriptome to survive in human amniotic fluid (AF) and cause disease. To identify genes that were up or down regulated in response to growth in AF, GAS was grown in human AF or standard laboratory media (THY) and samples for expression microarray analysis were collected during mid-logarithmic, late-logarithmic, and stationary growth phases. Microarray analysis was performed using a custom Affymetrix chip and normalized hybridization values derived from three biological replicates were collected at each growth point. Ratios of AF/THY above a 2-fold change and P-value <0.05 were considered significant. Principal Findings The majority of changes in the GAS transcriptome involved down regulation of multiple adhesins and virulence factors and activation of the stress response. We observed significant changes in genes involved in the arginine deiminase pathway and in the nucleotide de novo synthesis pathway. Conclusions/Significance Our work provides new insight into how pathogenic bacteria respond to their environment to establish infection and cause disease. PMID:20352104
Rota, Cinzia; Imberti, Barbara; Pozzobon, Michela; Piccoli, Martina; De Coppi, Paolo; Atala, Anthony; Gagliardini, Elena; Xinaris, Christodoulos; Benedetti, Valentina; Fabricio, Aline S C; Squarcina, Elisa; Abbate, Mauro; Benigni, Ariela; Remuzzi, Giuseppe; Morigi, Marina
Human amniotic fluid stem (hAFS) cells, a novel class of broadly multipotent stem cells that share characteristics of both embryonic and adult stem cells, have been regarded as promising candidate for cell therapy. Taking advantage by the well-established murine model of acute kidney injury (AKI), we studied the proregenerative effect of hAFS cells in immunodeficient mice injected with the nephrotoxic drug cisplatin. Infusion of hAFS cells in cisplatin mice improved renal function and limited tubular damage, although not to control level, and prolonged animal survival. Human AFS cells engrafted injured kidney predominantly in peritubular region without acquiring tubular epithelial markers. Human AFS cells exerted antiapoptotic effect, activated Akt, and stimulated proliferation of tubular cells possibly via local release of factors, including interleukin-6, vascular endothelial growth factor, and stromal cell-derived factor-1, which we documented in vitro to be produced by hAFS cells. The therapeutic potential of hAFS cells was enhanced by cell pretreatment with glial cell line-derived neurotrophic factor (GDNF), which markedly ameliorated renal function and tubular injury by increasing stem cell homing to the tubulointerstitial compartment. By in vitro studies, GDNF increased hAFS cell production of growth factors, motility, and expression of receptors involved in cell homing and survival. These findings indicate that hAFS cells can promote functional recovery and contribute to renal regeneration in AKI mice via local production of mitogenic and prosurvival factors. The effects of hAFS cells can be remarkably enhanced by GDNF preconditioning.
Rota, Cinzia; Imberti, Barbara; Pozzobon, Michela; Piccoli, Martina; De Coppi, Paolo; Atala, Anthony; Gagliardini, Elena; Xinaris, Christodoulos; Benedetti, Valentina; Fabricio, Aline S.C.; Squarcina, Elisa; Abbate, Mauro; Benigni, Ariela; Remuzzi, Giuseppe
Human amniotic fluid stem (hAFS) cells, a novel class of broadly multipotent stem cells that share characteristics of both embryonic and adult stem cells, have been regarded as promising candidate for cell therapy. Taking advantage by the well-established murine model of acute kidney injury (AKI), we studied the proregenerative effect of hAFS cells in immunodeficient mice injected with the nephrotoxic drug cisplatin. Infusion of hAFS cells in cisplatin mice improved renal function and limited tubular damage, although not to control level, and prolonged animal survival. Human AFS cells engrafted injured kidney predominantly in peritubular region without acquiring tubular epithelial markers. Human AFS cells exerted antiapoptotic effect, activated Akt, and stimulated proliferation of tubular cells possibly via local release of factors, including interleukin-6, vascular endothelial growth factor, and stromal cell–derived factor-1, which we documented in vitro to be produced by hAFS cells. The therapeutic potential of hAFS cells was enhanced by cell pretreatment with glial cell line–derived neurotrophic factor (GDNF), which markedly ameliorated renal function and tubular injury by increasing stem cell homing to the tubulointerstitial compartment. By in vitro studies, GDNF increased hAFS cell production of growth factors, motility, and expression of receptors involved in cell homing and survival. These findings indicate that hAFS cells can promote functional recovery and contribute to renal regeneration in AKI mice via local production of mitogenic and prosurvival factors. The effects of hAFS cells can be remarkably enhanced by GDNF preconditioning. PMID:22066606
Li, Liru; Wang, Dejun; Zhou, Jun; Cheng, Yan; Liang, Tian; Zhang, Guangmei
The mesenchymal stem cells (MSCs) derived from amniotic fluid (AF) have become an attractive stem cells source for cell-based therapy because they can be harvested at low cost and avoid ethical disputes. In human research, stem cells derived from AF gradually became a hot research direction for disease treatment, specifically for their plasticity, their reduced immunogenicity and their tumor tropism regardless of the tumor size, location and source. Our work aimed to obtain and characterize human amniotic fluid mesenchymal stem cells (AFMSCs) and detect their ovarian cancer tropsim in nude mice model. Ten milliliters of twenty independent amniotic fluid samples were collected from 16-20 week pregnant women who underwent amniocentesis for fetal genetic determination in routine prenatal diagnosis in the first affiliated hospital of Harbin medical university. We successfully isolated the AFMSCs from thirteen of twenty amniotic fluid samples. AFMSCs presented a fibroblastic-like morphology during the culture. Flow cytometry analyses showed that the cells were positive for specific stem cell markers CD73,CD90, CD105, CD166 and HLA-ABC (MHC class I), but negative for CD 45,CD40, CD34, CD14 and HLA-DR (MHC class II). RT-PCR results showed that the AFMSCs expressed stem cell marker OCT4. AFMSCs could differentiate into bone cells, fat cells and chondrocytes under certain conditions. AFMSCs had the high motility to migrate to ovarian cancer site but didn't have the tumorigenicity. This study enhances the possibility of AFMSCs as drug carrier in human cell-based therapy. Meanwhile, the research emphasis in the future can also put in targeting therapy of ovarian cancer.
Dennis, D'Antonio C.; Turnock, Adam R.; Sutton, Collin; Chastant, Bradley; Vanderlan, Wesley B.
Variations in skin thickness and contours pose significant challenges to reconstruction of the lower third of the nose. Human amniotic membrane allograft offers a potential alternative to tissue transfer in reconstruction of the lower third of the nose. We reviewed the procedure and photographs of a healthy 56-year-old male with a 22 × 18 mm lower third nasal defect involving full thickness skin and subcutaneous tissue. Following preparation for grafting, dehydrated human amniotic membrane was fashioned to the dimensions of the defect and applied. No further surgical intervention was provided for 3 months. Complete re-epithelialization of the nasal and adjacent defects was achieved with minimal scar formation. Human amniotic membrane allograft provides an efficacious and cosmetically acceptable alternative to local and regional tissue transfer.
Baumer, Alexa; Lehn, Andrea; Leftwich, Megan
This study seeks to understand the fundamental fluid dynamic processes involved in human birth. We begin by examining the importance of amniotic fluid. This is done using two experimental techniques that approximate the laboring human uterus to different degrees of anatomical correctness. The first, in which a latex uterus is filled with fluid and a solid fetus is extracted, investigates the importance of both amniotic fluid properties and fetal position in the force required to remove a fetus. The second experiment simplifies the geometry of birth even more. In this case, a solid cylindrical rod is pulled through a highly flexible outer tube. The force to pull the inner cylinder as a function of the gap fluid properties is measured. By carefully controlling the fluid properties of the experiment, the study will provide further insight into the roles of amniotic fluid in human birth.
Wagner, Darcy E.; Bonenfant, Nicholas R.; Sokocevic, Dino; DeSarno, Michael; Borg, Zachary; Parsons, Charles; Brooks, Elice M.; Platz, Joseph; Khalpey, Zain; Hoganson, David M.; Deng, Bin; Lam, Ying Wai; Oldinski, Rachael A.; Ashikaga, Takamaru; Weiss, Daniel J.
Acellular scaffolds from complex whole organs such as lung are being increasingly studied for ex vivo organ generation and for in vitro studies of cell-extracellular matrix interactions. We have established effective methods for efficient de- and recellularization of large animal and human lungs including techniques which allow multiple small segments (∼1–3cm3) to be excised that retain 3-dimensional lung structure. Coupled with the use of a synthetic pleural coating, cells can be selectively physiologically inoculated via preserved vascular and airway conduits. Inoculated segments can be further sliced for high throughput studies. Further, we demonstrate thermography as a powerful noninvasive technique for monitoring perfusion decellularization and for evaluating preservation of vascular and airway networks following human and porcine lung decellularization. Collectively, these techniques are a significant step forward as they allow high throughput in vitro studies from a single lung or lobe in a more biologically relevant, three-dimensional acellular scaffold. PMID:24411675
Zajicek, Robert; Mandys, Vaclav; Mestak, Ondrej; Sevcik, Jan; Königova, Radana; Matouskova, Eva
A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7-10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs), CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing.
Zhu, Shuang; Zhu, Qingtang; Liu, Xiaolin; Yang, Weihong; Jian, Yutao; Zhou, Xiang; He, Bo; Gu, Liqiang; Yan, Liwei; Lin, Tao; Xiang, Jianping; Qi, Jian
The exact inner 3D microstructure of the human peripheral nerve has been a mystery for decades. Therefore, it has been difficult to solve several problems regarding peripheral nerve injury and repair. We used high-resolution X-ray computed microtomography (microCT) to scan a freeze-dried human acellular nerve allograft (hANA). The microCT images were then used to reconstruct a 3D digital model, which was used to print a 3D resin model of the nerve graft. The 3D digital model of the hANA allowed visualization of all planes. The magnified 3D resin model clearly showed the nerve bundles and basement membrane tubes of the hANA. Scanning electron microscopy (SEM) was used to analyse the microstructure of the hANA. Compared to the SEM images, the microCT image clearly demonstrated the microstructure of the hANA cross section at a resolution of up to 1.2 μm. The 3D digital model of the hANA facilitates a clear and easy understanding of peripheral nerve microstructure. Furthermore, the enlarged 3D resin model duplicates the unique inner structure of each individual hANA. This is a crucial step towards achieving 3D printing of a hANA or nerve that can be used as a nerve graft. PMID:27476584
Ferrando, Pietro Maria; Balmativola, Davide; Cambieri, Irene; Scalzo, Maria Stella; Bergallo, Massimiliano; Annaratone, Laura; Casarin, Stefania; Fumagalli, Mara; Stella, Maurizio; Sapino, Anna; Castagnoli, Carlotta
Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco’s Modified Eagle’s Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties. PMID:26918526
Sipahi, Mesut; Şahin, Sevinç; Arslan, Ergin; Börekci, Hasan; Metin, Bayram; Cantürk, Nuh Zafer
Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations. PMID:26457000
Ueno, Morio; Matsumura, Michiru; Watanabe, Kiichi; Nakamura, Takahiro; Osakada, Fumitaka; Takahashi, Masayo; Kawasaki, Hiroshi; Kinoshita, Shigeru; Sasai, Yoshiki
Here we report a human-derived material with potent inductive activity that selectively converts ES cells into neural tissues. Both mouse and human ES cells efficiently differentiate into neural precursors when cultured on the matrix components of the human amniotic membrane in serum-free medium [amniotic membrane matrix-based ES cell differentiation (AMED)]. AMED-induced neural tissues have regional characteristics (brainstem) similar to those induced by coculture with mouse PA6 stromal cells [a common method called stromal cell-derived inducing activity (SDIA) culture]. Like the SDIA culture, the AMED system is applicable to the in vitro generation of various CNS tissues, including dopaminergic neurons, motor neurons, and retinal pigment epithelium. In contrast to the SDIA method, which uses animal cells, the AMED culture uses a noncellular inductive material derived from an easily available human tissue; therefore, AMED should provide a more suitable and versatile system for generating a variety of neural tissues for clinical applications. PMID:16766664
Hattori, Yuka; Mukaide, Takahiro; Jiang, Li; Kotani, Tomomi; Tsuda, Hiroyuki; Mano, Yukio; Sumigama, Seiji; Hirayama, Tasuku; Nagasawa, Hideko; Kikkawa, Fumitaka; Toyokuni, Shinya
Amniotic fluid contains numerous biomolecules derived from fetus and mother, thus providing precious information on pregnancy. Here, we evaluated oxidative stress of human amniotic fluid and measured the concentration of catalytic Fe(II). Amniotic fluid samples were collected with consent from a total of 89 subjects in Nagoya University Hospital, under necessary medical interventions: normal pregnancy at term, normal pregnancy at the 2nd trimester, preterm delivery with maternal disorders but without fetal disorders, congenital diaphragmatic hernia, fetal growth restriction, pregnancy-induced hypertension, gestational diabetes mellitus, Down syndrome and trisomy 18. Catalytic Fe(II) and oxidative stress markers (8-hydroxy-2'-deoxyguanosine, 8-OHdG; dityrosine) were determined with RhoNox-1 and specific antibodies, respectively, using plate assays. Levels of 8-OHdG and dityrosine were higher in the 3rd trimester compared with the 2nd trimester in normal subjects, and the abnormal groups generally showed lower levels than the controls, thus suggesting that they represent fetal metabolic activities. In contrast, catalytic Fe(II) was higher in the 2nd trimester than the 3rd trimester in the normal subjects, and overall the abnormal groups showed higher levels than the controls, suggesting that high catalytic Fe(II) at late gestation reflects fetal pathologic alterations. Notably, products of H2O2 and catalytic Fe(II) remained almost constant in amniotic fluid.
Gholipourmalekabadi, M; Bandehpour, M; Mozafari, M; Hashemi, A; Ghanbarian, H; Sameni, M; Salimi, M; Gholami, M; Samadikuchaksaraei, A
Human amniotic membranes (HAMs) have attracted the attention of burn surgeons for decades due to favorable properties such as their antibacterial activity and promising support of cell proliferation. On the other hand, as a major implication in the health of burn patients, the prevalence of bacteria resistant to multiple antibiotics is increasing due to overuse of antibiotics. The aim of this study was to investigate whether HAMs (both fresh and acellular) are an effective antibacterial agent against antibiotic-resistant bacteria isolated from burn patients. Therefore, a HAM was decellularized and tested for its antibacterial activity. Decellularization of the tissue was confirmed by hematoxylin and eosin (H&E) and 4,6-diamidino-2-phenylindole (DAPI) staining. In addition, the cyto-biocompatibility of the acellular HAM was proven by the cell viability test (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT) and scanning electron microscopy (SEM). The resistant bacteria were isolated from burns, identified, and tested for their susceptibility to antibiotics using both the antibiogram and polymerase chain reaction (PCR) techniques. Among the isolated bacteria, three blaIMP gene-positive Pseudomonas aeruginosa strains were chosen for their high resistance to the tested antibiotics. The antibacterial activity of the HAM was also tested for Klebsiella pneumoniae (American Type Culture Collection (ATCC) 700603) as a resistant ATCC bacterium; Staphylococcus aureus (mecA positive); and three standard strains of ATCC bacteria including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27833), and S. aureus (ATCC 25923). Antibacterial assay revealed that only the latter three bacteria were susceptible to the HAM. All the data obtained from this study suggest that an alternative strategy is required to complement HAM grafting in order to fully protect burns from nosocomial infections.
Ye, Ken; Traianedes, Kathy; Choong, Peter F. M.; Myers, Damian E.
Acellular dermal matrix (ADM) has been in clinical use for decades in numerous surgical applications. The ability for ADM to promote cellular repopulation, revascularisation and tissue regeneration is well documented. Adipose stem cells have the ability to differentiate into mesenchymal tissue types, including bone and cartilage. The aim of this study was to investigate the potential interaction between ADM and adipose stem cells in vitro using TGFβ3 and BMP6. Human infrapatellar fat pad-derived adipose stem cells (IPFP-ASC) were cultured with ADM derived from rat dermis in chondrogenic (TGFβ3 and BMP6) medium in vitro for 2 and 4 weeks. Histology, qPCR, and immunohistochemistry were performed to assess for markers of chondrogenesis (collagen Type II, SOX9 and proteoglycans). At 4 weeks, cell-scaffold constructs displayed cellular changes consistent with chondrogenesis, with evidence of stratification of cell layers and development of a hyaline-like cartilage layer superficially, which stained positively for collagen Type II and proteoglycans. Significant cell–matrix interaction was seen between the cartilage layer and the ADM itself with seamless integration between each layer. Real time qPCR showed significantly increased COL2A1, SOX9, and ACAN gene expression over 4 weeks when compared to control. COL1A2 gene expression remained unchanged over 4 weeks. We believe that the principles that make ADM versatile and successful for tissue regeneration are applicable to cartilage regeneration. This study demonstrates in vitro the ability for IPFP-ASCs to undergo chondrogenesis, infiltrate, and interact with ADM. These outcomes serve as a platform for in vivo modelling of ADM for cartilage repair. PMID:26858950
Carman, Claire M.; Tobin, Chase; Chase, Serena A.; Rossmeier, Kerri A.
Background: Human acellular dermal matrix (HADM) is commonly used to provide coverage and support for breast reconstruction. The primary purpose of this study was to evaluate the complication rates associated with breast reconstruction procedures when performed in conjunction with multiple types of HADM in a consecutive series. Methods: After receiving institutional review board approval, medical records from a single surgeon were retrospectively reviewed for 126 consecutive patients (170 breasts and 211 procedures) who received a breast reconstruction or revision with implantation of HADM between 2012 and 2014. Patient demographics, surgical technique, and the complication profile of 4 major types of HADM were evaluated by procedure. Complication data were primarily evaluated for infection, seroma formation, necrosis, and other complications requiring additional surgery. Results: The total complication rate was 19.4%. The complication rates were not statistically different between all 4 types of HADM: Alloderm (n = 143); Alloderm RTU (n = 19); FlexHD (n = 18); hMatrix (n = 32) (P > 0.05). Smokers and large-breasted women (≥500 g) had a significantly higher complication rate than the rest of the population (P < 0.01 and P < 0.03, respectively). The complication rates associated with all other patient cohorts analyzed (age, body mass index, comorbid conditions, cancer diagnosis, prepectoral technique) showed no influence on complication rates (P > 0.05). Conclusions: In characteristically similar cohorts, there was no statistically significant difference in complication rates based on type of HADM; however, certain risk factors and anatomy should be considered before HADM-assisted breast reconstruction. PMID:27975023
Tancharoen, Waleephan; Aungsuchawan, Sirinda; Pothacharoen, Peraphan; Markmee, Runchana; Narakornsak, Suteera; Kieodee, Junjira; Boonma, Nonglak; Tasuya, Witoon
Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering.
Gholizadeh-Ghaleh Aziz, Shiva; Pashaei-Asl, Fatima; Fardyazar, Zahra; Pashaiasl, Maryam
Human stem cells and progenitor cells can be used to treat cancer and replace dysfunctional cells within a tissue or organ. The objective of this study was to identify the appropriate cells type in regenerative medicine and targeted therapy. As an alternative to embryonic and bone marrow stem cells, we examined human amniotic fluid stem cells (hAFSCs), one of the potential source of multipotent stem cells isolated from both cell pellet (using single-stage method), and supernatant of human amniotic fluid. Source of isolation and unique property of the cells emphasize that these cells are one of the promising new tools in therapeutic field. Double sources for isolation and availability of the left over samples in diagnostic laboratory at the same time have less legal and ethical concerns compared with embryonic stem cell studies. Cells were isolated, cultured for 18th passage for 6 months and characterized using qPCR and flow cytometry. Cells showed good proliferative ability in culture condition. The cells successfully differentiated into the adipogenic and osteogenic lineages. Based on these findings, amniotic fluid can be considered as an appropriate and convenient source of human amniotic fluid stem cells. These cells provide potential tools for therapeutic applications in the field of regenerative medicine. To get a better understanding of crosstalk between Oct4/NANOG with osteogenesis and adipogenesis, we used network analysis based on Common Targets algorithm and Common Regulators algorithm as well as subnetwork discovery based on gene set enrichment. Network analysis highlighted the possible role of MIR 302A and MIR let-7g. We demonstrated the high expression of MIR 302A and low expression of MIR let7g in hAFSCs by qPCR. PMID:27434028
Wang, Han; Chen, Shuai; Cheng, Xiang; Dou, Zhongying; Wang, Huayan
To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer, hASCs at 10-15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, zeta-globin and alpha-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing alpha-actin positive and Tbx5, Nkx2.5, GATA4 and alpha-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies.
Acar, M S; Bulut, Z B; Ateş, A; Nami, B; Koçak, N; Yıldız, B
Titanium dioxide (TiO2) nanoparticles (NPs) are commonly used materials present in many consumables for which most people are exposed to. The biological hazards of the NPs on human health have been demonstrated previously. In this study, we aimed to assess the cytotoxicity potency of TiO2 NPs on the primary human amniotic fluid cells. The cells derived from amniotic fluid were treated with different dosages of TiO2 NPs for some periods. Cell adhesion status was assessed using a light microscopic observation. Cell proliferation and cell death rates were determined using trypan blue staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Also, mitotic index was determined using fluorescence in situ hybridization with chromosome 8 centromer-specific DNA probe. Disrupted cell adhesion, decreased proliferation, and increased mortality rates were detected in the cells that were treated with TiO2 NPs depending on the dosage (p < 0.001). Also, reduced mitotic index was determined in the cells depending on the time and TiO2 dosage when compared with the controls (p < 0.0001). These results showed that TiO2 NPs have high cytotoxicity for amniotic fluid-derived cells. Therefore, different products containing TiO2 NPs should be used with care, especially for pregnant women.
Shohat, B; Faktor, J M
Twenty specimens of amniotic fluid (AF) obtained between week 16 and 18 of gestation from normal pregnant women and six specimens from pregnant women in which trisomia of chromosome 21 was found were tested for immunosuppressive activity. Incubation of normal human donor lymphocytes with 0.2-1 mL of AF from normal pregnant women for one hour at 37 degrees C was sufficient for induction of significant inhibition of the ability of these cells to induce a local xenogeneic graft-versus-host reaction (GVHR) as well as inhibition of E and E-active rosette formation, the GVHR being the most sensitive test. On the other hand, amniotic fluid obtained from the six pregnant women in which trisomia of chromosome 21 was found showed no inhibitory activity in either the E or E-active rosette formation, nor in the local xenogeneic graft-versus-host reaction. AF from all the women tested was found to have no effect on phenotype expression of the lymphocytes, as tested by the monoclonal antibodies OKT4+ and OKT8+, nor on B-lymphocytes, as tested by surface immunoglobulins. No correlation was found between the alpha-fetoprotein levels in the sera of those women and the immunosuppressive activity. These findings indicate that genetic defects of the conceptus are not limited to the embryo but may affect the composition of immunosuppressive components present in normal amniotic fluid.
Sanluis-Verdes, Anahí; Sanluis-Verdes, Namibia; Manso-Revilla, María Jesús; Castro-Castro, Antonio Manuel; Pombo-Otero, Jorge; Fraga-Mariño, María; Sanchez-Ibañez, Jacinto; Doménech, Nieves; Rendal-Vázquez, María Esther
Regenerative medicine, based on the use of stem cells, scaffolds and growth factors, has the potential to be a good approach for restoring damaged tissues of the central nervous system. This study investigated the use of human amniotic mesenchymal stem cells (hAMSC), human amniotic epithelial stem cells (hAESC), and human Wharton's jelly mesenchymal stem cells (hWJMSC) derived from human umbilical cord as a source of stem cells, and the potential of the human amniotic membrane (HAM) as a scaffold and/or source of growth factors to promote nerve regeneration. The hAMSC and hAESC obtained from HAM and the hWJMSC from umbilical cords were cultured in induction medium to obtain neural-like cells. The morphological differentiation of hAMSC, hAESC and hWJMSC into neural-like cells was evident after 4-5 days, when they acquired an elongated and multipolar shape, and at 21 days, when they expressed neural and glial markers. On other way, the HAM was completely decellularized without affecting the components of the basement membrane or the matrix. Subsequently, hAMSC, hAESC and hWJMSC differentiated into neural-like cells were seeded onto the decellularized HAM, maintaining their morphology. Finally, conditioned media from the HAM allowed proliferation of hAMSC, hAESC and hWJMSC differentiated to neural-like cells. Both HAM and umbilical cord are biomaterials with great potential for use in regenerative medicine for the treatment of neurodegenerative diseases.
Netea, M G; Selzman, C H; Kullberg, B J; Galama, J M; Weinberg, A; Stalenhoef, A F; Van der Meer, J W; Dinarello, C A
Accumulating evidence suggest that infection with Chlamydia pneumoniae is associated with atherosclerosis, but the mechanisms involved remain unclear. Inflammation is important in the initial phase of atherogenesis, and cytokines are important in the initiation and progression of inflammation. The aim of this study was to assess the capacity of acellular components of C. pneumoniae to stimulate the production of pro-inflammatory cytokines and chemokines. Peripheral blood mononuclear cells were stimulated in vitro with sonicated C. pneumoniae. Significant amounts of TNF-alpha, IL-1, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) were produced. Inhibition of endotoxin using polymyxin B revealed that chlamydial endotoxin plays a minor role in the cytokine induction. Neutralization of TNF by TNF-binding protein and blockade of IL-1 receptors by IL-1 receptor antagonist revealed that TNF, IL-1 and IL-6 production was independent from each other, whereas IL-8 synthesis was strongly dependent on endogenous TNF and IL-1. In contrast, synthesis of MCP-1 and MIP-1alpha was dependent on endogenous TNF, but not IL-1. In conclusion, acellular components of C. pneumoniae are a potent stimulus for cytokine production, and this mechanism may have an important role in the inflammatory aspects of atherogenesis.
Xinaris, Christodoulos; Benedetti, Valentina; Novelli, Rubina; Abbate, Mauro; Rizzo, Paola; Conti, Sara; Tomasoni, Susanna; Corna, Daniela; Pozzobon, Michela; Cavallotti, Daniela; Yokoo, Takashi; Morigi, Marina; Benigni, Ariela; Remuzzi, Giuseppe
Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact three-dimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research.
Fini, Jean-Baptiste; Mughal, Bilal B.; Le Mével, Sébastien; Leemans, Michelle; Lettmann, Mélodie; Spirhanzlova, Petra; Affaticati, Pierre; Jenett, Arnim; Demeneix, Barbara A.
Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.
Fini, Jean-Baptiste; Mughal, Bilal B; Le Mével, Sébastien; Leemans, Michelle; Lettmann, Mélodie; Spirhanzlova, Petra; Affaticati, Pierre; Jenett, Arnim; Demeneix, Barbara A
Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.
Fini, Jean-Baptiste; Mughal, Bilal B.; Le Mével, Sébastien; Leemans, Michelle; Lettmann, Mélodie; Spirhanzlova, Petra; Affaticati, Pierre; Jenett, Arnim; Demeneix, Barbara A.
Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development. PMID:28266608
Chang, Yu-Jen; Su, Hong-Lin; Hsu, Lee-Feng; Huang, Po-Jui; Wang, Tzu-Hao; Cheng, Fu-Chou; Hsu, Li-Wen; Tsai, Ming-Song; Chen, Chih-Ping; Chang, Yao-Lung; Chao, An-Shine; Hwang, Shiaw-Min
Human neural stem cells (NSCs) are particularly valuable for the study of neurogenesis process and have a therapeutic potential in treating neurodegenerative disorders. However, current progress in the use of human NSCs is limited due to the available NSC sources and the complicated isolation and culture techniques. In this study, we describe an efficient method to isolate and propagate human NSCs from the amniotic fluid with diagnosed neural tube defects (NTDs), specifically, anencephaly. These amniotic fluid-derived NSCs (AF-NSCs) formed neurospheres and underwent long-term expansion in vitro. In addition, these cells showed normal karyotypes and telomerase activity and expressed NSC-specific markers, including Nestin, Sox2, Musashi-1, and the ATP-binding cassette G2 (ABCG2). AF-NSCs displayed typical morphological patterns and expressed specific markers that were consistent with neurons, astrocytes, oligodendrocytes, and dopaminergic neurons after proper induction conditions. Furthermore, grafted AF-NSCs improved the physiological functions in a rat stroke model. The ability to isolate and bank human NSCs from this novel source provides a unique opportunity for translational studies of neurological disorders.
Sulcová, J; Jirásek, J E; Stárka, L
3 beta-Hydroxysteroid dehydrogenase / delta 5-4 isomerase activity was demonstrated in the human amniotic epithelium from the first trimester of pregnancy. The evidence was based on the in vitro formation of [4-14C] testosterone and [4-14C] androstenedione from [4-14C] 5-androstenediol and [4-14CA1 DEHYDROEPIANDROSTERONE, RESPECTIVELY. The activity of the enzyme studied in age dependent, reaching a maximum in the 8th-9th week of pregnancy and decreasing to negligible values at the end of the second trimester of gestation.
Chang, H C; Jones, O W; Masui, H
A new supplemented medium has been developed to improve human amniotic fluid cell growth and to reduce the dependence on exogenously added serum. The medium consists of a mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with Hepes, antibiotics, and 10 growth-promoting factors at 4% fetal bovine serum. Good clonal growth is achieved consistently in 8--13 days and is associated with large numbers of metaphase cells. Primary clones may be analyzed directly, thereby reducing difficulty with interpretation of chromosomal mosaicism. This medium could also be used for cultivation of fetal solid tissues and peripheral blood cultures of lymphocytes. Images PMID:6956891
... carefully. Removing a sample of the fluid through amniocentesis can provide information about the sex, health, and development of the fetus. Images Amniocentesis Amniotic fluid Polyhydramnios Amniotic fluid References Cunningham FG, ...
Antonucci, Ivana; Provenzano, Martina; Rodrigues, Melissa; Pantalone, Andrea; Salini, Vincenzo; Ballerini, Patrizia; Borlongan, Cesar V; Stuppia, Liborio
In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers "in vitro". In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases.
Antonucci, Ivana; Provenzano, Martina; Rodrigues, Melissa; Pantalone, Andrea; Salini, Vincenzo; Ballerini, Patrizia; Borlongan, Cesar V.; Stuppia, Liborio
In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers “in vitro”. In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases. PMID:27110774
Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián
Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.
Neeper, I D; Patton, D L; Kuo, C C
Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450
Dizaji Asl, Khadijeh; Shafaei, Hajar; Soleimani Rad, Jafar; Nozad, Hojjat Ollah
BACKGROUND Mesenchymal stem cells (MSCs) are ideal candidates for treatment of diseases. Amniotic membranes are an inexpensive source of MSCs (AM-MSC) without any donor site morbidity in cell therapy. Adipose tissue derived stem cells (ASCs) are also suitable cells for cell therapy. There is discrepancy in CD271 expression among MSCs from different sources. In this study, the characteristics of AM-MSC and ASCs and CD271 expression were compared. METHODS Adult adipose tissue samples were obtained from patients undergoing elective surgical procedure, and samples of amniotic membrane were collected immediately after caesarean operation. After isolation and expansion of MSCs, the proliferation rate and viability of cells were evaluated through calculating DT and MTT assay. Expression of routine mesenchymal specific surface antigens of MSCs and CD271 was evaluated by flow cytometry for both types of cells. RESULTS The growth rate and viability of the MSCs from the amniotic membrane was significantly higher compared with the ASCs. The low expression of CD14 and CD45 indicated that AM-MSC and ASCs are non hematopoietic cells, and both cell types expressed high percentages of CD44, CD105. The results revealed that AM-MSC and ASCs expressed no CD271 on their surfaces. CONCLUSION This study showed that amniotic membrane is a suitable cell source for cell therapy, and CD271 is a negative marker for MSCs identification from amniotic membrane and adipose tissue. PMID:28289611
Chen, Qian; Xiao, Pan; Chen, Jia-Nan; Cai, Ji-Ye; Cai, Xiao-Fang; Ding, Hui; Pan, Yun-Long
Amniotic fluid-derived stem cells (AFSCs) are becoming an important source of cells for regenerative medicine given with apparent advantages of accessibility, renewal capacity and multipotentiality. In this study, the mechanical properties of human amniotic fluid-derived stem cells (hAFSCs), such as the average Young's modulus, were determined by atomic force microscopy (3.97 ± 0.53 kPa for hAFSCs vs. 1.52 ± 0.63 kPa for fully differentiated osteoblasts). These differences in cell elasticity result primarily from differential actin cytoskeleton organization in these two cell types. Furthermore, ultrastructures, nanostructural details on the surface of cell, were visualized by atomic force microscopy (AFM). It was clearly shown that surface of osteoblasts were covered by mineralized particles, and the histogram of particles size showed that most of the particles on the surface of osteoblasts distributed from 200 to 400 nm in diameter, while the diameter of hAFSCs particles ranged from 100 to 200 nm. In contrast, there were some dips on the surface of hAFSCs, and particles were smaller than that of osteoblasts. Additionally, as osteogenic differentiation of hAFSCs progressed, more and more stress fibers were replaced by a thinner actin network which is characteristic of mature osteoblasts. These results can improve our understanding of the mechanical properties of hAFSCs during osteogenic differentiation. AFM can be used as a powerful tool for detecting ultrastructures and mechanical properties.
Kim, S S; Sohn, S K; Lee, K Y; Lee, M J; Roh, M S; Kim, C H
The object of this experimental study was to assess the effect of wrapping human amniotic membrane around a repaired ulnar nerve in a rabbit model of perineural adhesion. Ulnar nerves from 10 white New Zealand rabbits were exposed bilaterally, dissected and repaired. Human amniotic membrane was then wrapped around the repair site in one limb with no such wrap in the neurorrhaphy of the contralateral limb. Three months later, the same nerves were re-explored and removed using microsurgical external neurolysis. Perineural adhesion around the ulnar nerve was evaluated by blinded surgical dissection and scored using a visual 4-point qualitative scale. Extent and grade of fibrosis around repair sites were measured microscopically (x 200) after Masson trichrome staining using measure of the depth of fibrosis and the grading criteria of adhesion. Quantitative morphometric analysis was also performed under light microscopy (x 200) with the aid of a digital counter and virtual slide imaging software (ScanScope T2, Vista, CA, USA). Human amniotic membrane wrapped nerves showed significantly less perineural adhesion and fibrosis than controls (P < 0.05). No nerve healing problems were encountered. This study suggests that human amniotic membrane application can reduce fibrosis and adhesion around neurorrhaphy sites in this animal model.
Wu, Sing-Yung ); Huang, Wen-Sheng; Chen, Wei-Lian ); Polk, D.; Reviczky, A.; Williams, J. III; Chopra, I.J.; Fisher, D.A. )
Sulfated iodothyronines including T[sub 4]-sulfate (T[sub 4]S) and T[sub 3]-sulfate (T[sub 3]S) have been identified in human serum and amniotic fluid. Little is know, however, about the existence of sulfate conjugation of reverse T[sub 3] (rT[sub 3]S) in man. In this report, the authors employed a novel, sensitive, and specific rT[sub 3]S RIA to address this question. The rabbit antiserum to rT[sub 3]S was highly specific; T[sub 4], T[sub 3], rT[sub 3], and 3,3'-T[sub 2] showed less than 0.002% cross-reaction with the antiserum. Only T[sub 4]S and T[sub 3]S cross-reacted significantly (0.3% and 0.01%, respectively); other analogs cross-reacted less than 0.0001%. The detection threshold of the RIA was 14 pmol/L (1.0 ng/dL). The mean serum rT[sub 3]S concentration (pmol/L) was 40 in euthyroid subjects. Values were similar in hypothyroid patients (38) and pregnant women (52) but significantly (P < 0.01) elevated to 176 in hyperthyroid patient, 74 in patients with nonthyroid illnesses, and 684 in cord sera of newborns. Serum rT[sub 3]S increased significantly in hyperthyroid patients 1 day after administration of 1 g sodium ipodate orally. Reverse T[sub 3]S was detected consistently in amniotic fluid at 14 to 22 weeks of gestation and showed a marked rise 1-3 weeks after intraamniotic administration of 500-1000 [mu]g T[sub 4]. The various data suggest that : (1) rT[sub 3]S is a normal component of human serum and amniotic fluid; (2) it is derived from metabolism of T[sub 4] or rT[sub 3]; (3) circulating rT[sub 3]S increases in hyperthyroidism and in circumstances where type I 5'-monodeiodinating activity is low, e.g. nonthyroid illnesses, fetal life, and after administration of ipodate. 20 refs., 4 figs.
Zhao, Bin; Zhang, Yijie; Han, Shichao; Zhang, Wei; Zhou, Qin; Guan, Hao; Liu, Jiaqi; Shi, Jihong; Su, Linlin; Hu, Dahai
Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.
Hansen, T L; Andersen, H
Through a methodological evaluation, reliable histochemical and biochemical methods for succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells were developed. The histochemical method includes a cleaning of the cultured cells in 1 mM malonate in 0.9% NaCl, air-drying and fixation in acetone (5 min at -20 degrees C), coating of cells with CoQ10 (0.2 mg/ml in ether/acetone) and incubation for 1 h at 37 degrees C in 50 mM succinate and 0.5 mg/ml Nitro BT in 200 mM phosphate buffer, pH 7.6 PMS as an intermediate electron carrier was found inferior to exogenous CoQ10. Both types of cells exhibit equal activity. In the biochemical method homogenizing was performed in 50 mM Tris-HCl buffer, pH 7.5, and 200 mM sucrose. The standard incubation was 2.0 mM INT and 10 mM succinate in 10 mM Tris-HCl buffer, pH 7.5 for 1 h at 37 degrees C. The apparent Km values for INT and succinate were estimated to 0.39 mM and 0.13 mM, respectively, while I0.5 for malonate was 0.46 mM. Activity in amniotic fluid cells was 18.1 pkat/mg protein and in human skin fibroblasts 20.3 pkat/mg protein. Specificity of the methods was tested by use of a Chinese hamster fibroblast strain B9 known to be succinate dehydrogenase deficient in addition to various control experiments. Congruent results were obtained with the two methods.
Godin, Lindsay M.; Sandri, Brian J.; Wagner, Darcy E.; Meyer, Carolyn M.; Price, Andrew P.; Akinnola, Ifeolu; Weiss, Daniel J.; Panoskaltsis-Mortari, Angela
The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases. PMID:26954258
Godin, Lindsay M; Sandri, Brian J; Wagner, Darcy E; Meyer, Carolyn M; Price, Andrew P; Akinnola, Ifeolu; Weiss, Daniel J; Panoskaltsis-Mortari, Angela
The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.
Chang, Chih-Hung; Chen, Chia-Chun; Liao, Cheng-Hao; Lin, Feng-Huei; Hsu, Yuan-Ming; Fang, Hsu-Wei
In our previous study, we found that cartilage fragments from osteoarthritic knee promoted chondrogenesis of mesenchymal stem cells. In this study, we further transformed the cartilage tissues into acellular cartilage matrix (ACM) and explored the feasibility of using ACM as a biological scaffold. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery and were successfully fabricated into ACM powders. The ACM powders and human synovium-derived mesenchymal stem cells (SMSCs) were mixed into collagen gel for in vitro culture. Histological results showed a synergistic effect of ACM powders and chondrogenic growth factors in the formation of engineered cartilage. The findings of real-time polymerase chain reaction (PCR) suggested that ACM powders had the potential of promoting type II collagen gene expression in the growth factors-absent environment. Moreover, with growth factors induction, the ACM powders could reduce the hypertrophy in chondrogenesis of SMSCs. In summary, ACM powders could serve as a functional scaffold that benefited the chondrogenesis of SMSCs for cartilage tissue engineering.
Wang, Ting-gang; Xu, Jie; Zhu, Ai-hua; Lu, Hua; Miao, Zong-ning; Zhao, Peng; Hui, Guo-zhen; Wu, Wei-jiang
Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithelial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the transplant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial cells combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells. PMID:27904501
Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo
The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680
Elwan, Mohamed A; Ishii, Takashi; Sakuragawa, Norio
In this study we sought to investigate whether the dopamine transporter, DAT, and its binding sites are expressed in the human amniotic epithelial cells (HAEC) using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding studies, respectively. The RT-PCR findings showed that HAEC expressed DAT mRNA with 100% homology to the human brain DAT. Saturation binding studies using [3H]mazindol showed a high affinity DAT binding site with K(D) and B(max) values of 12.32+/-1.67 nM and 82.7+/-9.74 fmol/mg protein, respectively. Competition experiments showed that selective DAT blockers are potent displacers of [3H]mazindol binding. The rank order of potency of the competing drugs is consistent with the pharmacology of the DAT. The present results provide compelling evidence that HAEC natively express the DAT mRNA and binding sites. More importantly, these results may suggest that HAEC is an appropriate human cell model for studying dopamine release and uptake processes and potential ligands at these sites.
Yusoff, Nurul Hidayat; Alshehadat, Saaid Ayesh; Azlina, Ahmad; Kannan, Thirumulu Ponnuraj; Hamid, Suzina Sheikh Abdul
In the past decade, the field of stem cell biology is of major interest among researchers due to its broad therapeutic potential. Stem cells are a class of undifferentiated cells that are able to differentiate into specialised cell types. Stem cells can be classified into two main types: adult stem cells (adult tissues) and embryonic stem cells (embryos formed during the blastocyst phase of embryological development). This review will discuss two types of adult mesenchymal stem cells, dental stem cells and amniotic stem cells, with respect to their differentiation lineages, passage numbers and animal model studies. Amniotic stem cells have a greater number of differentiation lineages than dental stem cells. On the contrary, dental stem cells showed the highest number of passages compared to amniotic stem cells. For tissue regeneration based on animal studies, amniotic stem cells showed the shortest time to regenerate in comparison with dental stem cells.
Booth, Carol W.; And Others
Prenatal monitoring for metachromatic leukodystrophy (a fatal inherited metabolic disorder) suggested that the determination of levels of cerebroside sulfatase in the amniotic fluid helped in the prenatal detection of this disorder. (DB)
Dai, Miao; Xu, Peirong; Hou, Min; Teng, Yincheng; Feng, Jie
Tissue-engineered biologic products may be a viable option in the reconstruction of pelvic organ prolapse (POP). This study was based on the hypothesis that human adipose-derived stem cells (hASCs) are viable in acellular bovine pericardium (ABP), when reseeded by two different techniques, and thus, aid in the reconstruction. To investigate the reseeding of hASCs on ABP grafts by using non-invasive bioluminescence imaging (BLI), and to identify the effective hASCs–scaffold combinations that enabled regeneration. Thirty female athymic nude mice were randomly divided into three groups: In the VIVO group, ABPs were implanted in the subcutaneous pockets and enhanced green fluorescent protein luciferase (eGFP·Luc)-hASCs (1 × 106 cells/50 µL) were injected on the ABP at the same time. In the VITRO group, the mice were implanted with grafts that ABP were co-cultured with eGFP·Luc-hASCs in vitro. The BLANK group mice were implanted with ABP only. The eGFP·Luc-hASCs reseeded on ABP were analyzed by BLI, histology, and immunohistochemistry. The eGFP·Luc-hASCs reseeded on ABP could be visualized at 12 weeks in vivo. Histology revealed that the VIVO group displayed the highest cell ingrowths, small vessels, and percent of collagen content per unit area. Desmin and α-smooth muscle actin were positive at the same site in the VIVO group cells. However, few smooth muscles were observed in the VITRO and BLANK groups. These results suggest that hASCs reseeded on ABP in vivo during surgery may further enhance the properties of ABP and may promote regeneration at the recipient site, resulting in a promising treatment option for POP. PMID:26253192
Wu, Qingkai; Dai, Miao; Xu, Peirong; Hou, Min; Teng, Yincheng; Feng, Jie
Tissue-engineered biologic products may be a viable option in the reconstruction of pelvic organ prolapse (POP). This study was based on the hypothesis that human adipose-derived stem cells (hASCs) are viable in acellular bovine pericardium (ABP), when reseeded by two different techniques, and thus, aid in the reconstruction. To investigate the reseeding of hASCs on ABP grafts by using non-invasive bioluminescence imaging (BLI), and to identify the effective hASCs-scaffold combinations that enabled regeneration. Thirty female athymic nude mice were randomly divided into three groups: In the VIVO group, ABPs were implanted in the subcutaneous pockets and enhanced green fluorescent protein luciferase (eGFP·Luc)-hASCs (1 × 10(6) cells/50 µL) were injected on the ABP at the same time. In the VITRO group, the mice were implanted with grafts that ABP were co-cultured with eGFP·Luc-hASCs in vitro. The BLANK group mice were implanted with ABP only. The eGFP·Luc-hASCs reseeded on ABP were analyzed by BLI, histology, and immunohistochemistry. The eGFP·Luc-hASCs reseeded on ABP could be visualized at 12 weeks in vivo. Histology revealed that the VIVO group displayed the highest cell ingrowths, small vessels, and percent of collagen content per unit area. Desmin and α-smooth muscle actin were positive at the same site in the VIVO group cells. However, few smooth muscles were observed in the VITRO and BLANK groups. These results suggest that hASCs reseeded on ABP in vivo during surgery may further enhance the properties of ABP and may promote regeneration at the recipient site, resulting in a promising treatment option for POP.
Malhotra, Chintan; Jain, Arun K
The amniotic membrane (AM) is the inner layer of the fetal membranes and consist of 3 different layers: the epithelium, basement membrane and stroma which further consists of three contiguous but distinct layers: the inner compact layer, middle fibroblast layer and the outermost spongy layer. The AM has been shown to have anti-inflammatory, anti-fibrotic, anti-angiogenic as well as anti-microbial properties. Also because of its transparent structure, lack of immunogenicity and the ability to provide an excellent substrate for growth, migration and adhesion of epithelial corneal and conjunctival cells, it is being used increasingly for ocular surface reconstruction in a variety of ocular pathologies including corneal disorders associated with limbal stem cell deficiency, surgeries for conjunctival reconstruction, as a carrier for ex vivo expansion of limbal epithelial cells, glaucoma surgeries and sceral melts and perforations. However indiscriminate use of human AM needs to be discouraged as complications though infrequent can occur. These include risk of transmission of bacterial, viral or fungal infections to the recipient if the donors are not adequately screened for communicable diseases, if the membrane is not processed under sterile conditions or if storage is improper. Optimal outcomes can be achieved only with meticulous case selection. This review explores the ever expanding ophthalmological indications for the use of human AM. PMID:25032100
Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G.; Enríquez-Jiménez, Juana; Alcántara-Quintana, Luz E.; Fuentes-Mera, Lizeth; Piña-Barba, María C.; Zepeda-Rodríguez, Armando; and others
Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.
Maraldi, Tullia; Guida, Marianna; Zavatti, Manuela; Resca, Elisa; Bertoni, Laura; La Sala, Giovanni B.; De Pol, Anto
Human amniotic fluid stem cells (AFSC) are an attractive source for cell therapy due to their multilineage differentiation potential and accessibility advantages. However the clinical application of human stem cells largely depends on their capacity to expand in vitro, since there is an extensive donor-to-donor heterogeneity. Reactive oxygen species (ROS) and cellular oxidative stress are involved in many physiological and pathophysiological processes of stem cells, including pluripotency, proliferation, differentiation, and stress resistance. The mode of action of ROS is also dependent on the localization of their target molecules. Thus, the modifications induced by ROS can be separated depending on the cellular compartments they affect. NAD(P)H oxidase family, particularly Nox4, has been known to produce ROS in the nucleus. In the present study we show that Nox4 nuclear expression (nNox4) depends on the donor and it correlates with the expression of transcription factors involved in stemness regulation, such as Oct4, SSEA-4, and Sox2. Moreover nNox4 is linked with the nuclear localization of redox sensitive transcription factors, as Nrf2 and NF-κB, and with the differentiation potential. Taken together, these results suggest that nNox4 regulation may have important effects in stem cell capability through modulation of transcription factors and DNA damage. PMID:26273418
Osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells (AF-MSCs) has been widely studied in vitro and in vivo as a potential tool for regenerative medicine and tissue engineering. While most of the studies analyze changes in transcriptional profile during differentiation to date there is not much information regarding epigenetic changes in AF-MSCs during differentiation. The aim of our study was to evaluate epigenetic changes during osteogenic differentiation of AF-MS cells. Isolated AF-MSCs were characterized morphologically and osteogenic differentiation was confirmed by cell staining and determining expression of alkaline phosphatase and osteopontin by RT-qPCR. Variation in gene expression levels of pluripotency markers and specific microRNAs were also evaluated. Analysis of epigenetic changes revealed that levels of chromatin modifying enzymes such as Polycomb repressive complex 2 (PRC2) proteins (EZH2 and SUZ12), DNMT1, HDAC1, and HDAC2 were reduced after osteogenic differentiation of AF-MSCs. We demonstrated that the level of specific histone markers keeping active state of chromatin (H3K4me3, H3K9Ac, and others) increased and markers of repressed state of chromatin (H3K27me3) decreased. Our results show that osteogenic differentiation of AF-MSCs is conducted by various epigenetic alterations resulting in global chromatin remodeling and provide insights for further epigenetic investigations in human AF-MSCs. PMID:27818691
Verma, Prashant K; Humphrey, Victor F; Duck, Francis A
The frequency dependence of attenuation coefficient in amniotic fluid, urine and 4.5% and 20% human serum albumin solutions over the frequency range 5 MHz to 25 MHz was measured at both room temperature and physiological temperature using a variable path length technique. A 15 MHz (13 mm diameter) transducer was used to produce a broadband single-cycle pulse and a 4 mm diameter bilaminar polyvinylidene difluoride membrane hydrophone was used to detect the attenuated pulse. Standard time-of-flight measurement techniques were used to measure the acoustic velocity in the same fluid samples. At physiological temperature, the attenuation coefficients in amniotic fluid, urine and 4.5% and 20% human albumin solution were found to be 0.0053 f(1.65), 0.0047 f(1.67), 0.019 f(1.57) and 0.167 f(1.27) dB cm(-1), respectively, where f is in MHz. The velocities in amniotic fluid, urine and 4.5% human albumin solution at physiological temperature were found to be 1541.1 m s(-1) +/- 1.3 m s(-1), 1551.3 m s(-1) +/- 1.3 ms(-1) and 1547.3 m s(-1) +/- 1.0 m s(-1), respectively. The results provide unique data over the diagnostic and therapeutic ultrasonic frequency range that can be used as input data for theoretical models that attempt to simulate nonlinear pressure fields and temperature rises from medical ultrasonic transducers.
Ay, Birol; Karaoz, Erdal; Kesemenli, Cumhur C; Kenar, Halime
The reconstruction of skeletal muscle tissue is currently performed by transplanting a muscle tissue graft from local or distant sites of the patient's body, but this practice leads to donor site morbidity in case of large defects. With the aim of providing an alternative treatment approach, skeletal muscle tissue formation potential of human myoblasts and human menstrual blood derived mesenchymal stem cells (hMB-MSCs) on synthetic [poly(l-lactide-co-caprolactone), 70:30] scaffolds with oriented microfibers, human muscle extracellular matrix (ECM), and their hybrids was investigated in this study. The reactive muscle ECM pieces were chemically crosslinked to the synthetic scaffolds to produce the hybrids. Cell proliferation assay WST-1, scanning electron microscopy (SEM), and immunostaining were carried out after culturing the cells on the scaffolds. The ECM and the synthetic scaffolds were effective in promoting spontaneous myotube formation from human myoblasts. Anisotropic muscle patch formation was more successful when human myoblasts were grown on the synthetic scaffolds. Nonetheless, spontaneous differentiation could not be induced in hMB-MSCs on any type of the scaffolds. Human myoblast-synthetic scaffold combination is promising as a skeletal muscle patch, and can be improved further to serve as a fast integrating functional patch by introducing vascular and neuronal networks to the structure. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 879-890, 2017.
Jiao, Hongliang; Shi, Ke; Zhang, Weijie; Yang, Liang; Yang, Lu; Guan, Fangxia; Yang, Bo
Growing evidence indicates that the presence of extensive oxidative stress plays an essential role in the initiation and progression of Alzheimer's disease (AD). Amyloid-β (Aβ) aggregation is involved in the elevation of oxidative stress, contributing to mitochondrial dysfunction and lipid peroxidation. In the present study, human placenta amniotic membrane-derived mesenchymal stem cells (hAMMSCs) were intravenously injected into C57BL/6J-APP transgenic mice. hAMMSCs significantly ameliorated spatial learning and memory function, and were associated with a decreased amount of amyloid plaques of the brain. The correlation of oxidative stress with Aβ levels was lower in the hAMMSCs-injected group than in the phosphate-buffered saline (PBS)-injected group, as indicated by the increased level of antioxidative enzymes and the decreased level of lipid peroxidation product. The glutathione (GSH) level and ratio of GSH to glutathione disulfide were higher in the hAMMSC group than in the PBS group. The superoxide dismutase activity and malonaldehyde level were improved significantly as the level of Aβ decreased, but there was no such trend in the PBS group. As a result, our findings represent evidence that hAMMSC treatment might improve the pathology of AD and memory function through the regulation of oxidative stress. PMID:27588134
Jiawen, Si; Jianjun, Zhang; Jiewen, Dai; Dedong, Yu; Hongbo, Yu; Jun, Shi; Xudong, Wang; Shen, Steve G.F.
The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect. PMID:25368378
Sánchez, Pedro L; Fernández-Santos, M Eugenia; Costanza, Salvatore; Climent, Andreu M; Moscoso, Isabel; Gonzalez-Nicolas, M Angeles; Sanz-Ruiz, Ricardo; Rodríguez, Hugo; Kren, Stefan M; Garrido, Gregorio; Escalante, Jose L; Bermejo, Javier; Elizaga, Jaime; Menarguez, Javier; Yotti, Raquel; Pérez del Villar, Candelas; Espinosa, M Angeles; Guillem, María S; Willerson, James T; Bernad, Antonio; Matesanz, Rafael; Taylor, Doris A; Fernández-Avilés, Francisco
The best definitive treatment option for end-stage heart failure currently is transplantation, which is limited by donor availability and immunorejection. Generating an autologous bioartificial heart could overcome these limitations. Here, we have decellularized a human heart, preserving its 3-dimensional architecture and vascularity, and recellularized the decellularized extracellular matrix (dECM). We decellularized 39 human hearts with sodium-dodecyl-sulfate for 4-8 days. Cell removal and architectural integrity were determined anatomically, functionally, and histologically. To assess cytocompatibility, we cultured human cardiac-progenitor cells (hCPC), bone-marrow mesenchymal cells (hMSCs), human endothelial cells (HUVECs), and H9c1 and HL-1 cardiomyocytes in vitro on dECM ventricles up to 21 days. Cell survival, gene expression, organization and/or electrical coupling were analyzed and compared to conventional 2-dimensional cultures. Decellularization removed cells but preserved the 3-dimensional cardiac macro and microstructure and the native vascular network in a perfusable state. Cell survival was observed on dECM for 21 days. hCPCs and hMSCs expressed cardiocyte genes but did not adopt cardiocyte morphology or organization; HUVECs formed a lining of endocardium and vasculature; differentiated cardiomyocytes organized into nascent muscle bundles and displayed mature calcium dynamics and electrical coupling in recellularized dECM. In summary, decellularization of human hearts provides a biocompatible scaffold that retains 3-dimensional architecture and vascularity and that can be recellularized with parenchymal and vascular cells. dECM promotes cardiocyte gene expression in stem cells and organizes existing cardiomyocytes into nascent muscle showing electrical coupling. These findings represent a first step toward manufacturing human heart grafts or matrix components for treating cardiovascular disease.
Orczyk-Pawiłowicz, Magdalena; Augustyniak, Daria; Hirnle, Lidia; Kątnik-Prastowska, Iwona
The sugar moiety of IgA is known to provide a link between the innate and adaptive immune systems. Terminally located glycotopes on IgA are potential ligands engaged in the interactions which may modulate the biological activities of IgA. In the present work the expressions of Maackia amurensis (MAA), Sambucus nigra (SNA), Lens culinaris (LCA), Tetragonolobus purpureus (LTA), and Ulex europaeus (UEA) reactive glycotopes on maternal plasma and amniotic IgA were evaluated in relation to the progression of a normal human pregnancy, from the 2nd trimester, throughout the 3rd trimester, perinatal period, post-date pregnancy and delivery, by lectin-IgA-ELISA, using specific biotinylated lectins. The amniotic and maternal plasma IgA concentrations and a degree of SNA and LCA reactivity of maternal plasma IgA were almost unaltered during the normal pregnancy. The amniotic IgA from the 2nd trimester was decorated by MAA-, SNA-reactive and LCA-, LTA-, and UEA-reactive glycotopes. At the turn of the 2nd and 3rd trimesters the expression of MAA-, SNA-, LTA-, and UEA-reactive glycotopes, except for LCA-reactive, increased and remained almost at unaltered levels throughout the perinatal period and delivery. However, in the post-date pregnancy the expression of LCA-, LTA-, and UEA-reactive and SNA-reactive glycotopes were significantly higher. The unique fucosylated and sialylated glycovariants of amniotic IgA associated with the progression of the normal pregnancy may illustrate a general importance of carbohydrate-lectin receptor interactions in the control and modulation of biological events to ensuring homeostasis during pregnancy, protection and well-being of fetus.
Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min
Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies. PMID:27294211
Background Human amniotic fluid cells (hAFCs) may differentiate into multiple cell lineages and thus have a great potential to become a donor cell source for regenerative medicine. The ability of hAFCs to differentiate into germ cell and oocyte-like cells has been previously documented. Herein we report the potential use of hAFCs to help restore follicles in clinical condition involving premature ovarian failure. Results Human amniotic fluid was obtained via amniocentesis, yielding a subpopulation of cloned hAFCs that was able to form embryoid bodies (EBs) and differentiate into three embryonic germ layers. Moreover, culture of EBs in medium containing human follicular fluid (HFF) or a germ cell maturation factor cocktail (FAC), expressed germ cells markers such as BLIMP1, STELLA, DAZL, VASA, STRA8, SCP3, SCP1, and GDF9. Furthermore, one cell line was grown from clone cells transfected with lentivirus-GFP and displaying morphological characteristics of mesenchymal cells, had the ability to restore ovarian morphology following cell injection into the ovaries of mice sterilized by intraperitoneal injection of cyclophosphamide and busulphan. Restored ovaries displayed many follicle-enclosed oocytes at all stages of development, but no oocytes or follicles were observed in sterilized mice whose ovaries had been injected with medium only (control). Notably, identification of GFP-labeled cells and immunostaining with anti–human antigen-specific antibodies demonstrated that grafted hAFCs survived and differentiated into granulosa cells which directed oocyte maturation. Furthermore, labeling of ovarian tissue for anti-Müllerian hormone expression, a functional marker of folliculogenesis, was strong in hAFCs-transplanted ovaries but inexistent in negative controls. Conclusion These findings highlight the possibility of using human amniotic fluid-derived stem cells in regenerative medicine, in particular in the area of reproductive health. PMID:24006896
Razavi Tousi, Seyed Mohammad Taghi; Faghihi, Mahdieh; Nobakht, Maliheh; Molazem, Mohammad; Kalantari, Elham; Darbandi Azar, Amir; Aboutaleb, Nahid
Background: Recently, stem cells have been considered for the treatment of heart diseases, but no marked improvement has been recorded. This is the first study to examine the functional and histological effects of the transplantation of human amniotic mesenchymal stromal cells (hAMSCs) in rats with heart failure (HF). Methods: This study was conducted in the years 2014 and 2015. 35 male Wistar rats were randomly assigned into 5 equal experimental groups (7 rats each) as 1- Control 2- Heart Failure (HF) 3- Sham 4- Culture media 5- Stem Cell Transplantation (SCT). Heart failure was induced using 170 mg/kg/d of isoproterenol subcutaneously injection in 4 consecutive days. The failure confirmed by the rat cardiac echocardiography on day 28. In SCT group, 3×106 cells in 150 µl of culture media were transplanted to the myocardium. At the end, echocardiographic and hemodynamic parameters together with histological evaluation were done. Results: Echocardiography results showed that cardiac ejection fraction in HF group increased from 58/73 ± 9% to 81/25 ± 6/05% in SCT group (p value < 0.001). Fraction shortening in HF group was increased from 27/53 ± 8/58% into 45/55 ± 6/91% in SCT group (p value < 0.001). Furthermore, hAMSCs therapy significantly improved mean diastolic blood pressure, mean arterial pressure, left ventricular systolic pressure, rate pressure product, and left ventricular end-diastolic pressure compared to those in the HF group, with the values reaching the normal levels in the control group. A marked reduction in fibrosis tissue was also found in the SCT group (p value < 0.001) compared with the animals in the HF group. Conclusion: The transplantation of hAMSCs in rats with heart failure not only decreased the level of fibrosis but also conferred significant improvement in heart performance in terms of echocardiographic and hemodynamic parameters. PMID:27956912
Fatimah, Simat Siti; Ng, Sook Luan; Chua, Kien Hui; Hayati, Abdul Rahman; Tan, Ay Eeng; Tan, Geok Chin
Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.
