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Fritscher-Ravens A.,University of Kiel | Milla P.,University College London | Ellrichmann M.,University of Kiel | Hellwig I.,University of Kiel | And 3 more authors.
Gastrointestinal Endoscopy | Year: 2013

Background: Many GI motility disorders are associated with underlying GI neuromuscular pathology, which requires full-thickness biopsies (FTB) for histopathologic diagnosis. Currently, none of the endoscopy-based attempts to obtain FTB specimens have proven suitable for routine use. This study evaluated a novel endoscopic prototype device (ED) for this purpose. Objective: To determine (1) the ability of the ED to obtain suitable FTB specimens, (2) associated complications, (3) feasibility of reliable defect closure, and (4) ability to evaluate intramural neuromuscular components. Design: Preclinical proof-of-concept study in 30 pigs. Setting: Animal laboratory. Intervention: Gastric FTB specimens were obtained with a circular cutter and anchor. The defect was closed by over-the-scope clips/T-tags. The resection site was inspected via laparoscopy. After 2 to 4 weeks, necropsy was carried out to evaluate late complications. Main Outcome Measurements: Feasibility, safety, and closure rate of the procedure. FTB specimens were assessed by histology/immunohistochemistry to visualize enteric neuromusculature. Results: A total of 29 of 30 procedures were successfully performed; one hemorrhage required endoscopic treatment. A total of 29 of 30 FTB specimens (mean diameter 9.1 mm) were retrieved in 7.1 ± 0.4 minutes (range 3.0-12.5 minutes), displaying optimal tissue quality. Defect closure took 10.8 ± 0.9 minutes (range 7.2-32 minutes). Laparoscopy did not reveal damage to adjacent organs. Necropsy showed well-healed scars at the resection site and no complications, peritonitis, or abscess formation. Histology showed smooth muscle layers and submucosal and myenteric ganglia. Limitations: Survival animal pilotstudy, no patients. Conclusion: The novel ED enabled safe harvesting of well-preserved FTB specimens. Defect closure proved to be reliable. All neuromuscular structures relevant for histopathologic evaluation of GI neuromuscular pathology were demonstrated. Further studies are needed to verify the efficacy of this prototype device in the entire gut and in humans. © 2013 American Society for Gastrointestinal Endoscopy.

Ulbrich S.E.,TU Munich | Meyer S.U.,TU Munich | Zitta K.,Ludwig Maximilians University of Munich | Hiendleder S.,Ludwig Maximilians University of Munich | And 8 more authors.
Molecular and Cellular Endocrinology | Year: 2011

Early embryonic development is critically dependent on both maternal preparation and embryonic signalling of pregnancy. Matrix metallopeptidases (MMP) contribute to spatial and temporal matrix remodeling in the bovine endometrium. In this study we observed distinct changes in expression of MMP2, MMP14, and the metallopeptidase inhibitor TIMP2 between different phases of the estrous cycle indicating an endocrine regulation. An increase of TIMP2 protein abundance was ascertained in the uterine lumen during the time of embryo elongation. The expression pattern and cellular localization correlate well with the assumed effects of MMPs on release and activation of cytokines and growth factors directing cell migration, differentiation, and vascularization during this pivotal period of development. Specifically, active MMP2 in the endometrium may determine the allocation of growth factors supporting conceptus development. The presence of a day 18 conceptus in vivo and day 8 blastoysts i. n vitro induced endometrial TIMP2 mRNA expression. The results imply that TIMP2 is involved in very early local maternal recognition of pregnancy. Matrix metallopeptidases are likely to participate in remodeling processes preparing a receptive endometrium for a timely and precise regulation of embryo development. © 2010 Elsevier Ireland Ltd.

Groebner A.E.,TU Munich | Rubio-Aliaga I.,TU Munich | Schulke K.,TU Munich | Reichenbach H.D.,Institute of Animal Breeding | And 4 more authors.
Reproduction | Year: 2011

Amino acids (AAs) are crucial for the developing conceptus prior to implantation. To provide insights into the requirements of the bovine embryo, we determined the AA composition of the uterine fluid. At days 12, 15, and 18 post-estrus, the uteri of synchronized pregnant and non-pregnant Simmental heifers were flushed for the analysis of 41 AAs and their derivatives by liquid chromatography-tandem mass spectrometry. The ipsilateral endometrium was sampled for quantitative PCR. In addition to a pregnancy-dependent increase of the essential AAs (P<0.01), we detected elevated concentrations for most non-essential proteinogenic AAs. Histidine (His) and the expression of the His/peptide transporter solute carrier 15A3 (SLC15A3) were significantly increased at day 18 of pregnancy in vivo. In addition, SLC15A3 was predominantly stimulated by trophoblast-derived interferon-τ in stroma cells of an in vitro co-culture model of endometrial cells. Our results show an increased concentration of AAs most likely to optimally provide the elongating pre-attachment conceptus with nutrients. © 2011 Society for Reproduction and Fertility.