García-Castro, Irma Lydia; García-López, Guadalupe; Ávila-González, Daniela; Flores-Herrera, Héctor; Molina-Hernández, Anayansi; Portillo, Wendy; Ramón-Gallegos, Eva; Díaz, Néstor Fabián
Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC. PMID:26720151
Papanna, Ramesha; Mann, Lovepreet K; Tseng, Scheffer C.G.; Stewart, Russell J; Kaur, Sarbjit S; Swindle, M Michael; Kyriakides, Themis R; Tatevian, Nina; Moise, Kenneth J
Introduction We investigated the ability of cryopreserved human amniotic membrane (hAM) scaffold sealed with an underwater adhesive, bio-inspired by marine sandcastle worms to promote healing of iatrogenic fetal membrane defects in a pregnant swine model. Methods Twelve Yucatan miniature pigs underwent laparotomy under general anesthesia at 70 days gestation (term = 114 days). The gestational sacs were assigned to uninstrumented (n=24) and instrumented with 12 Fr trocar, which was further randomized into four different arms-no hAM patch, (n=22), hAM patch secured with suture (n=16), hAM patch with no suture (n=14), and hAM patch secured with adhesive (n=9). The animals were euthanized 20 days after the procedure. Gross and histological examination of the entry site was performed for fetal membrane healing. Results There were no differences in fetal survival, amniotic fluid levels, or dye-leakage from the amniotic cavity between the groups. The fetal membranes spontaneously healed in instrumented sacs without hAM patches. In sacs with hAM patches secured with sutures, the patch was incorporated into the swine fetal membranes. In sacs with hAM patches without sutures, 100% of the patches were displaced from the defect site, whereas in sacs with hAM patches secured with adhesive 55% of the patches remained in place and showed complete healing (p=0.04). Discussion In contrast to humans, swine fetal membranes heal spontaneously after an iatrogenic injury and thus not an adequate model. hAM patches became incorporated into the defect site by cellular ingrowth from the fetal membranes. The bioinspired adhesive adhered the hAM patches within the defect site. PMID:26059341
de Meringo, A; Morscheidt, C; Thélohan, S; Tiesler, H
The assessment of biodurability of man-made vitreous fibers is essential to the limitation of health hazards associated with human exposure to environments in which respirable fibers are present. In vitro acellular systems provide effective test methods of measuring fiber solubility provided care is taken to select the most suitable solvent and test conditions for the specific fiber type and dimension. PMID:7882955
Zeng, Wei; Li, Yanwei; Zeng, Guangwei; Yang, Bo; Zhu, Yu
Amniotic membranes (AM) have been used in a wide range of clinical applications. We successfully extracted mesenchymal stem cells (MSCs) from human AM, but little is known about the use and efficacy of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) for the treatment of alkali burns. We utilized hAM-dMSCs transplantation, AM grafting, and their combined use in the treatment of alkali burns. An experimental model in rabbits was devised to analyze the use of these techniques with immunocytochemistry and ELISA. The survival and migration of hAM-dMSCs labeled by SPION in the host were assessed with Prussian blue staining. Compared with the control group, the treated groups demonstrated faster reconstruction of the corneal epithelium, and lower levels of corneal opacification and neovascularization within corneal alkali burns. Furthermore, dark blue-stained particles were detected in the limbus corneae at day 28. These results demonstrated the ability of hAM-dMSCs to enhance epithelial healing and reduce corneal opacification and neovascularization in corneal alkali wounds.
Maddipati, Krishna Rao; Romero, Roberto; Chaiworapongsa, Tinnakorn; Zhou, Sen-Lin; Xu, Zhonghui; Tarca, Adi L; Kusanovic, Juan Pedro; Munoz, Hernan; Honn, Kenneth V
Lipid mediators play an important role in reproductive biology, especially, in parturition. Enhanced biosynthesis of eicosanoids, such as prostaglandin E2 (PGE2) and PGF2α, precedes the onset of labor as a result of increased expression of inducible cyclooxygenase 2 (COX-2) in placental tissues. Metabolism of arachidonic acid results in bioactive lipid mediators beyond prostaglandins that could significantly influence myometrial activity. Therefore, an unbiased lipidomic approach was used to profile the arachidonic acid metabolome of amniotic fluid. In this study, liquid chromatography-mass spectrometry was used for the first time to quantitate these metabolites in human amniotic fluid by comparing patients at midtrimester, at term but not in labor, and at term and in spontaneous labor. In addition to exposing novel aspects of COX pathway metabolism, this lipidomic study revealed a dramatic increase in epoxygenase- and lipoxygenase-pathway-derived lipid mediators in spontaneous labor with remarkable product selectivity. Despite their recognition as anti-inflammatory lipid mediators and regulators of ion channels, little is known about the epoxygenase pathway in labor. Epoxygenase pathway metabolites are established regulators of vascular homeostasis in cardiovascular and renal physiology. Their presence as the dominant lipid mediators in spontaneous labor at term portends a yet undiscovered physiological function in parturition.
De Gemmis, Paola; Lapucci, Cristina; Bertelli, Matteo; Tognetto, Anna; Fanin, Erika; Vettor, Roberto; Pagano, Claudio; Pandolfo, Massimo; Fabbri, Andrea
Regulation of adipocyte differentiation is an important process in the control of adipose tissue development. So far, adipogenesis has been investigated through the use of various experimental models. In this work, we used human mesenchymal stem cells (hMSCs) obtained from amniotic fluid (AF) as an alternative model more representative of what naturally happens in vivo. In our opinion, these hMSCs are still not influenced by differentiation stimuli and could act in a way more correspondent to the physiological process of adipogenesis, representing also an ethically acceptable alternative to totipotent human embryonic stem cells (ES). Adipocyte differentiation was monitorated following the expressions of key genes. We measured the expression levels of PPARgamma2, PPARgamma-C1alpha, UCP-1, adipsin, and leptin genes using quantitative real-time PCR. We tested our experimental model with two different media. Understanding in vivo adipogenesis mechanisms will shed light on the pathophysiology of many diseases.
Nerve Allografts plus amniotic Fluid Derived Stem Cells (AFS). PRINCIPAL INVESTIGATOR: Li, Zhongyu CONTRACTING ORGANIZATION: Wake Forest...Gap Peripheral Nerve Injuries Using 5a. CONTRACT NUMBER Acellular Nerve Allografts plus amniotic Fluid Derived Stem Cells (AFS). 5b. GRANT NUMBER...Major accomplishments this year include successful seeding of AFS into ANA. This accomplishment also documented that these cells remained viable up
Knickmeyer, Rebecca Christine; Wheelwright, Sally; Taylor, Kevin; Raggatt, Peter; Hackett, Gerald; Baron-Cohen, Simon
Sex differences in play are apparent in a number of mammalian species, including humans. Prenatal testosterone may contribute to these differences. The authors report the first attempt to correlate gender-typed play in a normative sample of humans with measurements of amniotic testosterone (aT). Testosterone was measured in the amniotic fluid of…
Rosner, Margit; Dolznig, Helmut; Schipany, Katharina; Mikula, Mario; Brandau, Oliver; Hengstschläger, Markus
Besides their putative usage for therapies, stem cells are a promising tool for functional studies of genes involved in human genetic diseases or oncogenesis. For this purpose induced pluripotent stem (iPS) cells can be derived from patients harbouring specific mutations. In contrast to adult stem cells, iPS cells are pluripotent and can efficiently be grown in culture. However, iPS cells are modulated due to the ectopic induction of pluripotency, harbour other somatic mutations accumulated during the life span of the source cells, exhibit only imperfectly cleared epigenetic memory of the source cell, and are often genomically instable. In addition, iPS cells from patients only allow the investigation of mutations, which are not prenatally lethal. Embryonic stem (ES) cells have a high proliferation and differentiation potential, but raise ethical issues. Human embryos, which are not transferred in the course of in vitro fertilization, because of preimplantation genetic diagnosis of a genetic defect, are still rarely donated for the establishment of ES cell lines. In addition, their usage for studies on gene functions for oncogenesis is hampered by the fact the ES cells are already tumorigenic per se. In 2003 amniotic fluid stem (AFS) cells have been discovered, which meanwhile have been demonstrated to harbour the potential to differentiate into cells of all three germ layers. Monoclonal human AFS cell lines derived from amniocenteses have a high proliferative potential, are genomically stable and are not associated with ethical controversies. Worldwide amniocenteses are performed for routine human genetic diagnosis. We here discuss how generation and banking of monoclonal human AFS cell lines with specific chromosomal aberrations or monogenic disease mutations would allow to study the functional consequences of disease causing mutations. In addition, recently a protocol for efficient and highly reproducible siRNA-mediated long-term knockdown of endogenous gene
Briese, V; Straube, W; Brock, J; Lorenz, U
Secretory immunoglobulin A (S-IgA) was estimated in amniotic fluid samples by means of the single radial immunodiffusion according to Mancini. A monospecific antiserum against human secretory component was used. 163 amniotic fluid samples from normal pregnancies and risk pregnancies respectively were investigated. Within the 3rd trimenon the S-IgA content in amniotic fluid increased significantly. With respect to literature and examinations performed previously a connection between S-IgA content in amniotic fluid and fetal lung maturity seems to be possible.
Bryant, E M; Crouch, E; Bornstein, P; Martin, G M; Johnston, P; Hoehn, H
Pure populations of proliferating synkaryons were obtained from polyethylene glycol-mediated crosses between diploid human foreskin fibroblasts and epithelioid amniotic fluid cells. These hybrids proved to be chromosomally stable tetraploids. They continuously produced heteropolymeric G6PD and showed strictly additive patterns of silver staining of both parental sets of nucleolar organizing chromosomes. Collagenous proteins characteristic of the fibroblast parent were synthesized, while fibronectin production appeared to be directed by the epithelioid portion of the genome. Even though these heterotypic hybrids proliferated at a reduced rate and achieved fewer population doublings relative to homotypic (fibroblast X fibroblast) crosses, they survived passage by trypsinization better than pure populations of epithelioid cells. These observations suggest a concerted action of both parental genomes with respect to proteins responsible for "household" functions, but complementation and possibly modulation of gene action with respect to "luxury" protein synthesis and cell growth. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:717401
Zibara, Kazem; Hamdan, Rima; Dib, Leila; Sindet-Pedersen, Steen; Kharfan-Dabaja, Mohamed; Bazarbachi, Ali; El-Sabban, Marwan
Hematopoietic stem cells (HSC) derived from cord blood (CB), bone marrow (BM), or mobilized peripheral blood (PBSC) can differentiate into multiple lineages such as lymphoid, myeloid, erythroid cells and platelets. The local microenvironment is critical to the differentiation of HSCs and to the preservation of their phenotype in vivo. This microenvironment comprises a physical support supplied by the organ matrix as well as tissue specific cytokines, chemokines and growth factors. We investigated the effects of acellular bovine bone marrow extracts (BME) on HSC in vitro and in vivo. We observed a significant increase in the number of myeloid and erythroid colonies in CB mononuclear cells (MNC) or CB CD34+ cells cultured in methylcellulose media supplemented with BME. Similarly, in xeno-transplantation experiments, pretreatment with BME during ex-vivo culture of HSCs induced a significant increase in HSC engraftment in vivo. Indeed, we observed both an increase in the number of differentiated myeloid, lymphoid and erythroid cells and an acceleration of engraftment. These results were obtained using CB MNCs, BM MNCs or CD34+ cells, transplanted in immuno-compromised mice (NOD/SCID or NSG). These findings establish the basis for exploring the use of BME in the expansion of CB HSC prior to HSC Transplantation. This study stresses the importance of the mechanical structure and soluble mediators present in the surrounding niche for the proper activity and differentiation of stem cells. PMID:22768336
Balbi, Carolina; Piccoli, Martina; Barile, Lucio; Papait, Andrea; Armirotti, Andrea; Principi, Elisa; Reverberi, Daniele; Pascucci, Luisa; Becherini, Pamela; Varesio, Luigi; Mogni, Massimo; Coviello, Domenico; Bandiera, Tiziano; Pozzobon, Michela; Cancedda, Ranieri; Bollini, Sveva
Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT(+) hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT(+) hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn(F7/F7) mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT(+) hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. © Stem Cells Translational Medicine 2017.
Naseroleslami, Maryam; Parivar, Kazem; Khoei, Samideh; Aboutaleb, Nahid
Objective The label and detection of cells injected into target tissues is an area of focus for researchers. Iron oxide nanoparticles can be used to label cells as they have special characteristics. The purpose of this study is to examine the effects of iron oxide nanoparticles on human-derived amniotic membrane stem cell (hAMCs) survival and to investigate the magnetic properties of these nanoparticles with increased contrast in magnetic resonance imaging (MRI). Materials and Methods In this experimental study, we initially isolated mesenchymal stem cells from amniotic membranes and analyzed them by flow cytometry. In addition, we synthesized superparamagnetic iron oxide nanoparticles (SPIONs) and characterized them by various methods. The SPIONs were incubated with hAMCs at concentrations of 25-800 μg/mL. The cytotoxicity of nanoparticles on hAMCs was measured by the MTT assay. Next, we evaluated the effectiveness of the magnetic nanoparticles as MRI contrast agents. Solutions of SPION were prepared in water at different iron concentrations for relaxivity measurements by a 1.5 Tesla clinical MRI instrument. Results The isolated cells showed an adherent spindle shaped morphology. Polyethylene glycol (PEG)-coated SPIONs exhibited a spherical morphology. The average particle size was 20 nm and magnetic saturation was 60 emu/g. Data analysis showed no significant reduction in the percentage of viable cells (97.86 ± 0.41%) after 72 hours at the 125 μg/ml concentration compared with the control. The relaxometry results of this SPION showed a transverse relaxivity of 6.966 (μg/ml.s)-1 Conclusion SPIONs coated with PEG used in this study at suitable concentrations had excellent labeling efficiency and biocompatibility for hAMCs. PMID:27602314
Sandor, Maryellen; Leamy, Patrick; Assan, Pearl; Hoonjan, Amardeep; Huang, Li-Ting; Edwards, Marianne; Zuo, Wenqi; Li, Hui; Xu, Hui
Objective: Benchtop methods were evaluated for preclinical inflammation/capsule formation correlation following implantation of human acellular dermal matrices. Methods: Dermal matrices were compared with native dermis for structure (histology, scanning electron microscopy), collagen solubility (hydroxyproline), enzymatic susceptibility (collagenase), and thermal stability (differential scanning calorimetry). Results were compared with implantation outcomes in a primate tissue expander model. Results: Native dermis, electron beam-sterilized, and freeze-dried human acellular dermal matrices had equivalent morphology, acid-soluble collagen (60.5% ± 6.3%, 65.3% ± 3.2%, and 63.3% ± 2.4%, respectively), and collagenase resistance. Implant results showed minimal inflammation/matrix degradation, lack of capsule formation, insignificant elastic modulus change (57.65 ± 20.24 MPa out-of-package/44.84 ± 23.87 MPa in vivo), and low antibody induction (2- to 8-fold increase) for electron beam-sterilized matrix. Similar results for freeze-dried dermal matrix were previously observed. γ-Irradiated, γ-irradiated/freeze-dried, and ethanol-stored dermal matrices were statistically different from native dermis for acid-soluble collagen (82.4% ± 5.8%, 72.2% ± 6.2%, and 76.8% ± 5.0%, respectively) and collagenase digestion rate, indicating matrix damage. γ-Irradiated matrix-implanted animals demonstrated elevated inflammatory response, foreign body giant cells, capsule formation at the tissue expander junction, and robust matrix metalloproteinase-1 staining with significant elastic modulus decrease (37.43 ± 7.52 MPa out-of-package/19.58 ± 1.16 MPa in vivo). Antibody increase (32- to 128-fold) was observed 6 to 10 weeks following γ-irradiated matrix implantation. Ethanol-stored dermal matrix elicited an acute antibody response (4- to 128-fold increase, 2-4 weeks) and macrophage-concentrated synovial-like hyperplasia at the tissue expander junction, moderate matrix
Leamy, Patrick; Assan, Pearl; Hoonjan, Amardeep; Huang, Li-Ting; Edwards, Marianne; Zuo, Wenqi; Li, Hui; Xu, Hui
Objective: Benchtop methods were evaluated for preclinical inflammation/capsule formation correlation following implantation of human acellular dermal matrices. Methods: Dermal matrices were compared with native dermis for structure (histology, scanning electron microscopy), collagen solubility (hydroxyproline), enzymatic susceptibility (collagenase), and thermal stability (differential scanning calorimetry). Results were compared with implantation outcomes in a primate tissue expander model. Results: Native dermis, electron beam–sterilized, and freeze-dried human acellular dermal matrices had equivalent morphology, acid-soluble collagen (60.5% ± 6.3%, 65.3% ± 3.2%, and 63.3% ± 2.4%, respectively), and collagenase resistance. Implant results showed minimal inflammation/matrix degradation, lack of capsule formation, insignificant elastic modulus change (57.65 ± 20.24 MPa out-of-package/44.84 ± 23.87 MPa in vivo), and low antibody induction (2- to 8-fold increase) for electron beam–sterilized matrix. Similar results for freeze-dried dermal matrix were previously observed. γ-Irradiated, γ-irradiated/freeze-dried, and ethanol-stored dermal matrices were statistically different from native dermis for acid-soluble collagen (82.4% ± 5.8%, 72.2% ± 6.2%, and 76.8% ± 5.0%, respectively) and collagenase digestion rate, indicating matrix damage. γ-Irradiated matrix-implanted animals demonstrated elevated inflammatory response, foreign body giant cells, capsule formation at the tissue expander junction, and robust matrix metalloproteinase-1 staining with significant elastic modulus decrease (37.43 ± 7.52 MPa out-of-package/19.58 ± 1.16 MPa in vivo). Antibody increase (32- to 128-fold) was observed 6 to 10 weeks following γ-irradiated matrix implantation. Ethanol-stored dermal matrix elicited an acute antibody response (4- to 128-fold increase, 2-4 weeks) and macrophage-concentrated synovial-like hyperplasia at the tissue expander junction, moderate matrix
Background The use of human acellular dermis (hAD) to close open abdomen in the treatment process of severe peritonitis might be an alternative to standard care. This paper describes an investigation of the effects of fluids simulating an open abdomen environment on the biomechanical properties of Epiflex® a cell-free human dermis transplant. Methods hAD was incubated in Ringers solution, blood, urine, upper gastrointestinal (upper GI) secretion and a peritonitis-like bacterial solution in-vitro for 3 weeks. At day 0, 7, 14 and 21 breaking strength was measured, tensile strength was calculated and standard fluorescence microscopy was performed. Results hAD incubated in all five of the five fluids showed a decrease in mean breaking strength at day 21 when compared to day 0. However, upper GI secretion was the only incubation fluid that significantly reduced the mechanical strength of Epiflex after 21days of incubation when compared to incubation in Ringer’s solution. Conclusion hAD may be a suitable material for closure of the open abdomen in the absence of upper GI leakage and pancreatic fistulae. PMID:24468201
Janz, Felipe de Lara; Debes, Adriana de Aguiar; Cavaglieri, Rita de Cássia; Duarte, Sérgio Aloísio; Romão, Carolina Martinez; Morón, Antonio Fernandes; Zugaib, Marcelo; Bydlowski, Sérgio Paulo
Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me2SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me2SO and glycerol presented workable viability ratios. PMID:22665987
Moorefield, Emily C; McKee, Elizabeth E; Solchaga, Luis; Orlando, Guisseppe; Yoo, James J; Walker, Steve; Furth, Mark E; Bishop, Colin E
Amniotic fluid stem (AFS) cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC) show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR) and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family members as well as interleukin-6 (IL-6). AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α), MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and absence of
Chiang, Chien-Yi; Liu, Shih-An; Sheu, Meei-Ling; Chen, Fu-Chou; Chen, Chun-Jung; Su, Hong-Lin; Pan, Hung-Chuan
Purpose The neurobehavior of neuropathic pain by chronic constriction injury (CCI) of sciatic nerve is very similar to that in humans, and it is accompanied by a profound local inflammation response. In this study, we assess the potentiality of human amniotic fluid derived mesenchymal stem cells (hAFMSCs) for alleviating the neuropathic pain in a chronic constriction nerve injury model. Methods and Methods This neuropathic pain animal model was conducted by four 3–0 chromic gut ligatures loosely ligated around the left sciatic nerve in Sprague—Dawley rats. The intravenous administration of hAFMSCs with 5x105 cells was conducted for three consecutive days. Results The expression IL-1β, TNF-α and synaptophysin in dorsal root ganglion cell culture was remarkably attenuated when co-cultured with hAFMSCs. The significant decrease of PGP 9.5 in the skin after CCI was restored by administration of hAFMSCs. Remarkably increased expression of CD 68 and TNF-α and decreased S-100 and neurofilament expression in injured nerve were rescued by hAFMSCs administration. Increases in synaptophysin and TNF-α over the dorsal root ganglion were attenuated by hAFMSCs. Significant expression of TNF-α and OX-42 over the dorsal spinal cord was substantially attenuated by hAFMSCs. The increased amplitude of sensory evoked potential as well as expression of synaptophysin and TNF-α expression was alleviated by hAFMSCs. Human AFMSCs significantly improved the threshold of mechanical allodynia and thermal hyperalgesia as well as various parameters of CatWalk XT gait analysis. Conclusion Human AFMSCs administration could alleviate the neuropathic pain demonstrated in histomorphological alteration and neurobehavior possibly through the modulation of the inflammatory response. PMID:27441756
Loeffelbein, Denys J.; Rohleder, Nils H.; Eddicks, Matthias; Baumann, Claudia M.; Stoeckelhuber, Mechthild; Wolff, Klaus-D.; Drecoll, Enken; Steinstraesser, Lars; Hennerbichler, Simone; Kesting, Marco R.
Human amniotic membrane (HAM) has been used as a biomaterial in various surgical procedures and exceeds some qualities of common materials. We evaluated HAM as wound dressing for split-thickness skin-graft (STSG) donor sites in a swine model (Part A) and a clinical trial (Part B). Part A: STSG donor sites in 4 piglets were treated with HAM or a clinically used conventional polyurethane (PU) foil (n = 8 each). Biopsies were taken on days 5, 7, 10, 20, 40, and 60 and investigated immunohistochemically for alpha-smooth muscle actin (αSMA: wound contraction marker), von Willebrand factor (vWF: angiogenesis), Ki-67 (cell proliferation), and laminin (basement membrane integrity). Part B: STSG donor sites in 45 adult patients (16 female/29 male) were treated with HAM covered by PU foam, solely by PU foam, or PU foil/paraffin gauze (n = 15 each). Part A revealed no difference in the rate of wound closure between groups. HAM showed improved esthetic results and inhibitory effects on cicatrization. Angioneogenesis was reduced, and basement membrane formation was accelerated in HAM group. Part B: no difference in re-epithelialization/infection rate was found. HAM caused less ichor exudation and less pruritus. HAM has no relevant advantage over conventional dressings but might be a cost-effective alternative. PMID:25003117
Lv, Xiaokui; Guo, Qianping; Han, Fengxuan; Chen, Chunyang; Ling, Christopher; Chen, Weiguo; Li, Bin
Tissue engineering-based urethral replacement holds potential for repairing large segmental urethral defects, which remains a great challenge at present. This study aims to explore the potential of combining biodegradable poly(l-lactide) (PLLA)/poly(ethylene glycol) (PEG) scaffolds and human amniotic mesenchymal cells (hAMSCs) for repairing urethral defects. PLLA/PEG fibrous scaffolds with various PEG fractions were fabricated via electrospinning. The scaffolds were then seeded with hAMSCs prior to implantation in New Zealand male rabbits that had 2.0 cm-long defects in the urethras. The rabbits were randomly divided into three groups. In group A, hAMSCs were grown on PLLA/PEG scaffolds for two days and then implanted to the urethral defects. In group B, only the PLLA/PEG scaffolds were used to rebuild the rabbit urethral defect. In group C, the urethral defect was reconstructed using a regular urethral reparation technique. The repair efficacy was compared among the three groups by examining the urethral morphology, tissue reconstruction, luminal patency, and complication incidence (including calculus formation, urinary fistula, and urethral stricture) using histological evaluation and urethral radiography methods. Findings from this study indicate that hAMSCs-loaded PLLA/PEG scaffolds resulted in the best urethral defect repair in rabbits, which predicts the promising application of a tissue engineering approach for urethral repair. PMID:27517902
Fleury, Vincent; Murukutla, Ameya Vaishnavi; Chevalier, Nicolas R.; Gallois, Benjamin; Capellazzi-Resta, Marina; Picquet, Pierre; Peaucelle, Alexis
We present a detailed study of the formation of the amniotic sac in the avian embryo, and a comparison with the crocodile amniotic sac. We show that the amniotic sac forms at a circular line of stiffness contrast, separating rings of cell domains. Cells align at this boundary, and this in turn orients and concentrates the tension forces. The tissue fold which forms the amniotic sac is locked exactly along this line due to the colocalization of the stiffness contrast and of the tensile force. In addition, the tensile force plays a regenerative role when the amniotic sac is cut. The fold forming the ventral side of the embryo displays the same characteristics. This work shows that amniote embryogenesis consists of a cascade of buckling events taking place at the boundaries between regions of differing mechanical properties. Hence, amniote embryogenesis relies on a simple and robust biomechanical scheme used repeatedly, and selected ancestrally.
Ma, Xiaorong; Li, Huanqi; Xin, Shujia; Ma, Yueting; Ouyang, Tianxiang
Human embryonic stem cells (hESCs) are pluripotent cells which can give rise to almost all adult cell lineages. Culture system of hESCs is complex, requiring exogenous b-FGF and feeder cell layer. Human mesenchymal stem cells (MSCs) not only synthesize soluble cytokines or factors such as b-FGF, but also provide other mechanism which might play positive role on sustaining hESCs propagation and pluripotency. Human amniotic fluid stem (AFS) cells, which share characteristics of both embryonic and adult stem cells, have been regarded as promising cells for regenerative medicine. Taking advantage by AFS cells, we studied the ability of AFS cells in supporting undifferentiated propagation and pluripotency of Chinese population derived X-01 hESCs. Human AF-type amniotic fluid stem cells (hAF-AFSCs) transcribed genes including Activin A, TGF-β1, Noggin and b-FGF, which involved in maintaining pluripotency and self-renewal of hESCs. Compared to mouse embryonic fibroblasts (MEFs), hAF-AFSCs secreted higher concentration of b-FGF which was important in hESCs culture (P < 0.05). The hESCs were propagated more than 30 passages on hAF-AFSCs layer with exogenous b-FGF supplementation, keeping undifferentiated status. While exogenous b-FGF was obviated, propagation of hESCs with undifferentiated status was dependent on density of hAF-AFSC feeder layer. Lower density of hAF-AFSCs resulted in rapid decline in undifferentiated clone number, while higher ones hindered the growth of colonies. The most appropriate hAF-AFSCs feeder density to maintain the X-01 hESC line without exogenous b-FGF was 15-20×10(4)/well. To the best of our knowledge, this is the first study demonstrating that hAF-AFSCs could support undifferentiated propagation and pluripotency of Chinese population derived hESCs without exogenous b-FGF supplementation.
Li, Xueyong; Meng, Xianghai; Wang, Xiaolin; Li, Yuejun; Li, Wangzhou; Lv, Xiaoxing; Xu, Xiaoli; Lei, Zhanjun; Li, Jinqing
The potential of acellular dermal matrix (ADM) to improve cosmetic and functional outcomes has been demonstrated; however, there have been few clinical comparative studies assessing the long-term morphological, histological and functional changes after ADM placement. This study was designed to retrospectively evaluate the long-term outcomes of the cograft acellular dermal matrix with autologous thin split-thickness skin for the coverage of wounds in extensively burned patients. Thirty burn patients treated with a composite graft of ADM with autologous split-thickness skin from January 2007 to December 2009 were enrolled in this study. Another group of thirty patients who received only an autogenous split-thickness skin implant served as the control. Our study revealed that the collagen in the dermis treated with ADM were ordered, and the proportion of collagen III/I was much higher in the control group than in the ADM group. The basement membrane was prominent and continuous. Meanwhile, the VBSS (Vancouver Burn Skin Score) was used to evaluate skin quality, which shows a significant differences between the two group (P<0.001). Then the functional level was evaluated by the BI (Barthel Index), and the ADM group was much better than the control group (P=0.005). Based on these results, we concluded that the composite graft of ADM with autologous thin split-thickness skin was suitable for repairing the defects in functional areas after a burn. This technique might facilitate wound management with acceptable esthetic outcomes, good functional recovery and less scar hyperplasia at the donor site.
Moslemi, Sam; Joraghi, Sajjad Ahmadi; Roshanravan, Reza; Ghahramani, Leila; Mohammadianpanah, Mohammad; Hosseinzadeh, Masood; Rezaianzadeh, Abbas; Hussein, Ahmed Mohammed Ali; Najibpour, Neda; Hosseini, Seyed Vahid
Background: Radiotherapy is one of the most important factors which results in negative effects on wound healing and increases anastomosis leakage. Diverting loop ileostomy has been usually performed after colorectal anastomosis in cases of colorectal cancer with a history of neoadjuvant radiotherapy to decrease the chance of leakage. Considering the side effects of diverting loop ileostomy, the objective of the present study is to investigate the effect of human amniotic membrane (HAM) on colorectal anastomosis leakage after neo-adjuvant radiotherapy. Methods: In this experimental animal study, 20 crossbreed rabbits were randomly divided into two groups (case group: 13 rabbits, control group: 7 rabbits) after receiving an equal dose of external beam radiation. Four weeks after irradiation, resection of 4 cm of colorectal segment and end-to-end single layer anastomosis were conducted. In the case group, a 2×2 cm wrap of HAM applied around the site of anastomosis. Eight weeks later, all the survived rabbits were sacrificed. A segment of anastomotic sites was resected in all expired and survived rabbits and sent for pathological evaluation. Mann-Whitney U Test (SPSS for Windows, Ver. 16, Chicago, IL) was applied to analyze healing scores between the two groups. Results: Due to anastomosis dehiscence, 5 rabbits expired in the control group, but all the 13 rabbits (case group) survived after 8 weeks and showed no leakage. In addition, pathological evaluation revealed significant epithelialization and neovascularization in the case group. Statistically, healing score was higher in the case group rather than the control group (P<0.001). Conclusion: To prevent post irradiation colorectal anastomosis leakage, the use of HAM might play a significant role and a feasible technical approach. PMID:27853330
Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh
Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.
Bolat, Elif; Kocamaz, Erdoğan; Kulahcilar, Zeki; Yilmaz, Ali; Topcu, Abdullah; Coskun, Mehmet Erdal
Study Design A retrospective study. Purpose The aim of this study was to evalute the effects of mitomycin-C, sodium hyaluronate and human amniotic fluid on preventing spinal epidural fibrosis. Overview of Literature The role of scar tissue in pain formation is not exactly known, but it is reported that scar tissue causes adhesions between anatomic structures. Intensive fibrotic tissue compresses on anatomic structures and increases the sensitivity of the nerve root for recurrent herniation and lateral spinal stenosis via limiting movements of the root. Also, neuronal atrophy and axonal degeneration occur under scar tissue. Methods The study design included 4 groups of rats: group 1 was the control group, groups 2, 3, and 4 receieved antifibrotic agents, mitomycin-C (group 2), sodium hyaluronate (group 3), and human amniotic fluid (group 4). Midline incision for all animals were done on L5 for total laminectomy. Four weeks after the surgery, the rats were sacrificed and specimens were stained with hematoxylin-eosin and photos of the slides were taken for quantitive assesment of the scar tissue. Results There was no significant scar tissue in the experimental animals of groups 2, 3, and 4. It was found that there was no significant difference between drug groups, but there was a statistically significant difference between the drug groups and the control group. Conclusions This experimental study shows that implantation of mitomycin-C, sodium hyaluronate and human amniotic fluid reduces epidural fibrosis and adhesions after spinal laminectomy in rat models. Further studies in humans are needed to determine the complications of the agents researched. PMID:24353840
Wu, Qianqian; Fang, Tao; Lang, Hongxin; Chen, Min; Shi, Ping; Pang, Xining; Qi, Guoxian
In vivo studies have shown that amnion-produced growth factors participate in many diseases that involve angiogenesis, re-epithelialization and immunomodulation. Although human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs) can be obtained from amniotic membranes, there is little information regarding their biological differences. The aim of the present study was to isolate and characterize cells from human amnions, to investigate the biological potential and behavior of these cells on the function of endothelial cells in vivo and in vitro and to examine variations in the expression profile of growth factors in different human amnion-derived cell types. Amnion fragments were enzymatically digested into two cell fractions, which were analyzed by mesenchymal and epithelial cell markers. Human aortic endothelial cells (hAoECs) were cultured with conditioned medium (CdM) collected from hAECs or hAMSCs. We used scratch and Transwell assays to evaluate migration ability; Cell Counting Kit-8 (CCK-8) and cell cycle analysis to evaluate proliferation ability; and a Matrigel tube formation assay to evaluate angiogenesis ability. To detect expression of angiogenesis-related genes, qPCR and enzyme-linked immunosorbent assay (ELISA) analyses were conducted. As stem cells, hAECs and hAMSCs all expressed the stem cell markers SSEA-4, OCT-4 and SOX-2. CdM obtained from hAECs promoted cell migration; CdM obtained from hAMSCs promoted cell proliferation; CdM obtained from hAECs and hAMSCs both promoted angiogenesis in hAoECs. Amnion-derived cells secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, EGF, HB-EGF and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Our results highlight that human amniotic epithelial and mesenchymal stem cells, which showed differences in their soluble factor secretion and angiogenic functions, could be ideal cell sources for
Collado, Maria Carmen; Rautava, Samuli; Aakko, Juhani; Isolauri, Erika; Salminen, Seppo
Interaction with intestinal microbes in infancy has a profound impact on health and disease in later life through programming of immune and metabolic pathways. We collected maternal faeces, placenta, amniotic fluid, colostrum, meconium and infant faeces samples from 15 mother-infant pairs in an effort to rigorously investigate prenatal and neonatal microbial transfer and gut colonisation. To ensure sterile sampling, only deliveries at full term by elective caesarean section were studied. Microbiota composition and activity assessment by conventional bacterial culture, 16S rRNA gene pyrosequencing, quantitative PCR, and denaturing gradient gel electrophoresis revealed that the placenta and amniotic fluid harbour a distinct microbiota characterised by low richness, low diversity and the predominance of Proteobacteria. Shared features between the microbiota detected in the placenta and amniotic fluid and in infant meconium suggest microbial transfer at the foeto-maternal interface. At the age of 3–4 days, the infant gut microbiota composition begins to resemble that detected in colostrum. Based on these data, we propose that the stepwise microbial gut colonisation process may be initiated already prenatally by a distinct microbiota in the placenta and amniotic fluid. The link between the mother and the offspring is continued after birth by microbes present in breast milk. PMID:27001291
Lim, Kyung Sup; Wee, Sung Wook
A myxoma is a benign tumor found in the heart and in various soft tissues; however, a corneal myxoma is rare. A mucinous mass of unknown etiology was observed on the left cornea of a 32-year-old male patient. We performed deep anterior lamellar keratoplasty using acellular corneal tissue and concurrent amniotic membrane transplantation. Hematoxylin and eosin staining revealed vacuolation of the parenchyma and myxoid change in the corneal tissue that occurred in the anterior half of the corneal parenchyma. We identified a myxoid stroma by Alcian blue staining and observed collagen fibers with denatured stroma by Masson trichrome staining. The patient's visual acuity improved from light perception to 20 / 200, and the intraocular pressure remained within the normal range for one year after surgery. The transplanted cornea survived successfully with well-maintained transparency, and recurrence was not observed one year after surgery. PMID:24505204
Spectroscopic characterization by photodiode array detection of human urinary and amniotic protein HC subpopulations fractionated by anion-exchange and size-exclusion high-performance liquid chromatography.
Calero, M; Escribano, J; Soriano, F; Grubb, A; Brew, K; Méndez, E
A procedure for spectroscopic characterization and partial fractionation of human protein HC populations by high-performance liquid chromatography-photodiode array ultraviolet-visible detection is reported. Human protein HC from urine or amniotic fluid fractionated by anion-exchange HPLC in a protein Pak DEAE 5PW appeared to be heterogeneous as judged by the asymmetric elution pattern, consisting of a continuous irregular broad peak with several shoulders distributed along the whole chromatogram. Selected fractions containing shoulders were rechromatographed and finally six symmetrical homogeneous peaks with different retention times were obtained from each protein HC preparation. The direct automatic absorption spectra analyses at each peak maximum, indicated that all of the homogeneous peaks seemed to be protein HC, all of them associated to the same chromophore although with different stoichiometry ratios. Isoelectric focusing showed that each peak was composed of a limited number of subpopulations of protein HC with different isoelectric points. Size microheterogeneity has been also demonstrated in both urinary and amniotic protein HC preparations by a combination of size-exclusion HPLC on a TSK 3000 SW6 column and photodiode array detection. Partial fractionation of human albumin on an analytical anion-exchange Mono-Q PC 1.6/5 column, has allowed the identification of heterogeneous chromophore-containing populations displaying significant absorption in the visible region in resemblance to that of protein HC.
Benavides, Omar M; Brooks, Abigail R; Cho, Sung Kyung; Petsche Connell, Jennifer; Ruano, Rodrigo; Jacot, Jeffrey G
One of the greatest challenges in regenerative medicine is generating clinically relevant engineered tissues with functional blood vessels. Vascularization is a key hurdle faced in designing tissue constructs larger than the in vivo limit of oxygen diffusion. In this study, we utilized fibrin-based hydrogels to serve as a foundation for vascular formation, poly(ethylene glycol) (PEG) to modify fibrinogen and increase scaffold longevity, and human amniotic fluid-derived stem cells (AFSC) as a source of vascular cell types (AFSC-EC). AFSC hold great potential for use in regenerative medicine strategies, especially those involving autologous congenital applications, and we have shown previously that AFSC-seeded fibrin-PEG hydrogels have the potential to form three-dimensional vascular-like networks in vitro. We hypothesized that subcutaneously injecting these hydrogels in immunodeficient mice would both induce a fibrin-driven angiogenic host response and promote in situ AFSC-derived neovascularization. Two weeks postinjection, hydrogels were sectioned, and the following was demonstrated: the average maximum invasion distance of host murine cells into the subcutaneous fibrin/PEG scaffold was 147 ± 90 µm after 1 week and 395 ± 138 µm after 2 weeks; the average number of cell-lined lumen per square millimeter was significantly higher in hydrogels seeded with stem cells or cocultures containing stem cells (MSC, 36.5 ± 11.4; AFSC, 47.0 ± 18.9; AFSC/AFSC-EC, 32.8 ± 11.6; and MSC/HUVEC, 43.1 ± 25.1) versus endothelial cell types alone (AFSC-EC, 9.7 ± 6.1; HUVEC, 14.2 ± 8.8); and a subset of these lumen were characterized by the presence of red blood cells. Select areas of cell-seeded hydrogels contained CD31(+) lumen surrounded by α-smooth muscle cell support cells, whereas control hydrogels with no cells only showed infiltration of α-smooth muscle cell-positive host cells.
Benavides, Omar M; Quinn, Joseph P; Pok, Seokwon; Petsche Connell, Jennifer; Ruano, Rodrigo; Jacot, Jeffrey G
A major limitation in tissue engineering strategies for congenital birth defects is the inability to provide a significant source of oxygen, nutrient, and waste transport in an avascular scaffold. Successful vascularization requires a reliable method to generate vascular cells and a scaffold capable of supporting vessel formation. The broad potential for differentiation, high proliferation rates, and autologous availability for neonatal surgeries make amniotic fluid-derived stem cells (AFSC) well suited for regenerative medicine strategies. AFSC-derived endothelial cells (AFSC-EC) express key proteins and functional phenotypes associated with endothelial cells. Fibrin-based hydrogels were shown to stimulate AFSC-derived network formation in vitro but were limited by rapid degradation. Incorporation of poly(ethylene glycol) (PEG) provided mechanical stability (65%±9% weight retention vs. 0% for fibrin-only at day 14) while retaining key benefits of fibrin-based scaffolds-quick formation (10±3 s), biocompatibility (88%±5% viability), and vasculogenic stimulation. To determine the feasibility of AFSC-derived microvasculature, we compared AFSC-EC as a vascular cell source and AFSC as a perivascular cell source to established sources of these cell types-human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), respectively. Cocultures were seeded at a 4:1 endothelial-to-perivascular cell ratio, and gels were incubated at 37°C for 2 weeks. Mechanical testing was performed using a stress-controlled rheometer (G'=95±10 Pa), and cell-seeded hydrogels were assessed based on morphology. Network formation was analyzed based on key parameters such as vessel thickness, length, and area, as well as the degree of branching. There was no statistical difference between individual cultures of AFSC-EC and HUVEC in regard to these parameters, suggesting the vasculogenic potential of AFSC-EC; however, the development of robust vessels required the
Benavides, Omar M.; Quinn, Joseph P.; Pok, Seokwon; Petsche Connell, Jennifer; Ruano, Rodrigo
A major limitation in tissue engineering strategies for congenital birth defects is the inability to provide a significant source of oxygen, nutrient, and waste transport in an avascular scaffold. Successful vascularization requires a reliable method to generate vascular cells and a scaffold capable of supporting vessel formation. The broad potential for differentiation, high proliferation rates, and autologous availability for neonatal surgeries make amniotic fluid-derived stem cells (AFSC) well suited for regenerative medicine strategies. AFSC-derived endothelial cells (AFSC-EC) express key proteins and functional phenotypes associated with endothelial cells. Fibrin-based hydrogels were shown to stimulate AFSC-derived network formation in vitro but were limited by rapid degradation. Incorporation of poly(ethylene glycol) (PEG) provided mechanical stability (65%±9% weight retention vs. 0% for fibrin-only at day 14) while retaining key benefits of fibrin-based scaffolds—quick formation (10±3 s), biocompatibility (88%±5% viability), and vasculogenic stimulation. To determine the feasibility of AFSC-derived microvasculature, we compared AFSC-EC as a vascular cell source and AFSC as a perivascular cell source to established sources of these cell types—human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), respectively. Cocultures were seeded at a 4:1 endothelial-to-perivascular cell ratio, and gels were incubated at 37°C for 2 weeks. Mechanical testing was performed using a stress-controlled rheometer (G′=95±10 Pa), and cell-seeded hydrogels were assessed based on morphology. Network formation was analyzed based on key parameters such as vessel thickness, length, and area, as well as the degree of branching. There was no statistical difference between individual cultures of AFSC-EC and HUVEC in regard to these parameters, suggesting the vasculogenic potential of AFSC-EC; however, the development of robust vessels required the
A prospective, randomised, controlled, multicentre clinical trial examining healing rates, safety and cost to closure of an acellular reticular allogenic human dermis versus standard of care in the treatment of chronic diabetic foot ulcers.
Zelen, Charles M; Orgill, Dennis P; Serena, Thomas; Galiano, Robert; Carter, Marissa J; DiDomenico, Lawrence A; Keller, Jennifer; Kaufman, Jarrod; Li, William W
Acellular dermal matrices can successfully heal wounds. This study's goal was to compare clinical outcomes of a novel, open-structure human reticular acellular dermis matrix (HR-ADM) to facilitate wound closure in non-healing diabetic foot ulcers (DFUs) versus DFUs treated with standard of care (SOC). Following a 2-week screening period in which DFUs were treated with offloading and moist wound care, patients were randomised to either SOC alone or HR-ADM plus SOC applied weekly for up to 12 weeks. At 6 weeks, the primary outcome time, 65% of the HR-ADM-treated DFUs healed (13/20) compared with 5% (1/20) of DFUs that received SOC alone. At 12 weeks, the proportions of DFUs healed were 80% and 20%, respectively. Mean time to heal within 12 weeks was 40 days for the HR-ADM group compared with 77 days for the SOC group. There was no incidence of increased adverse or serious adverse events between groups or any adverse events related to the graft. Mean and median graft costs to closure per healed wound in the HR-ADM group were $1475 and $963, respectively. Weekly application of HR-ADM is an effective intervention for promoting closure of non-healing DFUs.
Ma, Xiaorong; Zhang, Shengli; Zhou, Junmei; Chen, Baisong; Shang, Yafeng; Gao, Tongbing; Wang, Xue; Xie, Hua; Chen, Fang
Stem cell-based therapy may be the most promising method to cure skeletal muscle degenerative diseases such as Duchenne muscular dystrophy (DMD) and trauma in the future. Human amniotic fluid is enriched with early-stage stem cells from developing fetuses and these cells have cardiomyogenic potential both in vitro and in vivo. In the present study, we investigated the characteristics of human amniotic fluid-derived AF-type stem (HAF-AFS) cells by flow cytometry, immunofluorescence staining, reverse-transcription polymerase chain reaction, and osteogenic and adipogenic differentiation analysis. After confirming the stemness of HAF-AFS cells, we tested whether HAF-AFS cells could differentiate into skeletal myogenic cells in vitro and incorporate into regenerating skeletal muscle in vivo. By temporary exposure to the DNA demethylation agent 5-aza-2'-deoxycytidine (5-Aza dC) or co-cultured with C2C12 myoblasts, HAF-AFS cells differentiated into skeletal myogenic cells, expressing skeletal myogenic cell-specific markers such as Desmin, Troponin I (Tn I) and α-Actinin. Four weeks after transplantation into cardiotoxin-injured and X-ray-irradiated tibialis anterior (TA) muscles of NOD/SCID mice, HAF-AFS cells survived, differentiated into myogenic precursor cells and fused with host myofibres. The findings that HAF-AFS cells differentiate into myogenic cells in vitro and incorporate in skeletal muscle regeneration in vivo hold the promise of HAF-AFS cell-based therapy for skeletal muscle degenerative diseases.
Liu, Te; Cheng, Weiwei; Huang, Yongyi; Huang, Qin; Jiang, Lizhen; Guo, Lihe
Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder
Guller, Anna; Trusova, Inna; Petersen, Elena; Shekhter, Anatoly; Kurkov, Alexander; Qian, Yi; Zvyagin, Andrei
Rationale: Tissue engineering (TE) is an emerging alternative approach to create models of human malignant tumors for experimental oncology, personalized medicine and drug discovery studies. Being the bottom-up strategy, TE provides an opportunity to control and explore the role of every component of the model system, including cellular populations, supportive scaffolds and signalling molecules. Objectives: As an initial step to create a new ex vivo TE model of cancer, we optimized protocols to obtain organ-specific acellular matrices and evaluated their potential as TE scaffolds for culture of normal and tumor cells. Methods and results: Effective decellularization of animals' kidneys, ureter, lungs, heart, and liver has been achieved by detergent-based processing. The obtained scaffolds demonstrated biocompatibility and growthsupporting potential in combination with normal (Vero, MDCK) and tumor cell lines (C26, B16). Acellular scaffolds and TE constructs have been characterized and compared with morphological methods. Conclusions: The proposed methodology allows creation of sustainable 3D tumor TE constructs to explore the role of organ-specific cell-matrix interaction in tumorigenesis.
Hosper, Nynke A; Bank, Ruud A; van den Berg, Paul P
In spina bifida, the neural tube fails to close during the embryonic period. Exposure of the neural tube to the amniotic fluid during pregnancy causes additional neural damage. Intrauterine tissue engineering using a biomaterial seeded with stem cells might prevent this additional damage. For this purpose, autologous cells from the amniotic fluid are an attractive source. To close the defect, it is important that these cells deposit an extracellular matrix. However, it is not known if amniotic fluid mesenchymal cells (AFMCs) from a fetus with a neural tube defect (NTD) share the same characteristics as AFMCs from a healthy fetus. We found that cells derived from fetuses with a NTD, in contrast to healthy human amniotic fluid cells, did not deposit collagen type I. Furthermore, the NTD cells showed, compared with both healthy amniotic fluid cells and fetal fibroblasts, much lower mRNA expression levels of genes that are involved in collagen biosynthesis [procollagen C-endopeptidase enhancer proteins (PCOLCE), PCOLCE2, ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2), ADAMTS14]. This indicates that NTD-AFMCs have different characteristics compared with healthy AFMCs and might not be suitable for fetal therapy to close the defect in spina bifida patients.
Barba, Marta; Pirozzi, Filomena; Saulnier, Nathalie; Vitali, Tiziana; Natale, Maria Teresa; Logroscino, Giandomenico; Robbins, Paul D.; Gambotto, Andrea; Neri, Giovanni; Michetti, Fabrizio; Pola, Enrico; Lattanzi, Wanda
Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs), can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC. PMID:23097599
... oxygen can cause permanent, severe neurological damage or brain death. Lengthy hospital stay. Women who survive an amniotic ... Infant death. Your baby is at risk of brain injury or death. Prompt evaluation and delivery of your baby improves ...
Reconstruction of the abdominal wall by using a combination of the human acellular dermal matrix implant and an interpositional omentum flap after extensive tumor resection in patients with abdominal wall neoplasm: A preliminary result
Gu, Yan; Tang, Rui; Gong, Ding-Quan; Qian, Yun-Liang
AIM: To present our trial using a combination of the human acellular dermal matrix (HADM) implant and an interpositional omentum flap to repair giant abdominal wall defects after extensive tumor resection. METHODS: Between February and October of 2007, three patients with giant defects of the abdominal wall after extensive tumor resection underwent reconstruction with a combination of HADM and omentum flap. Postoperative morbidities and signs of herniation were monitored. RESULTS: The abdominal wall reconstruction was successful in these three patients, there was no severe morbidity and no signs of herniation in the follow-up period. CONCLUSION: The combination of HADM and omentum flap offers a new, safe and effective alternative to traditional forms in the repair of giant abdominal wall defects. Further analysis of the long-term outcome and more cases are needed to assess the reliability of this technique. PMID:18205267
Yang, Shu; Sun, Hai-Mei; Yan, Ji-Hong; Xue, Hong; Wu, Bo; Dong, Fang; Li, Wen-Shuai; Ji, Feng-Qing; Zhou, De-Shan
Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD.
Guiet-Bara, A; Bara, M; Durlach, J
The ultrastructure of human amniotic epithelial cells from normal pregnancies, at term, was studied using transmission electron microscopy. The results were analysed by a stereological method which indicates the ratio between the volume of the intercellular space (R1, the microvilli (R2), and the podocytes (R3) versus the cell volume. At low concentration (2 mM), MgCl2 decreased R1 and R3 and had no significant effect on R2. In contrast, taurine (2 mM) increased R1 and had no significant effect on R2 and R3. There is no vicarious action between Mg and taurine. These data are in contrast to the results obtained after electrophysiological studies, which indicates that the structural targets for Mg and taurine are different from the targets responsible for ionic transfer.
Kaur, Kiranpreet; Bhardwaj, Mamta; Kumar, Prashant; Singhal, Suresh; Singh, Tarandeep; Hooda, Sarla
Amniotic fluid embolism (AFE) is one of the catastrophic complications of pregnancy in which amniotic fluid, fetal cells, hair, or other debris enters into the maternal pulmonary circulation, causing cardiovascular collapse. Etiology largely remains unknown, but may occur in healthy women during labour, during cesarean section, after abnormal vaginal delivery, or during the second trimester of pregnancy. It may also occur up to 48 hours post-delivery. It can also occur during abortion, after abdominal trauma, and during amnio-infusion. The pathophysiology of AFE is not completely understood. Possible historical cause is that any breach of the barrier between maternal blood and amniotic fluid forces the entry of amniotic fluid into the systemic circulation and results in a physical obstruction of the pulmonary circulation. The presenting signs and symptoms of AFE involve many organ systems. Clinical signs and symptoms are acute dyspnea, cough, hypotension, cyanosis, fetal bradycardia, encephalopathy, acute pulmonary hypertension, coagulopathy etc. Besides basic investigations lung scan, serum tryptase levels, serum levels of C3 and C4 complements, zinc coproporphyrin, serum sialyl Tn etc are helpful in establishing the diagnosis. Treatment is mainly supportive, but exchange transfusion, extracorporeal membrane oxygenation, and uterine artery embolization have been tried from time to time. The maternal prognosis after amniotic fluid embolism is very poor though infant survival rate is around 70%. PMID:27275041
Wiest, E; Armbruster, F P; Loeser, E L; Seeger, H; Voelter, W; Lippert, T H
Relaxin was measured in serum and amniotic fluid of 136 pregnant women between the 12th and 38th gestational week by means of a new human relaxin-RIA. The pregnancies consisted of 111 pathology-free single fetuses, 10 with rhesus incompatibility, 7 with chromosomal aberration and 8 with sonographic diagnosed abnormalities. Relaxin could be detected in all samples tested the levels being ten times lower in amniotic fluid compared to serum. Serum relaxin levels showed a slight but not statistically significant decrease with increasing gestational age, in amniotic fluid relaxin values were consistent over the course of pregnancy. The ratio of amniotic fluid to serum relaxin displayed a statistically significant increase from the 12th to 23rd week of pregnancy. Individual courses of relaxin concentration in amniotic fluid revealed only low intra-individual variations but distinct inter-individual differences.
Nassif, Joseph; Abbasi, Sehrish A.; Kechli, Mohamad Karim; Boutary, Suzan S.; Ghulmiyyah, Labib; Khalifeh, Ibrahim; Abou Ghaddara, Hussein; Nassar, Anwar H.
Adhesions after abdomino-pelvic surgery are a cause of morbidity and reoperations. The use of human amniotic membrane (HAM) for adhesion prevention has given controversial results. The mode of administration of the amniotic membrane has not been well studied. This study assessed the efficacy of two modes of application of cryopreserved HAM, patch or fragmented in Lactated Ringer (LR) solution, for the prevention of pelvic adhesion formation postabdomino-pelvic surgery in a mice model. After a midline laparotomy incision, a small cautery lesion was done on each side of the abdominal wall peritoneum in mice. In Group A (control; n = 42), the abdomen was closed directly, Group B (n = 42) received 2.5 ml of LR prior to closure. In Groups C (n = 42) and D (n = 42), a 2 cm × 2 cm patch of HAM and another one fragmented and dispersed in 2.5 ml of LR were applied prior to closure, respectively. Two weeks later, a laparotomy was performed, and gross and pathological evaluation of adhesions, fibrosis, angiogenesis, and inflammation were conducted. Group D exhibited a significantly lower rate of gross adhesion formation. Fibrosis was significantly lowest in Group C as compared to the control. Group B had the lowest vascular formation in the adhesions. The use of HAM fragmented in LR solution is associated with a significantly lower incidence of postoperative adhesions in mice when compared to LR alone, HAM patch, or control. The mechanism of action of this reduction needs to be elucidated by future studies. PMID:27066485
Nassif, Joseph; Abbasi, Sehrish A; Kechli, Mohamad Karim; Boutary, Suzan S; Ghulmiyyah, Labib; Khalifeh, Ibrahim; Abou Ghaddara, Hussein; Nassar, Anwar H
Adhesions after abdomino-pelvic surgery are a cause of morbidity and reoperations. The use of human amniotic membrane (HAM) for adhesion prevention has given controversial results. The mode of administration of the amniotic membrane has not been well studied. This study assessed the efficacy of two modes of application of cryopreserved HAM, patch or fragmented in Lactated Ringer (LR) solution, for the prevention of pelvic adhesion formation postabdomino-pelvic surgery in a mice model. After a midline laparotomy incision, a small cautery lesion was done on each side of the abdominal wall peritoneum in mice. In Group A (control; n = 42), the abdomen was closed directly, Group B (n = 42) received 2.5 ml of LR prior to closure. In Groups C (n = 42) and D (n = 42), a 2 cm × 2 cm patch of HAM and another one fragmented and dispersed in 2.5 ml of LR were applied prior to closure, respectively. Two weeks later, a laparotomy was performed, and gross and pathological evaluation of adhesions, fibrosis, angiogenesis, and inflammation were conducted. Group D exhibited a significantly lower rate of gross adhesion formation. Fibrosis was significantly lowest in Group C as compared to the control. Group B had the lowest vascular formation in the adhesions. The use of HAM fragmented in LR solution is associated with a significantly lower incidence of postoperative adhesions in mice when compared to LR alone, HAM patch, or control. The mechanism of action of this reduction needs to be elucidated by future studies.
Samoila, O; Lacramioara, Totu; Mihu, D
The basic principles of conjunctival reconstruction are similar in all pathologies that inflict conjunctival tissue loss. Large conjunctival defects are difficult to treat, with little conjunctival reserve that can be used to close the defect. The study had the objective to find alternatives to conjunctival autograft. Frozen amniotic membrane was investigated. From 27 cases, 22 cases were reconstructed using amniotic membrane alone, but in 5 cases conjunctival autograft was also needed. Bulbar conjunctiva was reconstructed in 23 cases, of which 19 with pterygium, 3 with conjunctival tumors, one operatory plague. Fornix reconstruction was carried out in 4 cases, one tumor and 3 symblepharons (2 after conjunctival burns and one pemfigus). The rate of pterygium recurrence was 7,14%. Amniotic membrane can heal large defects of the conjunctiva, either bulbar or forniceal.
Wasnik, Samiksha; Kantipudi, Suma; Kirkland, Mark A.; Pande, Gopal
The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion. PMID:26981135
Chun, So Young; Mack, David L; Moorefield, Emily; Oh, Se Heang; Kwon, Tae Gyun; Pettenati, Mark J; Yoo, James J; Coppi, Paolo De; Atala, Anthony; Soker, Shay
This study investigated the differentiation of human amniotic fluid-derived stem cells (hAFSCs) into insulin-producing clusters in vitro. Adenovirally-delivered mouse Pdx1 (Ad-Pdx1) induced human Pdx1 expression in hAFSCs and enhanced the coordinated expression of downstream β-cell markers. When Ad-Pdx1-transduced hAFSCs were sequentially treated with activin A, bFGF and nicotinamide and the culture plate surface coated with poly-l-ornithine, the expression of islet-associated human mRNAs for Pdx1, Pax6, Ngn3 and insulin was increased. C-peptide ELISA confirmed that Ad-Pdx1-transduced hAFSCs processed and secreted insulin in a manner consistent with that pathway in pancreatic β-cells. To sustain the β-cell-like phenotype and investigate the effect of three-dimensional (3D) conformation on the differentiation of hAFSCs, Pdx1-transduced cells were encapsulated in alginate and cultured long-term under serum-free conditions. Over 2 weeks, partially differentiated hAFSC clusters increased in size and increased insulin secretion. Taken together, these data demonstrate that ectopic Pdx1 expression initiates pancreatic differentiation in hAFSCs and that a β-cell-like phenotype can be augmented by culture conditions that mimic the stromal components and 3D geometry associated with pancreatic islets.
You, Qi; Yao, Yuan; Zhang, Yuanlong; Fu, Songbin; Du, Mei; Zhang, Guangmei
The aim of the present study was to investigate the effect of using amniotic fluid mesenchymal stem cells (AF-MSCs) in targeted ovarian cancer therapy in vivo. AF-MSCs were isolated from human second trimester AF and a plasmid, enhanced green fluorescent protein‑human interleukin‑2 (pEGFP‑hIL‑2) was formed. The plasmid was stably transfected into the AF‑MSCs and the cells were intravenously injected into ovarian cancer nude mice models. Following stable transfection of the vector, tumor formation, and the expression and activity of hIL‑2 were investigated, and microscopic pathological examinations of the tumor were performed. It was found that AF‑MSCs exhibited high motility during migration in vivo, and the vector, pEGFP‑hIL‑2 can be stably transfected into AF‑MSCs. Following stable transfection, this type of stem cell is able to successfully transport the therapeutic gene, IL-2, migrate to the ovarian cancer tumor site to secrete the functional IL-2 and treat the tumor. Thus, AF-MSCs may serve as transporters for therapeutic genes targeting ovarian tumor sites and, therefore, be involved in the treatment of tumors.
Solomon, A.; Rosenblatt, M.; Monroy, D.; Ji, Z.; Pflugfelder, S.; Tseng, S.
AIMS—Amniotic membrane (AM) transplantation reduces inflammation in a variety of ocular surface disorders. The aim of this study was to determine if AM stroma suppresses the expression of the IL-1 gene family in cultured human corneal limbal epithelial cells. METHODS—Human corneal limbal epithelial cells were cultured from limbocorneal explants of donor eyes on plastic or on the AM stroma. Transcript expression of IL-1α, IL-1β, IL-1 receptor antagonist (RA), and GAPDH was compared with or without addition of lipopolysaccharide to their serum-free media for 24 hours using RNAse protection assay (RPA). Their protein production in the supernatant was analysed by ELISA. RESULTS—Expression of IL-1α and IL-1β transcripts and proteins was significantly reduced by cells cultured on the AM stromal matrix compared with plastic cultures whether lipopolysaccharide was added or not. Moreover, expression of IL-1 RA by cells cultured in the lipopolysaccharide-free medium was upregulated by AM stromal matrix. The ratio between IL-1 RA and IL-1α protein levels in AM cultures was higher than in plastic cultures. CONCLUSIONS—AM stromal matrix markedly suppresses lipopolysaccharide induced upregulation of both IL-1α and IL-1β. These data may explain in part the effect of AM transplantation in reducing ocular surface inflammation, underscoring the unique feature of the AM as a substrate for tissue engineering. PMID:11264135
Galerne, D; Baudon, J; Bruhat, M; Dastugue, G
Quantitative analyses of 10 enzymes (LAP, GGTP, SGOT, SGPT. acid and alkaline phosphatases, 5' nucleotidase, amylase. beta-glucuronidase and aldolase) in a series of 50 samples of amniotic fluid gave widely-scattered results. In some cases, it was possible to relate high enzymatic activity to a pathological condition, in other cases, the amniotic fluid examined seemed to come from normal, full-term or almost full-term pregnancies without particular signs.
Bitsika, Vasiliki; Roubelakis, Maria G; Zagoura, Dimitra; Trohatou, Ourania; Makridakis, Manousos; Pappa, Kalliopi I; Marini, Frank C; Vlahou, Antonia; Anagnou, Nicholas P
Recent studies support cell-based therapies for cancer treatment. An advantageous cell type for such therapeutic schemes are the mesenchymal stem cells (MSCs) that can be easily propagated in culture, genetically modified to express therapeutic proteins, and exhibit an innate tropism to solid tumors in vivo. Recently, we successfully isolated and expanded MSCs from second-trimester amniotic fluid (AF-MSCs). The main characteristic of AF-MSCs is their efficient and rapid expansion in vitro. Herein, we investigated the AF-MSCs tropism and capability to transport interferon beta (IFNβ) to the region of neoplasia in a bladder tumor model. To this end, we used the T24M bladder cancer cell line, previously generated from our studies, and developed a disease progression model in immunosuppressed mice, that can recapitulate the molecular events of bladder carcinogenesis. Our results documented that AF-MSCs exhibited high motility, when migrated either to T24M cells or to T24M-conditioned medium, and we further identified and studied the secreted factors which may trigger these enhanced migratory properties. Further, lentivirus-transduced AF-MSCs, expressing green fluorescent protein (GFP) or IFNβ, were intravenously administered to T24M tumor-bearing animals at multiple doses to examine their therapeutic effect. GFP- and IFNβ-AF-MSCs successfully migrated and colonized at the tumor site. Notably, significant inhibition of tumor growth as well as prolonged survival of mice were observed in the presence of IFNβ-AF-MSCs. Collectively, these results document the great potential of AF-MSCs as anti-cancer vehicles, implemented by the targeting of the tumor site and further facilitated by their high proliferation rate and expansion efficiency in culture.
Mareschi, Katia; Castiglia, Sara; Sanavio, Fiorella; Rustichelli, Deborah; Muraro, Michela; Defedele, Davide; Bergallo, Massimiliano; Fagioli, Franca
Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications.
La Marca-Ghaemmaghami, Pearl; Dainese, Sara M; Stalla, Günter; Haller, Marina; Zimmermann, Roland; Ehlert, Ulrike
This study explored the association between the acute psychobiological stress response, chronic social overload and amniotic fluid corticotropin corticotropin-releasing hormone (CRH) and urocortin (UCN) in 34 healthy, second-trimester pregnant women undergoing amniocentesis. The study further examined the predictive value of second-trimester amniotic fluid CRH and UCN for fetal growth and neonatal birth outcome. The amniocentesis served as a naturalistic stressor, during which maternal state anxiety and salivary cortisol was measured repeatedly and an aliquot of amniotic fluid was collected. The pregnant women additionally completed a questionnaire on chronic social overload. Fetal growth parameters were obtained at amniocentesis using fetal ultrasound biometry and at birth from medical records. The statistical analyses revealed that the acute maternal psychobiological stress response was unassociated with the amniotic fluid peptides, but that maternal chronic overload and amniotic CRH were positively correlated. Moreover, amniotic CRH was negatively associated with fetal size at amniocentesis and positively with growth in size from amniocentesis to birth. Hardly any studies have previously explored whether acute maternal psychological stress influences fetoplacental CRH or UCN levels significantly. Our findings suggest that 1) chronic, but not acute maternal stress may affect fetoplacental CRH secretion and that 2) CRH is complexly involved in fetal growth processes as previously shown in animals.
Protzman, Nicole M; Brigido, Stephen A
The management of chronic wounds is a considerable challenge for foot and ankle surgeons. The well-established tenets of adequate vascular supply, debridement with eradication of infection, and offloading must be employed in the management of all extremity wounds. Regenerative scaffolds are a viable means of reestablishing a favorable wound environment. The matrix facilitates cell migration, chemoattraction, angiogenesis, wound bed granulation, and expedited wound closure. Although studies have demonstrated success with acellular matrices, a multimodal approach should always be employed to improve healing success. Negative pressure wound therapy, compression, offloading, and antibiotics are advocated to improve outcomes. Acellular graft selection requires a multifactorial analysis, taking into consideration the specific patient and wound characteristics as well as the differences between acellular matrices. Patient age, comorbidities, activity level, and ability to comply with protocol as well as wound etiology, duration, depth, surface area, exudate, bacterial burden, location, vascular status, ischemic status, and presentation are all critical components. To effectively choose a matrix, the clinician must have a comprehensive understanding of the products available and the data validating their use. The mechanisms by which the acellular matrix accelerates wound healing and increases the likelihood of wound healing continue to be investigated. However, it is clear that these acellular biologic tissue scaffolds are incorporating into the host tissue, with resultant revascularization and cellular repopulation. Moving forward, additional investigations examining the effectiveness of acellular biologic tissue scaffolds to improve healing in complex, nondiabetic wounds are warranted.
ROMERO, Roberto; SCHAUDINN, Christoph; KUSANOVIC, Juan Pedro; GORUR, Amita; GOTSCH, Francesca; WEBSTER, Paul; NHAN-CHANG, Chia-Ling; EREZ, Offer; KIM, Chong Jai; ESPINOZA, Jimmy; GONÇALVES, Luis F.; VAISBUCH, Edi; MAZAKI-TOVI, Shali; HASSAN, Sonia S.; COSTERTON, J. William
Objective Microbial biofilms are communities of sessile microorganisms formed by cells that are attached irreversibly to a substratum or interface or to each other and embedded in a hydrated matrix of extracellular polymeric substances. Microbial biofilms have been implicated in >80% of human infections such as periodontitis, urethritis, endocarditis, and device-associated infections. Thus far, intra-amniotic infection has been attributed to planktonic (free-floating) bacteria. A case is presented in which “amniotic fluid sludge” was found to contain microbial biofilms. This represents the first report of a microbial biofilm in the amniotic cavity. Study Design “Amniotic fluid sludge” was detected by transvaginal sonography, and retrieved by transvaginal amniotomy. Bacteria were identified using scanning electron microscope and fluorescence in situ hybridization for conserved regions of the microbial genome; and the exopolymeric matrix was identified by histochemistry using the wheat germ agglutinin lectin method. The structure of the biofilm was imaged with confocal laser scanning microscopy. Results “Amniotic fluid sludge” was imaged with scanning electron microscopy, which allowed identification of bacteria embedded in an amorphous material and inflammatory cells. Bacteria were demonstrated using fluorescent in situ hybredization using a eubacteria probe. Extracellular matrix was identified with the wheat germ agglutinin lectin stain. Confocal microscopy allowed three-dimensional visualization of the microbial biofilm. Conclusion Microbial biofilms have been identified in a case of intra-amniotic infection with “amniotic fluid sludge.” PMID:18166328
Cohen, Liat; Dean, Mason; Shipov, Anna; Atkins, Ayelet; Monsonego-Ornan, Efrat; Shahar, Ron
The histological diversity of the skeletal tissues of fishes is impressive compared with that of other vertebrate groups, yet our understanding of the functional consequences of this diversity is limited. In particular, although it has been known since the mid-1800s that a large number of fish species possess acellular bones, the mechanical advantages and consequences of this structural characteristic - and therefore the nature of the evolution of this feature - remain unclear. Although several studies have examined the material properties of fish bone, these have used a variety of techniques and there have been no direct contrasts of acellular and cellular bone. We report on a comparison of the structural and mechanical properties of the ribs and opercula between two freshwater fish - the common carp Cyprinus carpio (a fish with cellular bone) and the tilapia Oreochromis aureus (a fish with acellular bone). We used light microscopy to show that the bones in both fish species exhibit poor blood supply and possess discrete tissue zones, with visible layering suggesting differences in the underlying collagen architecture. We performed identical micromechanical testing protocols on samples of the two bone types to determine the mechanical properties of the bone material of opercula and ribs. Our data support the consensus of literature values, indicating that Young's moduli of cellular and acellular bones are in the same range, and lower than Young's moduli of the bones of mammals and birds. Despite these similarities in mechanical properties between the bone tissues of the fish species tested here, cellular bone had significantly lower mineral content than acellular bone; furthermore, the percentage ash content and bone mineral density values (derived from micro-CT scans) show that the bone of these fishes is less mineralized than amniote bone. Although we cannot generalize from our data to the numerous remaining teleost species, the results presented here suggest
Pan, Hung-Chuan; Yang, Dar-Yu; Ho, Shu-Peng; Sheu, Meei-Ling; Chen, Chung-Jung; Hwang, Shiaw-Min; Chang, Ming-Hong; Cheng, Fu-Chou
Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits. PMID:19698158
Carvalho, M. L.; Custódio, P. J.; Reus, U.; Prange, A.
This work is an attempt to evaluate the possible influence of the mother's age in trace element concentrations in human amniotic fluid and placenta and whether these concentrations are correlated to the weight of the newborn infants. Total-reflection X-ray fluorescence (TXRF) was used to analyze 16 amniotic fluid samples, and the placenta samples were analyzed by energy dispersive X-ray fluorescence (EDXRF). The whole samples were collected during delivery from healthy mothers and healthy infants and full-term pregnancies. According to the age of the mother, three different groups were considered: 20-25, 25-30 and 30-40 years old. Only two mothers were aged more than 35 years. The weight of the infants ranged from 2.56 to 4.05 kg and three groups were also considered: 2.5-3, 3-3.5 and 3.5-4 kg. The organic matrix of the amniotic fluid samples was removed by treatment with HNO 3 followed by oxygen plasma ashing. Yttrium was used as the internal standard for TXRF analysis. Placenta samples were lyophilized and analyzed by EDXRF without any chemical treatment. Very low levels of Ni and Sr were found in the amniotic fluid samples, and were independent of the age of the mother and weight of the child. Cr, Mn, Se and Pb were at the level of the detection limit. Zn, considered one of the key elements in neonatal health, was not significantly different in the samples analyzed; however, it was weakly related to birth weigh. The concentrations obtained ranged from 0.11 to 0.92 mg/l and 30 to 65 μg/g in amniotic fluid and placenta, respectively. The only two elements which seemed to be significantly correlated with mother's age and newborn weight were Ca and Fe for both types of sample: Ca levels were increased in heavier children and older mothers; however, Fe increased with increasing maternal age, but decreased for heavier babies. The same conclusions were obtained for placenta and amniotic fluid samples. Cu is closely associated with Fe in its function in the organism
Capobianco, Valentina; Caterino, Marianna; Iaffaldano, Laura; Nardelli, Carmela; Sirico, Angelo; Del Vecchio, Luigi; Martinelli, Pasquale; Pastore, Lucio; Pucci, Pietro; Sacchetti, Lucia
Maternal obesity increases the risk of obesity and/or obesity-related diseases in the offspring of animal models. The aim of this study was to identify metabolic dysfunctions that could represent an enhanced risk for human obesity or obesity-related diseases in newborn or in adult life, similar to what occurs in animal models. To this aim, we studied the proteome of 12 obese (Ob-) and 6 non-obese (Co-) human amniotic mesenchymal stem cells (hA-MSCs) obtained from women at delivery by cesarean section (pre-pregnancy body mass index [mean ± SD]: 42.7 ± 7.7 and 21.3 ± 3.3 kg/m2, respectively). The proteome, investigated by two-dimensional fluorescence difference gel electrophoresis/mass spectrometry, revealed 62 differently expressed proteins in Ob- vs Co-hA-MSCs (P < 0.05), nine of which were confirmed by western blotting. Bioinformatics analysis showed that these 62 proteins are involved in several statistically significant pathways (P < 0.05), including the stress response, cytoskeleton and metabolic pathways. Oxidative stress was shown to be an early triggering factor of tissue fat accumulation and obesity-related disorders in the offspring of obese animal models. Our finding of a reduced stress response in Ob-hA-MSCs suggests that a similar mechanism could occur also in humans. Long-term follow-up studies of newborns of obese mothers are required to verify this hypothesis. PMID:27125468
Capobianco, Valentina; Caterino, Marianna; Iaffaldano, Laura; Nardelli, Carmela; Sirico, Angelo; Del Vecchio, Luigi; Martinelli, Pasquale; Pastore, Lucio; Pucci, Pietro; Sacchetti, Lucia
Maternal obesity increases the risk of obesity and/or obesity-related diseases in the offspring of animal models. The aim of this study was to identify metabolic dysfunctions that could represent an enhanced risk for human obesity or obesity-related diseases in newborn or in adult life, similar to what occurs in animal models. To this aim, we studied the proteome of 12 obese (Ob-) and 6 non-obese (Co-) human amniotic mesenchymal stem cells (hA-MSCs) obtained from women at delivery by cesarean section (pre-pregnancy body mass index [mean ± SD]: 42.7 ± 7.7 and 21.3 ± 3.3 kg/m(2), respectively). The proteome, investigated by two-dimensional fluorescence difference gel electrophoresis/mass spectrometry, revealed 62 differently expressed proteins in Ob- vs Co-hA-MSCs (P < 0.05), nine of which were confirmed by western blotting. Bioinformatics analysis showed that these 62 proteins are involved in several statistically significant pathways (P < 0.05), including the stress response, cytoskeleton and metabolic pathways. Oxidative stress was shown to be an early triggering factor of tissue fat accumulation and obesity-related disorders in the offspring of obese animal models. Our finding of a reduced stress response in Ob-hA-MSCs suggests that a similar mechanism could occur also in humans. Long-term follow-up studies of newborns of obese mothers are required to verify this hypothesis.
The ideal technique to repair large hiatal and diaphragmatic defects remains controversial. Due to high recurrence rates with primary repair alone, attempts at crural reinforcement with various products has been investigated. Initial evaluation of synthetic mesh at the hiatus in retrospective studies led to the conclusion that there were too many serious complications with these products. The next step was to see how biologic grafts fared in this location. Beginning with porcine intestine submucosa in a laminated array and progressing through human and porcine acellular dermal matrices, multiple, retrospective studies looked at the efficacy and safety of these products. Unfortunately, most of these studies evaluated a small sample size with a relatively short follow-up period. The one study followed out to 5 years failed to show any benefit using the biologic (porcine intestinal submucosa) compared with the primary repair alone. Additional, prospective, randomized studies with ample numbers carried out for years will be necessary to see which biologic graft is not only safe but also successful in preventing recurrent herniations.
Human amniotic membrane and umbilical cord tissues (AM/UC) are fetal tissues that contain proteins, cytokines, and growth factors that, when transplanted, can modulate inflammation and promote healing. Lyophilized, particulate AM/UC tissues can be used as wound coverings for chronic dermal ulcers or defects to promote granulation tissue formation and rapid re-epithelialization. This study reviews a case series of 5 patients presenting with chronic nonhealing wounds that received particulate AM/UC tissues (NEOX® FLO, Amniox Medical, Atlanta, GA). For all cases, wounds were debrided in the office setting and a single application of lyophilized particulate was used with minimal additional dressings. The lyophilized AM/UC tissue was placed within the wound bed and a dressing consisting of Adaptic®, 2x2 or 4x4 (Systagenix, Quincy, MA), Kling® (Johnson & Johnson, New Brunswick, NJ), and ACE™ (3M, St. Paul, MN) wrap were applied. Dressings were kept in place until weekly follow-up appointments in which a new Adaptic, 2x2 and Kling were applied. Overall, healing of wounds was noted to have a mean of 5 weeks to complete epithelialization. Upon complete healing patients were able to return to planned postoperative care and rehabilitation. Wound complications occur despite the best standard of care. Chronic wounds that remain weeks after surgery inhibit patients from progressing to physical rehabilitation and significantly affect patients both physically and mentally. These case presentations demonstrate how use of human AM/UC tissue may help wounds heal quickly and help patients return to normal function.
In vivo Quantification of the Effects of Radiation and Presence of Hair Follicle Pores on the Proliferation of Fibroblasts in an Acellular Human Dermis in a Dorsal Skinfold Chamber: Relevance for Tissue Reconstruction following Neoadjuvant Therapy
Maier, Patrick; Hohenberger, Peter; Roessner, Eric Dominic
Introduction In neoadjuvant therapy, irradiation has a deleterious effect on neoangiogenesis. The aim of this study was to examine the post-implantation effects of neoadjuvant irradiation on the survival and proliferation of autologous cells seeded onto an acellular human dermis (hAD; Epiflex). Additionally, we examined the influence of dermal hair follicle pores on viability and proliferation. We used dorsal skinfold chambers implanted in rats and in-situ microscopy to quantify cell numbers over 9 days. Methods 24 rats received a skinfold chamber and were divided into 2 main groups; irradiated and unirradiated. In the irradiated groups 20Gy were applied epicutaneously at the dorsum. Epiflex pieces were cut to size 5x5mm such that each piece had either one or more visible hair follicle pores, or no such visible pores. Fibroblasts were transduced lentiviral with a fluorescent protein for cell tracking. Matrices were seeded statically with 2.5x104 fluorescent fibroblasts and implanted into the chambers. In each of the two main groups, half of the rats received Epiflex with hair follicle pores and half received Epiflex without pores. Scaffolds were examined in-situ at 0, 3, 6 and 9 days after transplantation. Visible cells on the surface were quantified using ImageJ. Results In all groups cell numbers were decreased on day 3. A treatment-dependent increase in cell numbers was observed at subsequent time points. Irradiation had an adverse effect on cell survival and proliferation. The number of cells detected in both irradiated and non-irradiated subjects was increased in those subjects that received transplants with hair follicle pores. Discussion This in-vivo study confirms that radiation negatively affects the survival and proliferation of fibroblasts seeded onto a human dermis transplant. The presence of hair follicle pores in the dermis transplants is shown to have a positive effect on cell survival and proliferation even in irradiated subjects. PMID:25955842
Zhu, Shuang; Li, Jiachun; Zhu, Qingtang; Dai, Ting; He, Bo; Zhou, Xiang; Xiang, Jianping; Liu, Xiaolin
Human amniotic epithelial cells (hAECs) exhibit multi‑lineage differentiation ability. The present study investigated the possibility that hAECs possess the potential to differentiate into Schwann‑like cells using an in vitro indirect co‑culture approach. hAECs were isolated via enzymatic digestion, and immunocytochemistry and flow cytometry were performed to identify the hAECs. The hAECs were co‑cultured with Schwann cells (SCs) to differentiate the hAECs into Schwann‑like cells via induced proximity. The expression of typical S‑100 SC markers in the co‑cultured hAECs was determined via immunocytochemistry. For the functional experiments, reverse transcription quantitative polymerase chain reaction (RT‑qPCR) was performed to measure the expression levels of nerve growth factor (NGF), brain‑derived neurotrophic factor (BDNF) and glial cell‑derived neurotrophic factor (GDNF) mRNA. In addition, neurite outgrowth was measured in PC12 cells following co‑culture with the differentiated hAECs. Subsequent to co‑culture with SCs for 21 days, the hAECs exhibited spindle‑like morphology. The immunocytochemistry results revealed that the co‑cultured hAECs expressed S‑100, indicating differentiation into Schwann‑like cells. RT‑qPCR revealed that NGF, BDNF and GDNF expression was upregulated upon differentiation. The average axon length of the PC12 cells increased from 21.32±5.45 to 51.32±8.56 µm subsequent to co‑culture with the differentiated hAECs. These results demonstrate that this indirect co‑culture microenvironment induced the hAECs to differentiate into Schwann‑like cells that exhibited the morphological, phenotypic and functional characteristics of SCs. Therefore, the use of differentiated hAECs that exhibit the characteristics of SCs provides a promising alternative to the present techniques used for peripheral nerve regeneration.
Vaghjiani, Vijesh; Vaithilingam, Vijayaganapathy; Saraswati, Indah; Sali, Adnan; Murthi, Padma; Kalionis, Bill; Tuch, Bernard E.
Placenta derived human amniotic epithelial cells (hAEC) are an attractive source of stem cells for the generation of hepatocyte-like cells (HLC) for therapeutic applications to treat liver diseases. During hAEC differentiation into HLC, they become increasingly immunogenic, which may result in immune cell-mediated rejection upon transplantation into allogeneic recipients. Placing cells within devices such as alginate microcapsules can prevent immune cell-mediated rejection. The aim of this study was to investigate the characteristics of HLC generated from hAEC and to examine the effects of encapsulation on HLC viability, gene expression, and function. hAEC were differentiated for 4 weeks and evaluated for hepatocyte-specific gene expression and function. Differentiated cells were encapsulated in barium alginate microcapsules and cultured for 7 days and the effect of encapsulation on cell viability, function, and hepatocyte related gene expression was determined. Differentiated cells performed key functions of hepatocytes including urea synthesis, drug-metabolizing cytochrome P450 (CYP)3A4 activity, indocyanine green (ICG) uptake, low-density lipoprotein (LDL) uptake, and exhibited glutathione antioxidant capacity. A number of hepatocyte-related genes involved in fat, cholesterol, bile acid synthesis, and xenobiotic metabolism were also expressed showing that the hAEC had differentiated into HLC. Upon encapsulation, the HLC remained viable for at least 7 days in culture, continued to express genes involved in fat, cholesterol, bile acid, and xenobiotic metabolism and had glutathione antioxidant capacity. CYP3A4 activity and urea synthesis by the encapsulated HLC were higher than that of monolayer HLC cultures. Functional HLC can be derived from hAEC, and HLC can be encapsulated within alginate microcapsules without losing viability or function in vitro. PMID:24295364
Smith, C V
The mysterious environment surrounding the fetus for much of his or her life is now being explored with increasing fervor. Assessment of amniotic fluid in the early portion of pregnancy is now possible for fetal karyotype determination. This may permit early diagnosis of abnormal fetuses, increasing the options for patients. As pregnancy progresses, high-resolution ultrasound assessment of amniotic fluid volume is integral to the management of pregnancies at risk for oligohydramnios. Such pregnancies include those who are postdate and those with suspected intrauterine growth retardation. Additional evaluation and ultrasonography are recommended for evaluation of the fetus in this clinical situation. With either hydramnios or oligohydramnios, careful ultrasound assessment of the fetus is essential to rule out significant congenital malformations. Finally, the use of ultrasound-directed amniocentesis in later pregnancy permits an assessment of fetal lung maturity and of the fetus at risk for Rhesus immunization. Attention to detail should increase chances of a successful pregnancy outcome while decreasing neonatal morbidity and mortality.
Colard, Thomas; Falgayrac, Guillaume; Bertrand, Benoit; Naji, Stephan; Devos, Olivier; Balsack, Clara; Delannoy, Yann; Penel, Guillaume
Acellular extrinsic fiber cementum is a mineralized tissue that covers the cervical half of the tooth root surface. It contains mainly extrinsic or Sharpey’s fibers that run perpendicular to the root surface to anchor the tooth via the periodontal ligament. Acellular cementum is continuously and slowly produced throughout life and exhibits an alternating bright and dark pattern under light microscopy. However, although a better understanding of the structural background of acellular cementum is relevant to many fields, such as cementochronology, periodontology and tissue engineering, acellular cementum remains rarely studied and poorly understood. In this work, we studied the acellular cementum at the incremental line scale of five human mandibular canines using polarized Raman spectroscopy. We provided Raman imaging analysis and polarized acquisitions as a function of the angular orientation of the sample. The results showed that mineral crystals were always parallel to collagen fibrils, and at a larger scale, we proposed an organizational model in which we found radial collagen fibers, “orthogonal” to the cementum surface, and “non-orthogonal” fibers, which consist of branching and bending radial fibers. Concerning the alternating pattern, we observed that the dark lines corresponded to smaller, more mineralized and probably more organized bands, which is consistent with the zoological assumption that incremental lines are produced during a winter rest period of acellular cementum growth. PMID:27936010
Kang, N-H; Hwang, K-A; Yi, B-R; Lee, H J; Jeung, E-B; Kim, S U; Choi, K-C
As human amniotic fluid-derived stem cells (hAFSCs) are capable of multiple lineage differentiation, extensive self-renewal and tumor targeting, they may be valuable for clinical anticancer therapies. In this study, we used hAFSCs as vehicles for targeted delivery of therapeutic suicide genes to breast cancer cells. hAFSCs were engineered to produce AF2.CD-TK cells in order to express two suicide genes encoding bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) that convert non-toxic prodrugs, 5-fluorocytosine (5-FC) and mono-phosphorylate ganciclovir (GCV-MP), into cytotoxic metabolites, 5-fluorouracil (5-FU) and triphosphate ganciclovir (GCV-TP), respectively. In cell viability test in vitro, AF2.CD-TK cells inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the 5-FC or GCV prodrugs, or a combination of these two reagents. When the mixture of 5-FC and GCV was treated together, an additive cytotoxic effect was observed in the cell viability. In animal experiments using female BALB/c nude mouse xenografts, which developed by injecting MDA-MB-231 cells, treatment with AF2.CD-TK cells in the presence of 5-FC and GCV significantly reduced tumor volume and weight to the same extent seen in the mice treated with 5-FU. Histopathological and fluorescent staining assays further showed that AF2.CD-TK cells were located exactly at the site of tumor formation. Furthermore, breast tissues treated with AF2.CD-TK cells and two prodrugs maintained their normal structures (for example, the epidermis and reticular layers) while breast tissue structures in 5-FU-treated mice were almost destroyed by the potent cytotoxicity of the drug. Taken together, these results indicate that AF2.CD-TK cells can serve as excellent vehicles in a novel therapeutic cell-based gene-directed prodrug system to selectively target breast malignancies.
Briese, V; Straube, W; Brock, J; Stark, K H; Lorenz, U
Amniotic fluid levels of secretory immunoglobulin A (S-AgA) were measured by simple radial immunodiffusion according to the method of Mancini using a monospecific antiserum against the human secretory component. 256 samples from healthy pregnant women were examined. Amniotic fluid S-IgA concentration increases significantly during normal pregnancy and shows a loose correlation to the phospholipid level.
Iaffaldano, Laura; Nardelli, Carmela; Raia, Maddalena; Mariotti, Elisabetta; Ferrigno, Maddalena; Quaglia, Filomena; Labruna, Giuseppe; Capobianco, Valentina; Capone, Angela; Maruotti, Giuseppe Maria; Pastore, Lucio; Di Noto, Rosa; Martinelli, Pasquale; Del Vecchio, Luigi
Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P=0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2=0.84, P<0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P=0.05 and P=0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P<0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers. PMID:23488598
Iaffaldano, Laura; Nardelli, Carmela; Raia, Maddalena; Mariotti, Elisabetta; Ferrigno, Maddalena; Quaglia, Filomena; Labruna, Giuseppe; Capobianco, Valentina; Capone, Angela; Maruotti, Giuseppe Maria; Pastore, Lucio; Di Noto, Rosa; Martinelli, Pasquale; Sacchetti, Lucia; Del Vecchio, Luigi
Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.
Bertin, E.; Piccoli, M.; Franzin, C.; Spiro, G.; Donà, S.; Dedja, A.; Schiavi, F.; Taschin, E.; Bonaldo, P.; Braghetta, P.; De Coppi, P.; Pozzobon, M.
Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells. PMID:27845396
Liu, Xian-Ning; Zhu, Xiu-Ping; Wu, Jie; Wu, Zheng-Jie; Yin, Yong; Xiao, Xiang-Hua; Su, Xin; Kong, Bin; Pan, Shi-Yin; Yang, Hua; Cheng, Yan; An, Na; Mi, Sheng-Li
AIM To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy (TEM) and hematoxylin and eosin (H&E) staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype. RESULTS The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process. In vitro, the methyl thiazolyl tetrazolium results revealed that extracts of the acellular ostrich corneas (AOCs) had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes. The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea (APC) transplantation. The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer. CONCLUSION We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft. PMID:27158598
Chaiworapongsa, Tinnakorn; Romero, Roberto; Espinoza, Jimmy; Kim, Yeon Mee; Edwin, Samuel; Bujold, Emmanuel; Gomez, Ricardo; Kuivaniemi, Helena
OBJECTIVE Macrophage migration inhibitory factor (MIF) has emerged as an important mediator of septic shock. The administration of MIF increases lethality during endotoxemia, whereas neutralization of this cytokine prevents endotoxic shock and death associated with bacterial infection. The objective of this study was to determine whether there is a change in the amniotic fluid concentration of MIF in intra-amniotic infection and human parturition. STUDY DESIGN A cross-sectional study was conducted in women in the following categories: 1) midtrimester (n=84); 2) preterm labor and intact membranes who delivered at term (n=33), who delivered preterm (n=53), and preterm labor with intra-amniotic infection (n=23); 3) preterm premature rupture of membranes (PROM) with (n=25) and without intra-amniotic infection (n=26); and 4) term with intact membranes, in labor (n=52), and not in labor (n=31). MIF concentrations in amniotic fluid were determined using a sensitive and specific immunoassay. MIF concentrations in maternal plasma were also determined in patients with preterm labor and intact membranes. Immunohistochemistry was conducted in chorioamniotic membranes obtained from a different set of patients presenting with preterm labor with (n=18) and without (n=20) histologic chorioamnionitis. Quantitative RT-PCR was used to measure MIF mRNA expression in chorioamniotic membranes of patients with preterm labor with (n=13) and without (n=13) histologic chorioamnionitis. Parametric and non-parametric, receiver-operating characteristic (ROC) curve, survival analysis, and Cox regression model were used for analysis. RESULTS Immunoreactive MIF was detectable in 96% (313/327) of amniotic fluid samples. The concentration of amniotic fluid MIF at term was higher than that in the midtrimester (p=0.004). Intra-amniotic infection in women with preterm labor and preterm PROM was associated with a significant increase in median amniotic fluid MIF concentration (p<0.001 and 0
Suh, Alexander; Weber, Claudia C; Kehlmaier, Christian; Braun, Edward L; Green, Richard E; Fritz, Uwe; Ray, David A; Ellegren, Hans
Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs) are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs) have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic "fossil" is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote-HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts.
Welch, Robert A; Shaw, Michael K; Welch, Kathryn C
Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is required in the biosynthesis of pulmonary surfactant. This short communication describes our assessment of LPCAT1 mRNA levels in human amniotic fluid. We found a direct correlation between LPCAT1 mRNA copies and the amniotic fluid lamellar body count (LBC). This finding corroborates an association between LPCAT1 and surfactant phospholipid biosynthesis in humans. It may provide a model for future research in perinatal medicine.
Miller, E; Ashworth, L A; Redhead, K; Thornton, C; Waight, P A; Coleman, T
The performance of four acellular pertussis vaccines containing between two and five pertussis antigens combined with diphtheria and tetanus toxoids was compared with that of British whole-cell diphtheria/tetanus/pertussis (DTP) vaccine both in laboratory assays for potency, toxicity and immunogenicity, and for reactogenicity and immunogenicity in infants. Clinical responses were evaluated in double blind randomized Phase II trials using 3/5/9 month and 2/3/4 month schedules. The acellular DTPs had much lower toxicity than whole-cell DTP in laboratory tests and were significantly less pyrogenic than whole-cell DTP under both schedules. Local reactions were not consistently lower in acellular than whole-cell vaccinees and varied with the source of the diphtheria and tetanus antigens used. Differences in endotoxin level and content of active pertussis toxin (PT) between acellular DTP vaccines were not clinically significant. The reactogenicity advantage of the acellular vaccines was substantially reduced under the 2/3/4 month schedule due to the reduced reactogenicity of the whole-cell DTP vaccine when given at a younger age. There was no relationship between antigen content measured in micrograms per dose and ELISA antibody responses to filamentous haemagglutinin (FHA) and PT in infants, nor was murine immunogenicity predictive of immunogenicity in humans. Antibody response to PT was attenuated in the whole-cell group under the 2/3/4 month schedule but was unaffected in the group receiving acellular vaccines with individually purified components; antibody response to pertactin (69 kDa antigen) was similar in recipients of the whole-cell and component acellular vaccines under the 2/3/4 month schedule. PT antibody persistence until 4-5 years of age was significantly better in recipients of the component acellular than either the whole-cell vaccine or the co-purified acellular vaccine under the 3/5/9 month schedule. However, diphtheria antitoxin levels were reduced in
Chen, C; Wang, Z Y; Weng, J; Wang, Z B; Mei, J; Du, X H; Wang, L
Objective: To explore the effect of acellular normal and fibrotic lung matrices on alpha smooth muscle actin (α-SMA) expression in human lung adenocarcinoma cell line A549. Methods: Twenty adult SD rats were randomly divided into normal group and idiopathic pulmonary fibrosis(IPF)group (n=10 each). The pulmonary fibrosis was induced by Bleomycin. Normal and fibrotic decellularized lungs were made, then sections with 500 μm thick were cut by a standard Vibratome. None scaffold was set as control group. A549 cells were seeded dropwise into different slices (normal and fibrotic scaffolds), and cultured for one week in vitro. The expression of α-SMA was measured by immunofluorescence staining and quantitative real time polymerase chain reaction (qRT-PCR). Results: In control group, the expression of α-SMA protein was positive in A549 cells by immunofluorescence staining. However, it expressed weakly both in normal and fibrotic scaffold group, and the fluorescence intensity in fibrotic scaffold group was significant lower than that in normal group (P<0.05). The relative expression amount of α-SMA mRNA in normal and fibrotic scaffold group were (0.70±0.11) and (0.55±0.12), which were significant lower than that of control group (1.28±0.21) (P<0.05). Moreover, the relative expression of α-SMA mRNA in fibrotic scaffold group was decreased compared to that in normal scaffold group (P<0.05). Conclusions: Acellular normal and fibrotic lung scaffold can downregulate the expression of α-SMA in human lung adenocarcinoma cell line A549. It may inhibit the movement of A549 cells in acellular normal and fibrotic lung matrices, especially in acellular fibrotic lung scaffold.
Briese, V; Straube, W; Brock, J; Stark, K H; Lorenz, U
Amniotic fluid levels of secretory immunoglobulin A (S-IgA) were measured by simple radial immunodiffusion according to the method of Mancini using a monospecific antiserum against the human secretory component. We examined 256 samples from healthy pregnant women and 149 samples from mothers suffering from rhesus isoimmunization. During normal pregnancy amniotic fluid S-IgA increased significantly and showed a loose correlation with the phospholipid levels. This was not observed in pregnancies complicated by rhesus isoimmunization.
A new method for immediate derivatization of hydroxyl groups by fluoroalkyl chloroformates and its application for the determination of sterols and tocopherols in human serum and amniotic fluid by gas chromatography-mass spectrometry.
Řimnáčová, L; Hušek, P; Šimek, P
A new method has been described for efficient derivatization of secondary alicyclic hydroxyl groups in steroids by alkyl chloroformates (RCFs). Cholesterol, an essential human sterol and a steroid precursor in eukaryotic cells, was used as a model for treatment with various RCFs in an aqueous and non-aqueous environment. While the cholesterol hydroxyl group did not react completely with any of the tested RCFs reagents in the former case, trifluoroethyl chloroformate (TFECF) or heptafluorobutyl chloroformate (HFBCF) fully converts cholesterol and related metabolites into the corresponding mixed carbonates under anhydrous conditions in seconds. The acylation reaction was combined with liquid-liquid microextraction (LLME) between isooctane and acetonitrile phase. The sample preparation requires just a stepwise addition of 50μl isooctane with 5μl of a pyridine catalyst, 100μl acetonitrile and 100μl isooctane with dissolved 5μl of the fluoroalkyl chloroformate reagent to a dried sample. The protocol developed in this study was successfully tested for GC-MS analysis of 12 important model steroids and four main tocopherols. Each analyte provided a single peak with excellent GC separation properties, well defined EI spectra containing diagnostic fragment ions suitable for their identification and quantitation. The new method was further validated for the determination of six diagnostic non-cholesterol sterols and four main tocopherols in human serum and in amniotic fluid. Satisfactory data were obtained in terms of calibration, quantitation limits (for sterols and tocopherols, 0.05 and 0.15μg/ml, respectively), within-run precision (0.9-19.5%) and between-run precision (0.2-19.0%), accuracy (82-115%) and recovery (90-110%). The validated method was successfully applied to GC-MS analysis of the analytes in woman sera and amniotic fluids and the results are well-comparable with those reported by other authors. The presented work demonstrates for the first time
Suh, Alexander; Weber, Claudia C.; Kehlmaier, Christian; Braun, Edward L.; Green, Richard E.; Fritz, Uwe; Ray, David A.; Ellegren, Hans
Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs) are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs) have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic “fossil” is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote–HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts. PMID:25501991
Slobozhanina, Ekaterina I.; Kozlova, Nataly M.; Kasko, Leonid P.; Mamontova, Marina V.; Chernitsky, Eugene A.
It is shown that the amniotic fluid has intensive ultra-violet luminescence caused by proteins. Along with it amniotic fluid radiated in the field of 380 - 650 nm with maxima at 430 - 450 nm and 520 - 560 nm. The first peak of luminescence ((lambda) exc equals 350 nm; (lambda) em equals 430 - 440 nm) is caused (most probably) by the presence in amniotic fluid of some hormones, NADH2 and NADPH2. A more long-wave component ((lambda) exc equals 460 nm; (lambda) em equals 520 - 560 nm) is most likely connected with the presence in amniotic fluid pigments (bilirubin connected with protein and other). It is shown that intensity and maximum of ultra-violet luminescence spectra of amniotic fluid in normality and at pathology are identical. However both emission spectra and excitation spectra of long-wave ((lambda) greater than 450 nm) luminescence of amniotic fluid from pregnant women with such prenatal abnormal developments of a fetus as anencephaly and spina bifida are too long-wave region in comparison with the norm. Results of research testify that spectral luminescent analysis of amniotic fluid can be used for screening of malformations of the neural tube. It is very difficult for a practical obstetrician to reveal pregnant women with a high risk of congenital malformations of the fetus. Apart from ultrasonic examination, cytogenetic examination of amniotic fluid and defumination of concentrations of alpha-fetoprotein and acetylcholin-esterases in the amniotic fluid and blood plasma are the most widely used diagnostic approaches. However, biochemical and cytogenetic diagnostic methods are time-consuming. In the present work spectral luminescence properties of the amniotic fluid are investigated to determine spectral parameters that can be used to reveal pregnant women with a high risk of congenital malformations of their offsprings.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Amniotic fluid sampler (amniocentesis tray). 884... Diagnostic Devices § 884.1550 Amniotic fluid sampler (amniocentesis tray). (a) Identification. The amniotic fluid sampler (amniocentesis tray) is a collection of devices used to aspirate amniotic fluid from...
... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Amniotic fluid sampler (amniocentesis tray). 884... Diagnostic Devices § 884.1550 Amniotic fluid sampler (amniocentesis tray). (a) Identification. The amniotic fluid sampler (amniocentesis tray) is a collection of devices used to aspirate amniotic fluid from...
... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Amniotic fluid sampler (amniocentesis tray). 884... Diagnostic Devices § 884.1550 Amniotic fluid sampler (amniocentesis tray). (a) Identification. The amniotic fluid sampler (amniocentesis tray) is a collection of devices used to aspirate amniotic fluid from...
... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Amniotic fluid sampler (amniocentesis tray). 884... Diagnostic Devices § 884.1550 Amniotic fluid sampler (amniocentesis tray). (a) Identification. The amniotic fluid sampler (amniocentesis tray) is a collection of devices used to aspirate amniotic fluid from...
... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Amniotic fluid sampler (amniocentesis tray). 884... Diagnostic Devices § 884.1550 Amniotic fluid sampler (amniocentesis tray). (a) Identification. The amniotic fluid sampler (amniocentesis tray) is a collection of devices used to aspirate amniotic fluid from...
Clark, Steven L; Romero, Roberto; Dildy, Gary A; Callaghan, William M; Smiley, Richard M; Bracey, Arthur W; Hankins, Gary D; D'Alton, Mary E; Foley, Mike; Pacheco, Luis D; Vadhera, Rakesh B; Herlihy, J Patrick; Berkowitz, Richard L; Belfort, Michael A
Amniotic fluid embolism is a leading cause of maternal mortality in developed countries. Our understanding of risk factors, diagnosis, treatment, and prognosis is hampered by a lack of uniform clinical case definition; neither histologic nor laboratory findings have been identified unique to this condition. Amniotic fluid embolism is often overdiagnosed in critically ill peripartum women, particularly when an element of coagulopathy is involved. Previously proposed case definitions for amniotic fluid embolism are nonspecific, and when viewed through the eyes of individuals with experience in critical care obstetrics, would include women with a number of medical conditions much more common than amniotic fluid embolism. We convened a working group under the auspices of a committee of the Society for Maternal-Fetal Medicine and the Amniotic Fluid Embolism Foundation whose task was to develop uniform diagnostic criteria for the research reporting of amniotic fluid embolism. These criteria rely on the presence of the classic triad of hemodynamic and respiratory compromise accompanied by strictly defined disseminated intravascular coagulopathy. It is anticipated that limiting research reports involving amniotic fluid embolism to women who meet these criteria will enhance the validity of published data and assist in the identification of risk factors, effective treatments, and possibly useful biomarkers for this condition. A registry has been established in conjunction with the Perinatal Research Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development to collect both clinical information and laboratory specimens of women with suspected amniotic fluid embolism in the hopes of identifying unique biomarkers of this condition.
Zhang, Liying; Zou, Dulei; Li, Sanming; Wang, Junqi; Qu, Yangluowa; Ou, Shangkun; Jia, Changkai; Li, Juan; He, Hui; Liu, Tingting; Yang, Jie; Chen, Yongxiong; Liu, Zuguo; Li, Wei
Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.
Zhang, Liying; Zou, Dulei; Li, Sanming; Wang, Junqi; Qu, Yangluowa; Ou, Shangkun; Jia, Changkai; Li, Juan; He, Hui; Liu, Tingting; Yang, Jie; Chen, Yongxiong; Liu, Zuguo; Li, Wei
Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering. PMID:26876685
Tramoni, G; Boisson, C; Gamerre, L; Clement, H-J; Bon, C; Rudigoz, R-C; Viale, J-P
Amniotic fluid embolism occurs rarely but is a leading cause of maternal mortality. It is a difficult and somewhat intangible diagnosis that warrants a high index of suspicion by physicians. AFE is an unpredictable, unpreventable, and, for the most part, an untreatable obstetric emergency. Management of this condition includes prompt recognition of the signs and symptoms, aggressive resuscitation efforts, and supportive therapy. Any delays in diagnosis and treatment can result in increased maternal and/or foetal impairment or death. Whereas once the invariable outcome of AFE was death of the mother, today the prognosis is somewhat brighter thanks to increased awareness of the syndrome and advances in intensive care medicine. No laboratory test is specific to attest the diagnosis and autopsy must to be realised in case of maternal death. Although non-specific, the diagnosis of AFE could be supported by the observation of amniotic fluid in the central venous blood as well as in the bronchoalveolar fluid. This easy and quick test will be helpful in decision-making. Prompt and aggressive supportive treatment is required to lessen an otherwise dismal outcome, which may include death and permanent disability. This article provides an account of the protean clinical features, pathogenesis, and principles involved in treatment.
Antonucci, Ivana; Di Pietro, Roberta; Alfonsi, Melissa; Centurione, Maria Antonietta; Centurione, Lucia; Sancilio, Silvia; Pelagatti, Francesca; D'Amico, Maria Angela; Di Baldassarre, Angela; Piattelli, Adriano; Tetè, Stefano; Palka, Giandomenico; Borlongan, Cesar V; Stuppia, Liborio
Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.
Richardson, Jo; Shono, Takanori; Okabe, Masataka; Graham, Anthony
The operculum is a large flap consisting of several flat bones found on the side of the head of bony fish. During development, the opercular bones form within the second pharyngeal arch, which expands posteriorly and comes to cover the gill-bearing arches. With the evolution of the tetrapods and the assumption of a terrestrial lifestyle, it was believed that the operculum was lost. Here, we demonstrate that an embryonic operculum persists in amniotes and that its early development is homologous with that of teleosts. As in zebrafish, the second pharyngeal arch of the chick embryo grows disproportionately and comes to cover the posterior arches. We show that the developing second pharyngeal arch in both chick and zebrafish embryos express orthologous genes and require shh signalling for caudal expansion. In amniotes, however, the caudal edge of the expanded second arch fuses to the surface of the neck. We have detailed how this process occurs and also demonstrated a requirement for thyroid signalling here. Our results thus demonstrate the persistence of an embryonic opercular flap in amniotes, that its fusion mirrors aspects of amphibian metamorphosis and gives insights into the origin of branchial cleft anomalies in humans. PMID:21632625
Gao, Yuhua; Zhu, Zhiqiang; Zhao, Yuhua; Hua, Jinlian; Ma, Yuehui; Guan, Weijun
The use of amnion and amniotic fluid (AF) are abundant sources of mesenchymal stem cells (MSCs) that can be harvested at low cost and do not pose ethical conflicts. In human and veterinary research, stem cells derived from these tissues are promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. This work aimed to obtain and characterize bovine amniotic fluid mesenchymal stem cells (AFMSC). The bovine AF from the amniotic cavity of pregnant gilts in the early stages of gestation (3- and 4-m-old bovine embryos) was collected. AFMSCs exhibit a fibroblastic-like morphology only starting from the fourth passage, being heterogeneous during the primary culture. Immunofluorescence results showed that AFMSCs were positive for β-integrin, CD44, CD73 and CD166, but negative for CD34, CD45. Meanwhile, AFMSCs expressed ES cell markers, such as Oct4, and when appropriately induced, are capable of differentiating into ectodermal and mesodermal lineages. This study reinforces the emerging importance of these cells as ideal tools in veterinary medicine; future studies aimed at a deeper evaluation of their immunological properties will allow a better understanding of their role in cellular therapy.
Conconi, Maria Teresa; Nico, Beatrice; Rebuffat, Piera; Crivellato, Enrico; Parnigotto, Pier Paolo; Nussdorfer, Gastone G; Ribatti, Domenico
We investigated the angiogenic response induced by acellular femoral matrices implanted in vivo on to the chick embryo chorioallantoic membrane (CAM), a useful model for such investigation. The results showed that acellular matrices were able to induce a strong angiogenic response, comparable with that of fibroblast growth factor-2 (FGF-2), a well-known angiogenic cytokine. The angiogenic response was further increased when exogenous FGF-2 or transforming growth factor beta-1 (TGF-β1) was added to the matrices and inhibited by the addition of anti-FGF-2 or anti-TGF-β1 antibodies. The response may be considered to be dependent on a direct angiogenic effect exerted by the matrices, and also in part by the presence of FGF-2 and TGF-β1 in the acellular matrices. PMID:16011546
Acellular dermal matrices have been used in breast surgery for a decade. They are widely used in implant-based breast reconstruction to provide coverage of the inferolateral aspects of the prosthesis. Numerous benefits have been reported with this approach including improved fold control, better support and control of the implant pocket with concomitant reduced risk of malposition, and improved lower pole expansion. Seroma, infection, mastectomy skin necrosis, and expander/implant loss are the most commonly reported complications with this approach, and the incidences vary widely among studies. Patient selection and adherence to established intraoperative technique principles related to acellular dermal matrix use are both critical to minimizing the risk of complications. Acellular dermal matrices are also being used in aesthetic breast surgery, revision breast surgery, and nipple reconstruction, but clinical experience is limited. This article reviews the complications associated with the use of matrices in breast surgery from the published literature.
Chávez-García, César; Jiménez-Corona, Aída; Graue-Hernández, Enrique O; Zaga-Clavellina, Verónica; García-Mejía, Mariana; Jiménez-Martínez, María Carmen; Garfias, Yonathan
Amniotic membrane, the inner layer of the placenta, has biological properties (e.g. promotes epithelization, reduces fibrosis, secretes antimicrobial products and inhibits immune responses) which make it a useful option for several ophthalmologic procedures, especially those involving the ocular surface. Its use in eye surgery has been reported by other authors. To our knowledge, there is a lack of descriptive studies on surgical indications using amniotic membrane in Mexican population. Here we describe the eight years Amniotic Membrane Bank experience in Mexico, including a detailed protocol of the donors selection, tissue harvesting, preparation, storage and distribution of amniotic membrane since its establishment in 2007. Moreover, we describe the Ophthalmological indications of amniotic membrane transplantation of the total of 1686 amniotic membranes fragments used during eight years. The five most common indications for amniotic membrane transplantation were pterygium (46 %), corneal ulcers (12.6 %), conjunctival surface repair (11.1 %), neoplasms (7.4 %), and persistent epithelial defects (7.3 %). In addition, we compared the indications of amniotic membrane use in two different types of Institutions: general hospitals and ophthalmologic reference hospitals. We found interesting differences between the indications and use rates between these institutions, although pterygium was the most frequent pathology that amniotic membrane fragments were used in both institutions, there was up to a five-fold increase in the use of amniotic membrane for correction of persistent epithelial defects in reference hospitals which could be explained due to the more complex and severe ophthalmological pathologies admitted in reference hospitals. In conclusion, Amniotic Membrane is used in a numerous ocular pathologies and especially on pterygium in our Mexican population.
Zou, Gang; Liu, Te; Guo, Lihe; Huang, Yongyi; Feng, Ya; Huang, Qin; Duan, Tao
In this study, we observed a great reduction in the expression of the endogenous long noncoding RNA ROR (lncRNA-ROR) and the stem cell transcription factor Sox2, in contrast to a marked increase in miR-145 expression, during the course of in vitro induced differentiation of human amniotic epithelial stem cells (HuAECs). Bioinformatics analysis and the luciferase reporter assay revealed binding of miR-145 to specific sites in lncRNA-ROR and Sox2, silencing their expression. Overexpression of a lncRNA-ROR-specific siRNA effectively downregulated the expression levels of Sox2 and other stem cell markers in HuAECs while weakening the efficiency of HuAEC differentiation into β islet-like cells. Moreover, the in vitro response of HuAEC-derived β islet-like cells to extracellular stimuli and C-peptide release by these cells were markedly weakened in the siRNA-ROR transfection group. Furthermore, the in vivo expression of β islet-like cell biomarkers was substantially reduced in HuAECs in the siRNA-ROR transfection group, and their in vivo β islet-like cell differentiation and insulin release capacities were reduced in a streptozocin-induced diabetic rat model. The experimental results indicate that lncRNA-ROR effectively maintains Sox2 gene expression through competitive binding to miR-145, achieving pluripotency maintenance in HuAECs and regulation of their directed β islet-like cell differentiation efficiency.
Cherubino, Mario; Pellegatta, Igor; Tamborini, Federico; Cerati, Michele; Sessa, Fausto; Valdatta, Luigi
Introduction: Much attention has been directed towards understanding the phenomena of angiogenesis and lymphangiogenesis in wound healing. Thanks to the manifold dermal substitute available nowadays, wound treatment has improved greatly. Many studies have been published about angiogenesis and cell invasion in INTEGRA®. On the other hand, the development of the lymphatic network in acellular dermal matrix (ADM) is a more obscure matter. In this article, we aim to characterize the different phases of host cell invasion in ADM. Special attention was given to lymphangiogenic aspects. Materials and Methods: Among 57 rats selected to analyse the role of ADM in lymphangiogenesis, we created four groups. We performed an excision procedure on both thighs of these rats: On the left one we did not perform any action except repairing the borders of the wound; while on the right one we used INTEGRA® implant. The excision biopsy was performed at four different times: First group after 7 days, second after 14 days, third after 21 days and fourth after 28 days. For our microscopic evaluation, we used the classical staining technique of haematoxylin and eosin and a semi-quantitative method in order to evaluate cellularity counts. To assess angiogenesis and lymphangiogenesis development we employed PROX-1 Ab and CD31/PECAM for immunohistochemical analysis. Results: We found remarkable wound contraction in defects that healed by secondary intention while minor wound contraction was observed in defects treated with ADM. At day 7, optical microscopy revealed a more plentiful cellularity in the granulation tissue compared with the dermal regeneration matrix. The immunohistochemical process highlighted vascular and lymphatic cells in both groups. After 14 days a high grade of fibrosis was noticeable in the non-treated group. At day 21, both lymphatic and vascular endothelial cells were better developed in the group with a dermal matrix application. At day 28, lymphatic endothelial
Xing, Dorothy; Asokanathan, Catpagavalli; Xu, Ying Hua; Bolgiano, Barbara; Douglas-Bardsley, Alex; Zhang, Shumin; Wang, Junzhi; Corbel, Michael
Comparison of the immunogenicity response and resistance to challenge in the modified intracerebral challenge assay induced by various acellular pertussis vaccines showed that these were not closely linked. The immunogenicity assay was effective for confirming the presence of specific antigenic components and was invaluable for detecting minor components present in co-purified vaccines. However, the magnitude of antibody responses was not consistently related to antigen concentration nor did it correlate with protection in the modified intracerebral challenge assay. The immunogenicity assay detected degradation of pertussis toxin and pertactin components but not of filamentous haemagglutinin or fimbriae 2 and 3 in denatured acellular pertussis vaccines. The modified intracerebral challenge assay was effective in detecting antigen degradation in all types of acellular pertussis vaccines including those of European/North American origin but was dominated by the response to pertussis toxin. Aerosol challenge was more sensitive in detecting denaturation of filamentous haemagglutinin or fimbriae. The modified intracerebral challenge assay was the only assay that provided a quantitative indication of protective activity. Both immunogenicity and challenge assays provided useful data on acellular pertussis vaccine properties but were complementary and not alternatives.
Xing, Dorothy; Asokanathan, Catpagavalli; Xu, Ying Hua; Bolgiano, Barbara; Douglas-Bardsley, Alex; Zhang, Shumin; Wang, Junzhi; Corbel, Michael
Comparison of the immunogenicity response and resistance to challenge in the modified intracerebral challenge assay induced by various acellular pertussis vaccines showed that these were not closely linked. The immunogenicity assay was effective for confirming the presence of specific antigenic components and was invaluable for detecting minor components present in co-purified vaccines. However, the magnitude of antibody responses was not consistently related to antigen concentration nor did it correlate with protection in the modified intracerebral challenge assay. The immunogenicity assay detected degradation of pertussis toxin and pertactin components but not of filamentous haemagglutinin or fimbriae 2 and 3 in denatured acellular pertussis vaccines. The modified intracerebral challenge assay was effective in detecting antigen degradation in all types of acellular pertussis vaccines including those of European/North American origin but was dominated by the response to pertussis toxin. Aerosol challenge was more sensitive in detecting denaturation of filamentous haemagglutinin or fimbriae. The modified intracerebral challenge assay was the only assay that provided a quantitative indication of protective activity. Both immunogenicity and challenge assays provided useful data on acellular pertussis vaccine properties but were complementary and not alternatives. PMID:25424817
Boyer, Richard B.; Sexton, Kevin W.; Rodriguez-Feo, Charles L.; Nookala, Ratnam; Pollins, Alonda C.; Cardwell, Nancy L.; Tisdale, Keonna Y.; Nanney, Lillian B.; Shack, R. Bruce; Thayer, Wesley P.