Groebner A.E.,TU Munich | Schulke K.,TU Munich | Unterseer S.,TU Munich | Reichenbach H.D.,Institute of Animal Breeding | And 5 more authors.
Placenta | Year: 2010

Bovine trophoblast cells release interferon-τ (IFNT), a type I IFN, as the pregnancy recognition signal. Since type I IFNs exert growth inhibitory and proapoptotic actions, the effect of the conceptus on components of the apoptosis pathways was determined in the bovine endometrium during the periimplantation period. Uteri of Simmental heifers were flushed post mortem at days 12, 15, and 18 of cycle or pregnancy for the recovery of conceptuses and the sampling of ipsilateral endometrial tissue at slaughter for quantitative RT-PCR, immunohistochemistry, caspase activity and TUNEL assays. Endometrium samples of pregnant animals revealed increased transcript levels for the proapoptotic genes XAF1 (day 15: 2.9-fold; day 18: 15.1-fold; p = 0.005) and CASP8 (day 18: 2.4-fold; p = 0.007). The mRNA expression increased significantly with the day of the cycle for the proapoptotic genes FASLG, TNFSF10, TNF and TNFSF1A (p = 0.004, p = 0.006, p = 0.001 and p = 0.007) and the antiapoptotic gene BIRC4 (p = 0.03). We detected high amounts of FASLG transcripts in day 18 conceptuses (16-fold higher than day 18 endometria). This finding was validated at the protein level by immunohistochemistry. To further analyse the endometrial activation of the caspase cascade, the activities of initiator caspase 8 and effector caspases 3/7 were determined luminometrically. No difference between pregnant and cyclic animals was found for either caspase activity. Additionally, a TUNEL assay showed no increase of apoptotic cells in the pregnant endometrium. In conclusion, although the bovine conceptus induces the expression of proapoptotic genes, neither an activation of a caspase cascade nor an increase of apoptotic cells was noticed. These results suggest inhibitory mechanisms preventing endometrial cells from programmed cell death. © 2009 Elsevier Ltd. All rights reserved.

Groebner A.E.,TU Munich | Schulke K.,TU Munich | Schefold J.C.,Charite - Medical University of Berlin | Fusch G.,University of Greifswald | And 5 more authors.
Reproduction, Fertility and Development | Year: 2011

A well-balanced immunological interaction between mother and the semi-allogenic embryo is of particular importance. The objective of the present study was to analyse mechanisms of immune tolerance in bovine pregnancy during peri-implantation. Simmental heifers inseminated with either cryopreserved spermatozoa or seminal plasma were killed 12, 15 or 18 days after oestrus. Uteri were flushed for the recovery of conceptuses and the ipsilateral intercaruncular endometrium was sampled for gene expression analysis. Indoleamine 2,3-dioxygenase (IDO) mRNA, coding for the initial enzyme of the kynurenine pathway, was 18-fold (P<0.001) more abundant in the endometrium of Day 18 pregnant v. non-pregnant animals. Tandem mass spectrometry revealed a decrease of endometrial l-tryptophan (P≤0.0008), but an increase of l-kynurenine concentration (P=0.005) from Day 12 to Day 18, suggesting increasing IDO activity (P<0.03). An in vitro coculture model of endometrial cells showed an induction of IDO expression following interferon- exposure primarily in stroma cells, which was confirmed by in situ hybridisation localising IDO mRNA mainly in deep stroma cells. Immunohistochemical analysis revealed fewer CD45-positive leucocytes in the zona basalis of pregnant animals. Elevated IDO activity may reduce the presence of leucocytes in the pregnant endometrium, providing a possible mechanism for protecting the semi-allogenic conceptus from maternal rejection. © 2011 CSIRO.

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