Background Acellular nerve allografts are now standard tools in peripheral nerve repair due to decreased donor site morbidity and operative time savings. Preparation of nerve allografts involves several steps of decellularization and modification of extracellular matrix to remove chondroitin sulfate proteoglycans (CSPGs), which have been shown to inhibit neurite outgrowth through a poorly understood mechanism involving RhoA and ECM-integrin interactions. Chondroitinase ABC (ChABC) is an enzyme that degrades CSPG molecules and has been shown to promote neurite outgrowth following injury of the central and peripheral nervous systems. Variable results following chondroitinase ABC treatment make it difficult to predict the effects of this drug in human nerve allografts, especially in the presence of native extracellular signaling molecules. Several studies have shown cross-talk between neurotrophic factor and CSPG signaling pathways, but their interaction remains poorly understood. In this study, we examined the adjuvant effects of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth post-injury in CSPG-reduced substrates and acellular nerve allografts. Materials and Methods E12 chicken DRG explants were cultured in medium containing ChABC, ChABC + NGF, ChABC + GDNF or control media. Explants were imaged at 3 d and neurite outgrowths measured. The rat sciatic nerve injury model involved a 1-cm sciatic nerve gap that was microsurgically repaired with ChABC pre-treated acellular nerve allografts. Prior to implantation, nerve allografts were incubated in NGF, GDNF or sterile water. Nerve histology was evaluated at 5d and 8wk post-injury. Results The addition of GDNF in vitro produced significant increase in sensory neurite length at 3 d compared to ChABC alone (P < 0.01), while NGF was not significantly different from control. In vivo adjuvant NGF produced increases in total myelinated axon count (P < 0.005) and motor axon
Briese, V; Brock, J; Stark, K H
Amniotic fluid levels of secretory immunoglobulin A(S-IgA) were measured by single radial immunodiffusion according to the method of Mancini using a monospecific antiserum against the human secretory component. 114 amniotic fluid samples were examined. The S-IgA values showed a correlation (r = 0.49) with the phospholipid levels. Additionally, it was observed no correlation between Serum and amniotic fluid S-IgA concentrations estimated as parallel investigations in 20 patients. S-IgA values greater than or equal to 82 mg/l were used to diagnose a sufficient fetal lung maturity. False negative values of S-IgA are possible. In 19 cases of newborns without signs of respiratory distress syndrome amniotic fluid S-IgA estimations were performed up to 32 hours before the delivery. False negative phospholipid levels were estimated in 2 cases, false negative S-IgA levels in 4 cases. The measurement of S-IgA in amniotic fluid will serve as an additional parameter for the estimation of fetal lung maturity.
Yang, Chenmin; Fan, Shuyue; Tang, Huixiang; Gong, Zhijuan; Gong, Xiuli; Ren, Zhaorui; Zeng, Fanyi
We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Managoli, Sanjeev; Chaturvedi, Pushpa; Vilhekar, Krishna Y; Iyenger, Janaki
An association of Amniotic Band Disruption Sequence and Mermaid Syndrome in a newborn having multiple congenital anomalies is being reported. The newborn had aberrant string like tissues attached to the amputed fingers and toes. Adhesions of amniotic bands had disrupted the fetal parts especially anteriorly in the midline, causing multiple anomalies. Apart from these features of Amniotic Band Disruption Sequence, the newborn had complete fusion of the lower limbs by cutaneous tissue, a characteristic of Mermaid Syndrome (Sirenomelia). Associated malformations were anal stenosis, rectal atresia, small horseshoe kidney, hypoplastic urinary bladder and a bicomuate uterus. The single umbilical artery had a high origin, arising directly from the aorta just distal to the celiac axis, which is unique to sirenomelia. Theories put forward regarding the etiopathogenesis of both the conditions are discussed.
Xing, Dorothy; Gaines Das, Rose; Douglas-Bardsley, Alex; Asokanathan, Catpagavalli; Corbel, Michael
Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose-response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.
Feng, Yuping; Wang, Jiao; Ling, Shixin; Li, Zhuo; Li, Mingsheng; Li, Qiongyi; Ma, Zongren; Yu, Sijiu
The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins, including βIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve. PMID:25598779
Simões, Irina N; Vale, Paulo; Soker, Shay; Atala, Anthony; Keller, Daniel; Noiva, Rute; Carvalho, Sandra; Peleteiro, Conceição; Cabral, Joaquim M S; Eberli, Daniel; da Silva, Cláudia L; Baptista, Pedro M
Patients with stress urinary incontinence mainly suffer from malfunction of the urethra closure mechanism. We established the decellularization of porcine urethras to produce acellular urethra bioscaffolds for future tissue engineering applications, using bioscaffolds or bioscaffold-derived soluble products. Cellular removal was evaluated by H&E, DAPI and DNA quantification. The presence of specific ECM proteins was assessed through immunofluorescence staining and colorimetric assay kits. Human skeletal muscle myoblasts, muscle progenitor cells and adipose-derived stromal vascular fractions were used to evaluate the recellularization of the acellular urethra bioscaffolds. The mechanochemical decellularization system removed ~93% of tissue's DNA, generally preserving ECM's components and microarchitecture. Recellularization was achieved, though methodological advances are required regarding cell seeding strategies and functional assessment. Through microdissection and partial digestion, different urethra ECM-derived coating substrates were formulated (i.e. containing smooth or skeletal muscle ECM) and used to culture MPCs in vitro. The skeletal muscle ECM substrates enhanced fiber formation leading to the expression of the main skeletal muscle-related proteins and genes, as confirmed by immunofluorescence and RT-qPCR. The described methodology produced a urethra bioscaffold that retained vital ECM proteins and was liable to cell repopulation, a crucial first step towards the generation of urethra bioscaffold-based Tissue Engineering products.
Simões, Irina N.; Vale, Paulo; Soker, Shay; Atala, Anthony; Keller, Daniel; Noiva, Rute; Carvalho, Sandra; Peleteiro, Conceição; Cabral, Joaquim M. S.; Eberli, Daniel; da Silva, Cláudia L.; Baptista, Pedro M.
Patients with stress urinary incontinence mainly suffer from malfunction of the urethra closure mechanism. We established the decellularization of porcine urethras to produce acellular urethra bioscaffolds for future tissue engineering applications, using bioscaffolds or bioscaffold-derived soluble products. Cellular removal was evaluated by H&E, DAPI and DNA quantification. The presence of specific ECM proteins was assessed through immunofluorescence staining and colorimetric assay kits. Human skeletal muscle myoblasts, muscle progenitor cells and adipose-derived stromal vascular fractions were used to evaluate the recellularization of the acellular urethra bioscaffolds. The mechanochemical decellularization system removed ~93% of tissue’s DNA, generally preserving ECM’s components and microarchitecture. Recellularization was achieved, though methodological advances are required regarding cell seeding strategies and functional assessment. Through microdissection and partial digestion, different urethra ECM-derived coating substrates were formulated (i.e. containing smooth or skeletal muscle ECM) and used to culture MPCs in vitro. The skeletal muscle ECM substrates enhanced fiber formation leading to the expression of the main skeletal muscle-related proteins and genes, as confirmed by immunofluorescence and RT-qPCR. The described methodology produced a urethra bioscaffold that retained vital ECM proteins and was liable to cell repopulation, a crucial first step towards the generation of urethra bioscaffold-based Tissue Engineering products. PMID:28165009
Although tetanus and diphtheria have become rare in developed countries, pertussis is still endemic in some developed countries. These are vaccine-preventable diseases and vaccination for adults is important to prevent the outbreak of disease. Strategies for tetanus, diphtheria, and pertussis vaccines vary from country to country. Each country needs to monitor consistently epidemiology of the diseases and changes vaccination policies accordingly. Recent studies showed that tetanus–diphtheria–acellular pertussis vaccine for adults is effective and safe to prevent pertussis disease in infants. However, vaccine coverage still remains low than expected and seroprevalence of protective antibodies levels for tetanus, diphtheria, and pertussis decline with aging. The importance of tetanus–diphtheria–acellular pertussis vaccine administration should be emphasized for the protection of young adult and elderly people also, not limited to children. PMID:28168170
Joo, Sunyoung; Ko, In Kap; Atala, Anthony; Yoo, James J; Lee, Sang Jin
The stem cells isolated from amniotic fluid present an exciting possible contribution to the field of regenerative medicine and amniotic fluid-derived stem (AFS) cells have significant potential for research and therapeutic applications. AFS cells are multipotent, showing the ability to differentiate into cell types from all three embryonic germ layers. They express both embryonic and adult stem cell markers, expand extensively without feeder cells, double in 36 h, and are not tumorigenic. The AFS cells can be maintained for over 250 population doublings and preserve their telomere length and a normal karyotype. They differentiate easily into specific cell lineages and do not require human embryo tissue for their isolation, thus avoiding the current controversies associated with the use of human embryonic stem (ES) cells. The discovery of the AFS cells has been recent, and a great deal of work remains to be performed on the characterization and use of these cells. This review describes the various differentiated lineages that AFS cells can form and the future of these promising new stem cells in regenerative medicine research.
Brace, Robert A; Cheung, Cecilia Y; Anderson, Debra F
Intramembranous absorption increases during intra-amniotic infusion of physiological saline solutions. The increase may be due partly to the concomitant elevation in fetal urine production as fetal urine contains a stimulator of intramembranous absorption. In this study, we hypothesized that the increase in intramembranous absorption during intra-amniotic infusion is due, in part, to dilution of a nonrenal inhibitor of intramembranous absorption that is present in amniotic fluid. In late-gestation fetal sheep, amniotic fluid volume and the four primary amniotic inflows and outflows were determined over 2-day intervals under three conditions: 1) control conditions when fetal urine entered the amniotic sac, 2) during intra-amniotic infusion of 2 l/day of lactated Ringer solution when urine entered the amniotic sac, and 3) during the same intra-amniotic infusion when fetal urine was continuously replaced with lactated Ringer solution. Amniotic fluid volume, fetal urine production, swallowed volume, and intramembranous absorption rate increased during the infusions independent of fetal urine entry into the amniotic sac or its replacement. Lung liquid secretion rate was unchanged during infusion. Because fetal membrane stretch has been shown not to be involved and because urine replacement did not alter the response, we conclude that the increase in intramembranous absorption that occurs during intra-amniotic infusions is due primarily to dilution of a nonrenal inhibitor of intramembranous absorption that is normally present in amniotic fluid. This result combined with our previous study suggests that a nonrenal inhibitor(s) together with a renal stimulator(s) interact to regulate intramembranous absorption rate and, hence, amniotic fluid volume.
Sarkar, P; Bergman, K; O'Connor, T G; Glover, V
Both animal and human studies have shown that maternal stress or anxiety during pregnancy is associated with increased risk of disturbance in offspring neurodevelopment and behaviour. In animal models, increased foetal exposure to glucocorticoids has been found to be one mechanism for such foetal programming. Little is understood of the mediating mechanisms in humans, and one aim of our research programme is to investigate this further. This review presents a synopsis of some of our recent results. We aimed to test the hypothesis that maternal anxiety was associated with raised maternal cortisol, and that this in turn was related to increased foetal exposure to cortisol. We studied this by recruiting women at amniocentesis, obtained their Spielberger State Anxiety scores, and assessed maternal plasma cortisol and amniotic fluid cortisol. We also examined maternal plasma and amniotic fluid testosterone levels. Awaiting amniocentesis was in general anxiogenic, but with a wide range of anxiety scores. Maternal anxiety was significantly associated with plasma cortisol before 17 weeks, albeit of modest magnitude (r = 0.0.23), and not after 17 weeks of gestation. This is probably due to the known attenuation of the maternal hypothalamic-pituitary-adrenal axis with increasing gestation. We found a strong correlation between maternal plasma and amniotic fluid cortisol levels, which increased with gestation and became robust after 18 weeks. This correlation increased with maternal anxiety, suggesting a possible effect of maternal mood on placental function. There was a positive correlation between cortisol and testosterone in amniotic fluid, in both male and female foetuses independent of maternal anxiety, plasma testosterone, gestational age, and time of collection. Foetal stress may be associated with increased foetal exposure to testosterone. However, maternal anxiety did not predict amniotic fluid cortisol or testosterone level. Thus, the role of these hormones in
Zhang, Yanru; Zhang, Hui; Katiella, Kaka; Huang, Wenhua
A chemically extracted acellular allogeneic nerve graft can reduce postoperative immune rejection, similar to an autologous nerve graft, and can guide neural regeneration. However, it remains poorly understood whether a chemically extracted acellular allogeneic nerve graft combined with neurotrophic factors provides a good local environment for neural regeneration. This study investigated the repair of injured rat sciatic nerve using a chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor. An autologous nerve anastomosis group and a chemical acellular allogeneic nerve bridging group were prepared as controls. At 8 weeks after repair, sciatic functional index, evoked potential amplitude of the soleus muscle, triceps wet weight recovery rate, total number of myelinated nerve fibers and myelin sheath thickness were measured. For these indices, values in the three groups showed the autologous nerve anastomosis group > chemically extracted acellular nerve graft + ciliary neurotrophic factor group > chemical acellular allogeneic nerve bridging group. These results suggest that chemically extracted acellular nerve grafts combined with ciliary neurotrophic factor can repair sciatic nerve defects, and that this repair is inferior to autologous nerve anastomosis, but superior to chemically extracted acellular allogeneic nerve bridging alone. PMID:25221592
Dulay, Antonette T.; Buhimschi, Catalin S.; Zhao, Guomao; Oliver, Emily; Abdel-Razeq, Sonya S.; Shook, Lydia L.; Bahtiyar, Mert O.; Buhimschi, Irina A.
Problem TLR4 mediates host responses to pathogens through a mechanism that involves protein myeloid differentiation-2 (MD-2) and its soluble form sMD-2. The role of sMD2 in intra-amniotic inflammation induced preterm birth has not been previously explored. Method of study Human amniotic fluid (AF) sMD-2 was studied by Western blotting in 152 AF samples of patients who had an amniocentesis to rule-out infection (yes infection, n=50; no infection, n=50) or women with normal pregnancy outcome (2nd trimester genetic karyotyping, n=26; 3rd trimester lung maturity testing, n=26). Histologic localization and mRNA expression of MD2 in fetal membranes were studied by immunohistochemistry and RT-PCR. The ability of fetal membrane to release sMD-2 and inflammatory cytokines was studied in-vitro. Results Human AF contains three sMD-2 proteoforms whose levels of expression were lower at term. Intra-amniotic infection up-regulated sMD-2. MD-2 mRNA and immunohistochemistry findings concurred. In vitro, LPS and monensin increased while cycloheximide decreased sMD-2 production. Recombinant sMD-2 modulated TNF-α and IL-6 levels in a dose and time-dependent fashion. Conclusion sMD2 proteoforms are constitutively present in human AF. The intensity of the intra-amniotic inflammatory response to bacteria or perhaps to other TLR4 ligands may be facilitated through synthesis and release of sMD2 by the amniochorion. PMID:25605324
Beling, C G; Cederqvist, L L
Amniotic fluid cells obtained by amnicentesis at 16-20 weeks' gestation were grown in culture until a confluent monolayer of cell had been formed. Radiolabeled pregnenolone, progesterone and 20 alpha-dihydroprogesterone were added to the cell cultures; steroid metabolites which formed after 24 and 48 hours of incubation were identified. Incubation of the cell cultures with pregnenolone-3H resulted in the formation of progesterone, 17alpha-progesterone and 20 alpha-dihydroprogesterone. A significant amount of progesterone was identified after incubating the cell cultures with 20 alpha-dihydroprogesterone. The results indicate that 3 beta-ol-dehydrogenase, 17 alpha-hydroxylase and 20 alpha-hydroxysteroid dehydrogenase enzymes are present in cultured amniotic fluid cells obtained at 16-20 weeks' gestation.
Greco, D; Esposito, S; Tozzi, A; Pandolfi, E; Icardi, G; Giammanco, A
Clinical pertussis resulting from infection with B. pertussis is a significant medical and public health problem, despite the huge success of vaccination that has greatly reduced its incidence. The whole cell vaccine had an undeniable success over the last 50 years, but its acceptance was strongly inhibited by fear, only partially justified, of severe side effects, but also, in the Western world, by the difficulty to enter in combination with other vaccines: today multi-vaccine formulations are essential to maintain a high vaccination coverage. The advent of acellular vaccines was greeted with enthusiasm by the public health world: in the Nineties, several controlled vaccine trials were carried out: they demonstrated a high safety and good efficacy of new vaccines. In fact, in the Western world, the acellular vaccines completely replaced the whole cells ones. In the last years, ample evidence on the variety of protection of these vaccines linked to the presence of different antigens of Bordetella pertussis was collected. It also became clear that the protection provided, on average around 80%, leaves every year a significant cohort of vaccinated susceptible even in countries with a vaccination coverage of 95%, such as Italy. Finally, it was shown that, as for the pertussis disease, protection decreases over time, to leave a proportion of adolescents and adults unprotected. Waiting for improved pertussis vaccines, the disease control today requires a different strategy that includes a booster at 5 years for infants, but also boosters for teenagers and young adults, re-vaccination of health care personnel, and possibly of pregnant women and of those who are in contact with infants (cocooning). Finally, the quest for better vaccines inevitably tends towards pertussis acellular vaccines with at least three components, which have demonstrated superior effectiveness and have been largely in use in Italy for fifteen years.
Farhat, Walid; Chen, Jun; Erdeljan, Petar; Shemtov, Oren; Courtman, David; Khoury, Antoine; Yeger, Herman
The objectives of this study are to examine the porosity of bladder acellular matrix (ACM) using deionized (DI) water as the model fluid and dextran as the indicator macromolecule, and to correlate the porosity to the ACM thickness. Porcine urinary bladders from pigs weighing 20-50 kg were sequentially extracted in detergent containing solutions, and to modify the ACM thickness, stretched bladders were acellularized in the same manner. Luminal and abluminal ACM specimens were subjected to fixed static DI water pressure (10 cm); and water passing through the specimens was collected at specific time interval. While for the macromolecule porosity testing, the diffusion rate and direction of 10,000 MW fluoroescein-labeled dextrans across the ACM specimens mounted in Ussing's chambers were measured. Both experiments were repeated on the thin stretched ACM. In both ACM types, the fluid porosity in both directions did not decrease with increased test duration (3 h); in addition, the abluminal surface was more porous to fluid than the luminal surface. On the other hand, when comparing thin to thick ACM, the porosity in either direction was higher in the thick ACM. Macromolecule porosity, as measured by absorbance, was higher for the abluminal thick ACM than the luminal side, but this characteristic was reversed in the thin ACM. Comparing thin to thick ACM, the luminal side in the thin ACM was more porous to dextran than in the thick ACM, but this characteristic was reversed for the abluminal side. The porcine bladder ACM possesses directional porosity and acellularizing stretched urinary bladders may increase structural density and alter fluid and macromolecule porosity.
Hedges, S Blair
The position of turtles among amniotes remains in dispute, with morphological and molecular comparisons giving different results. Morphological analyses align turtles with either lizards and their relatives, or at the base of the reptile tree, whereas molecular analyses, including a recent study by Chiari et al. in BMC Biology, place turtles with birds and crocodilians. Molecular studies have not wavered as the numbers of genes and species have increased, but morphologists have been reluctant to embrace the molecular tree.
Fracasso, Tony; Karger, Bernd; Vennemann, Mechtild; Bajanowski, Thomas; Golla-Schindler, Ute Maria; Pfeiffer, Heidi
The scope of this study was to evaluate the incidence and the eventual consequences of amniotic fluid aspiration (AFA) in cases of sudden infant death. Cases of sudden infant death syndrome (SIDS; n = 113: 39 females, 74 males; mean age 4.6 months) were compared to a control group of 39 cases of explained death (14 females, 25 males; mean age 5.6 months). In each case, sections of the lung stained with hematoxylin and eosin and with the immunohistochemical reaction 34BE12 specific for cytokeratins were available. The microscope slides were observed at x200 magnification and semi-quantitatively classified into four categories(-, +, ++, and +++). In both groups, rests of amniotic fluid could be observed up to the fourth month of life. The comparison between the two groups did not show any significant difference. In the SIDS group, immunohistochemical reactions with the antibodies CD68, MRP8, MRP14, 27E10, 25F9, CD3, CD20Cy, and CD45R0 were available for the lungs. Twelve cases with AFA were compared to a group of SIDS cases without AFA with similar age and pathological distribution to evaluate whether the presence of amniotic remnants induced inflammatory changes in the lungs. No differences emerged. This study shows that AFA is not a rare event. Even moderate to severe AFA does not necessary cause death. A correlation between AFA and SIDS could not be shown.
Onda, T; Fukushima, K; Tanaka, T; Sawa, R; Hayashi, Z; Tsutsumi, O; Takai, Y; Yoshida, K; Nakamura, Y; Hoshi, K; Fukada, Y; Okai, T; Sakai, M; Kitagawa, M; Akiyama, Y; Shimomura, K; Myrick, F; Dowman, A C; Grier, R E
Owing to differences in maternal serum alpha-fetoprotein, human chorionic gonadotrophin and oestriol levels between native Japanese and Caucasian women screened in this laboratory, a study was conducted to measure amniotic fluid alpha-fetoprotein (AFAFP) levels in native Japanese pregnancies. When the native Japanese AFAFP levels were compared with a United States (non-Black) population, the Japanese medians did not decrease as rapidly over the 14 to 22 weeks of gestation period investigated. At 14 weeks, the difference was negligible, graduating to a difference of 20 per cent by 22 weeks' gestation. Native Japanese pregnancy AFAFP levels should be interpreted based upon population data from that group alone. From these findings, prenatal screening laboratories should be encouraged to collect preliminary data for comparison before screening is initiated for a defined ethnic group.
Briese, V; Brock, J; Lorenz, U; Straube, W
S-IgA was estimated in amniotic fluid and in mucus samples of pharyngeal cavities and urine samples of newborns for assistance of the hypothesis that there is a connection between S-IgA content in amniotic fluid and fetal pulmonary maturity. It was used an antiserum against human secretory component and an S-IgA standard in the single radial immunodiffusion according to Mancini and coworkers. High S-IgA concentrations in the mucus samples of pharyngeal cavities refer to fetal bronchial-pulmonary system as the probable essential part in the synthesis of S-IgA established in amniotic fluid of the 3rd trimenon in pregnancy.
Rommel, Niklas; Rohleder, Nils H; Gabriel, Christian; Hennerbichler, Simone; Bauer, Florian; Mücke, Thomas; Kolk, Andreas; Loeffelbein, Denys J; Wolff, Klaus D; Kesting, Marco R
The objective of the study was to find out if human amniotic membrane could be used for corrective surgery after trauma to the orbital wall. Because of its proposed antiadhesive qualities, it seemed to be potentially suitable. We studied 8 men (mean age 37 (range 19-74) years) who had deficient ocular movement after fractures of the orbital floor. Five of them had already been operated on. Inclusion criteria were trauma dating back more than 4 months and a soft tissue stricture in the orbital floor diagnosed by magnetic resonance imaging. Patients were treated secondarily with lysis of adhesions and insertion of allogeneic human amniotic membrane laminated on to polyglactin 910/polydioxanone foil, which functioned as the carrier material. Patients were followed up for 3 months, by which time disorders of motility of the ocular bulb had disappeared completely in 5. Two patients had improved motility and a reduction in both their subjective and objective symptoms. One patient had no improvement. The considerable reduction in adhesions and scarring after insertion of the membrane confirms previous assumptions, according to which the epithelial side of the human amniotic membrane has an antiadhesive effect because of its smooth surface.
Kahramaner, Zelal; Cosar, Hese; Turkoglu, Ebru; Erdemir, Aydin; Kanik, Ali; Sutcuoglu, Sumer; Ozer, Esra Arun
Amniotic band sequence (ABS) is a rare cause of fetal disruptions associated with fibrous bands that entrap various fetal parts in utero and lead to abnormalities. Fetal disruptions of ABS are influenced by the timing of the amnion rupture and the site of amnion adherence. Herein we report an extreme case of ABS presented with dysmorphic face, amputation of four extremities and fusion of legs and genitalia with a fibrotic band. This is an extreme case of ABS characterized by an unusual combination of multiple fetal anomalies.
Maddipati, Krishna Rao; Romero, Roberto; Chaiworapongsa, Tinnakorn; Chaemsaithong, Piya; Zhou, Sen-Lin; Xu, Zhonghui; Tarca, Adi L; Kusanovic, Juan Pedro; Gomez, Ricardo; Docheva, Nikolina; Honn, Kenneth V
Clinical chorioamnionitis at term (TCC) is the most common obstetrical infliction diagnosed in labor and delivery units worldwide and is associated with a substantial increase in maternal and neonatal morbidity and mortality. This obstetrical complication is a heterogeneous condition, as only half of patients have detectable microorganisms in the amniotic cavity. Because bioactive lipids play a key role in the initiation and resolution of an inflammatory response, we aimed to characterize the amniotic fluid lipidome in patients with TCC. We studied the amniotic fluid of patients in the following groups: 1) spontaneous labor at term without clinical chorioamnionitis (TLB) and 2) spontaneous labor at term with clinical chorioamnionitis (TCC). The TCC group was subdivided into a) those with microbial invasion of the amniotic cavity (TCC-MIAC) and b) those without microbial invasion of the amniotic cavity (TCC-noMIAC). The amniotic fluid concentration of proinflammatory lipid mediators did not differ between patients in TLB with TCC. In contrast, concentration of lipids with anti-inflammatory/proresolution properties was significantly lower in all patients with TCC than in those with TLB. These results suggest that while proinflammatory lipid mediators are involved in infection-driven intra-amniotic inflammation, a relative deficiency of anti-inflammatory/proresolution lipid mediator biosynthesis is a characteristic of TCC.
Shah, Krupa Hitesh; Shah, Hitesh
Amniotic constriction bands and osteogenesis imperfecta are disorders arising from a collagen defect. We report a rare association of amniotic bands with osteogenesis imperfecta in a child. The child was born with multiple amniotic bands involving the right leg, both hands and both feet. Multiple fractures of long bones of lower limbs occurred in childhood due to trivial trauma. Deformities of the femur and tibia due to malunion with osteopenia and blue sclerae were present. The patient was treated with z plasty of constriction band of the right tibia and bisphosphonate for osteogenesis imperfecta. This rare association of both collagen diseases may provide further insight for the pathogenesis of these diseases.
Introduction. Allogeneic amniotic tissue and fluid may be used to treat chronic plantar fasciosis and Achilles tendinosis. This innovative approach involves delivering a unique allograft of live human cells in a nonimmunogenic structural tissue matrix to treat chronic tendon injury. These tissues convey very positive regenerative attributes; procurement is performed with maternal consent during elective caesarian birth. Materials and Methods. In the present investigation all patients were unresponsive to multiple standard therapies for a minimum of 6 months and were treated with one implantation of PalinGen SportFLOW around the plantar fascia and/or around the Achilles paratenon. The patients were given a standard protocol for postimplant active rehabilitation. Results. The analogue pretreatment pain score (VAS) of 8. By the fourth week after treatment, all patients had significantly reduced self-reported pain. Twelve weeks following the procedure the average pain level had reduced to only 2. No adverse reactions were reported in any of the patients. Conclusion. All patients in this study experienced heel or Achilles pain, unresponsive to standard therapy protocols. After treatment all patients noted significant pain reduction, indicating that granulized amniotic membrane and amniotic fluid can be successfully used to treat both chronic plantar fasciosis and Achilles tendinosis. PMID:26491722
Winsnes, R; Sesardic, D; Daas, A; Terao, E; Behr-Gross, M-E
An international collaborative study (coded BSP083) was performed under the aegis of the Biological Standardisation Programme supported by the Council of Europe and the European Commission, with the aim of replacing the in vivo challenge assays for potency determination of combined acellular pertussis (aP) vaccines by a refined procedure also allowing reduction of animal use. This study investigates whether the immunogenicity of aP vaccine components could be assayed in a guinea pig (gp) serology model, using the same vaccine immunising doses as for D and T components potency testing, instead of using separate animals as is currently done. The BSP83 project is a follow up of 3 former collaborative studies (coded BSP019, BSP034 and BSP035) on serological methods for the potency testing of tetanus (T) and diphtheria (D) vaccines for human use. The use of gp instead of mice serology has the advantage of providing a larger volume of good quality antiserum for the assay of several vaccine components in the same sample, hence providing the opportunity for animal sparing. The results of Phase I of the study demonstrated that gp serology may be a useful method for the immunogenicity assay of acellular pertussis vaccines. This was confirmed in Phase II of the study, using 7 different combined aP vaccines in an international collaborative study involving 17 laboratories from both public and private sectors. Clear dose-response relationships were observed for different vaccines by ELISA, for antibodies against aP antigens, i.e. pertussis toxin (PT), filamentous haemagglutinin (FHA), fimbrial agglutinogens-2/3 (Fim 2/3) and pertactin (PRN). Intra- and inter-laboratory variations of aP ELISA results were found to be within an acceptable range. For some combined vaccines, however, the range of vaccine dilutions for immunisation confirmed to be optimal for D and T potency testing may not provide optimal dose-response for all aP components. Method adjustments may thus be required
Ives, J. A.; Abbott, G. D.; Bailey, R. R.
Women with asymptomatic bacteriuria during pregnancy had sterile amniotic fluid at the time of delivery. There was no evidence that maternal urinary infection was associated with infection in the infant. PMID:5555492
Bujold, Emmanuel; Romero, Roberto; Kusanovic, Juan Pedro; Erez, Offer; Gotsch, Francesca; Chaiworapongsa, Tinnakorn; Gomez, Ricardo; Espinoza, Jimmy; Vaisbuch, Edi; Kim, Yeon Mee; Edwin, Samuel; Pisano, Mike; Allen, Beth; Podust, Vladimir N.; Dalmasso, Enrique A.; Rutherford, Jennifer; Rogers, Wade; Moser, Allan; Yoon, Bo Hyun; Barder, Tim
, IGFBP-1 fragments at about 13.5 kDa were present in patients with intra-amniotic IAI; 3) proteins which were over-expressed in group 1 included Von Ebner gland protein precursor, IL-7 precursor, apolipoprotein A1, tropomyosin sk1 (TPMsk1) fragment, ribosomal protein S6 kinase alpha-3 and alpha-1-microglobulin/bikunin precursor (AMBP); 4) proteins which were over-expressed in group 3 included fibrinopeptide B, transferrin, (MHC) class 1 chain-related A antigen fragment, transcription elongation factor A, sex-determining region Y (SRY) box 5 protein, Down syndrome critical region 2 protein (DSCR2), and human peptide 8 (HP8); and 5) one protein, retinol binding protein, was over-expressed in women who delivered preterm, regardless of the presence of IAI. Conclusions A combination of techniques involving 2D chromatography, mass spectrometry, and immunoassays allows identification of proteins that are differentially regulated in amniotic fluid of patients with preterm labor. Specifically, the amount of the IGFBP-1 fragments at approximately 13.5 kDa was found to be increased in patients with IAI, while the amount of the intact form of IGFBP-1 was decreased. PMID:19012186
Chaiworapongsa, Tinnakorn; Erez, Offer; Kusanovic, Juan Pedro; Vaisbuch, Edi; Mazaki-Tovi, Shali; Gotsch, Francesca; Than, Nandor Gabor; Mittal, Pooja; Kim, Yeon Mee; Camacho, Natalia; Edwin, Samuel; Gomez, Ricardo; Hassan, Sonia S.; Romero, Roberto
Objective Heat shock protein (HSP) 70, a conserved member of the stress protein family, is produced in almost all cell types in response to a wide range of stressful stimuli and their production has a survival value. Evidence suggests that extra-cellular HSP70 is involved in the activation of the innate and adaptive immune response. Furthermore, increased mRNA expression of HSP 70 was observed in human fetal membranes following endotoxin stimulation. This study was conducted to determine the changes in amniotic fluid HSP70 concentrations during pregnancy, term and preterm parturition, intra-amniotic infection (IAI), and histologic chorioamnionitis. Study design A cross-sectional study was conducted in 376 pregnant women in the following groups: 1) women with a normal pregnancy that were classified in the following categories: a) women in the mid-trimester (14–18 weeks) who underwent amniocentesis for genetic indications and delivered normal infants at term (n=72); b) women at term not in labor (n=23); and c) those at term in labor (n=48); 2) women with spontaneous preterm labor and intact membranes that were subdivided into the following categories: a) preterm labor who delivered at term without IAI (n=42), b) preterm labor who delivered preterm without IAI (n=57), and c) preterm labor and delivery with IAI (n=30); and 3) women with preterm prelabor rupture of membranes (PROM) with (n=50) and without (n=54) IAI. Among patients with preterm labor with intact membranes and preterm PROM who delivered within 72 hours of amniocentesis, placenta, umbilical cord and chorioamniotic membranes were collected and assessed for the presence or absence of acute inflammatory lesions in the extra-placental membranes (histologic chorioamnionitis) and/or umbilical cords (funisitis). HSP70 concentrations in amniotic fluid were determined using a sensitive and specific immunoassay. Non-parametric statistics were used for analysis. A p value <0.05 was considered statistically
Walter, R J; Matsuda, T; Reyes, H M; Walter, J M; Hanumadass, M
The efficacy of acellular dermal matrix (ADM) in the treatment of full-thickness skin injuries as a dermal substitute depends on its low antigenicity, capacity for rapid vascularization, and stability as a dermal template. These properties will be determined largely by the final composition of the ADM. We have treated human skin with either Dispase followed by Triton X-100 detergent or NaCl followed by SDS detergent, cryosectioned the resulting ADMs, and then characterized them immunohistochemically. Staining for cell-associated antigens (HLA-ABC, HLA-DR, vimentin, desmin, talin), extracellular matrix components (chondroitin sulfate, fibronectin, laminin, vitronectin, hyaluronic acid), elastin, and collagen type VII was dramatically reduced or absent from ADMs prepared by both methods. However, significant amounts of elastin, keratan sulfate, laminin, and collagen types III and IV were still observed in both ADMs. Both methods of ADM preparation resulted in extensive extraction of both cellular and extracellular components of the skin but retention of the basic dermal architecture. In general, ADM prepared by the NaCl-SDS method retained larger amounts of each antigen than did that prepared by the Dispase-Triton method. This was most evident for laminin and type VII collagen but larger amounts of type IV collagen, fibronectin, desmin, elastin, and HLA-DR were also evident in the NaCl-SDS ADM.
Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman
Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.
Balaji, V. R.; Ramakrishnan, T.; Manikandan, D.; Lambodharan, R.; Karthikeyan, B.; Niazi, Thanvir Mohammed; Ulaganathan, G.
Aims and Objectives: Obtaining root coverage has become an important part of periodontal therapy. The aims of this studyare to evaluate the clinical efficacy of acellular dermal matrix graft in the coverage of denuded roots and also to examine the change in the width of keratinized gingiva. Materials and Methods: A total of 20 sites with more than or equal to 2 mm of recession depth were taken into the study, for treatment with acellular dermal matrix graft. The clinical parameters such as recession depth, recession width, width of keratinized gingiva, probing pocket depth (PD), and clinical attachment level (CAL) were measured at the baseline, 8th week, and at the end of the study (16th week). The defects were treated with a coronally positioned pedicle graft combined with acellular dermal matrix graft. Results: Out of 20 sites treated with acellular dermal matrix graft, seven sites showed complete root coverage (100%), and the mean root coverage obtained was 73.39%. There was a statistically significant reduction in recession depth, recession width, and probing PD. There was also a statistically significant increase in width of keratinized gingiva and also gain in CAL. The postoperative results were both clinically and statistically significant (P < 0.0001). Conclusion: The results of this study were esthetically acceptable to the patients and clinically acceptable in all cases. From this study, it may be concluded that acellular dermal matrix graft is an excellent substitute for autogenous graft in coverage of denuded roots. PMID:27829749
Zhang, Jing-Kun; Du, Run-Xuan; Zhang, Lin; Li, Ya-Nan; Zhang, Ming-le; Zhao, Shuo; Huang, Xiang-Hua; Xu, Yan-Fang
Acellular matrix materials have been widely used to repair various tissues and organs. According to the plastic principle, when a part of the body is lost, it should be replaced with a similar material. Therefore, the use of a homologous organ-speciﬁc acellular vaginal tissue in vagina reconstruction repair surgery may show good results. However, the acellular vagina matrix (AVM) form large vertebrates is difficult to isolate. In this study, we described a multi-step method to prepare porcine AVM and evaluated the efﬁcacy of acellularization. We also investigated the biomechanical properties, biological activity elements and biocompatibility of the porcine AVM. We then used this material to reconstruct a rat vagina and performed further morphologic and functional analyses. Small intestinal submucosa (SIS), which is a commonly used acellular matrix material, was used in a control group. Histological examination, DNA content analysis and agarose gel electrophoresis revealed that the decellularization procedure was effective. The AVM had acceptable biomechanical properties and sufficient growth factor production (VEGF, FGF, TGF-β1 and PDGF-BB) compared with that of the SIS. Subcutaneous transplantation in rats showed that the AVM had good biocompatibility. The tissue-engineered vagina using the AVM more resembled normal-appearing tissue than did that using SIS following morphologic and functional analyses. The AVM has great potential for application in vaginal reconstructive surgery. This article is protected by copyright. All rights reserved.
Vácz, Gabriella; Cselenyák, Attila; Cserép, Zsuzsanna; Benkő, Rita; Kovács, Endre; Pankotai, Eszter; Lindenmair, Andrea; Wolbank, Susanne; Schwarz, Charlotte M.; Horváthy, Dénes B.; Kiss, Levente; Hornyák, István; Lacza, Zsombor
Purpose Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. Results Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs’ transplanted vessels. Conclusion We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration. PMID:28180006
Cruciani, Laura; Romero, Roberto; Vaisbuch, Edi; Kusanovic, Juan Pedro; Chaiworapongsa, Tinnakorn; Mazaki-Tovi, Shali; Mittal, Pooja; Ogge, Giovanna; Gotsch, Francesca; Erez, Offer; Kim, Sun Kwon; Dong, Zhong; Pacora, Percy; Lamont, Ronald F.; Yeo, Lami; Hassan, Sonia S.; Di Renzo, Gian Carlo
Objective Pentraxin 3 (PTX3) is a soluble pattern recognition receptor that has an important role in immunoregulation and vascular integrity. The aim of this study was to determine if PTX3 is present in amniotic fluid (AF) and if its concentration changes with gestational age, in the presence of labor, and in cases of intra-amniotic infection/inflammation (IAI) associated with spontaneous preterm labor (PTL) or preterm prelabor rupture of membranes (PPROM). Study design This was a cross-sectional study which included the following groups: 1) mid-trimester (n=45); 2) uncomplicated pregnancies at term with (n=48) and without (n=40) spontaneous labor; 3) women with PTL and intact membranes: a) who delivered at term (n=44); b) who delivered preterm without IAI (n=40); and c) who delivered preterm with IAI (n=62); 4) women with PPROM with (n=63) and without (n=36) IAI. Pentraxin-3 concentration in AF was determined by ELISA. Non-parametric statistics were used for analyses. Results 1) Among women in preterm labor with intact membranes, the median AF PTX3 concentration was significantly higher in women with IAI than in those without IAI (7.95 ng/mL vs. 0.38 ng/mL; p<0.001) and than in those who delivered at term (0.55 ng/mL; p<0.001); 2) women with PPROM and IAI had a higher median amniotic fluid PTX3 concentration than those without IAI (9.12 ng/mL vs. 0.76 ng/mL; p<0.001); 3) the median AF PTX3 concentration did not change with gestational age (mid-trimester: 0.79 ng/mL vs. term not in labor: 0.58 ng/mL; p=0.09); and 4) among women at term, no significant differences were observed in the median AF PTX3 concentration between women with spontaneous labor and those not in labor (0.54 ng/mL vs. 0.58 ng/mL, respectively; p=0.9). Conclusions PTX3 is a physiologic constituent of the AF, and its concentration is elevated in the presence of IAI, suggesting that PTX3 may play a role in the innate immune response against intra-amniotic infection. PMID:19792835
Romero, Roberto; Miranda, Jezid; Chaiworapongsa, Tinnakorn; Chaemsaithong, Piya; Gotsch, Francesca; Dong, Zhong; Ahmed, Ahmed I.; Yoon, Bo Hyun; Hassan, Sonia; Kim, Chong J.; Korzeniewski, Steven J.; Yeo, Lami
Objective The major challenges in using amniotic fluid (AF) cultivation techniques to diagnose microbial invasion of the amniotic cavity (MIAC) are: 1) several days are typically required to obtain results, and 2) many organisms implicated in the pathogenesis of human disease are difficult to culture. Here, we compare the performance of AF culture with a novel technique for the diagnosis of MIAC that can provide results within eight hours by combining broad-range real-time polymerase chain reaction with electrospray ionization mass spectrometry (PCR/ESI-MS) to identify and quantify genomic material from bacteria and viruses in AF. Methods AF samples obtained by transabdominal amniocentesis from 142 women with preterm labor (PTL) and intact membranes were analyzed using cultivation techniques (aerobic, anaerobic and genital mycoplasmas) as well as PCR/ESI-MS. The prevalence and relative magnitude of intra-amniotic inflammation [AF Interleukin 6 (IL-6) concentration ≥ 2.6 ng/mL], acute histologic chorioamnionitis, spontaneous preterm delivery, and perinatal mortality were examined according to the results of these two tests. Results 1) The prevalence of MIAC in patients with preterm labor and intact membranes was 7% using standard cultivation techniques and 12% using PCR/ESI-MS; 2) seven of ten patients with positive AF culture also had positive PCR/ESI-MS [≥17 genome equivalents per PCR reaction well (GE/well)] 3) patients with positive PCR/ESI-MS (≥17 GE/well) and negative AF cultures had significantly higher rates of intra-amniotic inflammation and histologic acute chorioamnionitis, shorter intervals to delivery [median (interquartile range-IQR)], and offspring at higher risk of perinatal mortality, than women with both tests negative [90% (9/10) vs. 32% (39/122); (p<0.001); 70% (7/10) vs. 35% (39/112); (p=0.04); 1 (IQR: <1 – 2) days vs. 25 (IQR: 5 – 51) days; (p=0.002); OR: 5.6; 95% CI: 1.4 – 22, respectively]; 5) there were no significant differences
Lee, Soong Deok; Kim, Mi Ran; Hwang, Pil Gyu; Shim, Soon-Sup; Yoon, Bo Hyun; Kim, Chong Jai
The present study was conducted to investigate the potential anatomical source of amniotic fluid neutrophils. Microdissection of neutrophils from the chorioamnion of the fetal membranes and the amnion of the chorionic plates of 10 preterm placentas with acute chorioamnionitis was performed and the genotypes of the neutrophils were compared with those of the mother and fetus using polymerase chain reaction of nine autosomal STR loci. In separate analyses, we reviewed eight cases of fetal autopsies with increased amniotic fluid neutrophils for the presence of neutrophils in the alveoli, and also analyzed the relationship between the amniotic fluid white blood cell (WBC) count and the histological pattern of placental inflammation. The genotypes of all of the neutrophils found in the chorioamnion of the fetal membrane matched those of the mother (n = 10). The genotypes of neutrophils found in the chorionic plate were of mixed maternal and fetal origin (n = 4). In the autopsy series of the fetuses with amniotic fluid WBC (n = 8), only five cases showed neutrophils in the alveolar space, while all the placentas had chorioamnionitis. There was no significant difference in amniotic fluid WBC count between the cases with or without acute membranitis, while among the cases with placental inflammation, those with inflammation of the chorionic plate had a significantly higher amniotic fluid WBC count than both the membranitis-only cases (P < 0.001) and the membranitis and funisitis cases (P < 0.05). These results imply that fetal vasculature at the chorionic plate is the main source of amniotic fluid neutrophils, especially in the cases without funisitis.
Hegerle, N; Dore, G; Guiso, N
Bordetella pertussis is the etiologic agent of whooping cough and has been the target of vaccination for over fifty years. The latest strategies include the use of acellular pertussis vaccines that induce specific immunity against few virulence factors amongst which pertactin is included in three and five component acellular pertussis vaccines. Recently, it has been reported that B. pertussis clinical isolates loose the production of this adhesin in regions reaching high vaccine coverage with vaccines targeting this virulence factor. We here demonstrate that isolates not producing pertactin are capable of sustaining longer infection as compared to pertactin producing isolates in an in vivo model of acellular pertussis immunization. Loosing pertactin production might thus provide a selective advantage to these isolates in this background, which could account for the upraise in prevalence of these pertactin deficient isolates in the population.
Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana
In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.
Peakman, D C; Moreton, M F; Corn, B J; Robinson, A
Over the past 6 years, using in situ processing methods, we have identified 32 cases of mosaicism in amniotic fluid cell cultures prepared from 1,100 samples. Two of these (45,X/46,XX and 46,XX/47,XX, + 21) were called true mosaics because multiple colonies demonstrated the same abnormal chromosome complement, and on subsequent evaluation of the newborn blood or fetal tissues, mosaicism was confirmed. Of the remaining cases, 29 were designated as pseudomosaics because only single or partial colonies exhibited an aberrant chromosome complement, 12 having a trisomy 2 line. In the final case, a double trisomy was demonstrated in only one of eight colonies in the first culture, but in the culture from a repeat sample an additional two colonies showed the same double trisomy. Since no abnormal cells were observed in infant blood, it was postulated that the mosaicism may only have been present in the extraembryonic tissues. It is our conviction that the use of these cloning methods should diminish the danger of misdiagnosis in genetic amniocentesis. PMID:453199
Anderson, S B; de Souza, R Ferreira; Hofmann-Rummelt, C; Seitz, B
Background/aims: Amniotic membrane transplantation (AMT) has become well established as a treatment for chronic epithelial defects, conjunctival reconstruction, and partial limbal cell deficiency. The aim of this study was to describe cases of corneal calcification following AMT and to search for risk factors that might predispose to this unusual finding. Methods: Details of 117 AMTs on 93 corneas of 91 patients with a follow up period of at least 1 month performed since 1999 were collected prospectively. In those with calcification clinical photographs were studied and the medical records retrospectively examined. Results: 15 calcifications in 117 AMTs (12.8%) were identified, occurring 3–17 (median 6.1) weeks after AMT, during a follow up period of 4–151 (median 25) weeks. Overall epithelial healing rate was 83%. Calcification covered a surface area between 0.7–40.5 mm2 maximum size with varied morphology. The primary diagnosis was diverse. Risk factors included the use of phosphate eye drops and pre-existing calcification in the operative or other eye. No patient with a “patch” AMT developed calcification. Conclusions: Corneal calcification occurs after some cases of AMT. A common risk factor was the postoperative use of phosphate containing eye drops. PMID:12714401
Lai, Po-Hong; Chang, Yen; Chen, Sung-Ching; Wang, Chung-Chi; Liang, Huang-Chien; Chang, Wei-Chun; Sung, Hsing-Wen
It was found in our previous study that acellular tissues derived from bovine pericardia consist primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans (GAGs). It is speculated that the inherent GAGs in acellular tissues may serve as a reservoir for loading basic fibroblast growth factor (bFGF) and promote angiogenesis and tissue regeneration. This study was therefore designed to investigate effects of the content of GAGs in acellular bovine pericardia on the binding of bFGF and its release profile in vitro while its stimulation in angiogenesis and tissue regeneration in vivo were evaluated subcutaneously in a rat model. To control the content of GAGs, acellular tissues were treated additionally with hyaluronidase for 1 (Hase-D1), 3 (Hase-D3), or 5 days (Hase-D5). The in vitro results indicated that a higher content of GAGs in the acellular tissue resulted in an increase in bFGF binding and in a more gradual and sustained release of the growth factor. The in vivo results obtained at 1 week postoperatively showed that the density and the depth of neo-vessels infiltrated into the acellular tissue loaded with bFGF (acellular/bFGF) were significantly greater than the other test samples. At 1 month postoperatively, vascularized neo-connective tissues were found to fill the pores within each test sample, particularly for the acellular/bFGF tissue. These results suggested that the sustained release of bFGF from the acellular/ bFGF tissue continued to be effective in enhancing angiogenesis and generation of new tissues. In conclusion, the inherent GAGs present in acellular tissues may be used for binding and sustained release of bFGF to enhance angiogenesis and tissue regeneration.
Horst, Maya; Milleret, Vincent; Noetzli, Sarah; Gobet, Rita; Sulser, Tullio; Eberli, Daniel
Poly(lactic-co-glycolic acid) (PLGA) based biomaterials for soft tissue engineering have inherent disadvantages, such as a relative rigidity and a limited variability in the mechanical properties and degradation rates. In this study, a novel electrospun biomaterial based on degradable polyesterurethane (PEU) (DegraPol(®) ) was investigated for potential use for bladder engineering in vitro and in vivo. Hybrid microfibrous PEU and PLGA scaffolds were produced by direct electrospinning of the polymer onto a bladder acellular matrix. The scaffold morphology of the scaffold was analyzed, and the biological performance was tested in vitro and in vivo using a rat cystoplasty model. Anatomical and functional outcomes after implantation were analyzed macroscopically, histologically and by cystometry, respectively. Scanning electron microscopy analysis showed that PEU samples had a lower porosity (p < 0.001) and were slightly thinner (p = 0.009) than the PGLA samples. Proliferation and survival of the seeded smooth muscle cells in vitro were comparable on PEU and PLGA scaffolds. After 8 weeks in vivo, the PEU scaffolds exhibited no shrinkage. However, cystometry of the reconstructed bladders exhibited a slightly greater functional bladder capacity in the PLGA group. Morphometric analyses revealed significantly better tissue healing (p < 0.05) and, in particular, better smooth muscle regeneration, as well as a lower rate of inflammatory responses at 8 weeks in the PEU group. Collectively, the results indicated that PEU-hybrid scaffolds promote bladder tissue formation with excellent tissue integration and a low inflammatory reaction in vivo. PEU is a promising biomaterial, particularly with regard to functional tissue engineering of the bladder and other hollow organs. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 658-667, 2017.
Loukogeorgakis, Stavros P; De Coppi, Paolo
Regenerative medicine has recently been established as an emerging field focussing on repair, replacement or regeneration of cells, tissues and whole organs. The significant recent advances in the field have intensified the search for novel sources of stem cells with potential for therapy. Recently, researchers have identified the amniotic fluid as an untapped source of stem cells that are multipotent, possess immunomodulatory properties and do not have the ethical and legal limitations of embryonic stem cells. Stem cells from the amniotic fluid have been shown to differentiate into cell lineages representing all three embryonic germ layers without generating tumours, which make them an ideal candidate for tissue engineering applications. In addition, their ability to engraft in injured organs and modulate immune and repair responses of host tissues suggest that transplantation of such cells may be useful for the treatment of various degenerative and inflammatory diseases affecting major tissues/organs. This review summarises the evidence on amniotic fluid cells over the past 15 years and explores the potential therapeutic applications of amniotic fluid stem cells and amniotic fluid mesenchymal stem cells.
Valdetaro, Gisele P.; Aldrovani, Marcela; Padua, Ivan R. M.; Cristovam, Priscila C.; Gomes, José A. P.; Laus, José L.
In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure–induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties. PMID:28018719
Sukumar, Neelima; Sloan, Gina Parise; Conover, Matt S.; Love, Cheraton F.; Mattoo, Seema; Kock, Nancy D.; Deora, Rajendar
The Bordetella species are Gram-negative bacterial pathogens that are characterized by long-term colonization of the mammalian respiratory tract and are causative agents of respiratory diseases in humans and animals. Despite widespread and efficient vaccination, there has been a world-wide resurgence of pertussis, which remains the leading cause of vaccine-preventable death in developed countries. It has been proposed that current acellular vaccines (Pa) composed of only a few bacterial proteins may be less efficacious because of vaccine-induced antigenic shifts and adaptations. To gain insight into the development of a newer generation of vaccines, we constructed a Bordetella bronchiseptica strain (LPaV) that does not express the antigenic homologs included in any of the Pa vaccines currently in use. This strain also lacks adenylate cyclase toxin, an essential virulence factor, and BipA, a surface protein. While LPaV colonized the mouse nose as efficiently as the wild-type strain, it was highly deficient in colonization of the lower respiratory tract and was attenuated in induction of inflammation and injury to the lungs. Strikingly, to our surprise, we found that in an intranasal murine challenge model, LPaV elicited cross-species protection against both B. bronchiseptica and Bordetella pertussis. Our data suggest the presence of immunogenic protective components other than those included in the pertussis vaccine. Combined with the whole-genome sequences of many Bordetella spp. that are available, the results of this study should serve as a platform for strategic development of the next generation of acellular pertussis vaccines. PMID:20176797
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lecithin/sphingomyelin ratio in amniotic fluid... Clinical Chemistry Test Systems § 862.1455 Lecithin/sphingomyelin ratio in amniotic fluid test system. (a) Identification. A lecithin/sphingomyelin ratio in amniotic fluid test system is a device intended to measure...
... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Lecithin/sphingomyelin ratio in amniotic fluid... Clinical Chemistry Test Systems § 862.1455 Lecithin/sphingomyelin ratio in amniotic fluid test system. (a) Identification. A lecithin/sphingomyelin ratio in amniotic fluid test system is a device intended to measure...
... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lecithin/sphingomyelin ratio in amniotic fluid... Clinical Chemistry Test Systems § 862.1455 Lecithin/sphingomyelin ratio in amniotic fluid test system. (a) Identification. A lecithin/sphingomyelin ratio in amniotic fluid test system is a device intended to measure...
... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Lecithin/sphingomyelin ratio in amniotic fluid... Clinical Chemistry Test Systems § 862.1455 Lecithin/sphingomyelin ratio in amniotic fluid test system. (a) Identification. A lecithin/sphingomyelin ratio in amniotic fluid test system is a device intended to measure...
... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lecithin/sphingomyelin ratio in amniotic fluid... Clinical Chemistry Test Systems § 862.1455 Lecithin/sphingomyelin ratio in amniotic fluid test system. (a) Identification. A lecithin/sphingomyelin ratio in amniotic fluid test system is a device intended to measure...
Dubil, Elizabeth A; Magann, Everett F
Introduction: Amniotic fluid, once thought to merely provide protection and room for necessary movement and growth for the fetus, is now understood to be a highly complex and dynamic system that is studied as a data point to interpret fetal wellbeing. Methods: Assessment of amniotic fluid volume is now routine when performing a sonographic evaluation of fetal status and is an important consideration in the assessment and management of perinatal morbidity and mortality.(1)(,)(2) In this review, we will cover the dynamics that affect amniotic fluid volume, review methods for measurement and quantification of volume, review definitions for normative data as related to neonatal outcomes, and provide evidence based guidance on the workup and management options for oligoydramnios and polyhydramnios in singleton and twin pregnancies. Conclusions: When abnormalities of fluid exist, appropriate workup to uncover the underlying etiology should be initiated as adverse fetal outcomes are sometimes associated with these variations from normalcy.
Gholizadeh-Ghaleh Aziz, Shiva; Fathi, Ezzatollah; Rahmati-Yamchi, Mohammad; Akbarzadeh, Abolfazl; Fardyazar, Zahra; Pashaiasl, Maryam
Recent studies have elucidated that cell-based therapies are promising for cancer treatments. The human amniotic fluid stem (AFS) cells are advantageous cells for such therapeutic schemes that can be innately changed to express therapeutic proteins. HAFSCs display a natural tropism to cancer cells in vivo. They can be useful in cancer cells targeting. Moreover, they are easily available from surplus diagnostic samples during pregnancy and less ethical and legal concern are associated with the collection and application than other putative cells are subjected. This review will designate representatives of amniotic fluid and stem cell derived from amniotic fluid. For this propose, we collect state of human AFS cells data applicable in cancer therapy by dividing this approach into two main classes (nonengineered and engineered based approaches). Our study shows the advantage of AFS cells over other putative cells types in terms differentiation ability to a wide range of cells by potential and effective use in preclinical studies for a variety of diseases. This study has shown the elasticity of human AFS cells and their favorable potential as a multipotent cell source for regenerative stem cell therapy and capable of giving rise to multiple lineages including such as osteoblasts and adipocyte.
Summary Stem cell-based therapies hold the potential to alleviate the burden of many serious diseases, including those of the liver. Among the different types of stem cells, human placenta-derived stem cells are potentially one of the most clinically applicable stem cells because of their tissue-specific advantages. They are a readily available cell source that can be procured in a noninvasive manner, and there are few ethical concerns regarding their use. Recent studies have demonstrated that the amniotic epithelium contains stem cells that possess four unique and advantageous properties; human amniotic epithelial cells (hAECs) have low immunogenicity, secrete several immune regulatory molecules, possess the potential to differentiate into all three germ layers, and contain abundant lysosomes allowing them to secrete lysosomal enzymes. This perspective article provides an overview of the beneficial properties of hAECs and proposes a rational strategy for translating placental stem cells toward clinical application for various liver diseases. Significance This article provides an overview of the beneficial properties of one type of human placental stem cell and proposes a rational strategy for translating placental stem cells toward clinical application for various liver diseases. PMID:26941361
Albergotti, Lori C.; Hamlin, Heather J.; McCoy, Michael W.; Guillette,, Louis J.
Background During development, all amniotes (mammals, reptiles, and birds) form extraembryonic membranes, which regulate gas and water exchange, remove metabolic wastes, provide shock absorption, and transfer maternally derived nutrients. In viviparous (live-bearing) amniotes, both extraembryonic membranes and maternal uterine tissues contribute to the placenta, an endocrine organ that synthesizes, transports, and metabolizes hormones essential for development. Historically, endocrine properties of the placenta have been viewed as an innovation of placental amniotes. However, an endocrine role of extraembryonic membranes has not been investigated in oviparous (egg-laying) amniotes despite similarities in their basic structure, function, and shared evolutionary ancestry. In this study, we ask whether the oviparous chorioallantoic membrane (CAM) of chicken (Gallus gallus) has the capability to synthesize and receive signaling of progesterone, a major placental steroid hormone. Methodology/Principal Findings We quantified mRNA expression of key steroidogenic enzymes involved in progesterone synthesis and found that 3β-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone exhibited a 464 fold increase in the CAM from day 8 to day 18 of embryonic development (F5, 68 = 89.282, p<0.0001). To further investigate progesterone synthesis, we performed explant culture and found that the CAM synthesizes progesterone in vitro in the presence of a steroid precursor. Finally, we quantified mRNA expression and performed protein immunolocalization of the progesterone receptor in the CAM. Conclusions/Significance Collectively, our data indicate that the chick CAM is steroidogenic and has the capability to both synthesize progesterone and receive progesterone signaling. These findings represent a paradigm shift in evolutionary reproductive biology by suggesting that endocrine activity of extraembryonic membranes is not a novel characteristic of placental
Stehr, K; Heininger, U; Uhlenbusch, R; Angersbach, P; Hackell, J; Eckhardt, T
Immunization against pertussis has been re-recommended for healthy children in Germany in 1991. In addition the former restriction of immunizing only in the first 2 years of life was abolished. In children born before 1991 immunization rates against pertussis were 15% or less. With the new recommendations physicians are now faced with an increasing demand of parents for catch-up vaccinations in these children. Since they were immunized against diphtheria and tetanus previously monovalent pertussis vaccines are needed for this indication. Therefore a monovalent, multicomponent acellular pertussis vaccine was studied in 249 German children 15 months to 6 years of age. Three doses were administered at 6-10 week intervals. Reactogenicity and antibody responses against the vaccine antigens pertussis toxin (PT), filamentous haemagglutinin (FHA), 69-kd antigen (pertactin) and fimbriae-2 (agglutinogen) were investigated. Local and systemic reactions were minimal in frequency and severity. Antibody responses against all vaccine antigens were pronounced with 93%-100% of vaccinees demonstrating at least four fold titre rises above pre-immunization after the third dose. These findings indicate that this monovalent, multicomponent acellular pertussis vaccine with excellent immunogenicity and low reactogenicity is an appropriate candidate for closing immunization gaps in older children in countries with previously low vaccination rates against pertussis. Based on the results of this study the monovalent acellular pertussis vaccine was licensed in Germany in January 1994.
Sandlin, Adam T; Chauhan, Suneet P; Magann, Everett F
Polyhydramnios is an excessive amount of amniotic fluid within the amniotic cavity. The etiology of polyhydramnios may be idiopathic, the consequence of fetal structural anomalies, or the consequence of various fetal and maternal conditions. The clinical importance of polyhydramnios is found in its association with adverse pregnancy outcomes and the risk of perinatal mortality. The antenatal management of polyhydramnios can be challenging as there are no formalized guidelines on the topic. The purpose of this review is to provide a literature-based overview on the subject of polyhydramnios in singleton pregnancies, demonstrate its clinical implications, and describe a practical approach to its management.
Stower, Matthew J; Bertocchini, Federica
In the animal kingdom, gastrulation, the process by which the primary germ layers are formed involves a dramatic transformation in the topology of the cells that give rise to all of the tissues of the adult. Initially formed as a mono-layer, this tissue, the epiblast, becomes subdivided through the internalization of cells, thereby forming a two (bi-laminar) or three (tri-laminar) layered embryo. This morphogenetic process coordinates the development of the fundamental body plan and the three-body axes (antero-posterior, dorso-ventral, and left-right) and begins a fundamental segregation of cells toward divergent developmental fates. In humans and other mammals, as well as in avians, gastrulating cells internalize along a structure, called the primitive streak, which builds from the periphery toward the center of the embryo. How these morphogenetic movements are orchestrated and evolved has been a question for developmental biologists for many years. Is the primitive streak a feature shared by the whole amniote clade? Insights from reptiles suggest that the primitive streak arose independently in mammals and avians, while the reptilian internalization site is a structure half-way between an amphibian blastopore and a primitive streak. The molecular machinery driving primitive streak formation has been partially dissected using mainly the avian embryo, revealing a paramount role of the planar cell polarity (PCP) pathway in streak formation. How did the employment of this machinery evolve? The reptilian branch of the amniote clade might provide us with useful tools to investigate the evolution of the amniote internalization site up to the formation of the primitive streak. WIREs Dev Biol 2017, 6:e262. doi: 10.1002/wdev.262 For further resources related to this article, please visit the WIREs website.
Shaw, S W Steven; Bollini, Sveva; Nader, Khalil Abi; Gastaldello, Annalisa; Gastadello, Annalisa; Mehta, Vedanta; Filppi, Elisa; Cananzi, Mara; Gaspar, H Bobby; Qasim, Waseem; De Coppi, Paolo; David, Anna L
Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep (n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital disease.
Korossis, Sotirios A.; Wilshaw, Stacy-Paul; Jennings, Louise M; Fisher, John; Ingham, Eileen
Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Immunohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrodynamic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of α-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure. PMID:24786313
Melo, Esther; Garreta, Elena; Luque, Tomas; Cortiella, Joaquin; Nichols, Joan; Navajas, Daniel; Farré, Ramon
Lung bioengineering, a novel approach to obtain organs potentially available for transplantation, is based on decellularizing donor lungs and seeding natural scaffolds with stem cells. Various physicochemical protocols have been used to decellularize lungs, and their performance has been evaluated in terms of efficient decellularization and matrix preservation. No data are available, however, on the effect of different decellularization procedures on the local stiffness of the acellular lung. This information is important since stem cells directly sense the rigidity of the local site they are engrafting to during recellularization, and it has been shown that substrate stiffness modulates cell fate into different phenotypes. The aim of this study was to assess the effects of the decellularization procedure on the inhomogeneous local stiffness of the acellular lung on five different sites: alveolar septa, alveolar junctions, pleura, and vessels' tunica intima and tunica adventitia. Local matrix stiffness was measured by computing Young's modulus with atomic force microscopy after decellularizing the lungs of 36 healthy rats (Sprague-Dawley, male, 250-300 g) with four different protocols with/without perfusion through the lung circulatory system and using two different detergents (sodium dodecyl sulfate [SDS] and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [CHAPS]). The local stiffness of the acellular lung matrix significantly depended on the site within the matrix (p<0.001), ranging from ∼ 15 kPa at the alveolar septum to ∼ 60 kPa at the tunica intima. Acellular lung stiffness (p=0.003) depended significantly, albeit modestly, on the decellularization process. Whereas perfusion did not induce any significant differences in stiffness, the use of CHAPS resulted in a ∼ 35% reduction compared with SDS, the influence of the detergent being more important in the tunica intima. In conclusion, lung matrix stiffness is considerably inhomogeneous, and
Chen, X.L.; Xia, Z.F.; Fang, L.S.; Wang, Y.J.; Wang, C.H.
Summary A 6-yr-old boy was the victim of a burns accident in a public bathhouse. The burns involved the face, neck, upper and lower extremities, anterior and posterior trunk, and both buttocks, covering 72% of the total body surface area (TBSA). The lesions in the lower extremities and parts of the right upper extremity were deep partial-thickness, comprising 40% TBSA. On day 5 post-burn, the lesions in both lower extremities were excised to the extent of the fascia under general anaesthesia. Meshed J1 Jayya Acellular Dermis®, a kind of acellular allodermal (ADM) matrix, was then placed on the left knee joint. The right knee joint served as control. The wounds in both lower extremities were then overlaid with microskin autografting. At 19 days post-application, the lesions in both lower extremities had almost completely resurfaced. Follow-up at six months revealed well-healed and stable skin of acellular ADM and microskin autografts on the left knee. However, the skin of the right knee was unstable and there was a chronic residual ulcer. Both legs showed some significant hypertrophic scars. The left knee joint (acellular ADM grafted site) showed mild contractures, while the right knee joint developed a significant contracture. The "skin" of the co-graft covered site appeared thicker and more elastic. The movement range of the left knee joint was much larger than that of the right knee joint. These results suggest that co-graft of acellular dermal matrix and autogenous microskin may be an effective way to repair this functional site in children with extensive burns and to improve the functional and cosmetic results. PMID:21991120
Uno, Yoshinobu; Nishida, Chizuko; Tarui, Hiroshi; Ishishita, Satoshi; Takagi, Chiyo; Nishimura, Osamu; Ishijima, Junko; Ota, Hidetoshi; Kosaka, Ayumi; Matsubara, Kazumi; Murakami, Yasunori; Kuratani, Shigeru; Ueno, Naoto; Agata, Kiyokazu; Matsuda, Yoichi
Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal
505. 2. Ma J, Smith BP, Smith TL, Walker FO, Rosencrance E, Koman LA. Juvenile and adult rat neuromuscular junctions: density, distribution, and...occur in neuromuscular junctions following delayed nerve injury/repair will be studied. If successful, the potential for the denervated muscle to...2002, 2007). In addition, axon morphology will be assessed and compared between treatment groups. Analysis of neuromuscular junction (NMJ) density
Wang, Feng; Guan, Xin; Wu, TianYi; Qiao, JianOu; Han, ZhaoQing; Wu, JinLong; Yu, XiaoWei; You, QingJun
We aimed to investigate whether acellular endocardium can be used as a useful biomaterial for the intima of engineered small-caliber vascular grafts. Fresh endocardium was harvested from the swine left atrium and was decellularized by digestion with the decellularization solution of Triton X-100 and SDS containing DNase I and RNase A. Surface morphological characteristics and Young's modulus were evaluated. To analyze the effect of mechanical characteristics on cell adhesion, the decellularized endocardium was stiffened with 2.5% glutaraldehyde. Small-caliber vascular grafts were constructed using decellularized endocardium treated with or without glutaraldehyde as the intima. CD34+ cells were seeded onto the luminal surface of the vascular grafts and linked to bioreactors that simulate a pulsatile blood stream. Acellular endocardium had distinct surface morphological characteristics, which were quite different from those of other materials. The compliance of acellular endocardium was higher than that of other materials tested by Young's modulus. CD34+ cells formed a monolayer structure and adhered to the inner face of the acellular endocardium. The glutaraldehyde treatment stiffened the acellular endocardium but had little impact on the surface morphological characteristics or static adhesiveness of the cells. Data from the bioreactor study showed that the detachment of the cells from the surface of glutaraldehyde-treated acellular endocardium increased dramatically when the pressure was equal or higher than 40 mm Hg, while the cells on the untreated acellular endocardium remained well and formed confluent monolayers and tight junctions under the same pressure. Acellular endocardium has distinct structures and mechanical characteristics that are beneficial for CD34+ cell adhesion and retention under dynamic fluid perfusion. Thus, it can be used as a useful biomaterial for the construction of the intima of engineered small-caliber vascular grafts.
Lim, Seok-Byung; Hong, Seung-Mo; Yu, Chang Sik; Hong, Yong Sang; Kim, Tae Won; Park, Jin-hong; Kim, Jong Hoon; Kim, Jin Cheon
Occasionally, patients with locally advanced rectal adenocarcinoma who receive preoperative chemoradiotherapy (CRT) show acellular mucin in resection specimens that had shown pathologic complete response (pCR), but the clinical and prognostic significance of this finding has been controversial. This study analyzed data from 217 consecutive patients showing pCR to preoperative CRT followed by resection to evaluate the clinicopathologic features and prognostic significance of acellular mucin. Patients were categorized according to the presence of acellular mucin, as identified by pathologic analysis. The clinicopathologic findings and oncologic results were compared. Acellular mucins were identified in 35 (16.1%) of 217 pCR patients. Acellular mucins were found predominantly in male patients (20.8% vs. 9.8%, P=0.039) and in those with mucinous/signet ring cell differentiation (66.7% vs. 15.1%, P=0.008). The presence of acellular mucin was more frequent in patients with a shorter (<42 d) CRT-operation interval (22.6% vs. 10.3%, P=0.017). With a mean follow-up of 41 months (range, 2 to 119 mo), the 3-year overall survival (96.8% with mucin vs. 95.9% without mucin, P=0.314) and the 3-year disease-free survival (97.0% with mucin vs. 93.0% without mucin, P=0.131) did not differ between the groups. The presence of acellular mucin in rectal cancer patients showing pCR to preoperative CRT is associated with male sex and mucinous differentiation and does not have a significant impact on oncologic outcomes. Acellular mucins are also associated with the CRT-operation interval as a phenomenon of time-dependent response to CRT.
Kramer, Boris W; Kallapur, Suhas G; Moss, Timothy J; Nitsos, Ilias; Newnham, John P; Jobe, Alan H
Epidemiological studies suggest that intra-uterine exposure to inflammation may prime postnatal immune responses. In fetal sheep, intra-amniotic injection of lipopolysaccharide (LPS) induced chorioamnionitis, lung inflammation and maturation, matured lung monocytes to macrophages and initiated systemic tolerance of fetal monocytes to subsequent challenge with LPS. We hypothesized that LPS-mediated chorioamnionitis altered the response of lung and blood monocytes to Toll-like receptor (TLR) ligands such as PamCysK4 (TLR2), flagellin (TLR5), and human CpG-DNA (TLR9). Time-mated ewes were given intra-amniotic injections of LPS or saline. Blood and lung monocytes were assessed after 2 days, 7 days and 2 days and 7 days repetitive LPS injections before delivery at 124 days gestational age (term 150 days). Responsiveness of blood and lung monocytes to TLR-ligands in vitro was assessed by interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and hydrogen peroxide. Monocytes from preterm controls had minimal responses. Lipopolysaccharide-mediated chorioamnionitis increased IL-6, TNF- alpha and hydrogen peroxide to all TLR agonists in blood and lung monocytes. Repetitive exposure to antenatal LPS reduced IL-6, TNF- alpha and hydrogen peroxide to TLR-ligands suggesting tolerance. Tolerance to TLR-ligands reduced IL-1 receptor associated kinase-4 expression. Thus, repeated fetal exposure to LPS induced tolerance to other TLR-ligands. These modulations of fetal innate immunity have implications for host defense and injury responses in preterm infants.
Hamid, Adila A; Joharry, Muhammad Khair; Mun-Fun, Hoo; Hamzah, Siti Nurusaadah; Rejali, Zulida; Yazid, Mohd Nazri; Karuppiah, Thilakavathy; Nordin, Norshariza
Amniotic fluid (AF) is now known to harbor highly potent stem cells, making it an excellent source for cell therapy. However, most of the stem cells isolated are from AF of mid-term pregnancies in which the collection procedure involves an invasive technique termed amniocentesis. This has limited the access in getting the fluid as the technique imposes certain level of risks to the mother as well as to the fetus. Alternatively, getting AF from full-term pregnancies or during deliveries would be a better resolution. Unfortunately, very few studies have isolated stem cells from AF at this stage of gestation, the fluid that is merely discarded. The question remains whether full-term AF harbors stem cells of similar potency as of the stem cells of mid-term AF. Here, we aim to review the prospect of having this type of stem cells by first looking at the origin and contents of AF particularly during different gestation period. We will then discuss the possibility that the AF, at full term, contains a population of highly potent stem cells. These stem cells are distinct from, and probably more potent than the AF mesenchymal stem cells (AF-MSCs) isolated from full-term AF. By comparing the studies on stem cells isolated from mid-term versus full-term AF from various species, we intend to address the prospect of having highly potent amniotic fluid stem cells from AF of full-term pregnancies in human and animals.
Boldenow, Erica; Gendrin, Claire; Ngo, Lisa; Bierle, Craig; Vornhagen, Jay; Coleman, Michelle; Merillat, Sean; Armistead, Blair; Whidbey, Christopher; Alishetti, Varchita; Santana-Ufret, Veronica; Ogle, Jason; Gough, Michael; Srinouanprachanh, Sengkeo; MacDonald, James W; Bammler, Theo K; Bansal, Aasthaa; Liggitt, H. Denny; Rajagopal, Lakshmi; Waldorf, Kristina M Adams
Preterm birth is a leading cause of neonatal morbidity and mortality. Although microbial invasion of the amniotic cavity (MIAC) is associated with the majority of early preterm births, the temporal events that occur during MIAC and preterm labor are not known. Group B Streptococci (GBS) are β-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur during MIAC, we utilized a unique chronically catheterized nonhuman primate model that closely emulates human pregnancy. This model allows monitoring of uterine contractions, timing of MIAC and immune responses during pregnancy-associated infections. Here, we show that adverse outcomes such as preterm labor, MIAC, and fetal sepsis were observed more frequently during infection with hemolytic GBS when compared to nonhemolytic GBS. Although MIAC was associated with systematic progression in chorioamnionitis beginning with chorionic vasculitis and progressing to neutrophilic infiltration, the ability of the GBS hemolytic pigment toxin to induce neutrophil cell death and subvert killing by neutrophil extracellular traps (NETs) in placental membranes in vivo facilitated MIAC and fetal injury. Furthermore, compared to maternal neutrophils, fetal neutrophils exhibit decreased neutrophil elastase activity and impaired phagocytic functions to GBS. Collectively, our studies demonstrate how a unique bacterial hemolytic lipid toxin enables GBS to circumvent neutrophils and NETs in placental membranes to induce fetal injury and preterm labor. PMID:27819066
Li, YongNan; Guo, Longzhe; Ahn, Hyun Sook; Kim, Moo Hyun; Kim, Sung-Whan
Recently, we reported that human amniotic membrane-derived mesenchymal stem cells (AMMs) possess great angiogenic potential. In this study, we determined whether local injection of AMMs ameliorates peripheral neuropathy. AMMs were transplanted into injured sciatic nerves. AMM injection promoted significant recovery of motor nerve conduction velocity and voltage amplitude compared to human adipose-derived mesenchymal stem cells. AMM implantation also augmented blood perfusion and increased intraneural vascularity. Whole-mount fluorescent imaging analysis demonstrated that AMMs exhibited higher engraftment and endothelial incorporation abilities in the sciatic nerve. In addition, the higher expression of pro-angiogenic factors was detected in AMMs injected into the peripheral nerve. Therefore, these data provide novel therapeutic and mechanistic insights into stem cell biology, and AMM transplantation may represent an alternative therapeutic option for treating peripheral neuropathy.
Guida, Maurizio; Guida, Marco; De Felice, Bruna; Santafede, Daniela; D'Alessandro, Raffaella; Di Spiezio Sardo, Attilio; Scognamiglio, Marianna; Ferrara, Cinzia; Bifulco, Giuseppe; Nappi, Carmine
The aim of this study was to assess the genotoxic effects of environmental chemicals on residents living near landfills. The study was based on samples of amniotic fluid from women living in the intensely polluted areas around the Campania region of Italy compared to a nonexposed control group. We evaluated the genetic effects that this amniotic fluids collected in contaminated sites had on Paracentrotus lividus embryos. DNA damage was detected through changes in RAPD (Random Amplified Polymorphism DNA) profiles. The absence of the amplified DNA fragments indicated deletions in Paracentrotus lividus DNA exposed to the contaminated amniotic fluids when compared to equal exposure to uncontaminated fluids. These results show the ability of RAPD-PCR to detect and isolate DNA sequences representing genetic alterations induced in P. lividus embryos. Using this method, we identified two candidate target regions for DNA alterations in the genome of P. lividus. Our research indicates that RAPD-PCR in P. lividus embryo DNA can provide a molecular approach for studying DNA damage from pollutants that can impact human health. To our knowledge, this is the first time that assessment of DNA damage in P. lividus embryos has been tested using the RAPD strategy after exposure to amniotic fluid from residents near waste landfill sites.
Guida, Maurizio; Guida, Marco; De Felice, Bruna; Santafede, Daniela; D'Alessandro, Raffaella; Di Spiezio Sardo, Attilio; Scognamiglio, Marianna; Ferrara, Cinzia; Bifulco, Giuseppe; Nappi, Carmine
The aim of this study was to assess the genotoxic effects of environmental chemicals on residents living near landfills. The study was based on samples of amniotic fluid from women living in the intensely polluted areas around the Campania region of Italy compared to a nonexposed control group. We evaluated the genetic effects that this amniotic fluids collected in contaminated sites had on Paracentrotus lividus embryos. DNA damage was detected through changes in RAPD (Random Amplified Polymorphism DNA) profiles. The absence of the amplified DNA fragments indicated deletions in Paracentrotus lividus DNA exposed to the contaminated amniotic fluids when compared to equal exposure to uncontaminated fluids. These results show the ability of RAPD-PCR to detect and isolate DNA sequences representing genetic alterations induced in P. lividus embryos. Using this method, we identified two candidate target regions for DNA alterations in the genome of P. lividus. Our research indicates that RAPD-PCR in P. lividus embryo DNA can provide a molecular approach for studying DNA damage from pollutants that can impact human health. To our knowledge, this is the first time that assessment of DNA damage in P. lividus embryos has been tested using the RAPD strategy after exposure to amniotic fluid from residents near waste landfill sites. PMID:20706694
Hartmann, K; Raabe, O; Wenisch, S; Arnhold, S
Amniotic fluid contains heterogeneous cell types and has become an interesting source for obtaining fetal stem cells. These stem cells have a high proliferative capacity and a good differentiation potential and may thus be suitable for regenerative medicine. As there is increasing evidence, that these stem cells are also able to be directed into the neural lineage, in our study we investigated the neuronal and glial differentiation potential of these cells, so that they may also be applied to cure degenerative diseases of the retina. Mesenchymal stem cells were isolated from routine prenatal amniocentesis at 15 to 18 weeks of pregnancy of human amniotic fluid and expanded in the cell culture. Cells were cultivated according to standard procedures for mesenchymal stem cells and were differentiated along the neural lineage using various protocols. Furthermore, it was also tried to direct them into cell types of the retina as well as into endothelial cells. Cells of more than 72 amniotic fluid samples were collected and characterized. While after induction neural-like phenotypes could actually be detected, which was confirmed using neural marker proteins such as GFAP and ßIII tubulina further differentiation into retinal like cells could not reliably be shown. These data suggest that amniotic fluid derived cells are an interesting cell source, which may also give rise to neural-like cells. However, a more specific differentiation into neuronal and glial cells could not unequivocally be shown, so that further investigations have to becarried out.
Romero, Roberto; Espinoza, Jimmy; Rogers, Wade T.; Moser, Allan; Nien, Jyh Kae; Kusanovic, Juan Pedro; Gotsch, Francesca; Erez, Offer; Gomez, Ricardo; Edwin, Sam; Hassan, Sonia S.
Objective Examination of the amniotic fluid proteome has been used to identify biomarkers for intra-amniotic inflammation, as well as those that may be useful in predicting the outcome of preterm labor. The purpose of this study was to combine a novel computational method of pattern discovery with mass spectrometric proteomic profiling of amniotic fluid to discover biomarkers of intra-amniotic infection/inflammation (IAI). Methods This cross-sectional study included patients with spontaneous preterm labor and intact membranes who delivered at term (n=59) and those who delivered preterm with IAI (n=60). Proteomic profiling was performed using SELDI mass spectrometry. A proteomic profile was acquired through multiple simultaneous SELDI conditions which were combined in a single proteomic “fingerprint” using a novel computational approach. Classification of patients based on their associated SELDI-TOF mass spectra as belonging to either the class of individuals with preterm delivery with IAI or term delivery was accomplished by constructing an empirical model. The first phase in the construction of this empirical model involved the selection of adjustable parameters utilizing a training/testing subset of data. The second phase tested the generalization of the model by utilizing a blinded validation set of patients who were not employed in parameter selection. Results Gestational age at amniocentesis was not significantly different between the groups. Thirty-nine unique mass spectrometric peaks discriminated patients with preterm labor/delivery with IAI from those with preterm labor and term delivery. In the testing/training dataset, the classification accuracies (averaged over 100 random draws) were: 91.4% (40.2/44) for patients with preterm delivery with IAI, and 91.2% (40.1/44) for term delivery. The overall accuracy of the classification of patients in the validation dataset was 90.3% (28/31). Conclusions Proteomic analysis of amniotic fluid allowed the
Du, Liqun; Wu, Xinyi
Our aim was to produce a natural, acellular matrix from porcine cornea for use as a scaffold in developing a tissue-engineered cornea replacement. Full-thickness, intact porcine corneas were decellularized by immersion in 0.5% (wt/vol) sodium dodecyl sulfate. The resulting acellular matrices were then characterized and examined specifically for completeness of the decellularization process. Histological analyses of decellularized corneal stromas showed that complete cell and α-Gal removal was achieved, while the major structural proteins including collagen type I and IV, laminin, and fibronectin were retained. DAPI staining did not detect any residual DNA within the matrix, and the DNA contents, which reflect the presence of cellular materials, were significantly diminished in the decellularized cornea. The collagen content of the decellularized cornea was well maintained compared with native tissues. Uniaxial tensile testing indicated that decellularization did not significantly compromise the ultimate tensile strength of the tissue (P > 0.05). In vitro cytotoxicity assays using rabbit corneal fibroblast cultures excluded the presence of soluble toxins in the biomaterial. In vivo implantation to rabbit interlamellar stromal pockets showed good biocompability. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the cornea has been successfully developed. The matrix is biocompatible with cornea-derived cells and has potential for use in corneal transplantation and tissue-engineering applications.
Uriarte, Juan J; Nonaka, Paula N; Campillo, Noelia; Palma, Renata K; Melo, Esther; de Oliveira, Luis V F; Navajas, Daniel; Farré, Ramon
Lung bioengineering using decellularized organ scaffolds is a potential alternative for lung transplantation. Clinical application will require donor scaffold sterilization. As gamma-irradiation is a conventional method for sterilizing tissue preparations for clinical application, the aim of this study was to evaluate the effects of lung scaffold sterilization by gamma irradiation on the mechanical properties of the acellular lung when subjected to the artificial ventilation maneuvers typical within bioreactors. Twenty-six mouse lungs were decellularized by a sodium dodecyl sulfate detergent protocol. Eight lungs were used as controls and 18 of them were submitted to a 31kGy gamma irradiation sterilization process (9 kept frozen in dry ice and 9 at room temperature). Mechanical properties of acellular lungs were measured before and after irradiation. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. After irradiation lungs presented higher values of resistance and elastance than before irradiation: RL increased by 41.1% (room temperature irradiation) and 32.8% (frozen irradiation) and EL increased by 41.8% (room temperature irradiation) and 31.8% (frozen irradiation). Similar increases were induced by irradiation in Est and Edyn. Scanning electron microscopy showed slight structural changes after irradiation, particularly those kept frozen. Sterilization by gamma irradiation at a conventional dose to ensure sterilization modifies acellular lung mechanics, with potential implications for lung bioengineering.
Goissis, Gilberto; Giglioti, Aparecida de Fátima; Braile, Domingo Marcolino
Major problems with biological heart valves post-implantation are associated with progressive structural deterioration and calcification attributed to glutaraldehyde processing, dead cells, and cell fragments present in the native tissue. In spite of these problems, glutaraldehyde still is the reagent of choice. The results with acellular matrix xenograft usually prepared by detergent treatment in association with enzymes are rather conflicting because while preserving mechanical properties, tissue morphology and collagen structure are process dependent. This work describes a chemical approach for the preparation of an acellular bovine pericardium matrix intended for the manufacture of heart valve bioprostheses. Cell removal was performed by an alkaline extraction in the presence of calcium salts for periods ranging from 6 to 48 h. The results showed that cell removal was achieved after 12 h, with swelling and negative charge increasing with processing time. Nevertheless, collagen fibril structure, ability to form fibrils, and stability to collagenase were progressive after 24-h processing. There was no denaturation of the collagen matrix. A process is described for the preparation of acellular bovine pericardium matrices with preserved fibril structure and morphology for the manufacture of cardiac valve bioprostheses and may be used in other applications for tissue reconstruction.
Alexander, G R; Hulsey, T C; Robillard, P Y; De Caunes, F; Papiernik, E
This study examines ethnic variations in meconium-stained amniotic fluid in term pregnancies, taking into account the role of gestational age, maternal sociodemographic characteristics, and medical risk factors. The study population included black and white singleton live births (N = 14,419) between 37 and 42 weeks' gestation, delivered vaginally at the Medical University of South Carolina from 1982 through 1990. Chi-square and logistic regression analysis were used to examine the association between the independent variables and meconium-stained amniotic fluid (MSAF). An increased risk of MSAF was found for advancing gestational age, indicators of fetal stress, fewer than five prenatal care visits, and > 15 hours labor. After controlling for demographic and clinical characteristics, the risk of MSAF in black patients was approximately 1.5 times that of white patients. The higher proportion of MSAF in blacks could not be explained with obvious risk factors.
Engels, Alexander C; Debeer, Anne; Russo, Francesca M; Aertsen, Michael; Aerts, Katleen; Miserez, Marc; Deprest, Jan; Lewi, Liesbeth; Devlieger, Roland
A 27-year-old woman, gravida 2, para 0, presented with an incomplete Pentalogy of Cantrell with an omphalocele, diaphragmatic hernia, and a pericardial defect at 32 weeks' gestation. A large pericardial effusion compressed the lungs and had led to a reduced lung growth with an observed-to-expected total lung volume of 28% as measured by MRI. The effusion disappeared completely after the insertion of a pericardio-amniotic shunt at 33 weeks. After birth, the newborn showed no signs of pulmonary hypoplasia and underwent a surgical correction of the defect. Protracted wound healing and a difficult withdrawal from opioids complicated the neonatal period. The child was discharged on postnatal day 105 in good condition. This case demonstrates that in case of Pentalogy of Cantrell with large pericardial effusion, the perinatal outcome might be improved by pericardio-amniotic shunting.
Siegel, L.; Walker, S.I.; Robin, N.I.
We describe an enzymic quantification of phosphatidylglycerol in amniotic fluid. Phosphatidylglycerol is hydrolyzed in alkali and the resulting glycerol is then enzymatically phosphorylated with adenosine 5'-(gamma-/sup 32/P)triphosphate to yield glycero(/sup 32/P)phosphate. After removal of excess (gamma-/sup 32/P)ATP by charcoal, the radioactivity of the glycerophosphate is measured in a liquid scintillation counter. Triglyceride in the amniotic fluid is hydrolyzed by lipase before extraction and thus does not interfere with the analysis. This method is specific for phosphatidylglycerol. Preliminary studies suggest that a phosphatidylglycerol value greater than or equal to 10 nmol/mL correlates with fetal lung maturity.
O'Meally, Denis; Ezaz, Tariq; Georges, Arthur; Sarre, Stephen D; Graves, Jennifer A Marshall
Several recent studies have produced comparative maps of genes on amniote sex chromosomes, revealing homology of gene content and arrangement across lineages as divergent as mammals and lizards. For example, the chicken Z chromosome, which shares homology with the sex chromosomes of all birds, monotremes, and a gecko, is a striking example of stability of genome organization and retention, or independent acquisition, of function in sex determination. In other lineages, such as snakes and therian mammals, well conserved but independently evolved sex chromosome systems have arisen. Among lizards, novel sex chromosomes appear frequently, even in congeneric species. Here, we review recent gene mapping data, examine the evolutionary relationships of amniote sex chromosomes and argue that gene content can predispose some chromosomes to a specialized role in sex determination.
Phosphatidylinositol and phosphatidylglycerol in amniotic fluid: Indicies of lung maturity. Am J Obstet Gynecol 1976; 125:613-17. 8. Coapman-Hankin RA, Kiechle FL...phosphatidylglycerol in the amniotic fluid in the prediction of respiratory distress syndrome. Obstet Gynecol 1981; 57:295-300. 12. McCulloch JC, Mendelsohn D...phosphatidylglycerol in amniotic fluid. Am J Obstet Gynecol 1983; 145:474-80. 15. Chapman JF, Phillips JC, Rosenthal MA, Herbert WNP. Evaluation of the
Chojnowski, Jena L; Braun, Edward L
Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also
Strasser, Bettina; Mlitz, Veronika; Hermann, Marcela; Rice, Robert H; Eigenheer, Richard A; Alibardi, Lorenzo; Tschachler, Erwin; Eckhart, Leopold
The evolution of amniotes has involved major molecular innovations in the epidermis. In particular, distinct structural proteins that undergo covalent cross-linking during cornification of keratinocytes facilitate the formation of mechanically resilient superficial cell layers and help to limit water loss to the environment. Special modes of cornification generate amniote-specific skin appendages such as claws, feathers, and hair. In mammals, many protein substrates of cornification are encoded by a cluster of genes, termed the epidermal differentiation complex (EDC). To provide a basis for hypotheses about the evolution of cornification proteins, we screened for homologs of the EDC in non-mammalian vertebrates. By comparative genomics, de novo gene prediction and gene expression analyses, we show that, in contrast to fish and amphibians, the chicken and the green anole lizard have EDC homologs comprising genes that are specifically expressed in the epidermis and in skin appendages. Our data suggest that an important component of the cornified protein envelope of mammalian keratinocytes, that is, loricrin, has originated in a common ancestor of modern amniotes, perhaps during the acquisition of a fully terrestrial lifestyle. Moreover, we provide evidence that the sauropsid-specific beta-keratins have evolved as a subclass of EDC genes. Based on the comprehensive characterization of the arrangement, exon-intron structures and conserved sequence elements of EDC genes, we propose new scenarios for the evolutionary origin of epidermal barrier proteins via fusion of neighboring S100A and peptidoglycan recognition protein genes, subsequent loss of exons and highly divergent sequence evolution.
Hortensius, Rebecca A.; Ebens, Jill H.; Harley, Brendan A. C.
Adult tendon wound repair is characterized by the formation of disorganized collagen matrix which leads to decreases in mechanical properties and scar formation. Studies have linked this scar formation to the inflammatory phase of wound healing. Instructive biomaterials designed for tendon regeneration are often designed to provide both structural and cellular support. In order to facilitate regeneration, success may be found by tempering the body’s inflammatory response. This work combines collagen-glycosaminoglycan scaffolds, previously developed for tissue regeneration, with matrix materials (hyaluronic acid and amniotic membrane) that have been shown to promote healing and decreased scar formation in skin studies. The results presented show that scaffolds containing amniotic membrane matrix have significantly increased mechanical properties and that tendon cells within these scaffolds have increased metabolic activity even when the media is supplemented with the pro-inflammatory cytokine interleukin-1 beta. Collagen scaffolds containing hyaluronic acid or amniotic membrane also temper the expression of genes associated with the inflammatory response in normal tendon healing (TNF-α, COLI, MMP-3). These results suggest that alterations to scaffold composition, to include matrix known to decrease scar formation in vivo, can modify the inflammatory response in tenocytes. PMID:26799369
Rahkonen, Leena; Nupponen, Irmeli; Pätäri-Sampo, Anu; Tikkanen, Minna; Sorsa, Timo; Juhila, Juuso; Andersson, Sture; Paavonen, Jorma; Stefanovic, Vedran
Introduction. Intra-amniotic infection (IAI) is a major cause of preterm labor and adverse neonatal outcome. We evaluated amniotic fluid (AF) proteolytic cascade forming biomarkers in relation to microbial invasion of the amniotic cavity (MIAC) and IAI in preterm pregnancies with intact membranes. Material and Methods. Amniocentesis was made to 73 women with singleton pregnancies; 27 with suspected IAI; and 46 controls. AF biomarkers were divided into three cascades: Cascade 1: matrix metalloproteinase-8 (MMP-8), MMP-9, myeloperoxidase (MPO), and interleukin-6; Cascade 2: neutrophil elastase (HNE), elafin, and MMP-9; Cascade 3: MMP-2, tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), MMP-8/TIMP-1 molar ratio, and C-reactive protein (CRP). MMP-8 was measured by an immunoenzymometric assay and the others were measured by ELISA. Standard biochemical methods, molecular microbiology, and culture techniques were used. Results. MMP-8, MMP-9, MPO, elafin, and TIMP-1 concentrations were higher in IAI suspected cases compared to controls and also in IAI suspected cases with MIAC compared to those without MIAC when adjusted by gestational age at amniocentesis. All biomarkers except elafin and MMP-2 had the sensitivity of 100% with thresholds based on ROC-curve. Odd ratios of biomarkers for MIAC were 1.2-38 and 95% confidential intervals 1.0-353.6. Conclusions. Neutrophil based AF biomarkers were associated with IAI and MIAC. PMID:28167848
Buhimschi, Irina A.; Nayeri, Unzila A.; Laky, Christine A.; Razeq, Sonya-Abdel; Dulay, Antonette T.; Buhimschi, Catalin S.
Introduction Intra-uterine infection is a global problem and a significant contributor to morbidity and perinatal death. The host response to infection causes an inflammatory state that acts synergistically with microbial insult to induce preterm birth and fetal damage. Prompt and accurate diagnosis of intra-amniotic infection in the asymptomatic stage of the disease is critical for improved maternal and neonatal outcomes. Areas Covered This article provides an overview of the most recent progress, challenges and opportunities for discovery and clinical implementation of various maternal serum, cervico-vaginal and amniotic fluid biomarkers in pregnancies complicated by intra-amniotic infection. Expert Opinion Clinically relevant biomarkers are critical to the accurate diagnostic of intra-uterine infection. Front end implementation of such biomarkers will also translate in lower incidence of early-onset neonatal sepsis which is an important determinant of neonatal morbidity and mortality associated with prematurity. However, of the hundreds of differentially expressed proteins, only few may have clinical utility and thus function as biomarkers. The small number of validation studies along with barriers to implementation of technological innovations in the clinical setting are current limitations. PMID:23530840
Hore, Timothy A.; Deakin, Janine E.; Marshall Graves, Jennifer A.
CTCF is an essential, ubiquitously expressed DNA-binding protein responsible for insulator function, nuclear architecture, and transcriptional control within vertebrates. The gene CTCF was proposed to have duplicated in early mammals, giving rise to a paralogue called “brother of regulator of imprinted sites” (BORIS or CTCFL) with DNA binding capabilities similar to CTCF, but testis-specific expression in humans and mice. CTCF and BORIS have opposite regulatory effects on human cancer-testis genes, the anti-apoptotic BAG1 gene, the insulin-like growth factor 2/H19 imprint control region (IGF2/H19 ICR), and show mutually exclusive expression in humans and mice, suggesting that they are antagonistic epigenetic regulators. We discovered orthologues of BORIS in at least two reptilian species and found traces of its sequence in the chicken genome, implying that the duplication giving rise to BORIS occurred much earlier than previously thought. We analysed the expression of CTCF and BORIS in a range of amniotes by conventional and quantitative PCR. BORIS, as well as CTCF, was found widely expressed in monotremes (platypus) and reptiles (bearded dragon), suggesting redundancy or cooperation between these genes in a common amniote ancestor. However, we discovered that BORIS expression was gonad-specific in marsupials (tammar wallaby) and eutherians (cattle), implying that a functional change occurred in BORIS during the early evolution of therian mammals. Since therians show imprinting of IGF2 but other vertebrate taxa do not, we speculate that CTCF and BORIS evolved specialised functions along with the evolution of imprinting at this and other loci, coinciding with the restriction of BORIS expression to the germline and potential antagonism with CTCF. PMID:18769711
Hore, Timothy A; Deakin, Janine E; Marshall Graves, Jennifer A
CTCF is an essential, ubiquitously expressed DNA-binding protein responsible for insulator function, nuclear architecture, and transcriptional control within vertebrates. The gene CTCF was proposed to have duplicated in early mammals, giving rise to a paralogue called "brother of regulator of imprinted sites" (BORIS or CTCFL) with DNA binding capabilities similar to CTCF, but testis-specific expression in humans and mice. CTCF and BORIS have opposite regulatory effects on human cancer-testis genes, the anti-apoptotic BAG1 gene, the insulin-like growth factor 2/H19 imprint control region (IGF2/H19 ICR), and show mutually exclusive expression in humans and mice, suggesting that they are antagonistic epigenetic regulators. We discovered orthologues of BORIS in at least two reptilian species and found traces of its sequence in the chicken genome, implying that the duplication giving rise to BORIS occurred much earlier than previously thought. We analysed the expression of CTCF and BORIS in a range of amniotes by conventional and quantitative PCR. BORIS, as well as CTCF, was found widely expressed in monotremes (platypus) and reptiles (bearded dragon), suggesting redundancy or cooperation between these genes in a common amniote ancestor. However, we discovered that BORIS expression was gonad-specific in marsupials (tammar wallaby) and eutherians (cattle), implying that a functional change occurred in BORIS during the early evolution of therian mammals. Since therians show imprinting of IGF2 but other vertebrate taxa do not, we speculate that CTCF and BORIS evolved specialised functions along with the evolution of imprinting at this and other loci, coinciding with the restriction of BORIS expression to the germline and potential antagonism with CTCF.
Rasier, Rifat; Gulsoy, Murat
AIM To avoid the side effects of the suture usage by welding amniotic membrane (AM) to contact lens (CL) with laser. METHODS AM was taken from pregnant women and cleaned from blood clots with sterile phosphate-buffered physiological saline solution which included antibiotics. Stromal side of the AM was spread inside of the CL and it was welded to CL by 1470 nm diode laser. 600 µm diameter fiber tip of the laser was contacted with the epithelial side of the AM from 4 separate points. After welding excess amniotic membrane around the CL was cut with a scalpel. RESULTS Stromal side of the AM was spread inside of the CL and then with laser fiber, different power levels and exposure times were applied on the epithelium of AM and 340 mW for seven seconds was found optimal. CL and AM attached with the spot welding effect in 4 points by touching fiber tip. CL-AM welded complex did not separated from each other while holding AM that extend beyond the CL with the help of two forceps. CONCLUSION As a conclusion, it was aimed in this study to achieve the success of the conventional amniotic membrane transplantation (AMT)with the easiness of applying a CL and to avoid risks and side effects of corneal or conjunctival suturing. The results showed that the application of the CL–AM complex will be as easy as the application of a CL and lasts shortly. PMID:25540753
Tajiri, Naoki; Acosta, Sandra; Portillo-Gonzales, Gabriel S.; Aguirre, Daniela; Reyes, Stephanny; Lozano, Diego; Pabon, Mibel; Dela Peña, Ike; Ji, Xunming; Yasuhara, Takao; Date, Isao; Solomita, Marianna A.; Antonucci, Ivana; Stuppia, Liborio; Kaneko, Yuji; Borlongan, Cesar V.
Accumulating preclinical evidence suggests the use of amnion as a source of stem cells for investigations of basic science concepts related to developmental cell biology, but also for stem cells’ therapeutic applications in treating human disorders. We previously reported isolation of viable rat amniotic fluid-derived stem (AFS) cells. Subsequently, we recently reported the therapeutic benefits of intravenous transplantation of AFS cells in a rodent model of ischemic stroke. Parallel lines of investigations have provided safety and efficacy of stem cell therapy for treating stroke and other neurological disorders. This review article highlights the need for investigations of mechanisms underlying AFS cells’ therapeutic benefits and discusses lab-to-clinic translational gating items in an effort to optimize the clinical application of the cell transplantation for stroke. PMID:25165432
Sasaki, Yuka; Tsuda, Hitoshi; Ueda, Shigeto; Asakawa, Hideki; Seki, Kunihiko; Murata, Tetsuya; Kuriki, Ken; Tamai, Seiichi; Matsubara, Osamu
Carcinoma with a large central acellular zone (central acellular carcinoma, CAC) and matrix-producing carcinoma (MPC) have been recently noted as basal-like-type breast cancers, but the two entities are often confused. To clarify their histological differences, the histopathological sections of 15 CAC and seven MPC were examined and the following features were compared by reviewing slides: (i) mode of invasion; (ii) alteration of cancer cell adhesion in the transitional area between cellular and acellular zones; (iii) staining of the stromal matrix; (iv) lymphocyte infiltration; and (v) tumor grade. Complete agreement was required between two observers for the assessments of these features. All CAC had relatively sharp margins but showed infiltrative growth accompanied by eosinophilic intercellular matrix. In CAC there was abrupt transition between peripheral cellular and central acellular zones without alteration of cancer cell adhesion. In contrast, all MPC showed expansive growth with a well circumscribed margin, accompanied by basophilic and myxoid intercellular matrix. In MPC there was gradual transition from cellular to acellular areas with gradual loss of cancer cell adhesion. Histological grade 3 and peripheral lymphocyte infiltration were common features. It is suggested that CAC and MPC are histologically distinct entities, and that the aforementioned features are helpful for differential diagnosis.
Becker, Simon; Steinmeyer, Jasmin; Avsar, Murat; Höffler, Klaus; Salman, Jawad; Haverich, Axel; Warnecke, Gregor; Ochs, Matthias; Schnapper, Anke
Normothermic ex vivo lung perfusion (EVLP) has developed as a powerful technique to evaluate particularly marginal donor lungs prior to transplantation. In this study, acellular and cellular perfusate compositions were compared in an identical experimental setting as no consensus has been reached on a preferred technique yet. Porcine lungs underwent EVLP for 12 h on the basis of an acellular or a cellular perfusate composition after 24 h of cold ischaemia as defined organ stress. During perfusion, haemodynamic and respiratory parameters were monitored. After EVLP, the lung condition was assessed by light and transmission electron microscopy. Aerodynamic parameters did not show significant differences between groups and remained within the in vivo range during EVLP. Mean oxygenation indices were 491 ± 39 in the acellular group and 513 ± 53 in the cellular group. Groups only differed significantly in terms of higher pulmonary artery pressure and vascular resistance in the cellular group. Lung histology and ultrastructure were largely well preserved after prolonged EVLP and showed only minor structural alterations which were similarly present in both groups. Prolonged acellular and cellular EVLP for 12 h are both feasible with lungs prechallenged by ischaemic organ stress. Physiological and ultrastructural analysis showed no superiority of either acellular or cellular perfusate composition.
Marlier, Luc; Schaal, Benoist; Soussignan, Robert
Studied head-orientation response of breast-feeding neonates in paired-choice odor tests. Found that 2-day olds detected amniotic fluid and colostrum, treating them as similar sensorily and/or hedonically. Four-day olds exhibited a preference for breast milk. Three-day olds oriented longer toward the odor of their own amniotic fluid than alien…
Gomez-Lopez, Nardhy; Romero, Roberto; Plazyo, Olesya; Panaitescu, Bogdan; Furcron, Amy E.; Miller, Derek; Roumayah, Tamara; Flom, Emily; Hassan, Sonia S.
Problem Sterile intra-amniotic inflammation is associated with spontaneous preterm labor. Alarmins are proposed to mediate this inflammatory process. The aim of this study was to determine whether intra-amniotic administration of an alarmin, HMGB1, could induce preterm labor/birth. Method of Study Pregnant B6 mice were intra-amniotically or intraperitoneally injected with HMGB1 or PBS (control). Following injection, the gestational age and the rates of preterm birth and pup mortality were recorded. Results Intra-amniotic injection of HMGB1 led to preterm labor/birth [HMGB1 57% (4/7) vs. PBS 0% (0/6); p=0.049], and a high rate of pup mortality at week one [HMGB1 60.9±11.7% (25/41) vs. PBS 28.9±12.6% (11/38); p=0.001]. Conclusion Intra-amniotic administration of HMGB1 induces preterm labor/birth. PMID:26781934
Amniotic fluid embolism was first recognized in 1926, in a Brazilian journal case report, on the basis of large amounts of fetal material in the maternal pulmonary vasculature at autopsy. The first English language description appeared in 1941 and consisted of eight parturients dying suddenly in which, once again, fetal material was seen in the pulmonary vasculature. A control group of 34 pregnant women dying of other recognized causes did not have fetal material in their lungs. The incidence of recognized, serious illness is on the order of two to eight per 100,000, with a mortality rate ranging from 13% to 35%. The diagnosis rests largely on one or more of four clinical signs: circulatory collapse, respiratory distress, coagulopathy, and seizures/ coma. The only confirmatory laboratory test remains autopsy findings although serum tests for fetal antigen, insulin-like growth factor binding protein-1, and complement are currently being investigated. One of the paradoxes of diagnosis is that fetal material in the pulmonary circulation at autopsy is specific for amniotic fluid embolism, while the same finding in the living is not. The mechanism of disease remains uncertain although the best available evidence suggests that complement activation might have a role. In contrast, mast cell degranulation probably is not a mechanism, so amniotic fluid embolism is not an anaphylaxis or anaphylactoid reaction as has been occasionally suggested. Perhaps the greatest unknown is not why 1 in 50,000 pregnant women develop what appears to be an immune response to their fetus, but rather why the other 49,999 do not? PMID:27512413
Jamal, Ashraf; Kazemi, Maryam; Marsoosi, Vajiheh; Eslamian, Laleh
Background: Normal amniotic fluid predicts normal placental function, fetal growth and fetal well-being. Objective: To determine adverse pregnancy outcomes in borderline amniotic fluid index (AFI). Materials and Methods: Pregnant women (37-40 wks) with diagnosis of borderline AFI between December 2012 and August 2014 were identified. Antepartum, intrapartum and neonatal data were collected and compared with those of pregnant women with normal AFI. An AFI less than 8 and more than 5 cm was defined for borderline AFI. Pregnancy outcomes included Cesarean section for non-reassuring fetal heart rate, meconium stained amniotic fluid, 5-min Apgar score <7, low birth weight, umbilical cord blood pH at term and NICU admission. Results: Gestational age at delivery in pregnancies with borderline AFI was significantly lower than normal AFI. Cesarean section rate for non-reassuring fetal heart rate in women of borderline AFI was significantly higher and there was an increased incidence of birth weight less than 10th percentile for gestation age in borderline AFI group. Incidence of low Apgar score and low umbilical artery pH in pregnancies with borderline AFI was significantly higher than women with normal AFI. There were no significant difference in the rate of NICU admission and meconium staining in both groups. Conclusion: There are significant differences for adverse pregnancy outcomes , such as Cesarean section due to non-reassuring fetal heart rate, birth weight less than 10th percentile for gestation age, low 5 min Apgar score and low umbilical artery pH between pregnancies with borderline and normal AFI. PMID:27981256
Prasongchean, Weerapong; Bagni, Marinella; Calzarossa, Cinzia; De Coppi, Paolo; Ferretti, Patrizia
Although amniotic fluid cells can differentiate into several mesenchymal lineages and have been proposed as a valuable therapeutic cell source, their ability to undergo terminal neuronal differentiation remains a cause of controversy. The aim of this study was to investigate the neuronal differentiation ability of the c-Kit-positive population from GFP-transgenic rat amniotic fluid, amniotic fluid stem (AFS) cells, and to assess how they affected injury response in avian embryos. AFS cells were found to express several neural stem/progenitor cell markers. However, no overt neuronal differentiation was apparent after either treatment with small molecules known to stimulate neuronal differentiation, attempts to differentiate AFS cell-derived embryoid body-like structures, or grafting AFS cells into environments known to support neuronal differentiation (organotypic rat hippocampal cultures, embryonic chick nervous system). Nonetheless, AFS cells significantly reduced hemorrhage and increased survival when grafted at the site of an extensive thoracic crush injury in E2.5 chick embryos. Increased embryo survival was induced neither by desmopressin treatment, which also reduced hemorrhage, nor by grafting other mesenchymal or neural cells, indicating a specific effect of AFS cells. This was found to be mediated by soluble factors in a transwell coculture model. Altogether, this study shows that AFS cells reduce tissue damage and increase survival in injured embryos, providing a potentially valuable tool as therapeutic agents for tissue repair, particularly prenatal/perinatal repair of defects diagnosed during gestation, but this effect is mediated via paracrine mechanisms rather than the ability of AFS cells to fully differentiate into neuronal cells.
Loganathan, Rajprasad; Little, Charles D; Joshi, Pranav; Filla, Michael B; Cheuvront, Tracey J; Lansford, Rusty; Rongish, Brenda J
Abstract The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ∼400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen's node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen's node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping
Barret, Maximilien; Pratico, Carlos Alberto; Camus, Marine; Beuvon, Frédéric; Jarraya, Mohamed; Nicco, Carole; Mangialavori, Luigi; Chaussade, Stanislas; Batteux, Frédéric; Prat, Frédéric
Background and Aims The prevention of esophageal strictures following circumferential mucosal resection remains a major clinical challenge. Human amniotic membrane (AM) is an easily available material, which is widely used in ophthalmology due to its wound healing, anti-inflammatory and anti-fibrotic properties. We studied the effect of AM grafts in the prevention of esophageal stricture after endoscopic submucosal dissection (ESD) in a swine model. Animals and Methods In this prospective, randomized controlled trial, 20 swine underwent a 5 cm-long circumferential ESD of the lower esophagus. In the AM Group (n = 10), amniotic membrane grafts were placed on esophageal stents; a subgroup of 5 swine (AM 1 group) was sacrificed on day 14, whereas the other 5 animals (AM 2 group) were kept alive. The esophageal stent (ES) group (n = 5) had ES placement alone after ESD. Another 5 animals served as a control group with only ESD. Results The prevalence of symptomatic strictures at day 14 was significantly reduced in the AM group and ES groups vs. the control group (33%, 40% and 100%, respectively, p = 0.03); mean esophageal diameter was 5.8±3.6 mm, 6.8±3.3 mm, and 2.6±1.7 mm for AM, ES, and control groups, respectively. Median (range) esophageal fibrosis thickness was 0.87 mm (0.78–1.72), 1.19 mm (0.28–1.95), and 1.65 mm (0.7–1.79) for AM 1, ES, and control groups, respectively. All animals had developed esophageal strictures by day 35. Conclusions The anti-fibrotic effect of AM on esophageal wound healing after ESD delayed the development of esophageal stricture in our model. However, this benefit was of limited duration in the conditions of our study. PMID:24992335
Shepherd, J; Sims, C; Craft, I
A small dose of prostaglandin E2 suspended in a viscous medium was instilled as a single application into the extra-amniotic space of patients with unfavourable induction features the day before planned induction in an attempt to improve the condition of the cervix. Two groups of 15 patients were studied, one receiving prostaglandin E2 250 mug suspended in methyl ethyl cellulose ('Tylose') 6% solution, and the other tylose alone. Cervical status did not change in those receiving tylose alone, whereas a significant improvement occurred in 14 out of 15 patients receiving the prostaglandin. Labour began before formal induction in 1 patient receiving tylose and in 8 receiving prostaglandin.
Del Toro-Valero, Azucena; Estrada-De la Fuente, Alejandro; Velázquez Santana, Héctor; Glicerio González, Jorge; Navarro Meza, María Cristina; Ortega-Hinojosa, Lilia; López-Cardona, María Guadalupe
Amniotic band sequence (ABS) is a group malformation that mainly affects limbs; clinically, constriction rings and lymphedema of the fingers, arms and legs, acrosyndactyly and pseudosyndactyly are observed; also there is congenital amputation of limbs due to distal swelling. Less frequently, craniofacial and trunk involvement are reported in some patients. Etiology is still unknown and most cases are isolated. In this report we present the case of a 45-day-old male with diagnosis of SBA and bilateral choanal atresia as attached finding, and review possible causes of SBA and associated alterations.
Kotomin, Ilya; Valtink, Monika; Hofmann, Kai; Frenzel, Annika; Morawietz, Henning; Werner, Carsten; Funk, Richard H. W.; Engelmann, Katrin
Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders. Trial Registration ClinicalTrials.gov NCT02168790 PMID:25955359
Suh, Alexander; Churakov, Gennady; Ramakodi, Meganathan P.; Platt, Roy N.; Jurka, Jerzy; Kojima, Kenji K.; Caballero, Juan; Smit, Arian F.; Vliet, Kent A.; Hoffmann, Federico G.; Brosius, Jürgen; Green, Richard E.; Braun, Edward L.; Ray, David A.; Schmitz, Jürgen
Chicken repeat 1 (CR1) retroposons are long interspersed elements (LINEs) that are ubiquitous within amniote genomes and constitute the most abundant family of transposed elements in birds, crocodilians, turtles, and snakes. They are also present in mammalian genomes, where they reside as numerous relics of ancient retroposition events. Yet, despite their relevance for understanding amniote genome evolution, the diversity and evolution of CR1 elements has never been studied on an amniote-wide level. We reconstruct the temporal and quantitative activity of CR1 subfamilies via presence/absence analyses across crocodilian phylogeny and comparative analyses of 12 crocodilian genomes, revealing relative genomic stasis of retroposition during genome evolution of extant Crocodylia. Our large-scale phylogenetic analysis of amniote CR1 subfamilies suggests the presence of at least seven ancient CR1 lineages in the amniote ancestor; and amniote-wide analyses of CR1 successions and quantities reveal differential retention (presence of ancient relics or recent activity) of these CR1 lineages across amniote genome evolution. Interestingly, birds and lepidosaurs retained the fewest ancient CR1 lineages among amniotes and also exhibit smaller genome sizes. Our study is the first to analyze CR1 evolution in a genome-wide and amniote-wide context and the data strongly suggest that the ancestral amniote genome contained myriad CR1 elements from multiple ancient lineages, and remnants of these are still detectable in the relatively stable genomes of crocodilians and turtles. Early mammalian genome evolution was thus characterized by a drastic shift from CR1 prevalence to dominance and hyperactivity of L2 LINEs in monotremes and L1 LINEs in therians. PMID:25503085
Young, Nathan M
Mammals, birds, and reptiles exhibit a remarkable diversity of limb proportions. These evolved differences are thought to reflect selection for biomechanical, postural, and locomotor requirements primarily acting on independent variation in later fetal and postnatal segmental growth. However, earlier conserved developmental events also have the potential to impact the evolvability of limb proportions by limiting or biasing initial variation among segments. Notably, proximo-distal patterning of the amniote limb through activation-inhibition dynamics predicts that initial proportions of segments should exhibit both tradeoffs between stylopod and autopod and a diagnostic reduction in variance of the zeugopod. Here it is demonstrated that this developmental "design rule" predicts patterns of macroevolutionary diversity despite the effects of variation in segmental growth over ontogeny, lineage-specific differences in phylogenetic history, or functional adaptation. These results provide critical comparative evidence of a conserved Turing-like mechanism in proximo-distal limb segmentation, and suggest that development has played a previously unrecognized role in the evolvability of limb proportions in a wide range of amniote taxa.
Yahaya, M I; Ogunfowokan, A O; Orji, E O
In this study concentration level of calcium, cadmium, copper, iron, magnesium, manganese, nickel, lead and zinc were determined in the amniotic fluid of pregnant women, aged 15 - 45 years enrolled at the Obafemi Awolowo University Teaching Hospitals Complex Ile - Ife. This was with a view to predict the body burden of the metals in the pregnant women and assess the health implications of the toxic elements to the pregnant women and their fetuses. Fifty samples of the amniotic fluid were collected from the pregnant women. The efficiency of extraction of trace metals using conventional wet acid digestion method (CDM) and microwave induced acid digestion method (MWD) was determined by recovery experiments. Levels of trace metals were determined using Atomic Absorption Spectrophotometry. The high percentage recoveries obtained from MWD made it a more efficient method than the CDM and hence its adoption for sample digestion. Statistical analysis of data using descriptive and inferential statistics revealed that age; education and profession have effects on the levels of the trace metals. The mean levels of most of the toxic metals obtained in this study were lower than the recommended limits of trace metals in women whole blood.
Rosenberg, Victor A.; Buhimschi, Irina A.; Dulay, Antonette T.; Abdel-Razeq, Sonya S.; Oliver, Emily A.; Duzyj, Christina M.; Lipkind, Heather; Pettker, Christian M.; Buhimschi, Catalin S.
PROBLEM Activins and inhibins are important modulators of inflammatory processes. We explored activation of amniotic fluid (AF) activin-A and inhibin-A system in women with intra-amniotic infection and preterm premature rupture of the membranes (PPROM). METHOD OF STUDY We analyzed 78 AF samples: “2nd trimester-control” (n=12), “3rd trimester-control” (n=14), preterm labor with intact membranes [positive-AF-cultures (n=13), negative-AF-cultures (n=13)] and PPROM [positive-AF-cultures (n=13), negative-AF-cultures (n=13)]. Activin-A levels were evaluated ex-vivo following incubation of amniochorion and placental villous explants with Gram-negative (LPS) or Gram-positive (Pam3Cys) bacterial mimics. Ability of recombinant activin-A and inhibin-A to modulate inflammatory reactions in fetal membranes was explored through explants’ IL-8 release. RESULTS Activin-A and inhibin-A were present in human AF and were gestational age-regulated. Activin-A was significantly upregulated by infection. Lower inhibin-A levels were seen in PPROM. LPS elicited release of activin-A from amniochorion, but not from villous explants. Recombinant activin-A stimulated IL-8 release from amniochorion, an effect that was not reversed by inhibin-A. CONCLUSION Human AF activin-A and inhibin-A are involved in biological processes linked to intra-amniotic infection/inflammation induced preterm birth. PMID:21992678
Xing, D; Maes, A; Behr-Gross, M-E; Costanzo, A; Daas, A; Buchheit, K-H
The European Pharmacopoeia (Ph. Eur.) and the World Health Organisation (WHO) require the performance of extensive quality and safety control testing before the release on the market of vaccine products for human use. Safety testing with regard to residual pertussis toxin (PT) in acellular pertussis combination vaccines is performed through assessment of fatal sensitisation of mice to histamine challenge by the vaccine product under test. Currently, use of different in-house procedures and no requirement for the inclusion of a standard reference in each assay render comparisons of results obtained for identical vaccine batches between different control laboratories very difficult. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM), an international collaborative study was organised for the standardization of the Histamine Sensitizing Test (HIST) in mice and the Chinese Hamster Ovary (CHO)-cell-based assay (performed at the bulk product level) for the residual toxicity testing of acellular pertussis vaccines or acellular pertussis-based combination vaccines. The study was run under the aegis of the Biological Standardisation Programme, jointly supported by the Council of Europe and the European Commission under the project code BSP076. Ten (10) laboratories participated in the study and were requested to perform 3 independent Histamine Sensitizing Tests in mice and to report results of the lethal end-point measurement as prescribed by the Ph. Eur. monographs. Some of them also reported data from an in-house validated CHO-cell-based assay. In addition, some of the laboratories reported concomitantly data obtained by measurement of the drop in temperature induced after the histamine challenge, a method currently under investigation to be added as an alternative end-point for the HIST in the Ph. Eur. monographs for acellular pertussis-based combination vaccines in order to alleviate animal suffering (in application of the 3
Vermeulen, Françoise; Verscheure, Virginie; Damis, Eliane; Vermeylen, Danièle; Leloux, Gaëlle; Dirix, Violette; Locht, Camille; Mascart, Françoise
Based on studies reporting specific antibody titers, it is recommended to vaccinate preterm infants against Bordetella pertussis according to their chronological age. However, as specific T-cell responses also are involved in the protection against B. pertussis, we have determined whether highly preterm infants (<31 weeks) are able to mount these immune responses during vaccination. Forty-eight premature infants were vaccinated at 2, 3, and 4 months of their chronological age with an acellular (Pa; n = 24) or a whole-cell (Pw; n = 24) tetravalent diphtheria-tetanus-pertussis-polio vaccine, and blood samples were collected at 2, 3, and 6 months of age. Most of the Pa- and Pw-vaccinated infants developed at 3 or 6 months of age a gamma interferon (IFN-γ) response to the B. pertussis antigens, accompanied by an interleukin-5 (IL-5) and IL-13 secretion for the Pa-vaccinated infants. No association was found between a very low infant birth weight, the occurrence of severe infections, and corticosteroid treatment or the administration of gammaglobulins with a low level of antigen-induced IFN-γ secretion. We conclude that like full-term infants, most preterm infants are able to mount a specific cellular immune response to the administration of the first doses of an acellular or a whole-cell pertussis vaccine. PMID:20016042
Peloso, Andrea; Ferrario, Jacopo; Maiga, Benedetta; Benzoni, Ilaria; Bianco, Carolina; Citro, Antonio; Currao, Manuela; Malara, Alessandro; Gaspari, Annalisa; Balduini, Alessandra; Abelli, Massimo; Piemonti, Lorenzo; Dionigi, Paolo; Orlando, Giuseppe; Maestri, Marcello
Abstract Kidney transplantation is the only potentially curative treatment for patient facing end-stage renal disease, and it is now routinely used. Its use is mainly limited by the supply of transplantable donor organs, which far exceeds the demand. Regenerative medicine and tissue engineering offer promising means for overcoming this shortage. In the present study, we developed and validated a protocol for producing acellular rat renal scaffolds. Left kidneys were removed from 26 male Lewis rats (weights: 250–350 g) and decellularized by means of aortic anterograde perfusion with ionic and anionic detergents (Triton X-100 1% and SDS 1%, respectively). 19 scaffolds thus obtained (and contralateral native kidneys as controls) were deeply characterized in order to evaluate the decellularization quality, the preservation of extracellular matrix components and resultant micro-angioarchitecture structure. The other 7 were transplanted into 7 recipient rats that had undergone unilateral nephrectomy. Recipients were sacrificed on post-transplantation day 7 and the scaffolds subjected to histologic studies. The dual-detergent protocol showed, with only 5 h of perfusion per organ, to obtain thoroughly decellularized renal scaffolds consisting almost exclusively of extracellular matrix. Finally the macro- and the microarchitecture of the renal parenchyma were well preserved, and the grafts were implanted with ease. Seven days after transplant, the scaffolds were morphologically intact although all vascular structures were obstructed with thrombi. Production and implantation of acellular rat renal scaffolds is a suitable platform for further studies on regenerative medicine and tissue engineering. PMID:26186418
Arciniega, J.; Wagner, L.; Prymula, R.; Sebo, P.; Isbrucker, R.; Descampe, B.; Chapsal, J.M.; Costanzo, A.; Hendriksen, C.; Hoonaker, M.; Nelson, S.; Lidster, K.; Casey, W.; Allen, D.
The ‘International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST’ was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9 th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials. PMID:27506225
Cheng, Hai-Ling Margaret; Loai, Yasir; Beaumont, Marine; Farhat, Walid A
Bladder acellular matrices (ACMs) derived from natural tissue are gaining increasing attention for their role in tissue engineering and regeneration. Unlike conventional scaffolds based on biodegradable polymers or gels, ACMs possess native biomechanical and many acquired biologic properties. Efforts to optimize ACM-based scaffolds are ongoing and would be greatly assisted by a noninvasive means to characterize scaffold properties and monitor interaction with cells. MRI is well suited to this role, but research with MRI for scaffold characterization has been limited. This study presents initial results from quantitative MRI measurements for bladder ACM characterization and investigates the effects of incorporating hyaluronic acid, a natural biomaterial useful in tissue-engineering and regeneration. Measured MR relaxation times (T(1), T(2)) and diffusion coefficient were consistent with increased water uptake and glycosaminoglycan content observed on biochemistry in hyaluronic acid ACMs. Multicomponent MRI provided greater specificity, with diffusion data showing an acellular environment and T(2) components distinguishing the separate effects of increased glycosaminoglycans and hydration. These results suggest that quantitative MRI may provide useful information on matrix composition and structure, which is valuable in guiding further development using bladder ACMs for organ regeneration and in strategies involving the use of hyaluronic acid.
Foster, B L; Soenjaya, Y; Nociti, F H; Holm, E; Zerfas, P M; Wimer, H F; Holdsworth, D W; Aubin, J E; Hunter, G K; Goldberg, H A; Somerman, M J
Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null ((-/-)) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp (-/-) mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp (-/-) mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp (-/-) mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function.
Foster, B.L.; Soenjaya, Y.; Nociti, F.H.; Holm, E.; Zerfas, P.M.; Wimer, H.F.; Holdsworth, D.W.; Aubin, J.E.; Hunter, G.K.; Goldberg, H.A.; Somerman, M.J.
Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null (-/-) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp-/- mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp-/- mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp-/- mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function. PMID:23183644
Kim, Eun Young; Lee, Kyung-Bon; Kim, Min Kyu
The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140] PMID:24499672
Gao, Liyang; Zhao, Mingyan; Ye, Wei; Huang, Jinzhi; Chu, Jiaqi; Yan, Shouquan; Wang, Chaojun; Zeng, Rong
The amniotic fluid has a heterogeneous population of cells. Some human amniotic fluid-derived stem (hAFS) cells have been shown to harbor the potential to differentiate into neural cells. However, the neural differentiation efficiency of hAFS cells remains low. In this study, we isolated CD117-positive hAFS cells from amniotic fluid and then examined the pluripotency of these cells through the formation of embryoid bodies (EBs). Additionally, we induced the neural differentiation of these cells using neuroectodermal medium. This study revealed that the GSK3-beta inhibitor SB216763 was able to stimulate the proliferation of CD117-positive hAFS cells without influencing their undifferentiated state. Moreover, SB216763 can efficiently promote the neural differentiation of CD117-positive hAFS cells towards neural progenitor cells in the presence of DMEM/F12 and N2 supplement. These findings provide an easy and low-cost method to maintain the proliferation of hAFS cells, as well as induce an efficacious generation of neural progenitor cells from hAFS cells. Such induction of the neural commitment of hAFS cells may provide an option for the treatment of neurodegenerative diseases by hAFS cells-based therapies.
Buhimschi, Irina A
Clinical proteomics encompasses a multitude of experimental approaches, tools, and techniques based on proteomics technology which are directly aimed to accelerate and improve diagnosis and treatment of human diseases. Surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry is a variant of matrix-enhanced laser desorption ionization (MALDI) that makes use of chemically-modified surfaces to reduce the complexity of biological samples prior to separation in the mass analyzer. Compared to other proteomic techniques, SELDI has several important advantages such as ability to analyze complex biological samples with minimal pre-processing, ease of handling and high throughput. Importantly, once the biomarker or combination of biomarkers with potential clinical value has been established, validation analyses can be conducted in close proximity to clinical settings which is important for establishing the utility of new diagnostics in clinical decision making and perhaps future theranostic interventions. This chapter provides protocols for experimental design and methodology aimed at (1) discovering biologically relevant biomarkers in amniotic fluid using SELDI-TOF; (2) validating the clinical utility of the biomarkers as new diagnostics; (3) translating the biomarker findings into pathophysiological phenomena to provide further insight and extend the current understanding of the disease process. Many of the principles described herein for amniotic fluid could be generalized to studies involving other types of biological samples and other clinical questions.
Benson, Michael D.
Amniotic fluid embolism (AFE) is one of the leading causes of maternal mortality and morbidity in developed countries. Current thinking about pathophysiology has shifted away from embolism toward a maternal immune response to the fetus. Two immunologic mechanisms have been studied to date. Anaphylaxis appears to be doubtful while the available evidence supports a role for complement activation. With the mechanism remaining to be elucidated, AFE remains a clinical diagnosis. It is diagnosed based on one or more of four key signs/symptoms: cardiovascular collapse, respiratory distress, coagulopathy, and/or coma/seizures. The only laboratory test that reliably supports the diagnosis is the finding of fetal material in the maternal pulmonary circulation at autopsy. Perhaps the most compelling mystery surrounding AFE is not why one in 20,000 parturients are afflicted, but rather how the vast majority of women can tolerate the foreign antigenic presence of their fetus both within their uterus and circulation? PMID:21969840
Dasgupta, Soham; Arya, Shreyas; Choudhary, Sanjeev; Jain, Sunil K
The gastrointestinal tract (GIT) is a complex system, which changes in response to requirements of the body. GIT represents a barrier to the external environment. To achieve this, epithelial cells must renew rapidly. This renewal of epithelial cells starts in the fetal life under the influence of many GIT peptides by swallowing amniotic fluid (AF). Development and maturation of GIT is a very complex cascade that begins long before birth and continues during infancy and childhood by breast-feeding. Many factors like genetic preprogramming, local and systemic endocrine secretions and many trophic factors (TF) from swallowed AF contribute and modulate the development and growth of the GIT. GIT morphogenesis, differentiation and functional development depend on the activity of various TF in the AF. This manuscript will review the role of AF borne TF in the development of GIT. PMID:26909227
Gogola, J; Hankins, G D
Amniotic fluid (AF) embolism is a rare but catastrophic complication of pregnancy. We present the first case where the debris was seen in the maternal uterine veins at the time of cesarean section. During a cesarean delivery performed for deteriorating fetal status and in conjunction with massive hydramnios; air bubbles and vernix were observed in the left uterine vein and in an area of Couvelaire appearance of the uterine fundus. As the patient was clinically stable and desired retained fertility, a decision was made to attempt to contain the debris in the uterine vasculature. The infundibulopelvic ligament and uterine arteries were ligated and the area of Couvelaire uterus was oversewn. With the exception of a mild laboratory coagulopathy, which required no specific treatment, the patient did well. The area of Couvelaire uterus is the likely portal for the debris seen in this patient's vasculature. Containment appears to have averted the AF embolism syndrome.
Joerger-Messerli, Marianne S; Marx, Caterina; Oppliger, Byron; Mueller, Martin; Surbek, Daniel V; Schoeberlein, Andreina
The discovery of mesenchymal stem cells (MSCs) in perinatal sources, such as the amniotic fluid (AF) and the umbilical connective tissue, the so-called Wharton's jelly (WJ), has transformed them into promising stem cell grafts for the application in regenerative medicine. The advantages of AF-MSCs and WJ-MSCs over adult MSCs, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), include their minimally invasive isolation procedure, their more primitive cell character without being tumourigenic, their low immunogenicity and their potential autologous application in congenital disorders and when cryopreserved in adulthood. This chapter gives an overview of the biology of AF-MSCs and WJ-MSCs, and their regenerative potential based on the results of recent preclinical and clinical studies. In the end, open questions concerning the use of WJ-MSCs and AF-MSCs in regenerative medicine will be emphasized.
Carrera González, Elier; Noa Hernández, Jose Eduardo; Marín Rojo, Carlos A
The application of amniotic membranes in patients diagnosed with skin damage is a valid treatment option. A care plan following the Virginia Henderson model and NANDA, NOC and NIC taxonomy was applied to 36 patients admitted to the Dr. Miguel Enríquez hospital with different cutaneous lesions. This membrane has already been used for years due to its healing properties. These are attributed to antimicrobial properties reducing infection risk and promoting epithelial activity. They can decrease the need for the use of antibiotics, expendable materials, and can be applied during long periods of healing. This decreases the cost of wide spectrum antibiotic treatments, as well as the time patients spend in hospital. We present the results of this application in cases with several types of skin lesions.
Su, C Y; Lin, C P
We report a new method combining the use of an amniotic membrane and cyanocrylate tissue adhesive to seal a corneal perforation. A 47-year-old male suffered from an alkali injury complicated with corneal melting and perforation in the left eye. We placed an amniotic membrane of optimal size in the anterior chamber directly under the corneal perforation lesion. The cyanocrylate tissue adhesive was then applied over the perforation site and sealed successfully. Three weeks later, the tissue adhesive had dislodged. The amniotic membrane had sealed the perforated lesion and was well adhered to the surrounding corneal tissue with complete epithelial covering. Vision was 20/25 six months after the operation. The combined use of an amniotic membrane and tissue adhesive is a promising method in the treatment of corneal perforation.
Janes, Daniel E.; Valenzuela, Nicole; Ezaz, Tariq; Amemiya, Chris; Edwards, Scott V.
Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently. These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200 kb of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced. With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries. PMID:20981143
The objective of the experiment is to investigate the effect of near weightlessness on a single cell. The test object is the acellular slime mold Physarum polycephalum. This cell is composed of a network of protoplastic strands which perform rhythmic contractions in the minute range. These contractions of the strands' ectoplastic walls generate the force to drive the vigorous shuttle streaming of fluid protoplasm inside the strands (hydrostatic pressure flow). A net transport of protoplasm in one direction determines the direction of the cell's locomotion itself. In this way, gravity modifies the contraction rhythm of the strands, the streaming velocity of protoplasm in the strands, and the direction of locomotion of the whole slime mold (geotaxis). The other parts of this experiment will address the major question of how this cell, which does not possess any specialized gravireceptors, gets the information about the direction of the gravity vector. Details of the experimental setup are given.
Belfort, Michael A.; Whitehead, William E.; Ball, Robert; Silver, Robert; Shamshirsaz, Alireza; Ruano, Rodrigo; Espinoza, Jimmy; Becker, Judith; Olutoye, Olutoyin; Hollier, Larry
Introduction Fetoscopic release of amniotic bands has proved its life- and limb-saving potential. Rupture of the amnion and separation of chorion from the amnion and uterine wall can however preclude the standard fetoscopic approach to release the amniotic bands using a single port. Methods and Materials A 28-year-old G1P0 woman was referred to our unit at 19 weeks due to amniotic band syndrome involving the left ankle, the infrapatellar region of the right leg, and the umbilical cord. Of note, part of the fetus was seen outside the amniotic cavity by ultrasonography and the left ankle and foot were severely swollen. The patient underwent a laparotomy and fetoscopic release of the amniotic bands as well as partial amnionectomy using two uterine ports and CO2 as distention. Results The surgery and postoperative recovery course were uneventful. At 341/7 weeks the patient went into labor, which was augmented resulting vaginal delivery of a 2,460-g male infant. The infant was noted to have a shallow skin indentation on the left lower extremity near the ankle. The infant was discharged in excellent condition. Conclusion In those cases where release of an amniotic band is impossible due to membrane separation, surgery in a CO2-filled uterus offers an option. PMID:27298754
Kuypers, Elke; Collins, Jennifer J. P.; Kramer, Boris W.; Ofman, Gaston; Nitsos, Ilias; Pillow, J. Jane; Polglase, Graeme R.; Kemp, Matthew W.; Newnham, John P.; Gavilanes, Antonio W. D.; Nowacki, Relana; Ikegami, Machiko; Jobe, Alan H.
The proinflammatory stimulus of chorioamnionitis is commonly associated with preterm delivery. Women at risk of preterm delivery receive antenatal glucocorticoids to functionally mature the fetal lung. However, the effects of the combined exposures of chorioamnionitis and antenatal glucocorticoids on the fetus are poorly understood. Time-mated ewes with singleton fetuses received an intra-amniotic injection of lipopolysaccharide (LPS) either preceding or following maternal intramuscular betamethasone 7 or 14 days before delivery, and the fetuses were delivered at 120 days gestational age (GA) (term = 150 days GA). Gestation matched controls received intra-amniotic and maternal intramuscular saline. Compared with saline controls, intra-amniotic LPS increased inflammatory cells in the bronchoalveolar lavage and myeloperoxidase, Toll-like receptor 2 and 4 mRNA, PU.1, CD3, and Foxp3-positive cells in the fetal lung. LPS-induced lung maturation measured as increased airway surfactant and improved lung gas volumes. Intra-amniotic LPS-induced inflammation persisted until 14 days after exposure. Betamethasone treatment alone induced modest lung maturation but, when administered before intra-amniotic LPS, suppressed lung inflammation. Interestingly, betamethasone treatment after LPS did not counteract inflammation but enhanced lung maturation. We conclude that the order of exposures of intra-amniotic LPS or maternal betamethasone had large effects on fetal lung inflammation and maturation. PMID:22160306
Köhler, E; Avenarius, S; Rabsilber, A; Gerloff, C; Jorch, G
Amniotic fluid was collected from 78 pregnant women at birth additionally with their urine prior to delivery as well as neonatal urine and meconium. The smoking markers, nicotine and its metabolites cotinine and trans-3'-hydroxycotinine (OH-cotinine), were determined using high-performance liquid chromatography (HPLC). The self-reported smoking status during pregnancy determined by means of a questionnaire was verified by measurement of maternal urine. In all smokers, nicotine metabolites were detected in amniotic fluid and in 80% of them nicotine as well. However, the sum of the nicotine metabolites (Sum(met)) was significantly lower (p < .001) in amniotic fluid (704 +/- 464 nmol/L) than in meconium (921 +/- 588 nmol/L), neonatal urine (1139 +/- 813 nmol/L) and maternal urine (4496 +/- 3535 nmol/L). Concentrations of nicotine metabolites in amniotic fluid correlated well (p < .001) with that in the other specimen types. After environmental tobacco smoke (ETS) exposure, no nicotine or nicotine metabolites were detectable in amniotic fluid but only in maternal and neonatal urine. Analysis of amniotic fluid at birth lends itself to verifying smoking habits during pregnancy and clearly discriminating from ETS exposure, but it is not a suitable approach to differentiating between ETS exposure and non-exposure.
Klein, Gabriel M.; Nasser, Ahmed E.; Phillips, Brett T.; Gersch, Robert P.; Fourman, Mitchell S.; Lilo, Sarit E.; Fritz, Jason R.; Khan, Sami U.; Dagum, Alexander B.
Introduction: Postoperative infections are a major complication associated with tissue-expander-based breast reconstruction. The use of acellular dermal matrix (ADM) in this surgery has been identified as a potential reservoir of infection, prompting the development of sterile ADM. Although aseptic and sterile ADMs have been investigated, no study has focused on the occurrence and clinical outcome of bacterial colonization before implantation. Methods: Samples of aseptic AlloDerm, sterile Ready-To-Use AlloDerm, and AlloMax were taken before implantation. These samples were incubated in Tryptic soy broth overnight before being streaked on Trypticase soy agar, MacConkey agar, and 5% blood agar plates for culture and incubated for 48 hours. Culture results were cross-referenced with patient outcomes for 1 year postoperatively. Results: A total of 92 samples of ADM were collected from 63 patients. There were 15 cases of postoperative surgical site infection (16.3%). Only 1 sample of ADM (AlloMax) showed growth of Escherichia coli, which was likely a result of contamination. That patient did not develop any infectious sequelae. Patient outcomes showed no difference in the incidence of seroma or infection between sterile and aseptic ADMs. Conclusions: This study evaluates the microbiology of acellular dermal matrices before use in breast reconstruction. No difference was found in the preoperative bacterial load of either aseptic or sterile ADM. No significant difference was noted in infection or seroma formation. Given these results, we believe aseptic processing used on ADMs is equivalent to sterile processing in our patient cohort in terms of clinical infection and seroma occurrence postoperatively. PMID:27482500
Kranenbarg, Sander; van Cleynenbreugel, Tim; Schipper, Henk; van Leeuwen, Johan
Mammalian bone is an active tissue in which osteoblasts and osteoclasts balance bone mass. This process of adaptive modelling and remodelling is probably regulated by strain-sensing osteocytes. Bone of advanced teleosts is acellular yet, despite the lack of osteocytes, it is capable of an adaptive response to physical stimuli. Strenuous exercise is known to induce lordosis. Lordosis is a ventrad curvature of the vertebral column, and the affected vertebrae show an increase in bone formation. The effects of lordosis on the strain distribution in sea bass (Dicentrarchus labrax L.) vertebrae are assessed using finite element modelling. The response of the local tissue is analyzed spatially and ontogenetically in terms of bone volume. Lordotic vertebrae show a significantly increased strain energy due to the increased load compared with normal vertebrae when loaded in compression. High strain regions are found in the vertebral centrum and parasagittal ridges. The increase in strain energy is attenuated by a change in architecture due to the increased bone formation. The increased bone formation is seen mainly at the articular surfaces of the vertebrae, although some extra bone is formed in the vertebral centrum. Regions in which the highest strains are found do not spatially correlate with regions in which the most extensive bone apposition occurs in lordotic vertebrae of sea bass. Mammalian-like strain-regulated bone modelling is probably not the guiding mechanism in adaptive bone modelling of acellular sea bass vertebrae. Chondroidal ossification is found at the articular surfaces where it mediates a rapid adaptive response, potentially attenuating high stresses on the dorsal zygapophyses.
Romero, Roberto; Miranda, Jezid; Chaemsaithong, Piya; Chaiworapongsa, Tinnakorn; Kusanovic, Juan P.; Dong, Zhong; Ahmed, Ahmed I.; Shaman, Majid; Lannaman, Kia; Yoon, Bo Hyun; Hassan, Sonia S.; Kim, Chong J.; Korzeniewski, Steven J.; Yeo, Lami; Kim, Yeon Mee
Objective The objectives of this study were to: 1) determine the amniotic fluid (AF) microbiology of patients with preterm prelabor rupture of membranes (PROM); and 2) examine the relationship between intra-amniotic inflammation with and without microorganisms (sterile inflammation) and adverse pregnancy outcomes in patients with preterm PROM. Methods AF samples obtained from 59 women with preterm PROM were analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital mycoplasmas) and with broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). AF concentration of interleukin-6 (IL-6) was determined using ELISA. Results of both tests were correlated with AF IL-6 concentrations, and the occurrence of adverse obstetrical/perinatal outcomes. Results 1) PCR/ESI-MS, AF culture, and the combination of these two tests, each identified microorganisms in 36% (21/59), 24% (14/59) and 41% (24/59) of women with preterm PROM, respectively; 2) the most frequent microorganisms found in the amniotic cavity were Sneathia species and Ureaplasma urealyticum; 3) the frequency of microbial-associated and sterile intra-amniotic inflammation was overall similar [ 29% (17/59)]: - however, the prevalence of each differed according to the gestational age when PROM occurred ; 4) the earlier the gestational age at preterm PROM, the higher the frequency of both microbial-associated and sterile intra-amniotic inflammation; 5) the intensity of the intra-amniotic inflammatory response against microorganisms is stronger when preterm PROM occurs early in pregnancy; and 6) the frequency of acute placental inflammation (histologic chorioamnionitis and/or funisitis) was significantly higher in patients with microbial-associated intra-amniotic inflammation than in those without intra-amniotic inflammation [93.3% (14/15) vs. 38% (6/16); p=0.001]. Conclusions 1) The frequency of microorganisms in preterm PROM is 40% using
Galende, Elisa; Karakikes, Ioannis; Edelmann, Lisa; Desnick, Robert J.; Kerenyi, Thomas; Khoueiry, Georges; Lafferty, James; McGinn, Joseph T.; Brodman, Michael; Fuster, Valentin; Hajjar, Roger J.
Abstract Recently, cultured human adult skin cells were reprogrammed to induced pluripotent stem (iPS) cells, which have characteristics similar to human embryonic stem (hES) cells. Patient-derived iPS cells offer genetic and immunologic advantages for cell and tissue replacement or engineering. The efficiency of generating human iPS cells has been very low; therefore an easily and efficiently reprogrammed cell type is highly desired. Here, we demonstrate that terminally differentiated human amniotic fluid (AF) skin cells provide an accessible source for efficiently generating abundant-induced pluripotent stem (AF-iPS) cells. By induction of pluripotency with the transcription factor quartet (OCT3/4, SOX2, KLF4, and c-MYC) the terminally differentiated, cultured AF skin cells formed iPS colonies approximately twice as fast and yielded nearly a two-hundred percent increase in number, compared to cultured adult skin cells. AF-iPS cells were identical to hES cells for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, telomerase activity, in vitro and in vivo differentiation into the three germ layers and for their capacity to form embryoid bodies (EBs) and teratomas. Our findings provide a biological interesting conclusion that these fetal AF cells are more rapidly, easily, and efficiently reprogrammed to pluripotency than neonatal and adult cells. AF-iPS cells may have a “young,” more embryonic like epigenetic background, which may facilitate and accelerate pluripotency. The ability to efficiently and rapidly reprogram terminally differentiated AF skin cells and generate induced pluripotent stem cells provides an abundant iPS cell source for various basic studies and a potential for future patient-specific personalized therapies. PMID:20677926
Xiao, Xinye; Luo, Pingping; Zhao, Hui; Chen, Jingyao; He, Hui; Xu, Yuxue; Lin, Zhirong; Zhou, Yueping; Xu, Jianjiang; Liu, Zuguo
Human amniotic membrane (AM) is avascular but contains various beneficial bioactive factors, its extract (AE) is also effective in treating many ocular surface disorders. In this study, we for the first time evaluated the therapeutic effects of AE on dry eye induced by benzalkonium chloride in a BALB/c mouse model. Topical application of AE (1.5 and 3 μg/eye/day) resulted in significantly longer tear break-up time on Day 3 and 6, lower fluorescein staining scores on Day 3, and lower inflammatory index on Day 6. AE reduced corneal epithelial K10 expression, inflammatory infiltration, and levels of TNF-α, IL-1β and IL-6 in BAC treated mice than that in the control mice. Moreover, decreased TUNEL positive cells in cornea and increased goblet cells in conjunctiva were also observed in AE treated corneas. Finally, AE induced more Ki-67 positive cells in corneal epithelium of dry eye mouse. Taken together, our data provide further support for BAC induced dry eye model as a valuable for dry eye study and suggest a great potential for AE as a therapeutic agent in the clinical treatment of dry eye.
Kim, Kyung-Sul; Kim, Hyun Sook; Park, Ji-Min; Kim, Han Wool; Park, Mi-Kyung; Lee, Hyun-Seob; Lim, Dae Seog; Lee, Tae Hee; Chopp, Michael; Moon, Jisook
Amyloid beta (Aβ) plays a major role in Alzheimer's disease (AD), and neuroinflammatory processes mediated by Aβ plaque-induced microglial cells and astrocytes contribute to AD pathogenesis. The present study examined human placenta amniotic membrane-derived mesenchymal stem cells (AMSCs), which have potent immunomodulatory and paracrine effects in a Tg2576 (APPswe) transgenic mouse model of AD. AMSCs secreted high levels of transforming growth factor-β under in vitro inflammatory environment conditions. Six weeks after the intravenous injection of AMSCs, APPswe mice showed evidence of improved spatial learning, which significantly correlated with the observation of fewer Aβ plaques in brain. The number of ED1-positive phagocytic microglial cells associated with Aβ plaques was higher in AMSC-injected mice than in phosphate-buffered saline-injected mice, and the level of Aβ-degrading enzymes (matrix metallopeptidase-9 and insulin-degrading enzyme) was also significantly higher. Furthermore, the level of proinflammatory cytokines, interleukin-1 and tumor necrosis factor-α, was lower and that of anti-inflammatory cytokines, interleukin-10 and transforming growth factor-β, was higher in AMSC-injected mice than phosphate-buffered saline-injected mice. These effects lasted until 12 weeks after AMSC injection. Taken together, these results collectively suggest that injection of AMSCs might show significant long-lasting improvement in AD pathology and memory function via immunomodulatory and paracrine mechanisms.
Cederqvist, L L; Ewool, L C; Bonsnes, R W; Litwin, S D
Immunoglobulin (Ig) M, IgA, and IgD class and IgA, and IgA2 subclass levels were detected in normal amniotic fluid throughout gestation with a hemagglutination-inhibition assay: IgG was measured by single radial immunodiffusion. In 161 tested samples, IgA, IgA, IgA2, and IgG were measurable in all cases; IgM was measurable in 99.4 per cent and IgD, in 90.6 per cent of the fluids. IgA, IgG, and IgD concentrations increased toward midpregnancy and thereafter decreased to term in a pattern similar to that for amniotic fluid total protein. IgM, on the other hand, remained relatively constant through week 35 of gestation; thereafter, it increased to term. There was no correlation in this normal group between amniotic fluid and cord blood levels of Ig class or subclass at term. Mean IgA values were 2.2 times higher in amniotic fluid than in cord serum. This was in sharp contrast to IgM, which was 16 times higher, IgG, which was 66 times higher, and IgD, which was 2.4 times higher in cord serum than in amniotic fluid. The inverse data for IgA as compared to other Ig classes suggest that amniotic fluid IgA may be partially derived from IgA in fetal gastrointestinal and pulmonary secretions. Determination of the concentration of the different Ig classes (and eventually subclasses) in amniotic fluid may be useful in diagnosis of intrauterine infections, malformations, and immunodeficiencies.
Baiguera, Silvia; Gonfiotti, Alessandro; Jaus, Massimo; Comin, Camilla E; Paglierani, Milena; Del Gaudio, Costantino; Bianco, Alessandra; Ribatti, Domenico; Macchiarini, Paolo
To date, only two human laryngeal allotransplants have been reported and, although they were successful, both patients required life-long immunosuppression. A bioengineered human larynx could represent a possible alternative to allotransplantation. Human larynxes were decellularized enzymatically to obtain acellular matrices. Histological and molecular analysis demonstrated that all cellular components and nuclear material were removed. SEM showed that decellularized matrices retained the hierarchical structures of the native larynx, and mechanical tests demonstrated that the decellularization did not significantly impaired the biomechanically properties of the obtained matrices. Immunohistochemical staining found residual angiogenic factors after decellularization, and CAM analysis demonstrated that acellular laryngeal scaffolds induce a strong in vivo angiogenic response. Using a decellularization method, we are now able to obtain, in a short and clinically useful time, natural bioengineered laryngeal scaffolds which could be use for partial or total implantation in humans.
Mahbod, Mirgholamreza; Shahhoseini, Saied; Khabazkhoob, Mehdi; Beheshtnejad, Amir-Houshang; Bakhshandeh, Haleh; Atyabi, Fatemeh; Hashemi, Hassan
Purpose: To compare different preparation methods for a suitable amniotic membrane (AM) extract containing a given amount of growth factors. Methods: In this interventional case series, we dissected the AM from eight placentas within 24 hours after delivery, under clean conditions. After washing and mixing, AM extracts (AMEs) were prepared using pulverization and homogenization methods, and different processing and storing conditions. Main outcome measures were the amount of added protease inhibitor (PI), the relative centrifugal force (g), in-process temperature, repeated extraction times, drying percentage, repeated pulverization times, and the effect of filtering with 0.2 μm filters. Extract samples were preserved at different temperature and time parameters, and analyzed for hepatic growth factor (HGF) and total protein using ELISA and calorimetric methods, respectively. Results: The extracted HGF was 20% higher with pulverization as compared to homogenization, and increased by increasing the PI to 5.0 μl/g of dried AM. Repeating centrifugation up to 3 times almost doubled the extracted HGF and protein. Storing the AME at −170° for 6 months caused a 50% drop in the level of HGF and protein. Other studied parameters showed no significant effect on the extracted amount of HGF or total protein. Conclusion: Appropriate extraction methods with an adequate amount of PI increases the level of extractable components from harvested AMs. To achieve the maximal therapeutic effects of AMEs, it is necessary to consider the half-life of its bioactive components. PMID:25667731
Figueroa-Damián, R; Garduño-Espinosa, J
The diagnosis of intraamniotic infection (IAI) is not difficult when clinical manifestations are present, but there are patients with subclinical infections, in these cases the examination of the amniotic fluid is the most important diagnostic procedure. We made a critical review of the medical literature of diagnostic tests of IAI, according to the analysis criterion of the medical articles of the Department of Clinical Epidemiology of the McMaster University. The articles were identified looking for in the MEDLINE-CD ROOM and INDEX MEDICUS from 1991 to 1995. We identified 19 articles, none of them complied with all of the analysis criterion, none of the studies were blinded nor independently compared with a gold standard test and only five articles studied a full spectrum of patients. The articles with better methodologic design were those that studied the interleukin-6 role as diagnostic test for IAI; they showed a sensibility between 75 to 89% and a specificity of 97 to 100%; nevertheless it is still necessary to standardize the cut-off point of the interleukin-6 levels.
Costantini, Maria; Greif, Gonzalo; Alvarez-Valin, Fernando; Bernardi, Giorgio
Two articles published 5 years ago concluded that the genome of the lizard Anolis carolinensis is an amniote genome without isochores. This claim was apparently contradicting previous results on the general presence of an isochore organization in all vertebrate genomes tested (including Anolis). In this investigation, we demonstrate that the Anolis genome is indeed heterogeneous in base composition, since its macrochromosomes comprise isochores mainly from the L2 and H1 families (a moderately GC-poor and a moderately GC-rich family, respectively), and since the majority of the sequenced microchromosomes consists of H1 isochores. These families are associated with different features of genome structure, including gene density and compositional correlations (e.g., GC3 vs flanking sequence GC and intron GC), as in the case of mammalian and avian genomes. Moreover, the assembled Anolis chromosomes have an enormous number of gaps, which could be due to sequencing problems in GC-rich regions of the genome. In conclusion, the Anolis genome is no exception to the general rule of an isochore organization in the genomes of vertebrates (and other eukaryotes). PMID:26992416
Noureddin, Gelareh S; Yeung, Sonia N
Pterygium is a fibrovascular growth of the bulbar conjunctiva that crosses the limbus and extends over the peripheral cornea, in some cases resulting in significant visual morbidity. When treatment is indicated, surgery is necessary, and several management options exist. These include excision, conjunctival autografting, and the use of adjuvant therapies. This paper reviews the incidence and prevalence of pterygia and also describes the various techniques currently used to treat this condition. These management options are compared to the use of dry amniotic membrane grafting (AMG), specifically with regard to recurrence rates, time to recurrence, safety and tolerability, as well as patient factors including cosmesis and quality of life. AMG has been used in the treatment of ocular surface disease due to a variety of benefits, including its anti-inflammatory properties, as well as its ability to promote epithelial growth and suppress transforming growth factor-β signaling and fibroblast proliferation. However, rates of recurrence for AMG following pterygium excision still surpass other commonly used techniques, including conjunctival and limbal autografting. Nevertheless, there are circumstances in which AMG may be most beneficial to the patient, such as when preexisting conjunctival scarring is present, when the conjunctiva must be spared for future glaucoma filtering surgery, or in cases of large or double-headed pterygia. Therefore, surgeons should be prepared to offer this procedure as an option to their patients for the treatment of pterygia. PMID:27143848
Miller, I.; Hsieh, C.L.; Songster, G.
Extra material was identified on the distal long arm of a chromosome 4 in an amniotic fluid specimen sampled at 16.6 weeks of gestational age. There was no visible loss of material from chromosome 4, and no evidence for a balanced rearrangement. The primary counseling issue in this case was advanced maternal age. Ultrasound findings were normal, and family history was unremarkable. The identical 4qs chromosome was observed in cells from a paternal peripheral blood specimen and appeared to be an unbalanced rearrangement. This extra material was NOR positive in lymphocytes from the father, but was negative in the fetal amniocytes. Father`s relatives were studied to verify the familial origin of this anomaly. In situ hybridization with both exon and intron sequences of ribosomal DNA demonstrated that ribosomal DNA is present at the terminus of the 4qs chromosome in the fetus, father, and paternal grandmother. This satellited 4q might have been derived from a translocation event that resulted in very little or no loss from the 4q and no specific phenotype. This derivative chromosome 4 has been inherited through at least 3 generations of phenotypically normal individuals. 8 refs., 3 figs.
Marongiu, Michela; Serra, Maria Paola; Contini, Antonella; Sini, Marcella; Strom, Stephen C; Laconi, Ezio; Marongiu, Fabio
Amniotic epithelial cells (AEC) derived from human placenta represent a useful and noncontroversial source for liver-based regenerative medicine. Previous studies suggested that human- and rat-derived AEC differentiate into hepatocyte-like cells upon transplantation. In the retrorsine (RS) model of liver repopulation, clusters of donor-derived cells engrafted in the recipient liver and, importantly, showed characteristics of mature hepatocytes. The aim of the current study was to investigate the possible involvement of cell fusion in the emergence of hepatocyte clusters displaying a donor-specific phenotype. To this end, 4-week-old GFP(+)/DPP-IV(-) rats were treated with RS and then transplanted with undifferentiated AEC isolated from the placenta of DPP-IV(+) pregnant rats at 16-19 days of gestational age. Results indicated that clusters of donor-derived cells were dipeptidyl peptidase type IV (DPP-IV) positive, but did not express the green fluorescent protein (GFP), suggesting that rat amniotic epithelial cells (rAEC) did not fuse within the host parenchyma, as no colocalization of the two tags was observed. Moreover, rAEC-derived clusters expressed markers of mature hepatocytes (eg, albumin, cytochrome P450), but were negative for the expression of biliary/progenitor markers (eg, epithelial cell adhesion molecule [EpCAM]) and did not express the marker of preneoplastic hepatic nodules glutathione S-transferase P (GST-P). These results extend our previous findings on the potential of AEC to differentiate into mature hepatocytes and suggest that this process can occur in the absence of cell fusion with host-derived cells. These studies support the hypothesis that amnion-derived epithelial cells can be an effective cell source for the correction of liver disease.
Faye, Justin; Chadee, Annika; Gottimukkala, Sri; Upadhyay, Ruchi; Lara, Carla; Rajegowda, Benamanahalli H.; Corwin, Andrew D.; Lala, Rasila V.; Vernon, Jessica; Nuritdinova, Dilfuza; Chasen, Stephen
Introduction. Amniotic band syndrome and sequence are a relatively rare condition in which congenital anomalies occur as a result of the adherence and entrapment of fetal parts with coarse fibrous bands of the amniotic membrane. A large percentage of reported cases have an atypical gestational history. The frequency of this obstetric complication is not affected by fetal gender, genetic abnormality, or prenatal infection. Case. A 21-year-old, G1P0 female parturient at 18 weeks and 5 days with a single intrauterine gestation during a routine ultrasound evaluation was noted to have amniotic band sequence. The pregnancy was subsequently complicated by preterm premature rupture of membranes with oligohydramnios, resulting in a surviving neonate scheduled for rehabilitative treatment. Conclusion. Amniotic band syndrome is an uncommon congenital anomaly resulting in multiple disfiguring and disabling manifestations. Several theories are proposed with most involving early rupture of the amnion and entanglement of fetal parts by amniotic bands. This syndrome can be manifested by development of multiple malformations, with the majority of the defects being limb abnormalities of a disorganized nature, as in the case we present. In the absence of a clear etiology of consequential congenital abnormalities, obstetric management guidelines should use shared decision models to focus on the quality of life for the offspring. PMID:28025631
Ma, D. H.; See, L.; Liau, S.; Tsai, R. J.
AIM—To study the efficacy and safety of amniotic membrane graft as an adjunctive therapy after removal of primary pterygium, and to compare the clinical outcome with conjunctival autograft and topical mitomycin C. METHODS—80 eyes of 71 patients with primary pterygia were treated with excision followed by amniotic membrane graft. The result was compared retrospectively with 56 eyes of 50 patients receiving conjunctival autograft, and 54 eyes of 46 patients receiving topical mitomycin C. Patients were followed for at least 6 months, and the averaged follow up periods for the three groups were 13.8, 22.8, and 18.4 months, respectively. RESULTS—There were three recurrences (3.8%) in the amniotic membrane graft group, three recurrences (5.4%) in the conjunctival autograft group, and two recurrences (3.7%) in the topical mitomycin C group. There was no significant difference in recurrence rate among the three groups (p = 0.879). No major complications occurred in the amniotic membrane graft group or the conjunctival autograft group. One case of infectious scleritis due to scleral ischaemia occurred in the topical mitomycin C group. CONCLUSION—This study showed that amniotic membrane graft was as effective as conjunctival autograft and mitomycin C in preventing pterygium recurrence, and can be considered as a preferred grafting procedure for primary pterygium. PMID:10966947
Rama, P; Giannini, R; Bruni, A; Gatto, C; Tiso, R; Ponzin, D
Objective: To define the best conditions for amniotic membrane preparation, storage and banking in its use for corneal reconstruction. Methods: Amniotic membrane pieces were prepared under sterile conditions from placentas selected on the basis of donor medical and social history, serology, microbiological tests and histology. The pieces were kept at -140 degrees C but before grafting they were thawed and stored at 4 degrees C in RPMI medium, to have a preparation usable within 72 h. This procedure was validated by testing its therapeutic effectiveness in 25 patients 13 of which had corneal ulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 band keratopathy and 6 corneal stem cell deficiency due to chemical or thermal burns. Results: The preparation showed appreciable anti-inflammatory and analgesic effects. In the absence of corneal stem cell deficiency a stable re-epithelialisation was achieved in 15 out of 19 patients. When the limbus was lesioned, the amniotic membrane decreased vascularization and increased the number of corneal epithelial cells only in 1 of the 6 patients. No adverse reactions attributable to the tissue were recorded. Conclusions: A ready-to-use amniotic membrane preparation stored at 4 degrees C after cryopreservation has been tested in corneal reconstruction. Like the amniotic membrane thawed immediately before grafting, this preparation displayed full therapeutic effect in epithelial defects with stromal ulceration but without severe limbal stem cell deficiency. In two years banking activity 463 pieces of the preparation were successfully distributed to 90 Italian hospitals.
Hawken, Steven; Manuel, Douglas G; Deeks, Shelley L; Kwong, Jeffrey C; Crowcroft, Natasha S; Wilson, Kumanan
The population-level safety benefits of the acellular pertussis vaccine may have been underestimated because only specific adverse events were considered, not overall impact on health services utilization. Using the Vaccine and Immunization Surveillance in Ontario (VISION) system, the authors analyzed data on 567,378 children born between April 1994 and March 1996 (before introduction of acellular pertussis vaccine) and between April 1998 and March 2000 (after introduction of acellular pertussis vaccine) in Ontario, Canada. Using the self-controlled case series study design, they examined emergency room visits and hospital admissions occurring after routine pediatric vaccinations. The authors determined the relative incidence of events taking place before introduction of the acellular vaccine versus after introduction by calculating relative incidence ratios (RIRs). The observed RIRs demonstrated a highly statistically significant reduction in relative incidence after introduction of the acellular vaccine. RIRs for vaccine administered at ages 2, 4, 6, and 18 months were 1.82 (95% confidence interval (CI): 1.64, 2.01), 1.91 (95% CI: 1.71, 2.13), 1.54 (95% CI: 1.38, 1.72), and 1.51 (95% CI: 1.34, 1.69), respectively, comparing event rates before the introduction of acellular vaccine with those after introduction. The authors estimated that approximately 90 emergency room visits and 9 admissions per month were avoided by switching to the acellular vaccine, which is a 38-fold higher impact than when they considered only admissions for febrile and afebrile convulsions. Future analyses comparing vaccines for safety should examine specific endpoints and general health services utilization.
Aghayan, Hamid Reza; Goodarzi, Parisa; Baradaran-Rafii, Alireza; Larijani, Bagher; Moradabadi, Leila; Rahim, Fakher; Arjmand, Babak
Human Amniotic Membrane (AM) transplantation can promote tissue healing and reduce inflammation, tissue scarring and neovascularization. Homa Peyvand Tamin (HPT) tissue bank has focused on manufacturing human cell and tissue based products including AM. The purpose of this study is to evaluate and identify bacterial contamination of AMs that is produced by HPT for several ophthalmic applications. From July 2006 to April 2011, 122 placentas from cesarean sections were retrieved by HPT after obtaining informed consent from the donors. Besides testing donor's blood sample for viral markers, microbiological evaluation was performed pre and post processing. During tissue processing, decontamination was performed by an antibiotic cocktail including; Gentamicin, Ceftriaxone and Cloxacillin. Of 271 cesarean section AM donors who were screened as potential donors, 122 were accepted for processing and assessed for microbiological contamination. Donors' age were between 21 and 41 years (Mean = 27.61 ± 0.24). More than 92% of mothers were in their first or second gravidity with full term pregnancies. The most prevalent organisms were Staphylococci species (72.53%). After processing, contamination rates markedly decreased by 84.62% (p value = 0.013). According to our results, most of bacterial contaminations were related to donation process and the contamination pattern suggests procurement team as a source. Therefore we recommend that regular training programs should be implemented by tissue banks for procurement staff. These programs should focus on improved donor screening and proper aseptic technique for tissue retrieval. We also suggest that tissue banks should periodically check the rate and types of tissue contaminations. These data help them to find system faults and to update processing methods.
Ramachandra, Durrgah L.; Shaw, Steven S. W.; Shangaris, Panicos; Loukogeorgakis, Stavros; Guillot, Pascale V.; Coppi, Paolo De; David, Anna L.
Congenital diseases are responsible for over a third of all pediatric hospital admissions. Advances in prenatal screening and molecular diagnosis have allowed the detection of many life-threatening genetic diseases early in gestation. In utero transplantation (IUT) with stem cells could cure affected fetuses but so far in humans, successful IUT using allogeneic hematopoietic stem cells (HSCs), has been limited to fetuses with severe immunologic defects and more recently IUT with allogeneic mesenchymal stem cell transplantation, has improved phenotype in osteogenesis imperfecta. The options of preemptive treatment of congenital diseases in utero by stem cell or gene therapy changes the perspective of congenital diseases since it may avoid the need for postnatal treatment and reduce future costs. Amniotic fluid stem (AFS) cells have been isolated and characterized in human, mice, rodents, rabbit, and sheep and are a potential source of cells for therapeutic applications in disorders for treatment prenatally or postnatally. Gene transfer to the cells with long-term transgenic protein expression is feasible. Recently, pre-clinical autologous transplantation of transduced cells has been achieved in fetal sheep using minimally invasive ultrasound guided injection techniques. Clinically relevant levels of transgenic protein were expressed in the blood of transplanted lambs for at least 6 months. The cells have also demonstrated the potential of repair in a range of pre-clinical disease models such as neurological disorders, tracheal repair, bladder injury, and diaphragmatic hernia repair in neonates or adults. These results have been encouraging, and bring personalized tissue engineering for prenatal treatment of genetic disorders closer to the clinic. PMID:25566071
Maayan-Metzger, Ayala; Leibovitch, Leah; Schushan-Eisen, Irit; Strauss, Tzipora; Kuint, Jacob
To evaluate whether meconium-stained amniotic fluid (MSAF) is a risk factor for neonatal hypoglycemia. Retrospective recording of medical charts of full-term infants born following observation of meconium-stained amniotic fluid to examine glucose levels in the first hours of life. Out of 803 infants of the study group, 68 (8.5%) had glucose levels lower than 47 mg/dl. Most (6.7%) had mild hypoglycemia, and 14 (1.8%) had moderate or severe hypoglycemia (1.4% and 0.4% respectively). No infant developed clinical signs clearly related to hypoglycemia. Low-risk infants born following meconium-stained amniotic fluid are not at increased risk for neonatal hypoglycemia.
Okpere, E; Okorodudu, A; Gbinigie, O
Estimation of the alkaline phosphates isoenzymes in paired maternal serum and amniotic fluids in term uncomplicated pregnancies and in patients with pre-eclampsia, showed poor correlation coefficients between the levels of both heat stable and heat labile isoenzymes. There was a statistically significant fall in AF (P less than .05) HSAP in pre-eclampsia and a highly significant rise of HLAP in meconial liquor. It is concluded that the poor correlation between the levels of HSAP in maternal serum and amniotic fluid (despite their common source of origin), the normal levels of HLAP in maternal serum in the presence of significantly high levels of HSAP in maternal serum in the presence of significantly diminished levels in amniotic fluid point to a state of relatively diminished permeability of the chorioamniotic membranes to the alkaline phosphatase isoenzymes in Nigerians.
Amniotic Fluid Embolism (AFE) is a catastrophic complication of pregnancy with high mortality rate. The most common clinical presentation is an abrupt onset of cardiopulmonary collapse. Here, we present an uncommon variant involving isolated disseminated intravascular coagulation that developed without antecedent cardiopulmonary disturbances. Both patients developed symptoms soon after delivery. Blood test was sent at 14 minutes postpartum for the second patient due to suspected amniotic fluid embolism. Fetal components were observed in the uterine veins of the lower uterine segments in both cases. Amniotic fluid embolism with disseminated intravascular coagulopathy typically progresses faster than disseminated intravascular coagulopathy associated with other causes and symptoms. It usually develops within two hours of delivery. Prompt recognition and treatment of this entity is crucial to survival. PMID:27891406
Filisetti, Denis; Yera, Hélène; Villard, Odile; Escande, Benoît; Wafo, Estelle; Houfflin-Debarge, Véronique; Delhaes, Laurence; Bastien, Patrick
We evaluated the molecular diagnosis of congenital toxoplasmosis (CT) on neonatal amniotic fluid samples from 488 mother-child pairs. Maternal infection during pregnancy was diagnosed and dated or could not be ruled out. Forty-six cases of CT were defined according to the European Research Network on CT classification system and case definitions. Neonatal amniotic fluid testing had an overall sensitivity of 54% (95% confidence interval [95% CI], 39 to 69%) and a specificity of 100% (95% CI, 99 to 100%). Its sensitivity was 33% (95% CI, 13 to 59%) when antenatal diagnosis was positive and 68% (95% CI, 48 to 84%) when antenatal diagnosis was negative or lacking. This difference in sensitivity may have been due to treatment of antenatally diagnosed cases. Relative to postnatal serology, neonatal amniotic fluid testing allowed an earlier diagnosis to be made in 26% of the cases (95% CI, 9 to 51%).
Filisetti, Denis; Villard, Odile; Escande, Benoît; Wafo, Estelle; Houfflin-Debarge, Véronique; Delhaes, Laurence; Bastien, Patrick
We evaluated the molecular diagnosis of congenital toxoplasmosis (CT) on neonatal amniotic fluid samples from 488 mother-child pairs. Maternal infection during pregnancy was diagnosed and dated or could not be ruled out. Forty-six cases of CT were defined according to the European Research Network on CT classification system and case definitions. Neonatal amniotic fluid testing had an overall sensitivity of 54% (95% confidence interval [95% CI], 39 to 69%) and a specificity of 100% (95% CI, 99 to 100%). Its sensitivity was 33% (95% CI, 13 to 59%) when antenatal diagnosis was positive and 68% (95% CI, 48 to 84%) when antenatal diagnosis was negative or lacking. This difference in sensitivity may have been due to treatment of antenatally diagnosed cases. Relative to postnatal serology, neonatal amniotic fluid testing allowed an earlier diagnosis to be made in 26% of the cases (95% CI, 9 to 51%). PMID:25694528
Loukogeorgakis, Stavros P; De Coppi, Paolo
The amniotic fluid has been identified as an untapped source of cells with broad potential, which possess immunomodulatory properties and don't have the ethical and legal limitations of embryonic stem cells. CD117(c-Kit)+ cells selected from amniotic fluid have been shown to differentiate into cell lineages representing all three embryonic germ layers without generating tumours, making them ideal candidates for regenerative medicine applications. Moreover, their ability to engraft in injured organs and modulate immune and repair responses of host tissues, suggest that transplantation of such cells may be useful for the treatment of various degenerative and inflammatory diseases. Although significant questions remain regarding the origin, heterogeneous phenotype and expansion potential of amniotic fluid stem cells, evidence to date supports their potential role as a valuable stem cell source for the field of regenerative medicine. This article is protected by copyright. All rights reserved.
Background. While the benefits of using acellular dermal matrices (ADMs) in breast reconstruction are well described, their use has been associated with additional complications. The purpose of this study was to determine if ADM thickness affects complications in breast reconstruction. Methods. A retrospective chart review was performed including all tissue expander based breast reconstructions with AlloDerm (LifeCell, Branchburg, NJ) over 4 years. We evaluated preoperative characteristics and assessed postoperative complications including seroma, hematoma, infection, skin necrosis, and need for reintervention. We reviewed ADM thickness and time to Jackson-Pratt (JP) drain removal. Results. Fifty-five patients underwent 77 ADM-associated tissue expander based breast reconstructions, with average age of 48.1 years and average BMI of 25.9. Average ADM thickness was 1.21 mm. We found higher complication rates in the thick ADM group. Significant associations were found between smokers and skin necrosis (p < 0.0001) and seroma and prolonged JP drainage (p = 0.0004); radiated reconstructed breasts were more likely to suffer infections (p = 0.0085), and elevated BMI is a significant predictor for increased infection rate (p = 0.0037). Conclusion. We found a trend toward increased complication rates with thicker ADMs. In the future, larger prospective studies evaluating thickness may provide more information. PMID:27190645
Collis, George N; TerKonda, Sarvam P; Waldorf, James C; Perdikis, Galen
Acellular dermal matrix (ADM) slings in breast reconstruction are increasingly used but are not yet validated. This study compares immediate, expander-based breast reconstruction with and without the use of inferolateral ADM slings. There were 63 patients (106 breasts) in the ADM group and 42 patients (68 breasts) in the control group. Initial intraoperative fill volumes were significantly greater in the ADM group, median 69% full (250 mL) versus 50% full (180 mL; P < 0.001). However, the number of days to complete expansion between the 2 groups was similar. One less office visit was required to complete the fills in the ADM group (P < 0.01). Drains were removed 3 days later in the ADM group (P < 0.01). Overall complication rate was greater in the ADM group (18.9% vs. 7.4%, P < 0.05), with a slightly higher percentage of expanders requiring removal due to infection in the ADM group (5.7% vs. 4.4%, P = NS). This study suggests inferolateral ADM slings in expander-based breast reconstruction allow for significantly increased initial fill volumes and may offer an aesthetic advantage; however, its use is costly and increases complications.
Judith, Rangasamy; Nithya, Mariappan; Rose, Chellan; Mandal, Asit Baran
Biological grafts have drawbacks such as donor scarcity, disease transmission, tissue infection, while the scaffolds of either collagen or chitosan fabrics fail to become part of the tissue at the wound site, though they favor the formation of connective tissue matrix. This study developed a novel composite consisting of the combination of atelocollagen and chitosan in order to provide a biodegradable molecular matrix in gel form as a biomimetic surface for cell attachment, to promote the wound healing in excision wounds. We found that the topical application of biopolymer composite on the wound promoted cell proliferation, migration and collagen deposition overtime. The enhanced cellular activity in the collagen-chitosan treated wound tissue was also assed by increased levels of Platelet derived growth factor (PDGF) and Nerve growth factor (NGF) associated with elevated levels of antioxidants and decreased level of lipid peroxidation. The acellular matrix-like topical application material is designed to guide the eventual re-establishment of an anatomically normal skin. The results of this study demonstrate the feasibility of multi-cell regeneration on a molecular system that mimics tissue engineering in vivo.
Liu, Xinhua; Dan, Nianhua; Dan, Weihua
The objective of this study was to extract and characterize an advanced collagen aggregate (Ag-col) from porcine acellular dermal matrix (pADM). Based on histological examination, scanning electron microscopy (SEM) and atomic force microscope (AFM), Ag-col was composed of the D-periodic cross-striated collagen fibrils and thick collagen fiber bundles with uneven diameters and non-orientated arrangement. Fourier transform infrared (FTIR) spectra of pADM, Ag-col and Col were similar and revealed the presence of the triple helix. Circular dichroism (CD) analysis exhibited a slightly higher content of α-helix but inappreciably less amount of random coil structure in Ag-col compared to Col. Moreover, imino acid contents of pADM, Ag-col and Col were 222.43, 218.30 and 190.01 residues/1000 residues, respectively. From zeta potential analysis, a net charge of zero was found at pH 6.45 and 6.11 for Ag-col and Col, respectively. Differential scanning calorimetry (DSC) study suggested that the Td of Ag-col was 20°C higher than that of Col as expected, and dynamic mechanical analysis (DMA) indicated that Ag-col possessed a higher storage modulus but similar loss factor compared to Col. Therefore, the collagen aggregate from pADM could serve as a better alternative source of collagens for further applications in food and biological industries.
Pawloski, Lucia C; Kirkland, Kathryn B; Baughman, Andrew L; Martin, Monte D; Talbot, Elizabeth A; Messonnier, Nancy E; Tondella, Maria Lucia
An anti-pertussis toxin (PT) IgG enzyme-linked immunosorbent assay (ELISA) was analytically validated for the diagnosis of pertussis at a cutoff of 94 ELISA units (EU)/ml. Little was known about the performance of this ELISA in the diagnosis of adults recently vaccinated with tetanus-diphtheria-acellular pertussis (Tdap) vaccine, which contains PT. The goal of this study was to determine when the assay can be used following Tdap vaccination. A cohort of 102 asymptomatic health care personnel (HCP) vaccinated with Tdap (Adacel; Sanofi Pasteur) were aged 19 to 79 years (median, 47 years) at vaccination. For each HCP, specimens were available for evaluation at 2 to 10 time points (prevaccination to 24 months postvaccination), and geometric mean concentrations (GMC) for the cohort were calculated at each time point. Among 97 HCP who responded to vaccination, a mixed-model analysis with prediction and tolerance intervals was performed to estimate the time at which serodiagnosis can be used following vaccination. The GMCs were 8, 21, and 9 EU/ml at prevaccination and 4 and 12 months postvaccination, respectively. Eight (8%) of the 102 HCP reached antibody titers of ≥94 EU/ml during their peak response, but none had these titers by 6 months postvaccination. The calculated prediction and tolerance intervals were <94 EU/ml by 45 and 75 days postvaccination, respectively. Tdap vaccination 6 months prior to testing did not confound result interpretation. This seroassay remains a valuable diagnostic tool for adult pertussis.
Latty, Tanya; Beekman, Madeleine
Speed-accuracy trade-offs (SATs) are thought to be a fundamental feature of biological information processing, yet most evidence of SATs comes from animals. Here, we examine SATs in the foraging decisions of an acellular, amoeboid organism: the slime mould Physarum polycephalum. Slime moulds were given a simple discrimination task: selecting the highest-quality food item from a set of three options. We investigated the effect of two stressors, light exposure and hunger, on the speed and accuracy of decision-making. We also examined the effect of task difficulty. When given a difficult discrimination task, stressed individuals tend to make faster decisions than non-stressed individuals. This effect was reversed in plasmodia given easy discrimination tasks, where stressed individuals made slower decisions than non-stressed individuals. We found evidence of SATs, such that individuals who made fast decisions were more likely to make costly errors by selecting the worst possible food option. Our results suggest that SATs occur in a wider range of taxa than previously considered.
O'Halloran, Alissa C; Lu, Peng-Jun; Williams, Walter W; Ding, Helen; Meyer, Sarah A
The incidence of pertussis in the United States has increased since the 1990s. Tetanus, diphtheria, and acellular pertussis (Tdap) vaccination of pregnant women provides passive protection to infants. Tdap vaccination is currently recommended for pregnant women during each pregnancy, but coverage among pregnant women and women of childbearing age has been suboptimal. Data from the 2013 Behavioral Risk Factor Surveillance System (BRFSS) and 2013 National Health Interview Survey (NHIS) were used to determine national and state-specific Tdap vaccination coverage among women of childbearing age by self-reported pregnancy status at the time of the survey. Although this study could not assess coverage of Tdap vaccination received during pregnancy because questions on whether Tdap vaccination was received during pregnancy were not asked in BRFSS and NHIS, demographic and access-to-care factors associated with Tdap vaccination coverage in this population were assessed. Tdap vaccination coverage among all women 18-44 years old was 38.4% based on the BRFSS and 23.3% based on the NHIS. Overall, coverage did not differ by pregnancy status at the time of the survey. Coverage among all women 18-44 years old varied widely by state. Age, race and ethnicity, education, number of children in the household, and access-to-care characteristics were independently associated with Tdap vaccination in both surveys. We identified associations of demographic and access-to-care characteristics with Tdap vaccination that can guide strategies to improve vaccination rates in women during pregnancy.
Ibrahim, Ahmed M. S.; Koolen, Pieter G. L.; Ashraf, Azra A.; Kim, Kuylhee; Mureau, Marc A. M.; Lee, Bernard T.
Background: Acellular dermal matrices (ADMs) in plastic surgery have become increasingly popular particularly for breast reconstruction. Despite their advantages, questions exist regarding their association with a possible increased incidence of complications. We describe a collective experience of plastic surgeons’ use of ADMs in reconstructive breast surgery using an internet-based survey. Methods: Members of the American Society of Plastic Surgeons were recruited through voluntary, anonymous participation in an online survey. The web-based survey garnered information about participant demographics and their experience with ADM use in breast reconstruction procedures. After responses were collected, all data were anonymously processed. Results: Data were ascertained through 365 physician responses of which 99% (n = 361) completed the survey. The majority of participants were men (84.5%) between 51 and 60 years (37.4%); 84.2% used ADM in breast reconstruction, including radiated patients (79.7%). ADM use was not favored for nipple reconstruction (81.5%); 94.6% of participants used drains, and 87.8% administered antibiotics postoperatively. The most common complications were seroma (70.9%) and infection (16%), although 57.4% claimed anecdotally that overall complication rate was unchanged after incorporating ADM into their practice. High cost was a deterrent for ADM use (37.5%). Conclusions: Plastic surgeons currently use ADM in breast reconstruction for both immediate and staged procedures. Of those responding, a majority of plastic surgeons will incorporate drains and use postoperative antibiotics for more than 48 hours. PMID:25973359
Liu, Zhao; Zhou, Qiang; Zhu, Jixiang; Xiao, Jianhui; Wan, Pengxia; Zhou, Chenjing; Huang, Zheqian; Qiang, Na; Zhang, Wei; Wu, Zheng; Quan, Daping; Wang, Zhichong
Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4 h at 37 °C, followed by lyophilization at -10 °C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7 ± 4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7 ± 4.6% and 23.01 ± 3.45 m(2)/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5 min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.
Li, Jia; Wang, Wenguang; Li, Jiuzhi; Rexiati, Mulati; An, Henqing; Wang, Feng; Wang, Yujie
Abstract Background: Hair follicle stem cells (HFSCs) were reported to have multidirectional differentiation ability and could be differentiated into melanocytes, keratin cells, smooth muscle cells, and neurons. However, the functionality of HFSCs in bladder tissue regeneration is unknown. Methods: This study was conducted to build HFSCs vs bladder acellular matrix (BAM) complexes (HFSCs–BAM complexes) in vitro and evaluated whether HFSCs have well biocompatibility with BAM. HFSCs were separated from SD rats. BAM scaffold was prepared from the submucosa of rabbit bladder tissue. Afterwards, HFSCs were inoculated on BAM. Results: HFSCs–BAM complexes grew rapidly through inverted microscope observation. Cell growth curve showed the proliferation was in stagnate phase at 7th and 8th day. Cytotoxicity assay showed the toxicity grading of BAM was 0 or 1. Scanning electron microscopy, HE staining, and masson staining showed that cells have germinated on the surface of scaffold. Conclusion: The results provide evidence that HFSCs–BAM complexes have well biocompatibility and accumulate important experimental basis for clinical applying of tissue engineering bladder. PMID:27828841
Stapleton, Thomas W; Ingram, Joanne; Katta, Jaynath; Knight, Richard; Korossis, Sotirios; Fisher, John; Ingham, Eileen
The objectives of this study were to characterize fresh porcine menisci and develop a decellularization protocol with a view to the generation of a biocompatible and biomechanically functional scaffold for use in tissue engineering/regeneration of the meniscus. Menisci were decellularized by exposing the tissue to freeze-thaw cycles, incubation in hypotonic tris buffer, 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors, nucleases, hypertonic buffer followed by disinfection using 0.1% (v/v) peracetic acid and final washing in phosphate-buffered saline. Histological, immunohistochemical, and biochemical analyses of the decellularized tissue confirmed the retention of the major structural proteins. There was, however, a 59.4% loss of glycosaminoglycans. The histoarchitecture was unchanged, and there was no evidence of the expression of the major xenogeneic epitope, galactose-alpha-1,3-galactose. Biocompatibility of the acellular scaffold was determined by using contact cytotoxicity and extract cytotoxicity tests. Decellularized tissue and extracts were not cytotoxic to cells. Biomechanical properties were determined by indentation and tensile tests, which confirmed the retention of biomechanical properties following decellularization. In conclusion, this study has generated data on the production of a biocompatible, biomechanically functional scaffold for use in meniscal repair.
Di Liddo, Rosa; Aguiari, Paola; Barbon, Silvia; Bertalot, Thomas; Mandoli, Amit; Tasso, Alessia; Schrenk, Sandra; Iop, Laura; Gandaglia, Alessandro; Parnigotto, Pier Paolo; Conconi, Maria Teresa; Gerosa, Gino
Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality. PMID:27789941
Notifications of infant deaths, assumed to be related to the introduction of new pentavalent DTwP-Hib-HBV childhood vaccines, caused, during 2008-2010 in few Asian countries, temporary interruptions of the respective vaccination programs. The sudden appearance of fatal cases was due to increased awareness/publicity and improved safety monitoring/reporting in countries with relatively high background infant mortalities. WHO investigations could not establish any causal relationships and vaccinations were again resumed. Recently, questions were raised in one concerned country as to why not to change to less reactogenic acellular pertussis (aP)-containing vaccines that are available in private practice and are generally perceived as 'better'. For resource-poor countries, the financial impacts render such a switch impossible and would also not be supported by external funding. Furthermore, it would be a disservice to the children, as in recent years evidence of inferior long-term efficacy of aP vaccines has accumulated. This report summarizes current knowledge on comparative whole-cell pertussis (wP) and aP vaccine performance, outlines the new July 2014 WHO guidance on the choice of pertussis vaccines and presents recent data on outbreak protection, antibody waning, long-term protection, wP-priming, pathogen adaptation, transmission and herd immunity.
Hata, Toshiyuki; Tanaka, Hirokazu; Noguchi, Junko
We present two cases of amniotic band syndrome diagnosed using two-dimensional (2D) ultrasound with three-dimensional (3D)/four-dimensional (4D) ultrasound in early pregnancy. In Case 1, at 13 weeks' gestation, multiple amniotic bands, acrania, the absence of fingers and amputation of the toes bilaterally were clearly shown using transvaginal 3D/4D ultrasound. In Case 2, at 15 weeks' gestation, several amniotic bands, acrania and a cleft lip were depicted with transabdominal 3D/4D ultrasound. The spatial relationship between the amniotic bands and the fetus was clearly visualized and easily discernible by 3D/4D ultrasound. The parents and families could readily understand the fetal conditions and undergo counseling; they then choose the option of termination of pregnancy. 3D/4D ultrasound has the potential to be a supplement to conventional 2D ultrasound in evaluating amniotic band syndrome.
Baulier, Edouard; Favreau, Frederic; Le Corf, Amélie; Jayle, Christophe; Schneider, Fabrice; Goujon, Jean-Michel; Feraud, Olivier; Bennaceur-Griscelli, Annelise; Hauet, Thierry; Turhan, Ali G
It is well known that ischemia/reperfusion injuries strongly affect the success of human organ transplantation. Development of interstitial fibrosis and tubular atrophy is the main deleterious phenomenon involved. Stem cells are a promising therapeutic tool already validated in various ischemic diseases. Amniotic fluid-derived mesenchymal stem cells (af-MSCs), a subpopulation of multipotent cells identified in amniotic fluid, are known to secrete growth factors and anti-inflammatory cytokines. In addition, these cells are easy to collect, present higher proliferation and self-renewal rates compared with other adult stem cells (ASCs), and are suitable for banking. Consequently, af-MSCs represent a promising source of stem cells for regenerative therapies in humans. To determine the efficiency and the safety of af-MSC infusion in a preclinical porcine model of renal autotransplantation, we injected autologous af-MSCs in the renal artery 6 days after transplantation. The af-MSC injection improved glomerular and tubular functions, leading to full renal function recovery and abrogated fibrosis development at 3 months. The strong proof of concept generated by this translational porcine model is a first step toward evaluation of af-MSC-based therapies in human kidney transplantation.
Jones, Janice; Gunst, Phillip R.; Kacerovsky, Marian; Fortunato, Stephen J.; Saade, George R.; Basraon, Sanmaan
Objective: To identify metabolic changes associated with early spontaneous preterm birth (PTB; <34 weeks) and term births, using high-throughput metabolomics of amniotic fluid (AF) in African American population. Method: In this study, AF samples retrieved from spontaneous PTB (<34 weeks [n = 25]) and normal term birth (n = 25) by transvaginal amniocentesis at the time of labor prior to delivery were subjected to metabolomics analysis. Equal volumes of samples were subjected to a standard solvent extraction method and analyzed using gas chromatography/mass spectrometry (MS) and liquid chromatography/MS/MS. Biochemicals were identified through matching of ion features to a library of biochemical standards. After log transformation and imputation of minimum observed values for each compound, t test, correlation tests, and false discovery rate corrections were used to identify differentially regulated metabolites. Data were controlled for clinical/demographic variables and medication during pregnancy. Results: Of 348 metabolites measured in AF samples, 121 metabolites had a gestational age effect and 116 differed significantly between PTB and term births. A majority of significantly altered metabolites could be classified into 3 categories, namely, (1) liver function, (2) fatty acid and coenzyme A (CoA) metabolism, and (3) histidine metabolism. The signature of altered liver function was apparent in many cytochrome P450-related pathways including bile acids, steroids, xanthines, heme, and phase II detoxification of xenobiotics with the largest fold change seen with pantothenol, a CoA synthesis inhibitor that was 8-fold more abundant in PTB. Conclusion: Global metabolic profiling of AF revealed alteration in hepatic metabolites involving xenobiotic detoxification and CoA metabolism in PTB. Maternal and/or fetal hepatic function differences may be developmentally related and its contribution PTB as a cause or effect of PTB is still unclear. PMID:24440995
Wu, Ping; Hou, Lianhai; Plikus, Maksim; Hughes, Michael; Scehnet, Jeffrey; Suksaweang, Sanong; Widelitz, Randall B.; Jiang, Ting-Xin; Chuong, Cheng-Ming
Integuments form the boundary between an organism and the environment. The evolution of novel developmental mechanisms in integuments and appendages allows animals to live in diverse ecological environments. Here we focus on amniotes. The major achievement for reptile skin is an adaptation to the land with the formation of a successful barrier. The stratum corneum enables this barrier to prevent water loss from the skin and allowed amphibian/reptile ancestors to go onto the land. Overlapping scales and production of β-keratins provide strong protection. Epidermal invagination led to the formation of avian feather and mammalian hair follicles in the dermis. Both adopted a proximal - distal growth mode that maintains endothermy. Feathers form hierarchical branches which produce the vane that makes flight possible. Recent discoveries of feathered dinosaurs in China inspire new thinking on the origin of feathers. In the laboratory, epithelial - mesenchymal recombinations and molecular mis-expressions were carried out to test the plasticity of epithelial organ formation. We review the work on the transformation of scales into feathers, conversion between barbs and rachis, and the production of “chicken teeth”. In mammals, tilting the balance of the BMP pathway in K14 noggin transgenic mice alters the number, size and phenotypes of different ectodermal organs, making investigators rethink the distinction between morpho-regulation and pathological changes. Models on the evolution of feathers and hairs from reptile integuments are discussed. A hypothetical Evo-Devo space where diverse integument appendages can be placed according to complex phenotypes and novel developmental mechanisms is presented. PMID:15272390
HASSAN, SONIA; ROMERO, ROBERTO; HENDLER, ISRAEL; GOMEZ, RICARDO; KHALEK, NAHLA; ESPINOZA, JIMMY; NIEN, JYH KAE; BERRY, STANLEY M.; BUJOLD, EMMANUEL; CAMACHO, NATALIA; SOROKIN, YORAM
OBJECTIVE A sonographically short cervix is a powerful predictor of spontaneous preterm delivery. However, the etiology and optimal management of a patient with a short cervix in the mid-trimester of pregnancy remain uncertain. Microbial invasion of the amniotic cavity (MIAC) and intra-amniotic inflammation are frequently present in patients with spontaneous preterm labor or acute cervical insufficiency. This study was conducted to determine the rate of MIAC and intra-amniotic inflammation in patients with a cervical length <25 mm in the mid-trimester. STUDY DESIGN A retrospective cohort study was conducted of patients referred to our high risk clinic because of a sonographic short cervix or a history of a previous preterm birth. Amniocenteses were performed for the evaluation of MIAC and for karyotype analysis in patients with a short cervix. Fluid was cultured for aerobic and anaerobic bacteria, as well as genital mycoplasmas. Patients with MIAC were treated with antibiotics selected by their physician. RESULTS Of 152 patients with a short cervix at 14–24 weeks, 57 had amniotic fluid analysis. The prevalence of MIAC was 9% (5/57). Among these patients, the rate of preterm delivery (<32 weeks) was 40% (2/5). Microorganisms isolated from amniotic fluid included Ureaplasma urealyticum (n=4) and Fusobacterium nucleatum (n=1). Patients with a positive culture for Ureaplasma urealyticum received intravenous Azithromycin. Three patients with Ureaplasma urealyticum had a sterile amniotic fluid culture after treatment, and subsequently delivered at term. The patient with Fusobacterium nucleatum developed clinical chorioamnionitis and was induced. CONCLUSION 1) Sub-clinical MIAC was detected in 9% of patients with a sonographically short cervix (<25 mm); and 2) maternal parenteral treatment with antibiotics can eradicate MIAC caused by Ureaplasma urealyticum. This was associated with delivery at term in the three patients whose successful treatment was documented by
Barrett, Bridget M.
The purpose of this study was to examine the status of antioxidant vitamins in women with premature rupture of the fetal membranes. Specimens of blood and amniotic fluid were obtained from 80 pregnant subjects included both smokers and non-smokers during the third trimester. The concentrations of ascorbic acid (ASA), beta -carotene, retinol and alpha -tocopherol in serum and amniotic fluid were determined. The experimental group consisted of those subjects with PROM while the control subjects were those with normal pregnancy. No statistical differences were found between the PROM and control groups in retinol and vitamin E concentrations in amniotic fluid and serum. Serum ASA concentrations of PROM subjects were not different from controls, but the PROM subjects had significantly lower amniotic fluid ASA concentrations. However, in a study with fewer subjects a lower serum ASA concentration in the PROM subjects was observed. The ratio of amniotic fluid ASA concentration to ASA serum concentration was significantly lower in PROM patients than in controls in both studies. This suggests that low levels of ASA in the amniotic fluid, but not in serum is better associated with PROM. A low amniotic fluid concentration of ASA may reflect an inefficient transfer and/or increased fetal utilization. Alterations in ASA concentration in the amniotic fluid may affect the integrity of the chorioamnion leading to PROM. beta -Carotene was not found in the amniotic fluid. Serum beta-carotene levels were significantly lower in the PROM group compared to the control group. Low concentrations of beta-carotene in maternal serum in smokers not only associated with poor maternal outcome (PROM) but also compromised the fetal outcome (decreased birth weight). Maintenance of adequate serum beta-carotene concentration and amniotic fluid ASA in smokers may result in better maternal and fetal outcome. This study demonstrated that nutrition is an important factor in the prevention of PROM.
Cortez-Ortega, Carolina; Flores-Velázquez, Joselín; Ruiz-Rodríguez, Socorro; Noyola-Frías, Miguel Ángel; Santos-Díaz, Miguel Ángel
Amniotic Band Syndrome (ABS) is a group of congenital malformations that includes the majority of typical constriction rings and limb and digital amputations, together with major craniofacial, thoracic, and abdominal malformations. The syndrome is caused by early rupture of the amniotic sac. Some of the main oral manifestations include micrognathia, hyperdontia, and cleft lip with or without cleft palate, which is present in 14.6% of patients with this syndrome. The purpose of this report was to describe the clinical characteristics and the oral treatment provided to a 6-month-old male patient affected with ABS with cleft lip and palate. PMID:28246561
Allen, Claire L.; Clare, Gerry; Stewart, Elizabeth A.; Branch, Matthew J.; McIntosh, Owen D.; Dadhwal, Megha; Dua, Harminder S.; Hopkinson, Andrew
Purpose Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material. Methods AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays. Results Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM. Conclusions Our modified preservation process and our resultant optimised dried AM has enhanced structural properties
Phermthai, Tatsanee; Suksompong, Singpetch; Tirawanchai, Nednapis; Issaragrisil, Surapol; Julavijitphong, Suphakde; Wichitwiengrat, Suparat; Silpsorn, Decha; Pokathikorn, Puttachart
Amniotic fluid stem cells (AFSs) are interesting mesenchymal stem cells (MSCs) that are characterized by their great potential for cell proliferation and differentiation compared with other types of MSCs identified to date. However, MSCs in prolonged culture have been found to exhibit defects in genetic stability and differentiation capacity. Epigenetic anomalies have been hypothesized to be a cause of these defects. Here, we investigated the genomic methylation and genetic imprinting in AFSs during prolonged in vitro culture. Four human imprinted genes, insulin-like growth factor 2 (IGF2), H19, small nuclear ribonucleoprotein polypeptide N gene (SNRPN), and mesoderm-specific transcript (MEST), were evaluated for their expression levels and methylation statuses in AFS lines. The data revealed epigenetic instability in high passage number AFS cultures. The real-time polymerase chain reaction analysis showed that the expression levels of the imprinted genes gradually increased with increased time in culture. The loss of parental allele-specific imprinting for at least 1 gene among IGF2, H19, and SNRPN was observed in every AFS line after passage 8 using allelic expression analysis. The imprinting control regions (ICRs) of the IGF2 and H19 genes were assayed for site-specific methylation using bisulfite sequencing. This assay revealed a variable level of methylated CpG sites in the ICRs of IGF2 and H19. This variable level of CpG methylation is related to the aberrant expression of the IGF2 and H19 genes in late-passage AFSs. Our results did not reveal any irregularity in the epigenetic control system in the early-passage AFSs, indicating that the standard in vitro culturing of AFSs used in medical treatments should be limited to 8 passages.
The purpose of this study was to investigate the carbodiimide cross-linking of amniotic membrane (AM) in the presence of amino acid bridges. The biological tissues were treated with glycine, lysine, or glutamic acid and chemically cross-linked to examine the role of amino acid types in collagenous biomaterial processing. Results of zeta potential measurements showed that the use of uncharged, positively and negatively charged amino acids dictates the charge state of membrane surface. Tensile strength and water content measurements demonstrated that the addition of lysine molecules to the cross-linking system can increase the cross-linking efficiency and dehydration degree while the introduction of glutamic acid in the AM samples decreases the number of cross-links per unit mass of chemically modified tissue collagen. The differences in the cross-linking density further determined the thermal and biological stability by differential scanning calorimetry and in vitro degradation tests. As demonstrated in matrix permeability studies, the improved formation of covalent cross-linkages imposed by lysine facilitated construction of stronger cross-linking structures. In contrast, the added glycine molecules were insufficient to enhance the resistances of the proteinaceous matrices to thermal denaturation and enzymatic degradation. The cytocompatibility of these biological tissue membranes was evaluated by using human corneal epithelial cell cultures. Results of cell viability, metabolic activity, and pro-inflammatory gene expression level showed that the AM materials cross-linked with carbodiimide in the presence of different types of amino acids are well tolerated without evidence of detrimental effect on cell growth. In addition, the amino acid treated and carbodiimide cross-linked AM implants had good biocompatibility in the anterior chamber of the rabbit eye model. Our findings suggest that amino acid type is a very important engineering parameter to mediate
Acellular allogeneic nerve grafting combined with bone marrow mesenchymal stem cell transplantation for the repair of long-segment sciatic nerve defects: biomechanics and validation of mathematical models
Li, Ya-jun; Zhao, Bao-lin; Lv, Hao-ze; Qin, Zhi-gang; Luo, Min
We hypothesized that a chemically extracted acellular allogeneic nerve graft used in combination with bone marrow mesenchymal stem cell transplantation would be an effective treatment for long-segment sciatic nerve defects. To test this, we established rabbit models of 30 mm sciatic nerve defects, and treated them using either an autograft or a chemically decellularized allogeneic nerve graft with or without simultaneous transplantation of bone marrow mesenchymal stem cells. We compared the tensile properties, electrophysiological function and morphology of the damaged nerve in each group. Sciatic nerves repaired by the allogeneic nerve graft combined with stem cell transplantation showed better recovery than those repaired by the acellular allogeneic nerve graft alone, and produced similar results to those observed with the autograft. These findings confirm that a chemically extracted acellular allogeneic nerve graft combined with transplantation of bone marrow mesenchymal stem cells is an effective method of repairing long-segment sciatic nerve defects. PMID:27651781
Park, Gui-Yong; Cho, Hee-Eun; Lee, Byung-Il; Park, Seung-Ha
Background The objective of this paper was to describe a novel technique for improving the maintenance of nipple projection in primary nipple reconstruction by using acellular dermal matrix as a strut in one of three different configurations, according to the method of prior breast reconstruction. The struts were designed to best fill the different types of dead spaces in nipple reconstruction depending on the breast reconstruction method. Methods A total of 50 primary nipple reconstructions were performed between May 2012 and May 2015. The prior breast reconstruction methods were latissimus dorsi (LD) flap (28 cases), transverse rectus abdominis myocutaneous (TRAM) flap (10 cases), or tissue expander/implant (12 cases). The nipple reconstruction technique involved the use of local flaps, including the C-V flap or star flap. A 1×2-cm acellular dermal matrix was placed into the core with O-, I-, and L-shaped struts for prior LD, TRAM, and expander/implant methods, respectively. The projection of the reconstructed nipple was measured at the time of surgery and at 3, 6, and 9 months postoperatively. Results The nine-month average maintenance of nipple projection was 73.0%±9.67% for the LD flap group using an O-strut, 72.0%±11.53% for the TRAM flap group using an I-strut, and 69.0%±10.82% for the tissue expander/implant group using an L-strut. There were no cases of infection, wound dehiscence, or flap necrosis. Conclusions The application of an acellular dermal matrix with a different kind of strut for each of 3 breast reconstruction methods is an effective addition to current techniques for improving the maintenance of long-term projection in primary nipple reconstruction. PMID:27689049
Harrington, John P; Orlik, Kseniya; Orlig, Kseniya; Zito, Samantha L; Wollocko, Jacek; Wollocko, Hanna
A zero-linked polymeric hemoglobin (OxyVita Hb) has been developed for application as an acellular therapeutic hemoglobin-based-oxygen-carrier (HBOC). For effective and safe oxygen binding, transport and delivery, an HBOC must meet essential molecular requirements related to its structural integrity and redox stability. OxyVita is a super polymer possessing an average M.wt. of 17 x 10(6) Da. Structural integrity was determined by unfolding studies of OxyVita in the presence of increasing concentrations of urea. The unfolding midpoints (D(1/2)) of different preparations of OxyVita (solution and powder forms) were compared to Lumbricus Hb (LtHb) and Arenicola Hb (ArHb), natural acellular polymeric hemoglobins, which are serving as models for an effective and safe acellular HBOC. Reduction studies of OxyVita Hb using endogenous reducing agents were also investigated. Results from these studies indicate that: 1) OxyVita Hb exhibits greater resistance to conformational change than either LtHb or ArHb in the reduced (oxyHb) state; and 2) the reduction of met OxyVita Hb to oxyHb occurs slowly in the presence of either ascorbic acid (70% reduction in 560 min.) or beta-NADH (40% reduction in 90 min.). These studies provide consistent evidence that OxyVita Hb possesses physiochemical properties that exhibit structural integrity and redox behavior necessary for functioning as an effective and safe HBOC within clinical applications. These results are in agreement with observations made by other investigators as to the reduction in heme-loss of OxyVita Hb, essential for the reversible binding/release of molecular oxygen within the circulatory system.
Butterworth, James; Petty, Paul
Objective: This study seeks an alternative to acellular dermal matrix in 2-staged breast reconstruction while minimizing cost. It was hypothesized that use of a Gore DualMesh would allow for similar intraoperative tissue expander fill volumes, time to second-stage reconstruction, and number of postoperative fills compared with acellular dermal matrix at only a fraction of the expense. Methods: Retrospective analysis comparing Gore DualMesh (59 breasts, 34 patients), acellular dermal matrix (13 breasts, 8 patients), and total muscle coverage (25 breasts, 14 patients) for postmastectomy breast reconstruction was performed. Time to second-stage reconstruction, number of expansions, and relative initial fill volumes were compared between the 3 groups. Secondarily, complication rates were also considered, including seroma, infection, expander/implant explantation, removal of mesh, and capsular contracture. Statistical analysis was performed utilizing the Fisher exact test and the χ2 test for categorical variables and the Mann-Whitney U test for continuous variables. Results: Relative initial fill volumes, number of expansions, and time to second-stage reconstruction showed no statistical difference between the acellular dermal matrix and Gore DualMesh groups (P = .494, P = .146, and P = .539, respectively). Furthermore, the Gore DualMesh group underwent significantly fewer fills (P < .001) and had a higher relative initial fill volume (P < .001) than the total muscle coverage group. The additional cost per breast as a result of including DualMesh was on average $385 versus $4287 for acellular dermal matrix. Complication rates were similar between all 3 groups without statistically significant differences. Conclusions: Gore DualMesh represents a safe alternative to acellular dermal matrix for breast reconstruction with similar aesthetic results in certain patients at a fraction of the cost. PMID:28261372
Introduction Synthetic mesh has been used traditionally to repair abdominal wall defects, but its use is limited in the case of bacterial contamination. New biological materials are now being used successfully for delayed primary closure of contaminated abdominal wall defects. The costs of biological materials may prevent surgeons from using them. We compared the conventional staged repair of contaminated abdominal wall defects with a single-stage procedure using a non-crosslinked porcine acellular dermal matrix. Methods A total of 14 cases with Grade 3 contaminated abdominal wall defects underwent delayed primary closure of the abdomen using a non-crosslinked porcine acellular dermal matrix (Strattice™ Reconstructive Tissue Matrix, LifeCell Corp., Branchburg, NJ, USA). The results were compared with a group of 14 patients who had received conventional treatment for the repair of contaminated abdominal wall defects comprising a staged repair during two separate hospital admissions employing synthetic mesh. Treatment modalities, outcomes, and costs were compared. Results In all cases treated with delayed primary closure employing non-crosslinked porcine acellular dermal matrix, there were no complications related to its use. Two patients died due to unrelated events. Although treatment costs were estimated to be similar in the two groups, the patients treated with porcine acellular dermal matrix spent less time as an inpatient than those receiving conventional two-stage repair. Conclusions Delayed primary closure of contaminated abdominal wall defects using a non-crosslinked porcine acellular dermal matrix may be a suitable alternative to conventional staged repair. In our patients, it resulted in early restoration of abdominal wall function and shorter hospitalization. The costs for treating contaminated abdominal wall defects using porcine acellular dermal matrix during a single hospital admission were not higher than costs for conventional two-stage repair
Hadad, Ivan; Liu, Allen S.
Background: Acellular dermal matrices (ADMs) are often used to improve lower-pole contour, as well as allow for single-stage reconstruction, but numerous studies have shown an increased complication rate using ADM. As such, our group has developed a minimal-ADM-use technique to lower complications while effectively recreating lower-pole contour. Methods: A total of 380 postmastectomy prosthesis-based breast reconstructions were performed in 265 patients by a single surgeon. One hundred eight reconstructions were performed using the traditional ADM technique, with a large piece of ADM along the entire inferior and lateral borders. Two hundred twenty-five reconstructions were performed with the minimal-use technique, patching only the lateral area of the reconstruction. Thirty-five reconstructions were performed without the use of any ADM for high-risk reconstructions, most often in morbidly obese patients. Results: Comparing the traditional technique with the minimal-use technique, the seroma rate dropped from 3% to 0%. The rate of infection and reconstruction loss fell from 9% to 1%. Upon greatly reducing or eliminating the use of ADM use in obese patients, the seroma rate decreased from 15.4% to 5.7%, and the reconstruction loss rate decreased from 38% to 9%. Conclusions: This article describes a new surgical approach to minimize the amount of ADM necessary to create an aesthetically pleasing breast reconstruction. We believe that this approach helps avoid the complications of seroma, infection, and loss of the reconstruction. In certain obese patients, total avoidance of ADM may be the better choice. PMID:26301161
Poppler, Louis; Cohen, Justin; Dolen, Utku Can; Schriefer, Andrew E.; Tenenbaum, Marissa M.; Deeken, Corey; Chole, Richard A.; Myckatyn, Terence M.
Background Subclinical infections, manifest as biofilms, are considered an important cause of capsular contracture. Acellular dermal matrices (ADMs) are frequently used in revision surgery to prevent recurrent capsular contractures. Objective We sought to identify an association between capsular contracture and biofilm formation on breast prostheses, capsules, and ADMs in a tissue expander/implant (TE/I) exchange clinical paradigm. Methods Biopsies of the prosthesis, capsule, and ADM from patients (N = 26) undergoing TE/I exchange for permanent breast implant were evaluated for subclinical infection. Capsular contracture was quantified with Baker Grade and intramammary pressure. Biofilm formation was evaluated with specialized cultures, rtPCR, bacterial taxonomy, live:dead staining, and scanning electron microscopy (SEM). Collagen distribution, capsular histology, and ADM remodeling were quantified following fluorescent and light microscopy. Results Prosthetic devices were implanted from 91 to 1115 days. Intramammary pressure increased with Baker Grade. Of 26 patients evaluated, one patient had a positive culture and one patient demonstrated convincing evidence of biofilm morphology on SEM. Following PCR amplification 5 samples randomly selected for 16S rRNA gene sequencing demonstrated an abundance of suborder Micrococcineae, consistent with contamination. Conclusions Our data suggest that bacterial biofilms likely contribute to a proportion, but not all diagnosed capsular contractures. Biofilm formation does not appear to differ significantly between ADMs or capsules. While capsular contracture remains an incompletely understood but common problem in breast implant surgery, advances in imaging, diagnostic, and molecular techniques can now provide more sophisticated insights into the pathophysiology of capsular contracture. Level of Evidence PMID:26229126
Salibian, Ara A.; Frey, Jordan D.; Choi, Mihye
Background: The availability of acellular dermal matrix (ADM) and synthetic mesh products has prompted plastic surgeons to revisit subcutaneous implant-based breast reconstruction. The literature is limited, however, with regards to evidence on patient selection, techniques, and outcomes. Methods: A systematic review of the Medline and Cochrane databases was performed for original studies reporting breast reconstruction with ADM or mesh, and subcutaneous implant placement. Studies were analyzed for level of evidence, inclusion/exclusion criteria for subcutaneous reconstruction, reconstruction characteristics, and outcomes. Results: Six studies (186 reconstructions) were identified for review. The majority of studies (66.7%) were level IV evidence case series. Eighty percent of studies had contraindications for subcutaneous reconstruction, most commonly preoperative radiation, high body mass index, and active smoking. Forty percent of studies commenting on patient selection assessed mastectomy flap perfusion for subcutaneous reconstruction. Forty-five percent of reconstructions were direct-to-implant, 33.3% 2-stage, and 21.5% single-stage adjustable implant, with ADM utilized in 60.2% of reconstructions versus mesh. Pooled complication rates included: major infection 1.2%, seroma 2.9%, hematoma 2.3%, full nipple-areola complex necrosis 1.1%, partial nipple-areola complex necrosis 4.5%, major flap necrosis 1.8%, wound healing complication 2.3%, explantation 4.1%, and grade III/IV capsular contracture 1.2%. Conclusions: Pooled short-term complication rates in subcutaneous alloplastic breast reconstruction with ADM or mesh are low in preliminary studies with selective patient populations, though techniques and outcomes are variable across studies. Larger comparative studies and better-defined selection criteria and outcomes reporting are needed to develop appropriate indications for performing subcutaneous implant-based reconstruction. PMID:27975034
Qureshi, Ali A.; Broderick, Kristen; Funk, Susan; Reaven, Nancy; Tenenbaum, Marissa M.
Background: Current cost data on tissue expansion followed by exchange for permanent implant (TE/I) reconstruction lack a necessary assessment of the experience of a heterogenous breast cancer patient population and their multiple outcome pathways. We extend our previous analysis to that of direct hospital cost as bundling of payments is likely to follow the changing centralization of cancer care at the hospital level. Methods: We performed a retrospective analysis (2003–2009) of TE/I reconstructions with or without an acellular dermal matrix (ADM), namely Alloderm RTM. Postreconstructive events were analyzed and organized into outcome pathways as previously described. Aggregated and normalized inpatient and outpatient hospital direct costs and physician reimbursement were generated for each outcome pathway with or without ADM. Results: Three hundred sixty-seven patients were analyzed. The average 2-year hospital direct cost per TE/I breast reconstruction patient was $11,862 in the +ADM and $12,319 in the −ADM groups (P > 0.05). Initial reconstructions were costlier in the +ADM ($6,868) than in the −ADM ($5,615) group, but the average cost of subsequent postreconstructive events within 2 years was significantly lower in +ADM ($5,176) than −ADM ($6,704) patients (P < 0.05). When a complication occurred, but reconstruction was still completed within 2 years, greater costs were incurred in the −ADM than in the +ADM group for most scenarios, leading to a net equalization of cost between study groups. Conclusion: Although direct hospital cost is an important factor for resource and fund allocation, it should not remain the sole factor when deciding to use ADM in TE/I reconstruction. PMID:27622099
Daar, David A; Gandy, Jessica R; Clark, Emily G; Mowlds, Donald S; Paydar, Keyianoosh Z; Wirth, Garrett A
The last decade has ushered in a rapidly expanding global discussion regarding acellular dermal matrix (ADM) applications, economic analyses, technical considerations, benefits, and risks, with recent emphasis on ADM use in breast surgery. This study aims to evaluate global trends in ADM research using bibliometric analysis. The top nine Plastic Surgery journals were determined by impact factor (IF). Each issue of the nine journals between 1999 and 2013 was accessed to compile a database of articles discussing ADM. Publications were further classified by IF, authors’ geographic location, study design, and level of evidence (LOE, I-V). Productivity index and productivity share were calculated for each region. In total, 256 ADM articles were accessed. The annual global publication volume increased significantly by 4.2 (0.87) articles per year (p<0.001), with a mean productivity index of 36.3 (59.0). The mean impact factor of the nine journals increased significantly from 0.61 (0.11) to 2.47 (0.99) from 1993 to 2013 (p<0.001). Despite this increase in the global ADM literature, the majority of research was of weaker LOE (level I: 2.29% and level II: 9.17%). USA contributed the most research (87%), followed by Asia (4.76%) and Western Europe (4.71%). USA contributed the greatest volume of research. Regarding clinical application of ADM, the majority of publications focused on ADM use in breast surgery, specifically breast reconstruction (154 articles, 60.2%). The majority of research was of lower LOE; thus, efforts should be made to strengthen the body of literature, particularly with regard to cost analysis. PMID:27579264
Pokrywczynska, Marta; Gubanska, Iga; Drewa, Gerard; Drewa, Tomasz
Construction of the urinary bladder de novo using tissue engineering technologies is the "holy grail" of reconstructive urology. The search for the ideal biomaterial for urinary bladder reconstruction has been ongoing for decades. One of the most promising biomaterials for this purpose seems to be bladder acellular matrix (BAM). In this review we determine the most important factors, which may affect biological and physical properties of BAM and its regeneration potential in tissue engineered urinary bladder. We also point out the directions in modification of BAM, which include incorporation of exogenous growth factors into the BAM structure. Finally, we discuss the results of the urinary bladder regeneration with cell seeded BAM.
Thiery, M; Parewyck, W; de Gezelle, H; van Kets, H; Derom, R; Martens, G
In 22 normal term gravidas with unfavorable cervix, 5 mg PGF2alpha in Tylose gel was instilled into the extra-amniotic space. The treatment improved the cervical state so much that the women could be successfully induced by conventional methods. The procedure was well tolerated by the mother and it appeared to be perinatally safe.
Kim, Kyung Sook; Cho, Chang-Hoon; Kim, Young-Sun; Yoon, Kyung-Sik; Jung, Min-Hyung; Park, Hun-Kuk
The structural and morphological properties of the teratoma membrane were investigated to better understand the pathogenesis of ovarian teratomas. A mature cystic teratoma and amnion were obtained from patients who underwent laparoscopic cystectomy and uncomplicated delivery, respectively. The teratoma membrane was divided into three layers according to the results of the histological analysis. Each layer showed distinct morphological properties, including an outer layer that was uniformly arranged, a middle layer with an irregular pattern of fibers, and an inner layer that was structurally dense with a wavy pattern of fibers. The morphology of the layers of the amniotic membrane was the reverse that of the teratoma membrane. In the teratoma membrane, the outer layer was primarily composed of type III collagen and the inner layer had a large amount of type III and IV collagen. The amniotic membrane showed a small amount of type III collagen in the outer layer, whereas the inner layer had large amounts of type I, III, and IV collagen. In the teratoma membrane, the collagen fibrils were arranged regularly in the outer layer, but irregularly in the inner layer. In the amniotic membrane, the arrangement of collagen fibrils was the reverse that of the teratoma membrane. Additionally, the collagen fibrils in the teratoma membrane were thinner than those of the amniotic membrane and had slightly shorter d-spacing. Two membranes showed the differences in collagen fibril arrangement, which may caused by the different functional roles.
Lambertz, Markus; Grommes, Kristina; Kohlsdorf, Tiana; Perry, Steven F
We show-in contrast to the traditional textbook contention-that the first amniote lungs were complex, multichambered organs and that the single-chambered lungs of lizards and snakes represent a secondarily simplified rather than the plesiomorphic condition. We combine comparative anatomical and embryological data and show that shared structural principles of multichamberedness are recognizable in amniotes including all lepidosaurian taxa. Sequential intrapulmonary branching observed during early organogenesis becomes obscured during subsequent growth, resulting in a secondarily simplified, functionally single-chambered lung in lepidosaurian adults. Simplification of pulmonary structure maximized the size of the smallest air spaces and eliminated biophysically compelling surface tension problems that were associated with miniaturization evident among stem lepidosaurmorphs. The remaining amniotes, however, retained the multichambered lungs, which allowed both large surface area and high pulmonary compliance, thus initially providing a strong selective advantage for efficient respiration in terrestrial environments. Branched, multichambered lungs instead of simple, sac-like organs were part and parcel of the respiratory apparatus of the first amniotes and pivotal for their success on dry land, with the sky literally as the limit.
Barros, P.S.M.; Garcia, J.A.; Laus, J.L.; Ferreira, A.L.; Salles Gomes, T.L.
This study was performed to evaluate the use of glycerol-preserved equine amniotic membrane as replacement for full-thickness corneal defects in dogs. Eighteen mixed-breed dogs were used. A perilimbal, full-thickness, 5 mm square corneal defect was created surgically, and a donor implant of equine amniotic membrane of the same size and shape sutured in place with 10-0 nylon simple interrupted sutures. Corneal edema was observed near the implant 24 h after surgery, but was absent after 1 week. Granulation tissue and corneal vascularization superficial to the implant were noticed on postoperative day 7, but were absent on day 30. Corneal vascularization persisted until the end of the experiment. There was no fluorescein retention by postoperative day 30. There was slight clearing of the corneal implant by postoperative 30, and slight pigmentation of the donor implant observed at postoperative day 180. An acute inflammatory process as well as fibroblasts were present at early postoperative stages. At postoperative day 60 there was no inflammatory cellular infiltrate, but fibroblasts and fibrosis were present. Corneal architecture was restored at the end of the experiment, with a layering of the epithelium-stroma-debris of amniotic membrane-stroma-endothelium present, and pigmentation and vascularization present in the deep layers of the cornea. Although vascularization indicated some degree of graft rejection, the clinical and histological evidence indicates that the xenologous amniotic membrane can be useful as a tectonic graft in the repair of full-thickness lesions of the cornea of dogs.
Depreux, Frederic F.; Wang, Lingyan; Jiang, Han; Jodelka, Francine M.; Rosencrans, Robert F.; Rigo, Frank; Lentz, Jennifer J.; Brigande, John V.; Hastings, Michelle L.
Congenital diseases account for a large portion of pediatric illness. Prenatal screening and diagnosis permit early detection of many genetic diseases. Fetal therapeutic strategies to manage disease processes in utero represent a powerful new approach for clinical care. A safe and effective fetal pharmacotherapy designed to modulate gene expression ideally would avoid direct mechanical engagement of the fetus and present an external reservoir of drug. The amniotic cavity surrounding the fetus could serve as an ideal drug reservoir. Antisense oligonucleotides (ASOs) are an established tool for the therapeutic modulation of gene expression. We hypothesize that ASOs administered to the amniotic cavity will gain entry to the fetus and modulate gene expression. Here, we show that an ASO targeting MALAT1 RNA, delivered by transuterine microinjection into the mouse amniotic cavity at embryonic day 13-13.5, reduces target RNA expression for up to 4 weeks after birth. A similarly delivered ASO targeting a causal splice site mutation for Usher syndrome corrects gene expression in the inner ear, a therapeutically relevant target tissue. We conclude that intra-amniotic delivery of ASOs is well tolerated and produces a sustained effect on postnatal gene expression. Transuterine delivery of ASOs is an innovative platform for developing fetal therapeutics to efficaciously treat congenital disease. PMID:27683224
Adly, O A; Moghazy, A M; Abbas, A H; Ellabban, A M; Ali, O S; Mohamed, B A
As allograft and xenografts are not available in Islamic countries, amniotic membrane seems to be an effective alternative in the management of deep burns. Its proven bioactivities and modest price suggest that it might be superior to synthetic dressings. Forty-six patients were enrolled in this randomized, controlled clinical trial conducted in the Burn Unit at Suez Canal University Hospital, Ismailia, Egypt. All age groups and both gender were included in the study. Only patients with less than 50% total body surface area burned were included, thus minimizing the dropouts in both groups. All were either second or third degree. These patients were randomly assigned either to group I: amniotic membrane (Biomembrane) dressing, or group II: polyurethane membrane (Tegaderm) dressing. Those in group I demonstrated a significantly lower rate of infection and required less frequent dressing changes than those in group II. They also sustained less electrolyte and albumin loss. The rate of healing in the amniotic membrane group was significantly faster than in the polyurethane group. Furthermore, pain was significantly less when Biomembrane was used. Based on these findings, we recommend the use of lyophilized gamma-irradiated amniotic membrane as an effective alternative for allograft and xenografts in Islamic countries and the Jewish population.
Magann, Everett F.; Dinnel, Kinsey I.; Rabie, Nader Z.; Shoemaker, Amanda L.; Manning, Nirvana A.
Patient: Female, 33 Final Diagnosis: Intramamniotic hemorrhage Symptoms: Abdominal pain • uterine contractions • vaginal bleeding Medication: — Clinical Procedure: Cesarean delivery Specialty: Obstetrics and Gynecology Objective: Unusual clinical course Background: A spontaneous intra-amniotic hemorrhage is rarely encountered during pregnancy. The correct diagnosis and management are problematic because of the infrequency of this condition and the high likelihood of a misdiagnosis. Case Report: A primigravida with an uncomplicated pregnancy and a normal targeted ultrasound presented late in the second trimester of pregnancy with antepartum bleeding of unknown origin. A repeat ultrasound was suggestive of an abdominal wall defect (gastroschisis). The patient continued to have antepartum bleeding and developed uterine contractions and abdominal pain necessitating frequent visits to labor and delivery. An MRI ruled out gastroschisis and diagnosed intra-amniotic hematoma. The patient presented with acute abdominal pain and was clinically considered to be having an abruption, and was delivered by cesarean. Old blood was noted in the abdominal cavity and within the uterine cavity. At the time of the cesarean, an area of intra-amniotic hematoma was identified, as well as a retroplacental blood clot. Conclusions: An intra-amniotic hematoma is unusual and may be misdiagnosed. MRI may be helpful in determining the correct diagnosis and subsequent management. PMID:27760979
Mendz, George L.; Kaakoush, Nadeem O.; Quinlivan, Julie A.
Infection-related preterm birth is a leading cause of infant mortality and morbidity; knowledge of bacterial populations invading the amniotic cavity and the routes of invasion is required to make progress in the prevention of preterm birth. Significant advances have been made in understanding bacterial communities in the vagina, but much less studied are intra-uterine bacterial populations during pregnancy. A systematic review of data published on the intra-uterine microbiome was performed; molecular information and summaries of species found in healthy individuals and in women with diagnosed infections served to construct a database and to analyse results to date. Thirteen studies fulfilled the review's inclusion criteria. The data of various investigations were collated, organized, and re-analyzed to achieve a more comprehensive understanding of microbial populations in the intra-amniotic space. The most common intra-amniotic bacterial taxa were species that can colonies the vagina in health and disease; there were others associated with the habitats of the mouth, gastrointestinal tract, and respiratory tract. The results suggest a central role for the ascending route of infections during pregnancy, and point to a possible secondary contribution via haematogenous invasion of the intra-amniotic space. The complete census of the intra-uterine microbiome awaits completion. PMID:24137568
Leguy, Marie-Clémence; Tavares, Silvina Dos Reis; Tsatsaris, Vassili; Lewin, Fanny; Clauser, Eric; Guibourdenche, Jean
Ultrasound scanning is useful to detect neural tube defect (NTD) but scarcely distinguished between closed NTD and open NTD, which had very different prognosis. An amniotic fluid punction is thus mandatory to search for an increase in alpha foeto protein (AFP) levels and for the presence of acetylcholinesterase which identified open NTD. However, AFP levels fluctuate both with the gestational age and the assay used. Our aim was to establish normative values for AFP in amniotic fluid in the second half of pregnancy using three different immunoassays and to improve their clinical relevance. Amniotic fluid punctions were performed on 527 patients from 9 week of gestation (WG) to 37 WG either for maternal age, Trisomy 21 screening, increase in nucal translucency (control group, n = 527) or for suspicion of neural tube defect or abdominal defect (n = 5). AFP was measured using the immunoassay developed for serum AFP on the Access 2 system, the Immulite 2000 and the Advia Centaur. Results were expressed in ng/ml, multiple of the median (MoM) and percentiles. AFP decrease by 1.5 fold between 9 and 19 WG. When NTD was suspected, an increase in anmniotic AFP was observed (from 2.5 MoM to 9.3 MoM) confirming an open NTD. In conclusion, the assay developed on those 3 automates is suitable for the measurement of AFP in amniotic fluid.
Kruszka, Paul; Uwineza, Annette; Mutesa, Leon; Martinez, Ariel F; Abe, Yu; Zackai, Elaine H; Ganetzky, Rebecca; Chung, Brian; Stevenson, Roger E; Adelstein, Robert S; Ma, Xuefei; Mullikin, James C; Hong, Sung-Kook; Muenke, Maximilian
Limb body wall complex (LBWC) and amniotic band sequence (ABS) are multiple congenital anomaly conditions with craniofacial, limb, and ventral wall defects. LBWC and ABS are considered separate entities by some, and a continuum of severity of the same condition by others. The etiology of LBWC/ABS remains unknown and multiple hypotheses have been proposed. One individual with features of LBWC and his unaffected parents were whole exome sequenced and Sanger sequenced as confirmation of the mutation. Functional studies were conducted using morpholino knockdown studies followed by human mRNA rescue experiments. Using whole exome sequencing, a de novo heterozygous mutation was found in the gene IQCK: c.667C>G; p.Q223E and confirmed by Sanger sequencing in an individual with LBWC. Morpholino knockdown of iqck mRNA in the zebrafish showed ventral defects including failure of ventral fin to develop and cardiac edema. Human wild-type IQCK mRNA rescued the zebrafish phenotype, whereas human p.Q223E IQCK mRNA did not, but worsened the phenotype of the morpholino knockdown zebrafish. This study supports a genetic etiology for LBWC/ABS, or potentially a new syndrome. PMID:26436108
Menon, Ramkumar; Williams, Scott M; Fortunato, Stephen J
The purpose of this study is to examine the racial differences between interleukin (IL)-1beta and IL-8 concentrations in the amniotic fluid of black and white women with spontaneous preterm birth (PTB). In this study, 350 amniotic fluid samples were collected: 165 PTB cases (<36 weeks' gestation; 52 blacks and 113 whites) and 185 controls (normal term delivery >37 weeks' gestation; 87 blacks and 98 whites). Amniotic fluid IL-1beta and IL-8 concentrations were measured by immunoassay. Wilcoxon nonparametric test was performed for statistical analysis. In data stratified by race, the median IL-1beta concentration was significantly higher in black cases (80 pg/mL) compared to black controls (23.7 pg/mL; P < .0001), and the difference was nonsignificant in white cases (25.5 pg/mL) compared to white controls (21.3 pg/mL; P = .1). IL-8 concentration was not higher in black cases (742.2 pg/mL) compared to black controls (731.4 pg/mL; P = .9), whereas it was higher in white cases (1362.3 pg/mL) compared to white controls (533.5 pg/mL; P = .0005). Between races, IL-1beta was significantly higher in blacks (P < .0001) than in whites in PTB, whereas no significant difference was noticed in IL-8 concentration between races (P = .1). In PTB, the cytokine footprint differs in the amniotic fluid between racial groups. IL-1beta is higher in black and IL-8 in white PTB. These differences in the amniotic fluid cytokine concentration might not explain the racial disparity in the PTB rate, but they are suggestive of different processes of PTB in whites and blacks.
Rashmi; Pathak, Rekha; Amarpal; Aithal, H. P.; Kinjavdekar, P.; Pawde, A. M.; Tiwari, A. K.; Sangeetha, P.; Tamilmahan, P.; Manzoor, A. B.
Aim: The aim of this study was to generate composite bone graft and investigate the rabbit fetal osteoblasts adhesion, proliferation and penetration on acellular matrices of cancellous bone. Materials and Methods: Acellular cancellous bone was prepared and developed as in the previous study with little modification. These matrices were decellularized by rapid freeze and thaw cycle. To remove the cell debris, they were then treated with hydrogen peroxide (3%) and ethanol to remove antigenic cellular and nuclear materials from the scaffold. Primary osteoblast cells were harvested from 20 to 22 days old rabbit fetal long and calvarial bone. These cells were cultured and characterized using a specific marker. The third passaged fetal osteoblast cells were then seeded on the scaffold and incubated for 14 days. The growth pattern of the cells was observed. Scanning electron microscope and hematoxylin and eosin staining were used to investigate cells proliferation. Results: The cells were found to be growing well on the surface of the scaffold and were also present in good numbers with the matrix filopodial extensions upto inside of the core of the tissue. Conclusion: Thus, a viable composite scaffold of bone could be developed which has a great potential in the field of bone tissue engineering. PMID:28344398
Valdatta, Luigi; Scamoni, Stefano; Minuti, Anna; Cherubino, Mario
The increasing use of commercially available acellular dermis matrices for postmastectomy breast reconstruction seems to have simplified the surgical procedure and enhanced the outcome. These materials, generally considered to be highly safe or with only minor contraindications due to the necessary manipulation in preparatory phases, allow an easier one-phase surgical procedure, in comparison with autologous flaps, offering a high patient satisfaction. Unfortunately, the claim for a higher rate of complications associated with irradiation at the implant site, especially when the radiation therapy was given before the reconstructive surgery, suggested a careful behaviour when this technique is preferred. However, this hypothesis was never submitted to a crucial test, and data supporting it are often discordant or incomplete. To provide a comprehensive analysis of the field, we searched and systematically reviewed papers published after year 2005 and registered clinical trials. On the basis of a meta-analysis of data, we conclude that the negative effect of the radiotherapy on the breast reconstruction seems to be evident even in the case of acellular dermis matrices aided surgery. However, more trials are needed to make solid conclusions and clarify the poor comprehension of all the factors negatively influencing outcome. PMID:24987526
Consolo, F; Brizzola, S; Tremolada, G; Grieco, V; Riva, F; Acocella, F; Fiore, G B; Soncini, M
A combined physical-chemical protocol for whole full-thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non-ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole-organ mechanical response to a physiological-like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation.
Pogorelova, T N; Linde, V A; Gunko, V O; Selyutina, S N
The levels of zinc, copper, iron, and magnesium ions, and some of their binding proteins have been investigated in an amniotic fluid under the fetal growth retardation (FGR). FGR, developed under conditions of placental insufficiency, is characterized by a decrease in the content of zinc, iron, and magnesium ions and by an increase in the copper content in the amniotic fluid in the II and III trimesters of pregnancy. During these trimesters the levels of ceruloplasmin, ferritin, and Ca2+,Mg2+-ATPase were lower in FGR, while the level of zinc-a-2-glycoprotein was higher than during the same periods of normal pregnancy. Changes in the parameters studied in the amniotic fluid were associated with developmental disorders of the newborns. These changes obviously have a pathogenetic importance in the development of FGR, and the levels of metal ions and their ratio in the amniotic fluid can be used as markers of the pre- and postnatal pathology.
Vomachka, A J; Lisk, R D
Using radioimmunoassay we have measured the plasma and amniotic fluid levels of androgen and estradiol in male and female hamster fetuses nearing parturition. On Days 14 and 15 of gestation (day of birth = Day 16), plasma levels of androgen are higher in males than females while estradiol levels are equal. Amniotic fluid levels of these hormones, while lower than plasma, reflect the difference in androgen and the similarity in estradiol between sexes. Uterine position analysis on Day 14 suggests that female siblings located caudally suppress amniotic fluid androgen and elevate estradiol levels of male siblings. Comparison of Day 18 gestation male and female rat amniotic fluid androgen to Day 14 hamsters reveals that male rats are bathed in high levels of androgen. Female rats have lower levels which are not different from those of male hamsters. Female hamsters are exposed to little androgen. Relevance to behavioral sexual differentiation and the display of adult behavior is discussed.
Ho-Asjoe, Mark; Junge, Klaus; Farhadi, Jian
Background Irradiation of implant-based breast reconstructions (BR) is known to increase capsular contracture (CC) rates on average by 4-fold over non-irradiated reconstructions. The use of acellular dermal matrix (ADM) has been associated with lower CC rates in non-irradiated reconstructions (0-3%). Experimental and clinical studies suggest that ADM may also reduce CC rates in irradiated breasts. The aim of this study was to evaluate CC rates in non-irradiated and irradiated one- and two-stage BRs performed with the assistance of porcine ADM (PADM). Methods A single centre, retrospective, cohort study was designed from December 2008 to October 2012. A total of 200 immediate implant-based BRs were performed using PADM for inferior pole reinforcement. We included non-irradiated BR with a minimum follow up of 6 month from primary surgery (one stage) or from explantation of expander and implantation of the definitive implant (two stage). Of the postoperatively irradiated BR we included patients with 1 year or more follow up time from termination of radiotherapy. CC was graded using the conventional Spear-Baker classification and modified version for irradiated BR. According to the literature Grade III and IV CC were defined as clinically significant CC. Results Of 200 BRs with PADM, 122 were included in this study (84 non-irradiated and 38 irradiated). Sixty-five BR were one stage and 57 were two stage BR. Grade III/IV CC was remarkable low in non-irradiated (6%) and irradiated BR (13%). There was a non-significant trend to increased Grade III and IV CC in irradiated BR vs. non-irradiated BR (13% vs. 6%, P=0.216). In this study follow up time (P<0.001) and the stage of ADM reconstruction (two vs. one stage, P=0.022) were significant risk factors for occurrence of grade III/IV CC on univariate analysis and remained significant for the follow up time (P=0.013) and remarkable for the stages (P=0.093) in multivariate analysis. Conclusions Our data support the current
Shen, Jia; Xu, Zhi Wei
An experiment was designed to find the suitable acellular bovine pericardium (ABP) patch in pericardial cavity reconstruction and to evaluate the effect of sodium hyaluronic acid (NaHA) on inflammatory reaction in prevention of pericardial adhesions. The pericardial adhesion model was established in 20 rabbits, weighing from 3.2 to 3.6 kg. Groups were classified as follows: Group A (n = 5), the control group, the pericardium was directly closed; Group B (n = 5), 0.15% glutaraldehyde-treated ABP (low cross-link degree); Group C, 0.3% glutaraldehyde-treated ABP (middle cross-link degree); Group D, 0.15% glutaraldehyde-treated ABP + NaHA solution. Blood samples were collected at 6 h, 24 h, 3 days, and 5 days, to assay postoperative inflammatory reaction. The tenacity and severity of adhesions were evaluated 2 months after operation, by macroscopic and microscopic examinations, and Q-PCR (real-time quantitative polymerase chain reaction) test was used to quantitatively analyze the associated genes with adhesion. Pericardium regeneration was demonstrated by immunohistochemical technique to identify mesothelial cells. In Group D, the serum concentration of tumor necrosis factor-α (TNF-α) was significantly lower in the early postoperative period, and the mean adhesion score (adhesion between the epicardium and ABP) was significantly lower compared with the control group (Groups D vs. A: 0.20 ± 0.45 vs. 2.00 ± 0.71, P = 0.009*). The signs of degradation of the ABPs were observed 2 months postoperation in Groups D and B. Immunohistochemically, the positive cytokeratin AE1 staining results demonstrated the relatively total regeneration of the pericardium in Group D. Signs of regeneration were observed in Group D. Compared with the control group, the level of TGF-β2 in Group D was significantly lower (0.00132 ± 0.00114, P = 0.022*). The TGF-β3 level was statistically significant, being highest in Group D (0.00805 ± 0.00136, P = 0.029*). The mean quantity of Smad6 in
Romero, Roberto; Miranda, Jezid; Chaiworapongsa, Tinnakorn; Chaemsaithong, Piya; Gotsch, Francesca; Dong, Zhong; Ahmed, Ahmed I.; Yoon, Bo Hyun; Hassan, Sonia S.; Kim, Chong J.; Korzeniewski, Steven J.; Yeo, Lami; Kim, Yeon Mee
Objective To determine the frequency and clinical significance of sterile- and microbial-associated intra-amniotic inflammation in asymptomatic patients with a sonographic short cervix. Methods Amniotic fluid (AF) samples obtained by transabdominal amniocentesis from 231 asymptomatic women with a sonographic short cervix [cervical length (CL) ≤25 mm] were analyzed using cultivation techniques (for aerobic and anaerobic as well as genital mycoplasmas) and broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). The frequency and magnitude of intra-amniotic inflammation [defined as an AF interleukin (IL)-6 concentration ≥2.6 ng/mL], acute histologic placental inflammation, spontaneous preterm delivery, and the amniocentesis-to-delivery interval were examined according to the results of AF cultures, PCR/ESI-MS and AF IL-6 concentrations. Results Ten percent (24/231) of patients with a sonographic short cervix had sterile intra-amniotic inflammation (an elevated AF IL-6 concentration without evidence of microorganisms using cultivation and molecular methods). Sterile intra-amniotic inflammation was significantly more frequent than microbial-associated intra-amniotic inflammation [10.4% (24/231) vs. 2.2% (5/231); p<0.001]. Patients with sterile intra-amniotic inflammation had a significantly higher rate of spontaneous preterm delivery <34 weeks of gestation [70.8% (17/24) vs. 31.6% (55/174); p<0.001] and a significantly shorter amniocentesis-to-delivery interval than patients without intra-amniotic inflammation [median 35, (IQR: 10 – 70) vs. median 71, (IQR: 47 – 98) days, (p<0.0001)]. Conclusion Sterile intra-amniotic inflammation is more common than microbial-associated intra-amniotic inflammation in asymptomatic women with a sonographic short cervix, and is associated with increased risk of spontaneous preterm delivery (<34 weeks). Further investigation is required to determine the causes of sterile
Buhimschi, Irina A.; Zhao, Guomao; Rosenberg, Victor A.; Abdel-Razeq, Sonya; Thung, Stephen; Buhimschi, Catalin S.
Background Though recent advancement in proteomics has provided a novel perspective on several distinct pathogenetic mechanisms leading to preterm birth (inflammation, bleeding), the etiology of most preterm births still remains elusive. We conducted a multidimensional proteomic analysis of the amniotic fluid to identify pathways related to preterm birth in the absence of inflammation or bleeding. Methodology/Principal Findings A proteomic fingerprint was generated from fresh amniotic fluid using surface-enhanced laser desorbtion ionization time of flight (SELDI-TOF) mass spectrometry in a total of 286 consecutive samples retrieved from women who presented with signs or symptoms of preterm labor or preterm premature rupture of the membranes. Inflammation and/or bleeding proteomic patterns were detected in 32% (92/286) of the SELDI tracings. In the remaining tracings, a hierarchical algorithm was applied based on descriptors quantifying similarity/dissimilarity among proteomic fingerprints. This allowed identification of a novel profile (Q-profile) based on the presence of 5 SELDI peaks in the 10–12.5 kDa mass area. Women displaying the Q-profile (mean±SD, gestational age: 25±4 weeks, n = 40) were more likely to deliver preterm despite expectant management in the context of intact membranes and normal amniotic fluid clinical results. Utilizing identification-centered proteomics techniques (fluorescence two-dimensional differential gel electrophoresis, robotic tryptic digestion and mass spectrometry) coupled with Protein ANalysis THrough Evolutionary Relationships (PANTHER) ontological classifications, we determined that in amniotic fluids with Q-profile the differentially expressed proteins are primarily involved in non-inflammatory biological processes such as protein metabolism, signal transduction and transport. Conclusion/Significance Proteomic profiling of amniotic fluid coupled with non-hierarchical bioinformatics algorithms identified a subgroup of
and inflammatory course, an inspiring model for understanding failure of tissue regeneration in higher vertebrates and humans. The participation of 5-Bromo-deoxyuridine (5BrdU) long retention cells, indicated as putative stem cells, for the following regeneration is analyzed and it shows that different tissue sites of the original tail contain putative stem cells that are likely activated from the wounding signal. In particular, the permanence of stem cells in the central canal of the spinal cord can explain the limited but important neurogenesis present in the caudal but also in the lumbar-thoracic spinal cord. In the latter, the limited number of glial and neurons regenerated is however sufficient to recover some limited hind limb movement after injury or spinal transection. Finally, the presence of stem cells in the spinal cord, in the regenerative blastema and skin allow to use these organs as a source of cells for studies on gene activation during cell differentiation in the new spinal cord, tail and in the epidermis. The above information form the basic knowledge for the future molecular studies on the specific gene activation capable to determine tail regeneration in lizards, and more in general genes involved in the reactivation of regeneration process in amniotes and humans.
Hsi, B L; Yeh, C J; Faulk, W P
Antibodies produced in rabbits to detergent-solubilized human amnion were found to react with Hassall's corpuscles in human thymus. Following nomenclature for placental antigens, the immunogenic group responsible for these antibodies has been tentatively designated as amnion antigens 1 (AA1). The anti-AA1 antisera did not react with other thymic components, nor did they react with any other extra-embryonic tissues than amniotic epithelium. Some adult ectodermally derived tissues, such as breast ductal and corneal epithelium, reacted with anti-AA1, but others such as skin and vagina did not. These findings link an antigenic relationship between amniotic epithelium and certain ectodermal derivatives. Amnion exists long before these tissues are formed, raising the possibility that amniotic epithelium may play an inductive role in their development. Images Figure 1 Figure 2 PMID:6343232
DeLuca, F.; Cametti, C.; Zimatore, G.; Maraviglia, B.; Pachi', A.
In this report we propose a new method for an in vitro test of the foetal lung maturity based on the measurement of the electrical conductivity of the overall amniotic fluid obtained from transabdominal amniocentesis, since this quantity can be linked to a first approximation in a very simple way to the phospholipid content. We have carried out measurements of 85 different samples of amniotic fluid as a function of gestation weeks and we have observed a pronounced change of the electrical conductivity that reflects the increase in the phospholipid concentration occurring at the end of normal pregnancies. The method could be further developed to obtain similar information on in vivo experiments by means of bioelectric impedance tomography, taking advantage of the frequency dependence of the tissue electrical impedance.
Craft, I L; Evans, D V; Richfield, L B
Extra-amniotic prostaglandin E2 (PGE2) suspended in a slow release gel (Tylose) was instilled in 35 patients prior to a planned surgical termination in an attempt to dilate the cervix, minimize cervical trauma, and reduce the possible risk of cervical trauma, and reduce the possible risk of cervical incompetence and its sequelae. Dilatation occurred in all patients to a minimum of 8 mm and 74% aborted before surgical evacuation performed 6 to 24 hours after injection. No serious side effects occurred. Extra-amniotic PGE2 in gel should be considered as a primary procedure when the cervix is obviously immature on examination. If the cervix is found to be tight and unyielding at surgical dilatation, the latter procedure should be dicontinued and PGE2 in gel injected.
Pozzobon, Michela; Piccoli, Martina; De Coppi, Paolo
Stem cell therapy is in constant need of new cell sources to conceive regenerative medicine approaches for diseases that are still without therapy. Scientists drew the attention toward amniotic membrane and amniotic fluid stem cells, since these sources possess many advantages: first of all as cells can be extracted from discarded foetal material it is inexpensive, secondly abundant stem cells can be obtained and finally, these stem cell sources are free from ethical considerations. Many studies have demonstrated the differentiation potential in vitro and in vivo toward mesenchymal and non-mesenchymal cell types; in addition the immune-modulatory properties make these cells a good candidate for allo- and xenotransplantation. This review offers an overview on markers characterisation and on the latest findings in pre-clinical or clinical setting of the stem cell populations isolated from these sources.
Kanasugi, Tomonobu; Kikuchi, Akihiko; Haba, Gen; Sasaki, Yuri; Isurugi, Chizuko; Oyama, Rie; Sugiyama, Toru
Newborn cases of VACTERL association with lower urinary tract obstruction (LUTO) are rare and there have been no reports on those patients undergoing fetal therapy in English literature. We successfully performed vesico-amniotic shunting in a fetus having LUTO caused by abnormality of the external genitalia at 16 weeks' gestation. Although fetal karyotype was normal 46XY, follow-up fetal ultrasound examinations revealed ventriculomegaly in the brain, a small stomach and a right multicystic dysplastic kidney. MRI at 31 weeks' gestation suggested lobar type holoprosencephaly. Diagnosis of VACTERL association was confirmed postnatally. We consider that vesico-amniotic shunting is indicated for a fetus of VACTERL association with LUTO if the parents wish the procedure after genetic counseling and explanations about the fetal conditions.
Pogorelova, T N; Gunko, V O; Linde, V A
Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.
Tok, Ozlem Yalcin; Kocaoglu, Fatma Akbas; Tok, Levent; Burcu, Ayse; Ornek, Firdevs
Ligneous conjunctivitis (LC) is a rare form of bilateral chronic recurrent disease in which thick membranes form on the palpebral conjunctiva and other mucosal sites. We report the clinical features and describe the management of two cases. Case 1 was an 8-month-old patient with bilateral membranous conjunctivitis. Case 2 was a 5-year-old patient with unilateral membranous conjunctivitis, esotropia, mechanical ptosis and complicated cataract, and had been treated with a number of medications. Histological investigation of the membrane in both cases showed LC. Treatments with amniotic membrane transplantation and institution of topical cyclosporine have shown good response. There has been complete resolution of the membranes with no recurrence at the end of 40- and 28-month follow-ups, respectively. No treatment related side effects were seen. Thus, it appears that amniotic membrane transplantation and topical cyclosporine are effective alternatives for the treatment of LC.
In the field of germline development in amniote vertebrates, primordial germ cell (PGC) specification in birds and reptiles remains controversial. Avians are believed to adopt a predetermination or maternal specification mode of PGC formation, contrary to an inductive mode employed by mammals and, supposedly, reptiles. Here, we revisit and review some key aspects of PGC development that channelled the current subdivision, and challenge the position of birds and reptiles as well as the ‘binary’ evolutionary model of PGC development in vertebrates. We propose an alternative view on PGC specification where germ plasm plays a role in laying the foundation for the formation of PGC precursors (pPGC), but not necessarily of PGCs. Moreover, inductive mechanisms may be necessary for the transition from pPGCs to PGCs. Within this framework, the implementation of data from birds and reptiles could provide new insights on the evolution of PGC specification in amniotes. PMID:27273724
Jiong, Chen; Jiake, Chai; Chunmao, Han; Yingen, Pan; Qiuhe, Wu; Zhouxi, Fang; Xiangsheng, Feng
The purpose of this study was to investigate the clinical effects of porcine acellular dermal matrix combined with autoskin grafting on full-thickness skin defects using long-term clinical follow-up study and histologic examination. One hundred fifty-two patients with deep burn or trauma hospitalized from February 2000 to July 2003 were repaired with porcine acellular dermal matrix and split-thickness autoskin graft. Take rate of the grafts was calculated on 1 week after operation. Scar hyperplasia was examined on 1, 3, 6, and 12 months after operation. At the same time, the contracture rates of grafted areas were also calculated. Skin biopsy was performed on five patients for histologic examination, as well as transmission electron microscopy 78 months after operation. The take rate of grafts of 116 patients (76.3%) was 100%, and the take rate of the rest of the patients (36 patients, 23.7%) was more than 95%. No one needed skin transplantation for the second time. One hundred twenty-seven patients were followed up on 1 month after operation; grafts showed mild contraction. There was slight cicatricle at skin junction with tender texture. There was no obvious pruritus and blister. One hundred one patients were followed up on 3 months after operation. The graft contraction showed obvious relief with good articular function. Eighty-two patients were followed up on 6 months after operation. The color and texture of the grafts were similar to normal skin without obvious cicatricial hyperplasia. Fifty-eight patients were followed up on 12 months after operation. The grafts were similar to normal skin without obvious rejection. There were no significant differences between the contracture rates at 3, 6, and 12 months and 1 month after the second surgery. Sixteen patients were followed up on 78 months after operation. The appearance of grafts was slightly dry compared with normal skin. Tissue structure of grafts was similar to normal skin with sweat gland-like structure
Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W
Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.
Background Mooren’s ulcer is a severe ulcerative inflammation of the cornea. The exact pathogenesis remains unclear. Therefore many therapies of Mooren’s ulcer are recommended in literature. To shed more light on the ongoing question of optimal treatment of severe progressive Mooren’s ulcer, we here report on a retrospective case series of patients treated with systemic immunosuppressive therapy and additional amniotic membrane transplantation. Methods Medical records from seven patients (eleven eyes), 4 male and 3 female, with severe progressive Mooren’s ulcer were analysed retrospectively. The mean follow up was 88.4 ± 80.8 months (range 12–232 month). A HLA-typing was performed in all patients. A systemic immunosuppressive therapy was administered in all patients. The amniotic membrane was transplanted after the base of the ulcer was resected. Results Multiple amniotic membrane transplantations were necessary in six patients. The visual outcome of all patients was poor. No patient achieved a visual acuity better than 20/630 Snellen chart. Five patients were positive for HLA-DQ2 and four patients were positive for HLA-DR17(3). Conclusions The aggressive and highly inflammatory form of Mooren’s ulcer is difficult to treat and the progression of the disease is hard to influence positively even under systemic immunosuppressive therapy. Therefore, the main intention of therapy is to achieve a stable epithelialized corneal surface without the risk of perforation. Amniotic membrane transplantation is not able to cure severe forms of Mooren’s ulcer. However it supports the immunosuppressive therapy in acute situations as in critical corneal thinning. PMID:24345289
In 2006, the American Academy of Pediatrics and the American Heart Association published the 2005 guidelines on neonatal resuscitation. Before the 2005 guidelines, management of a newborn with meconium-stained amniotic fluid included suctioning of the oropharynx and nasopharynx on the perineum after the delivery of the head but before the delivery of the shoulders. The 2005 guidelines did not support this practice because routine intrapartum suctioning does not prevent or alter the course of meconium aspiration syndrome in vigorous newborns. However, the 2005 guidelines did support intubation of the trachea and suctioning of meconium or other aspirated material from beneath the glottis in nonvigorous newborns. In 2015, the guidelines were updated. Routine intubation and tracheal suctioning are no longer required. If the infant is vigorous with good respiratory effort and muscle tone, the infant may stay with the mother to receive the initial steps of newborn care. If the infant born through meconium-stained amniotic fluid presents with poor muscle tone and inadequate breathing efforts, the initial steps of resuscitation should be completed under the radiant warmer. Appropriate intervention to support ventilation and oxygenation should be initiated as indicated for each infant. Infants with meconium-stained amniotic fluid should no longer routinely receive intrapartum suctioning, whether they are vigorous or not. In addition, meconium-stained amniotic fluid is a condition that requires the notification and availability of an appropriately credentialed team with full resuscitation skills, including endotracheal intubation. Resuscitation should follow the same principles for infants with meconium-stained fluid as for those with clear fluid.
In 2006, the American Academy of Pediatrics and the American Heart Association published the 2005 guidelines on neonatal resuscitation. Before the 2005 guidelines, management of a newborn with meconium-stained amniotic fluid included suctioning of the oropharynx and nasopharynx on the perineum after the delivery of the head but before the delivery of the shoulders. The 2005 guidelines did not support this practice because routine intrapartum suctioning does not prevent or alter the course of meconium aspiration syndrome in vigorous newborns. However, the 2005 guidelines did support intubation of the trachea and suctioning of meconium or other aspirated material from beneath the glottis in nonvigorous newborns. In 2015, the guidelines were updated. Routine intubation and tracheal suctioning are no longer required. If the infant is vigorous with good respiratory effort and muscle tone, the infant may stay with the mother to receive the initial steps of newborn care. If the infant born through meconium-stained amniotic fluid presents with poor muscle tone and inadequate breathing efforts, the initial steps of resuscitation should be completed under the radiant warmer. Appropriate intervention to support ventilation and oxygenation should be initiated as indicated for each infant. Infants with meconium-stained amniotic fluid should no longer routinely receive intrapartum suctioning, whether they are vigorous or not. In addition, meconium-stained amniotic fluid is a condition that requires the notification and availability of an appropriately credentialed team with full resuscitation skills, including endotracheal intubation. Resuscitation should follow the same principles for infants with meconium-stained fluid as for those with clear fluid.
Sharma, Anamika; Yadav, Komal
Background: Periodontal plastic surgical procedures aimed at coverage of exposed root surface. Owing to the second surgical donor site and difficulty in procuring a sufficient graft for the treatment of root coverage procedures, various alternative additive membranes have been used. A recent resorbable amniotic membrane, not only maintains the structural and anatomical configuration of regenerated tissues, but also enhances gingival wound healing, provides a rich source of stem cells. Therefore, amniotic membrane is choice of material these days in augmenting the better results in various periodontal procedures. Aim: The aim of this observational case series was to evaluate the effectiveness, predictability and the use of a novel material, amniotic membrane in the treatment of shallow-to-moderate isolated recession defects. Materials and Methods: A total of three cases, showing Miller's Class I or Class II gingival recession, participated in this study. Recession depth, recession width, keratinized gingiva (KG) tissue width, clinical attachment level (CAL) were recorded at baseline, 3 and 6 months postoperatively. Results: Six months following root coverage procedures, the mean root coverage was found to be 70.2 ± 6.8%. CAL significantly decreased from 6.4 ± 0.54 mm preoperatively to 3.5 ± 0.9 mm postoperatively at 6 months while KG showed significant improvement from 3.2 ± 0.28 mm preoperatively to 5.9 ± 0.74 mm postoperatively at 6 months. Conclusion: Autogenous graft tissue procurement significantly increases patient morbidity while also lengthening the duration of surgery in placing the graft, while self-adherent nature of amniotic membrane significantly reduces surgical time and made the procedure easier to perform, making it membrane of choice. PMID:26392696
Janes, D E; Chapus, C; Gondo, Y; Clayton, D F; Sinha, S; Blatti, C A; Organ, C L; Fujita, M K; Balakrishnan, C N; Edwards, S V
Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (<0.0003% to <0.001%), differences that we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds) and that this may reflect differing roles and constraints in gene regulation.
Janes, D.E.; Chapus, C.; Gondo, Y.; Clayton, D.F.; Sinha, S.; Blatti, C.A.; Organ, C.L.; Fujita, M.K.; Balakrishnan, C.N.; Edwards, S.V.
Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (<0.0003% to <0.001%), differences that we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds) and that this may reflect differing roles and constraints in gene regulation. PMID:21183607
Nardozza, Luciano Marcondes Machado; Araujo, Edward; Caetano, Ana Carolina Rabachini; Moron, Antonio Fernandes
Amniotic band syndrome is characterized by a build-up of bands and strings of fibrous tissue that adhere to the fetus and can compress parts of the fetus, thus causing malformations and even limb amputation while the fetus is still in the uterus. The clinical manifestations are extremely variable and their extent may range from a single abnormality, like a constriction ring, to multiple abnormalities. Such abnormalities are generally diagnosed at the end of the first or the beginning of the second trimester using two-dimensional ultrasonography (2DUS). Three-dimensional ultrasonography (3DUS) in rendering mode allows spatial analysis of the fetus and amniotic band, thus enabling better comprehension of this pathological condition and better counseling for the parents. There has not previously been any evidence to show that 3DUS would be useful in cases of late diagnosis (third trimester) of amniotic band syndrome. In the present case, a primigravid woman underwent her second obstetric ultrasound scan in the 34th week, from which we observed two bands in contact with the right forearm, but with normal movement of this limb and its fingers. 3DUS made it possible to see the spatial relationship of these bands to the fetal body, thereby confirming their adherence to the limb. After the birth, the prenatal diagnosis of amniotic band syndrome without limb constriction was confirmed. A surgical procedure was carried out on the third day after birth to excise the bands, and the newborn was then discharged in a good general condition. PMID:22616039
Piñeiro, Graciela; Núñez Demarco, Pablo; Meneghel, Melitta D
The hypotheses about the origin of the primitive amniotic tarsus are very speculative. Early studies argued that the origin of the astragalus, one of the largest proximal bones in the tarsus of basal amniotes, was produced by either the fusion of two, three, or even four of the original tarsal bones, the intermedium, the tibiale and the proximal centralia (c4 and c3), or that the intermedium alone transforms into the primitive astragalus. More recent studies have shown that the structure of the tarsus in Captorhinus supports the former hypothesis about a fusion of the intermedium, the tibiale, the proximal centrale (c4) and eventually c3, producing a purportedly multipartite structure of the amniotic astragalus, but the issue remained contentious. Very well preserved tarsi of the Early Permian aquatic amniote Mesosaurus tenuidens Gervais, 1864-1865, which represent the most complete ontogenetic succession known for a basal amniote (the other exceptional one is provided by the Late Permian diapsid Hovasaurus boulei Piveteau, 1926), suggest that there is more than one ossification center for the astragalus and that these fuse during late embryonic stages or maybe early after birth. A non-hatched Mesosaurus in an advanced stage of development shows that the tarsus is represented by a single bone, most probably the astragalus, which seems to be formed by the suturing of three bones, here interpreted as being the intermedium, the tibiale, probably already integrated to the c4 in an earlier stage of the development, and the c3. An amniote-like tarsal structure is observed in very basal Carboniferous and Permian tetrapods such as Proterogyrinus, Gephyrostegus, the diadectids Diadectes and Orobates, some microsaurs like Tuditanus and Pantylus and possibly Westlothiana, taxa that were all considered as true amniotes in their original descriptions. Therefore, the structure of the amniotic tarsus, including the configuration of the proximal series formed by the astragalus and
Núñez Demarco, Pablo; Meneghel, Melitta D.
The hypotheses about the origin of the primitive amniotic tarsus are very speculative. Early studies argued that the origin of the astragalus, one of the largest proximal bones in the tarsus of basal amniotes, was produced by either the fusion of two, three, or even four of the original tarsal bones, the intermedium, the tibiale and the proximal centralia (c4 and c3), or that the intermedium alone transforms into the primitive astragalus. More recent studies have shown that the structure of the tarsus in Captorhinus supports the former hypothesis about a fusion of the intermedium, the tibiale, the proximal centrale (c4) and eventually c3, producing a purportedly multipartite structure of the amniotic astragalus, but the issue remained contentious. Very well preserved tarsi of the Early Permian aquatic amniote Mesosaurus tenuidens Gervais, 1864–1865, which represent the most complete ontogenetic succession known for a basal amniote (the other exceptional one is provided by the Late Permian diapsid Hovasaurus boulei Piveteau, 1926), suggest that there is more than one ossification center for the astragalus and that these fuse during late embryonic stages or maybe early after birth. A non-hatched Mesosaurus in an advanced stage of development shows that the tarsus is represented by a single bone, most probably the astragalus, which seems to be formed by the suturing of three bones, here interpreted as being the intermedium, the tibiale, probably already integrated to the c4 in an earlier stage of the development, and the c3. An amniote-like tarsal structure is observed in very basal Carboniferous and Permian tetrapods such as Proterogyrinus, Gephyrostegus, the diadectids Diadectes and Orobates, some microsaurs like Tuditanus and Pantylus and possibly Westlothiana, taxa that were all considered as true amniotes in their original descriptions. Therefore, the structure of the amniotic tarsus, including the configuration of the proximal series formed by the astragalus
Brace, Robert A; Anderson, Debra F; Cheung, Cecilia Y
Experimentation in late-gestation fetal sheep has suggested that regulation of amniotic fluid (AF) volume occurs primarily by modulating the rate of intramembranous transport of water and solutes across the amnion into underlying fetal blood vessels. In order to gain insight into intramembranous transport mechanisms, we developed a computer model that allows simulation of experimentally measured changes in AF volume and composition over time. The model included fetal urine excretion and lung liquid secretion as inflows into the amniotic compartment plus fetal swallowing and intramembranous absorption as outflows. By using experimental flows and solute concentrations for urine, lung liquid, and swallowed fluid in combination with the passive and active transport mechanisms of the intramembranous pathway, we simulated AF responses to basal conditions, intra-amniotic fluid infusions, fetal intravascular infusions, urine replacement, and tracheoesophageal occlusion. The experimental data are consistent with four intramembranous transport mechanisms acting in concert: 1) an active unidirectional bulk transport of AF with all dissolved solutes out of AF into fetal blood presumably by vesicles; 2) passive bidirectional diffusion of solutes, such as sodium and chloride, between fetal blood and AF; 3) passive bidirectional water movement between AF and fetal blood; and 4) unidirectional transport of lactate into the AF. Further, only unidirectional bulk transport is dynamically regulated. The simulations also identified areas for future study: 1) identifying intramembranous stimulators and inhibitors, 2) determining the semipermeability characteristics of the intramembranous pathway, and 3) characterizing the vesicles that are the primary mediators of intramembranous transport.
Hosoya, Yumiko; Watanabe, Masafumi; Terashima, Masahiro; Amiya, Eisuke; Nakao, Tomoko; Hasegawa, Akiko; Hyodo, Hironobu; Ando, Jiro; Fujii, Tomoyuki; Nagai, Ryozo; Komuro, Issei
Amniotic fluid embolism (AFE) is a rare but devastating complication of pregnancy. Acute circulatory failure and obstetric disseminated intravascular coagulopathy are often associated with AFE and lead to poor prognosis of this syndrome. Although many reports of AFE and its cardiopulmonary complications exist, their etiology remains unknown. Classically, it was believed that the fatal cardiopulmonary complication in AFE is due to acute and severe pulmonary hypertension caused by critical obstruction of the pulmonary vessels by embolized amniotic fluid. However, recent hypotheses are suggesting that anaphylactic reaction or a cytokine effect induced by amniotic fluid is the main pathophysiological mechanism. We report a case in which cardiac magnetic resonance imaging was performed at the chronic stage of AFE. Late gadolinium enhancement (LGE) was detected at the mid-wall of the left ventricle with no evidence of pulmonary hypertension. This finding suggests that the pathophysiological mechanism of severe cardiac complications in AFE may include direct left ventricular myocardial injury through an immune reaction or cytokine release, rather than pulmonary embolism.
Moreno, Nerea; González, Agustín
Numerous studies over the last few years have demonstrated that the amygdaloid complex in amniotes shares basic developmental, hodological and neurochemical features. Furthermore, homologous territories of all the main amygdaloid subdivisions have been recognized among amniotes, primarily highlighted by the common expression patterns for numerous developmental genes. Thus, derivatives from the lateral pallium, ventral pallium and subpallium constitute the fundamental parts of the amygdaloid complex. With the development of new technical approaches, study of the precise neuroanatomy of the telencephalon of the anuran amphibians (anamniotes) has been possible. Current embryological, hodological and immunohistochemical evidence strongly suggests that most of the structures present in amniotes are recognizable in these anamniotes. These investigations have yielded enough results to support the notion that the organization of the anuran amygdaloid complex includes subdivisions with their origin in ventral pallial and subpallial territories; a strong relationship with the vomeronasal and olfactory systems; abundant intra-amygdaloid connections; a main output centre involved in the autonomic system; recognizable amygdaloid fibre systems; and distinct chemoarchitecture. Therefore, the new ideas regarding the amygdaloid evolution based on the recent findings in anamniotes, and especially in anurans, strongly support the notion that basic amygdaloid structures were present at least in the brain of ancestral tetrapods organized following a basic plan shared by tetrapods.
Farjah, Gh. H.; Fazli, F.
The purpose of this experimental study was to evaluate the effect of chicken amniotic fluid (AF) on a cross section of rat sciatic nerves. Thirty adult male Sprague-Dawley rats weighing 275 to 300 g, were randomized into three groups treated with (1) amniotic fluid or AF (n=10), (2) normal saline or NS (n=10), and (3) sham surgery (n=10). The AF was aspirated from the amniotic cavity of incubating chick embryos at day 14. The sciatic nerve was exposed and sharply transected. Immediate epineurial repair was then performed. AF treated animals were given 2 ml/kg of the chick embryo AF subcutaneously, once daily, five times a week for up to 2 weeks. All animals were evaluated by sciatic functional index (SFI), electrophysiology, histology, and immunohistochemistry at days 28 and 56 after surgery. The SFI difference between AF and NS groups at days 21 and 28 after operation was statistically significant (P<0.05). The number of myelinated fibers in the AF group was significantly greater than that of the NS group at day 28 (P<0.05). At days 28 and 56 after operation, the nerve conduction velocity (NCV) mean of the AF group was faster than that of the NS group, but the difference was not statistically significant (P>0.05). The results of this study demonstrate that chick AF can enhance peripheral nerve regeneration. PMID:27175170
Suliburska, J; Kocyłowski, R; Komorowicz, I; Grzesiak, M; Bogdański, P; Barałkiewicz, D
The concentrations of various trace elements in amniotic fluid (AF) change over the course of pregnancy, with gestational age and fetus growth. The aim of the present study was to evaluate the concentrations of selected essential and toxic elements in AF and their relations to maternal and fetal parameters. The study was carried out in 39 pregnant women, aged 34.6 ± 4.7 years, between weeks 16 and 26 of gestation. Amniotic fluid samples were obtained during the standard procedure of amniocentesis in high-risk patients for chromosomal abnormalities. An inductively coupled plasma mass spectrometry (ICP-MS) technique was used to determine the levels of Al, As, Ba, Cd, Co, Cr, Cu, Mg, Mn, Ni, Sr, U, and V in AF. Body mass and blood pressure were measured in all the women. The basic parameters of fetal development were also assayed. It was found that the age of the mother, the gender of the fetus, and the week of the pregnancy may affect the concentrations of mineral in the amniotic fluid. Moreover, several significant correlations between the essential and toxic elements and maternal and fetal parameters were observed. In particular, negative and positive correlations between fetal parameters and magnesium and copper levels in AF, respectively, were seen. The present findings demonstrate the association between minerals in AF and fetal development.
Beckman, G; Beckman, L; Löfstrand, T
171 samples of amniotic fluid were obtained by abdominal amniocentesis from 67 women with complicated pregnancies (isoimmunization, diabetes mellitus or toxaemia). The levels of heat-labile alkaline phosphatase (HLAP), heat-stable alkaline phosphatase (HSAP) and acid phosphatase (AcP) were determined and compared to the enzyme levels in 179 samples from women with normal pregnancies of corresponding gestational ages. HLAP showed two "peaks" of activity, one in the 5th-22nd week and the other at term. HSAP and AcP showed increased activity at term. HSAP was decreased (p less than 0.01) in isoimmunization between the 36th and 40th week. 11 cases of toxaemia with placental insufficiency showed no differences in the levels of HLAP and HSAP compared with normal pregnancy. AcP showed no differences between normal and complicated pregnancy. Samples contaminated by blood showed no significant increase in the acid- and alkaline phosphatase levels. Samples contaminated by meconium showed a complex pattern. Some samples had normal enzyme levels, some had high levels of HLAP only and some had high levels of HSAP and AcP. The origin of the enzymes is not known with certainty. HSAP in amniotic fluid is most likely not of placental but intestinal origin. Determinations of acid- and alkaline phosphatase in amniotic fluid seem to be of little values in the clinical management of complicated pregnancy.
Maritati, Martina; Gonelli, Arianna; Greco, Pantaleo
Inflammation plays an important role in pregnancy, and cytokine and matrix metalloproteases (MMPs) imbalance has been associated with premature rupture of membranes and increased risk of preterm delivery. Previous studies have demonstrated that lactoferrin (LF), an iron-binding protein with anti-inflammatory properties, is able to decrease amniotic fluid (AF) levels of IL-6. Therefore, we aimed to evaluate the effect of vaginal LF administration on amniotic fluid PGE2 level and MMP-TIMP system in women undergoing genetic amniocentesis. One hundred and eleven women were randomly divided into controls (n = 57) or treated with LF 4 hours before amniocentesis (n = 54). Amniotic fluid PGE2, active MMP-9 and MMP-2, and TIMP-1 and TIMP-2 concentrations were determined by commercially available assays and the values were normalized by AF creatinine concentration. PGE2, active MMP-9, and its inhibitor TIMP-1 were lower in LF-treated group than in controls (p < 0.01, p < 0.005, and p < 0.001, resp.). Conversely, active MMP-2 (p < 0.0001) and MMP-2/TIMP-2 molar ratio (p < 0.001) were increased, whilst TIMP-2 was unchanged. Our data suggest that LF administration is able to modulate the inflammatory response following amniocentesis, which may counteract cytokine and prostanoid imbalance that leads to abortion. This trial is registered with Clinical Trial number NCT02695563. PMID:27872513
Munoz, Jessian L; Kessler, Alan A; Felig, Philip; Curtis, Jenifer; Evans, Mark I
Several isolated reports of fetal goiter treatment have shown limited generalizability of approaches and provide no real guidance for optimal timing, dosages, and treatment strategies. Graves' disease accounts for >60% of these cases. Maternal treatments of hyperthyroidism include antithyroid medications such as methimazole and more commonly propylthiouracil (PTU). Here, our management of a patient with a fetal thyroid goiter from maternal exposure to PTU diagnosed at 23.6 weeks' gestation and the management of other cases allow us propose a general strategy for treatment. Intrauterine therapy with 200 and then 400 μg of levothyroxine (3 weeks apart) showed an 85% reduction in fetal thyroid goiter volume. We collected amniotic fluid samples at the time of treatments and assayed thyroid hormones and associated antibodies which closely reflected the changes in thyroid goiter mass volume. Our observations suggest a weekly or biweekly therapeutic intervention schedule. Utilizing both goiter size as well as a novel approach in using amniotic fluid hormone levels to monitor therapy efficacy might improve the quality of treatments. Only with a standardized approach and collection of amniotic fluid thyroid panels do we have the opportunity to develop the database required to determine the number and timing of treatments needed.
Cubo, Jorge; Legendre, Pierre; de Ricqlès, Armand; Montes, Laëtitia; de Margerie, Emmanuel; Castanet, Jacques; Desdevises, Yves
The biological features observed in every living organism are the outcome of three sets of factors: historical (inherited by homology), functional (biological adaptation), and structural (properties inherent to the materials with which organs are constructed, and the morphogenetic rules by which they grow). Integrating them should bring satisfactory causal explanations of empirical data. However, little progress has been accomplished in practice toward this goal, because a methodologically efficient tool was lacking. Here we use a new statistical method of variation partitioning to analyze bone growth in amniotes. (1) Historical component. The variation of bone growth rates contains a significant phylogenetic signal, suggesting that the observed patterns are partly the outcome of shared ancestry. (2) Functional causation. High growth rates, although energy costly, may be adaptive (i.e., they may increase survival rates) in taxa showing short growth periods (e.g., birds). In ectothermic amniotes, low resting metabolic rates may limit the maximum possible growth rates. (3) Structural constraint. Whereas soft tissues grow through a multiplicative process, growth of mineralized tissues is accretionary (additive, i.e., mineralization fronts occur only at free surfaces). Bone growth of many amniotes partially circumvents this constraint: it is achieved not only at the external surface of the bone shaft, but also within cavities included in the bone cortex as it grows centrifugally. Our approach contributes to the unification of historicism, functionalism, and structuralism toward a more integrated evolutionary biology.
Pokorná, Martina; Altmanová, Marie; Kratochvíl, Lukáš
It is notable that the occurrence of multiple sex chromosomes differs significantly between major lineages of amniote vertebrates. In this respect, birds are especially conspicuous, as multiple sex chromosomes have not been observed in this lineage so far. On the other hand, in mammals, multiple sex chromosomes have evolved many times independently. We hypothesize that this contrast can be related to the different involvement of sex-specific sex chromosomes in female meiosis subjected to the female meiotic drive under male versus female heterogamety. Essentially, the male-specific Y chromosome is not involved in female meiosis and is therefore sheltered against the effects of the female meiotic drive affecting the X chromosome and autosomes. Conversely, the Z and W sex chromosomes are both present in female meiosis. Nonrandom segregation of these sex chromosomes as a consequence of their rearrangements connected with the emergence of multiple sex chromosomes would result in a biased sex ratio, which should be penalized by selection. Therefore, the emergence of multiple sex chromosomes should be less constrained in the lineages with male rather than female heterogamety. Our broader phylogenetic comparison across amniotes supports this prediction. We suggest that our results are consistent with the widespread occurrence of female meiotic drive in amniotes.
Johnson Pokorná, Martina; Kratochvíl, Lukáš
Amniote vertebrates, the group consisting of mammals and reptiles including birds, possess various mechanisms of sex determination. Under environmental sex determination (ESD), the sex of individuals depends on the environmental conditions occurring during their development and therefore there are no sexual differences present in their genotypes. Alternatively, through the mode of genotypic sex determination (GSD), sex is determined by a sex-specific genotype, i.e. by the combination of sex chromosomes at various stages of differentiation at conception. As well as influencing sex determination, sex-specific parts of genomes may, and often do, develop specific reproductive or ecological roles in their bearers. Accordingly, an individual with a mismatch between phenotypic (gonadal) and genotypic sex, for example an individual sex-reversed by environmental effects, should have a lower fitness due to the lack of specialized, sex-specific parts of their genome. In this case, evolutionary transitions from GSD to ESD should be less likely than transitions in the opposite direction. This prediction contrasts with the view that GSD was the ancestral sex-determining mechanism for amniote vertebrates. Ancestral GSD would require several transitions from GSD to ESD associated with an independent dedifferentiation of sex chromosomes, at least in the ancestors of crocodiles, turtles, and lepidosaurs (tuataras and squamate reptiles). In this review, we argue that the alternative theory postulating ESD as ancestral in amniotes is more parsimonious and is largely concordant with the theoretical expectations and current knowledge of the phylogenetic distribution and homology of sex-determining mechanisms.
Jacobo-Estrada, Tania; Cardenas-Gonzalez, Mariana; Santoyo-Sánchez, Mitzi; Parada-Cruz, Benjamín; Uria-Galicia, Esther; Arreola-Mendoza, Laura; Barbier, Olivier
Cadmium is a well-characterized nephrotoxic agent that is also capable of accumulating and diffusing across the placenta; however, only a few studies have addressed its effects over fetal kidneys and none of them has used a panel of sensitive and specific biomarkers for the detection of kidney injury. The goal of this study was to determine cadmium renal effects in rat fetuses by the quantification of early kidney injury biomarkers. Pregnant Wistar rats were exposed by inhalation to an isotonic saline solution or to CdCl2 solution (DDel =1.48 mg Cd kg(-1) day(-1) ) during gestational days (GD) 8-20. On GD 21, dams were euthanized and samples obtained. Kidney injury biomarkers were quantified in amniotic fluid samples and fetal kidneys were microscopically evaluated to search for histological alterations. Our results showed that cadmium exposure significantly raised albumin, osteopontin, vascular endothelial growth factor and tissue inhibitor of metalloproteinases-1 levels in amniotic fluid, whereas it decreased creatinine. Clusterin, calbindin and IFN-inducible protein 10 did not show any change. Accordingly, histological findings showed tubular damage and precipitations in the renal pelvis. In conclusion, gestational exposure to cadmium induces structural alterations in fetal renal tissue that can be detected by some kidney injury biomarkers in amniotic fluid samples. Copyright © 2016 John Wiley & Sons, Ltd.
Müller, Johannes; Scheyer, Torsten M.; Head, Jason J.; Barrett, Paul M.; Werneburg, Ingmar; Ericson, Per G. P.; Pol, Diego; Sánchez-Villagra, Marcelo R.
The development of distinct regions in the amniote vertebral column results from somite formation and Hox gene expression, with the adult morphology displaying remarkable variation among lineages. Mammalian regionalization is reportedly very conservative or even constrained, but there has been no study investigating vertebral count variation across Amniota as a whole, undermining attempts to understand the phylogenetic, ecological, and developmental factors affecting vertebral column variation. Here, we show that the mammalian (synapsid) and reptilian lineages show early in their evolutionary histories clear divergences in axial developmental plasticity, in terms of both regionalization and meristic change, with basal synapsids sharing the conserved axial configuration of crown mammals, and basal reptiles demonstrating the plasticity of extant taxa. We conducted a comprehensive survey of presacral vertebral counts across 436 recent and extinct amniote taxa. Vertebral counts were mapped onto a generalized amniote phylogeny as well as individual ingroup trees, and ancestral states were reconstructed by using squared-change parsimony. We also calculated the relationship between presacral and cervical numbers to infer the relative influence of homeotic effects and meristic changes and found no correlation between somitogenesis and Hox-mediated regionalization. Although conservatism in presacral numbers characterized early synapsid lineages, in some cases reptiles and synapsids exhibit the same developmental innovations in response to similar selective pressures. Conversely, increases in body mass are not coupled with meristic or homeotic changes, but mostly occur in concert with postembryonic somatic growth. Our study highlights the importance of fossils in large-scale investigations of evolutionary developmental processes. PMID:20080660
Abramyan, John; Richman, Joy Marion
The assembly of the upper jaw is a pivotal moment in the embryonic development of amniotes. The upper jaw forms from the fusion of the maxillary, medial nasal, and lateral nasal prominences, resulting in an intact upper lip/beak and nasal cavities; together called the primary palate. This process of fusion requires a balance of proper facial prominence shape and positioning to avoid craniofacial clefting, whilst still accommodating the vast phenotypic diversity of adult amniotes. As such, variation in craniofacial ontogeny is not tolerated beyond certain bounds. For clarity, we discuss primary palatogenesis of amniotes into in two categories, according to whether the nasal and oral cavities remain connected throughout ontogeny or not. The transient separation of these cavities occurs in mammals and crocodilians, while remaining connected in birds, turtles and squamates. In the latter group, the craniofacial prominences fuse around a persistent choanal groove that connects the nasal and oral cavities. Subsequently, all lineages except for turtles, develop a secondary palate that ultimately completely or partially separates oral and nasal cavities. Here, we review the shared, early developmental events and highlight the points at which development diverges in both primary and secondary palate formation. PMID:26293818
McQuilling, John P; Vines, Jeremy B; Mowry, Katie C
Chronic wounds require extensive healing time and place patients at risk of infection and amputation. Recently, a fresh hypothermically stored amniotic membrane (HSAM) was developed and has subsequently shown promise in its ability to effectively heal chronic wounds. The purpose of this study is to investigate the mechanisms of action that contribute to wound-healing responses observed with HSAM. A proteomic analysis was conducted on HSAM, measuring 25 growth factors specific to wound healing within the grafts. The rate of release of these cytokines from HSAMs was also measured. To model the effect of these cytokines and their role in wound healing, proliferation and migration assays with human fibroblasts and keratinocytes were conducted, along with tube formation assays measuring angiogenesis using media conditioned from HSAM. Additionally, the cell-matrix interactions between fibroblasts and HSAM were investigated. Conditioned media from HSAM significantly increased both fibroblast and keratinocyte proliferation and migration and induced more robust tube formation in angiogenesis assays. Fibroblasts cultured on HSAMs were found to migrate into and deposit matrix molecules within the HSAM graft. These collective results suggest that HSAM positively affects various critical pathways in chronic wound healing, lending further support to promising qualitative results seen clinically and providing further validation for ongoing clinical trials.
Maioli, Margherita; Contini, Giovanni; Santaniello, Sara; Bandiera, Pasquale; Pigliaru, Gianfranco; Sanna, Raimonda; Rinaldi, Salvatore; Delitala, Alessandro P; Montella, Andrea; Bagella, Luigi; Ventura, Carlo
Mouse embryonic stem cells were previously observed along with mesenchymal stem cells from different sources, after being treated with a mixed ester of hyaluronan with butyric and retinoic acids, to show a significant increase in the yield of cardiogenic and vascular differentiated elements. The aim of the present study was to determine if stem cells derived from primitive fetal cells present in human amniotic fluid (hAFSCs) and cultured in the presence of a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids show a higher yield of differentiation toward the cardiovascular phenotype as compared with untreated cells. During the differentiation process elicited by exposure to HA + BU + RA, genes controlling pluripotency and plasticity of stem cells, such as Sox2, Nanog, and Oct4, were significantly downregulated at the transcriptional level. At this point, a significant increase in expression of genes controlling the appearance of cardiogenic and vascular lineages in HA + BU + RA-treated cells was observed. The protein expression levels typical of cardiac and vascular phenotypes, evaluated by Western blotting, immunofluorescence, and flow cytometry, were higher in hAFSCs cultured in the presence of HA + BU + RA, as compared with untreated control cells. Appearance of the cardiac phenotype was further inferred by ultrastructural analysis using transmission and scanning electron microscopy. These results demonstrate that a mixture of HA + BU + RA significantly increased the yield of elements committed toward cardiac and vascular phenotypes, confirming what we have previously observed in other cellular types.
Zheng, Xiao-Yu; Wan, Qian-Quan; Zheng, Chuan-Yi; Zhou, Hong-Long; Dong, Xing-Yu; Deng, Qing-Shan; Yao, Hui; Fu, Qiang; Gao, Mou; Yan, Zhong-Jie; Wang, Shan-Shan; You, Yu; Lv, Jun; Wang, Xiang-Yu; Chen, Ke-En; Zhang, Mao-Ying; Xu, Ru-Xiang
Transplantation of human amniotic mesenchymal stem cells (hAM-MSCs) seems to be a promising strategy for the treatment of neurodegenerative disorders, including Alzheimer's disease (AD). However, the clinical therapeutic effects of hAM-MSCs and their mechanisms of action in AD remain to be determined. Here, we used amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic mice to evaluate the effects of hAM-MSC transplantation on AD-related neuropathology and cognitive dysfunction. We found that hAM-MSC transplantation into the hippocampus dramatically reduced amyloid-β peptide (Aβ) deposition and rescued spatial learning and memory deficits in APP/PS1 mice. Interestingly, these effects were associated with increasing in Aβ-degrading factors, elevations in activated microglia, and the modulation of neuroinflammation. Furthermore, enhanced hippocampal neurogenesis in the subgranular zone (SGZ) of the dentate gyrus (DG) and enhanced synaptic plasticity following hAM-MSC treatment could be another important factor in reversing the cognitive decline in APP/PS1 mice. Instead, the mechanism underlying the improved cognition apparently involves a robust increase in hippocampal synaptic density and neurogenesis that is mediated by brain-derived neurotrophic factor (BDNF). In conclusion, our data suggest that hAM-MSCs may be a new and effective therapy for the treatment of AD.
Amemiya, Takeshi; Nakamura, Takahiro; Yamamoto, Toshiro; Kinoshita, Shigeru; Kanamura, Narisato
The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects. PMID:25915046
Kim, Sung-Whan; Zhang, Hong-Zhe; Guo, Longzhe; Kim, Jong-Min; Kim, Moo Hyun
Although human amniotic mesenchymal stem cells (AMMs) have been recognised as a promising stem cell resource, their therapeutic potential for wound healing has not been widely investigated. In this study, we evaluated the therapeutic potential of AMMs using a diabetic mouse wound model. Quantitative real-time PCR and ELISA results revealed that the angiogenic factors, IGF-1, EGF and IL-8 were markedly upregulated in AMMs when compared with adipose-derived mesenchymal stem cells (ADMs) and dermal fibroblasts. In vitro scratch wound assays also showed that AMM-derived conditioned media (CM) significantly accelerated wound closure. Diabetic mice were generated using streptozotocin and wounds were created by skin excision, followed by AMM transplantation. AMM transplantation significantly promoted wound healing and increased re-epithelialization and cellularity. Notably, transplanted AMMs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, indicating a direct contribution to cutaneous closure. Taken together, these data suggest that AMMs possess considerable therapeutic potential for chronic wounds through the secretion of angiogenic factors and enhanced engraftment/differentiation capabilities.
Yuen, Chun-Ting; Horiuchi, Yoshinobu; Asokanathan, Catpagavalli; Cook, Sarah; Douglas-Bardsley, Alexandra; Ochiai, Masaki; Corbel, Michael; Xing, Dorothy
The histamine sensitisation test (HIST) for pertussis toxin is currently an official batch release test for acellular pertussis containing combination vaccines in Europe and North America. However, HIST, being a lethal endpoint assay, often leads to repeated tests due to large variations in test performance. Although a more precise HIST test based on measurement of temperature reduction after the histamine challenge is used in Asian countries, this test still uses animals. An in vitro test system based on a combination of enzyme coupled-HPLC and carbohydrate-binding assays with results analysed by a mathematical formula showed a good agreement with the in vivo HIST results based on measurement of temperature reduction after histamine challenge. The new in vitro test system was shown to be a potential alternative to the current in vivo HIST.
Vogel, Christoph F A; Charrier, Jessica G; Wu, Dalei; McFall, Alexander S; Li, Wen; Abid, Aamir; Kennedy, Ian M; Anastasio, Cort
While nanoparticles (NPs) are increasingly used in a variety of consumer products and medical applications, some of these materials have potential health concerns. Macrophages are the primary responders to particles that initiate oxidative stress and inflammatory reactions. Here, we utilized six flame-synthesized, engineered iron oxide NPs with various physicochemical properties (e.g. Fe oxidation state and crystal size) to study their interactions with RAW 264.7 macrophages, their iron solubilities, and their abilities to produce hydroxyl radical in an acellular assay. Both iron solubility and hydroxyl radical production varied between NPs depending on crystalline diameter and surface area of the particles, but not on iron oxidation state. Macrophage treatment with the iron oxide NPs showed a dose-dependent increase of heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO-1). The nuclear factor (NF)-erythroid-derived 2 (E2)-related factor 2 (Nrf2) modulates the transcriptional activity of antioxidant response element (ARE)-driven genes, such as HO-1 and NQO-1. Here, we show that the iron oxide NPs activate Nrf2, leading to its increased nuclear accumulation and enhanced Nrf2 DNA-binding activity in NP-treated RAW 264.7 macrophages. Iron solubility and acellular hydroxyl radical generation depend on the physical properties of the NPs, especially crystalline diameter; however, these properties are weakly linked to the activation of cellular signaling of Nrf2 and the expression of oxidative stress markers. Overall, our work shows for the first time that iron oxide nanoparticles induce cellular marker genes of oxidative stress and that this effect is transcriptionally mediated through the Nrf2-ARE signaling pathway in macrophages.
Kim, Myung-Sun; Yu, Ji Hea; Lee, Min-Young; Kim, Ah Leum; Jo, Mi Hyun; Kim, MinGi; Cho, Sung-Rae; Kim, Young-Han
Preeclampsia is a common disease that can occur during human pregnancy and is a leading cause of both maternal and neonatal morbidity and mortality. Inadequate trophoblast invasion and deficient remodeling of uterine spiral arteries are associated with preeclampsia (PE). The development of this syndrome is thought to be related to multiple factors. Recently, we isolated patient-specific human amniotic epithelial cells (AECs) from the placentas of 3 women with normal pregnancy and 3 with preeclamptic pregnancy. Since the characteristics of human AECs in PE are different from those in normal pregnancy, we sought to confirm the genes differentially expressed between preeclamptic pregnancy and normal pregnancy. Therefore, we performed transcriptome analysis to investigate the candidate genes associated with the possible pathophysiology of preeclampsia. Pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) online resource. In this study, we selected a total of 12 pathways and focused on extracellular matrix-related and biological adhesion molecules. Using RT-PCR array and real-time PCR, we confirmed that COL16A1, ITGB2, and LAMA3 were significantly up-regulated, but ITGA1, ITGA3, ITGA6, MMP1, MMP3, MMP10 and MMP11 were significantly down-regulated in preeclamptic fetal origin cells. Taken together, we suggest that the genes and pathways identified here may be responsible for the occurrence and development of PE, and controlling their expression may play a role in communication with fetal-maternal placenta to keep normal pregnancy. PMID:27218821
Carbone, Annalucia; Castellani, Stefano; Favia, Maria; Diana, Anna; Paracchini, Valentina; Di Gioia, Sante; Seia, Manuela; Casavola, Valeria; Colombo, Carla; Conese, Massimo
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.
Carbone, Annalucia; Castellani, Stefano; Favia, Maria; Diana, Anna; Paracchini, Valentina; Di Gioia, Sante; Seia, Manuela; Casavola, Valeria; Colombo, Carla; Conese, Massimo
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co-cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o- line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F-actin content was increased in co-cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co-cultures had a tendency of increased expression of occludin and ZO-1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC-CFBE co-cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC-CFBE co-cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co-culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype. PMID:24894806
Wan, Jin; Jiang, Fei; Zhang, Jiao; Xu, Qingsong; Chen, Daiwen; Yu, Bing; Mao, Xiangbing; Yu, Jie; Luo, Yuheng; He, Jun
Foetal loss and intrauterine growth restriction are major problems in mammals, but there are few effective ways in preventing it. Intriguingly, chitosan oligosaccharide (COS), a biomaterial derived from chitosan, can promote foetal survival and growth. Therefore, we have investigated how COS affects foetal survival and growth in a pig model. Fifty-two sows were divided into two treatment groups (n = 26) and fed either solely a control diet or a control diet that includes 100 mg/kg COS. Amniotic fluid and foetus samples from six sows that were of average body weight in each group were collected on gestation day 35. We applied a 1H NMR-based metabolomics approach combined with biochemistry analysis to track the changes that occurred in the amniotic fluid of pregnant sows after COS intervention. Maternal COS inclusion had enhanced (P < 0.05) the foetal survival rate and size at 35 days. COS supplementation had both increased (P < 0.05) SOD, CAT and T-AOC activities and elevated (P < 0.05) IL-10, IgG and IgM concentrations in the amniotic fluid. Moreover, COS had affected (P < 0.05) the amniotic fluid’s lysine, citrate, glucose and hypoxanthine levels. Overall, COS inclusion induced amniotic fluid antioxidant status and metabolic profiles modifications characterising improvements in foetal survival and growth in a pig model. PMID:28300194
Tanahashi, Hiroshi; Tian, Qing-Bao; Hara, Yoshinobu; Sakagami, Hiroyuki; Endo, Shogo; Suzuki, Tatsuo
Amniotic fluid volume during mid-to-late gestation depends mainly on the urine excretion from the foetal kidneys and partly on the fluid secretion from the foetal lungs during foetal breathing-like movements. Urine is necessary for foetal breathing-like movements, which is critical for foetal lung development. Bilateral renal agenesis and/or obstruction of the urinary tract lead to oligohydramnios, which causes infant death within a short period after birth due to pulmonary hypoplasia. Lrp4, which functions as an agrin receptor, is essential for the formation of neuromuscular junctions. Herein, we report novel phenotypes of Lrp4 knockout (Lrp4(-/-)) mice. Most Lrp4(-/-) foetuses showed unilateral or bilateral kidney agenesis, and Lrp4 knockout resulted in polyhydramnios. The loss of Lrp4 compromised foetal swallowing and breathing-like movements and downregulated the expression of aquaporin-9 in the foetal membrane and aquaporin-1 in the placenta, which possibly affected the amniotic fluid clearance. These results suggest that amniotic fluid removal was compromised in Lrp4(-/-) foetuses, resulting in polyhydramnios despite the impairment of urine production. Our findings indicate that amniotic fluid removal plays an essential role in regulating the amniotic fluid volume.
Kasper, David C; Mechtler, Thomas P; Reischer, Georg H; Witt, Armin; Langgartner, Michaela; Pollak, Arnold; Herkner, Kurt R; Berger, Angelika
Ureaplasma spp. are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), and preterm labor (PL). We analyzed 118 samples from amniotic fluid of preterm infants before 34 weeks of gestation by quantitative polymerase chain reaction (qPCR). Bacterial load, Ureaplasma biovar discrimination (Ureaplasma urealyticum and Ureaplasma parvum), and the level of inflammation were correlated with short-term clinical outcome. U. parvum was the predominant biovar, and increased bacterial load was significantly linked to histologic chorioamnionitis, PROM + PL, early-onset sepsis, and bronchopulmonary dysplasia. Furthermore, there was a positive correlation between the amount of U. parvum and the magnitude of inflammatory response inside the amniotic cavity observed by elevated interleukin 8 levels. We postulate that the bacterial load of Ureaplasma spp. measured by qPCR should be determined in studies investigating the potential clinical impact of intrauterine Ureaplasma spp. on the outcome of preterm infants.
Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M
The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.
Gupta, Kanika; Venkatesan, Bhuvaneswari; Chandra, Tushar; Rajeswari, Kathiah; Devi, Thangammal Kandasamy Renuka
Amniotic band syndrome is a rare congenital disorder caused by entrapment of fetal parts by fibrous amniotic bands in utero. The congenital anomalies seen in this syndrome vary widely and defects may be isolated or multiple and do not follow a specific pattern. Asymmetric distribution of defects is the hallmark of this syndrome. The diagnosis is difficult to make on ultrasound and relies on identification of amniotic bands. We report a case of amniotic band syndrome with sacral agenesis diagnosed on routine antenatal ultrasound scan in the second offspring of a recently diagnosed diabetic mother. The associated features were entrapment of umbilical cord, caudal adhesions and lower limb anomalies. Medical termination of pregnancy was done and all the fetal anomalies as well as umbilical cord abnormalities were confirmed. The importance of meticulous scanning to evaluate for amniotic bands and the umbilical cord in addition to the fetal structures is emphasized. PMID:25926929
Lenco, Juraj; Vajrychova, Marie; Link, Marek; Tambor, Vojtech; Liman, Victor; Bullarbo, Maria; Nilsson, Staffan; Tsiartas, Panagiotis; Cobo, Teresa; Kacerovsky, Marian; Jacobsson, Bo
Objective The aim of this study was to identify early proteomic biomarkers of spontaneous preterm delivery (PTD) in mid-trimester amniotic fluid from asymptomatic women. Methods This is a case-cohort study. Amniotic fluid from mid-trimester genetic amniocentesis (14–19 weeks of gestation) was collected from 2008 to 2011. The analysis was conducted in 24 healthy women with subsequent spontaneous PTD (cases) and 40 randomly selected healthy women delivering at term (controls). An exploratory phase with proteomics analysis of pooled samples was followed by a verification phase with ELISA of individual case and control samples. Results The median (interquartile range (IQR: 25th; 75th percentiles) gestational age at delivery was 35+5 (33+6–36+6) weeks in women with spontaneous PTD and 40+0 (39+1–40+5) weeks in women who delivered at term. In the exploratory phase, the most pronounced differences were found in C-reactive protein (CRP) levels, that were approximately two-fold higher in the pooled case samples than in the pooled control samples. However, we could not verify these differences with ELISA. The median (25th; 75th IQR) CRP level was 95.2 ng/mL (64.3; 163.5) in women with spontaneous PTD and 86.0 ng/mL (51.2; 145.8) in women delivering at term (p = 0.37; t-test). Conclusions Proteomic analysis with mass spectrometry of mid-trimester amniotic fluid suggests CRP as a potential marker of spontaneous preterm delivery, but this prognostic potential was not verified with ELISA. PMID:27214132
Neumann, Anne; Hoey, Robert F; Daigler, Lindsey B; Thompson, Alexis C; Kristal, Mark B
Previous research has shown that injection of morphine into the ventral tegmental area (VTA) facilitates the onset of maternal behavior in virgin female rats, and injection of the opioid antagonist naltrexone into the VTA disrupts the onset of maternal behavior in parturient rats. Placentophagia -- ingestion of placenta and amniotic fluid, usually at parturition -- modifies central opioid processes. Ingestion of the active substance in placenta and amniotic fluid, Placental Opioid-Enhancing Factor (POEF), enhances the hypoalgesic effect of centrally administered morphine, and more specifically, enhances delta- and kappa-opioid-receptor-mediated hypoalgesia and attenuates mu-opioid-receptor-mediated hypoalgesia. POEF (in placenta or amniotic fluid) ingestion does not, by itself, produce hypoalgesia. In the present study, we tested the hypothesis that ingestion of amniotic fluid enhances the facilitative effect of opioid activity (unilateral morphine injection) in the VTA on the rate of onset of maternal behavior. Virgin female Long-Evans rats were given one intra-VTA injection of morphine sulfate (0.0, 0.01, or 0.03 microg, in saline) and an orogastric infusion of 0.25 ml amniotic fluid or saline once each day of the first three days of the 10-day testing period. Subjects were continuously exposed to foster pups that were replaced every 12 h; replacement of pups was followed by a 15-min observation period. Maternal behavior latency was determined by the first of two consecutive tests wherein the subject displayed pup retrieval, pup licking in the nest, and crouching over all foster pups, during the 15-min observation. We confirmed the previous finding that the VTA injection, alone, of 0.03 microg morphine shortened the latency to show maternal behavior and that 0.0 microg and 0.01 microg morphine did not. Ingestion of amniotic fluid (and therefore POEF) facilitated the onset of maternal behavior in rats receiving an intra-VTA microinjection of an otherwise
Iravani, Kamyar; Hashemi, Seyed Basir; Tehrani, Maryam; Rashidi, Mohsen
Chondrosarcomas of the larynx are rare malignancies and frequently located in cricoid cartilage. They are characterized by a low tendency to metastasis (low grade type). The treatment of choice is surgery, which may be endoscopic or open partial surgery, if extension of the cancer is limited. Prognosis is generally good. In this report, a case of low grade chondrosarcoma of the larynx is presented, which was treated surgically with a combined use