Towards an in vitro fibrogenesis model of human vocal fold scarring.

BACKGROUND: Vocal fold (VF) scarring remains a therapeutic dilemma and challenge in modern laryngology. To facilitate corresponding research, we aimed to establish an in vitro fibrogenesis model employing human VF fibroblasts (hVFF) and the principles of macromolecular crowding (MMC). METHODS: Fibrogenesis was promoted by addition of transforming growth factor-ß1 to standard medium and medium containing inert macromolecules (MMC). Hepatocyte growth factor (HGF) and Botox type A were tested for their antifibrotic properties in various doses. Experiments were analyzed with respect to the biosynthesis of collagen, fibronectin, and α-smooth muscle actin using immunofluorescence, silver stain and western blot. RESULTS: MMC led to favourable enhanced deposition of collagen and other extracellular matrix components, reflecting fibrotic conditions. Low doses of HGF were able to dampen profibrotic effects. This could not be observed for higher HGF concentrations. Botox type A did not show any effects. CONCLUSION: Based on the principles of MMC we could successfully establish a laryngeal fibrogenesis model employing hVFF. Our finding of dose-dependent HGF effects is important before going into clinical trials in humans and has never been shown before. Our model provides a novel option to screen various potential antifibrotic compounds under standardized conditions in a short time.

Evidence from the very beginning: endoglandular trophoblasts penetrate and replace uterine glands in situ and in vitro.

STUDY QUESTION: How is histiotrophic nutrition of the embryo secured during the first trimester of pregnancy? SUMMARY ANSWER: Rather than specifically focusing on invasion into spiral arteries, extravillous trophoblasts also invade into uterine glands (endoglandular trophoblast) from the very beginning and open them toward the intervillous space. WHAT IS KNOWN ALREADY: Extravillous trophoblasts can be found in close contact and within the lumen of uterine glands, sometimes replacing glandular epithelial cells. STUDY DESIGN, SIZE, DURATION: As well as extensive screening of specimens from first trimester placentation sites in situ we used a previously established three-dimensional co-culture in vitro model system of first trimester villous explants with non-invaded decidua parietalis. PARTICIPANTS/MATERIALS, SETTING, METHODS: First trimester placentas were obtained from elective terminations of pregnancies (n = 48) at 5-11 weeks of gestational age. A subset was processed for confrontation co-culture (n = 31). Invaded decidua basalis was obtained from 20 placentas. All tissues were sectioned, subsequently immunostained and immunodoublestained with antibodies against keratin 7 (KRT7), major histocompatibility complex, class I, G (HLA-G), matrix metallopeptidase 9 (MMP9), von Willebrand factor (VWF) and the appropriate Immunoglobulin G (IgG) negative controls. Replacement of endothelial/epithelial cells by extravillous trophoblasts was quantified semi-quantitatively. Additionally, hematoxylin and eosin-stained archival specimens from early implantation sites were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: The earliest available specimen was from around Day 10 after conception; already at this stage trophoblasts had penetrated into uterine glands and had started to replace the epithelium of the glands. Endoglandular trophoblasts replaced uterine glands in vitro and in situ and could be found in the lumen of invaded glands. Quantitative analysis revealed significantly more replacement of epithelial cells in glands (63.8 ± 22.1%) compared with endothelial cells in vessels (26.4 ± 8.8%). Accumulated detached glandular epithelial cells could be repeatedly observed in the lumen of invaded glands. Additionally, in areas of trophoblast invasion the glandular epithelium seemed to be completely disintegrated compared with glandular epithelium in the non-invaded parts of the decidua. Whole tissue specimens were used in vitro and in situ instead of cell lines; these systems mostly maintain the context of the in vivo situation. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study supported by in vitro experiments. However, a histological section will always only be a snapshot and quantification from histological sections has its limitations. WIDER IMPLICATIONS OF THE FINDINGS: This study further strengthens the hypothesis of histiotrophic nutrition of the embryo prior to the establishment of the maternal blood flow toward the placenta. Invasion of uterine glands by endoglandular trophoblasts may have more impact on the outcome of early pregnancy than assumed up to now.

Distribution of decidual natural killer cells and macrophages in the neighbourhood of the trophoblast invasion front: a quantitative evaluation.

STUDY QUESTION: Do decidual natural killer (dNK) cells and decidual macrophages (dMph) become enriched in the vicinity of the trophoblast invasion front? SUMMARY ANSWER: Morphometric image analysis and areal cell density calculations, which excluded observer bias, showed an enrichment of decidual leukocytes in the neighbourhood of the trophoblast invasion front. WHAT IS KNOWN ALREADY: In previous studies, the number of decidual leukocytes was visually counted in medium- or high power fields. These methods, however, cannot reveal the exact spatial relationship between leukocytes and invasive trophoblast cells, and are therefore prone to subjective errors. Thus, a more objective approach is required. STUDY DESIGN, SIZE, DURATION: Applying a new method of morphometric image analysis, leukocyte populations were studied in human tissue fragments derived from first trimester placentation sites (n = 7) as well as in co-cultures of first trimester decidual tissue with placental villi of the same pregnancy representing an appropriate in vitro model of trophoblast invasion (n = 15). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: First trimester decidual tissue was obtained from women undergoing elective terminations of pregnancy at 7-10 weeks of gestational age. Tissue sections were double-stained immunohistochemically for markers of dNK cells or dMph on one hand, and for invasive extravillous trophoblast cells on the other. To analyse the distribution of leukocytes, distinct cell compartments as well as cell neighbourhood areas were defined. Finally, relative areal cell densities were calculated and these data were compared with those of an in vitro model of trophoblast invasion as well as with tissue fragments derived from decidua parietalis without trophoblast cells. MAIN RESULTS AND THE ROLE OF CHANCE: At first trimester placentation sites, a higher density of dNK cells as well as of dMph was found in close proximity to the invasive trophoblast (P ≤ 0.01), compared with the average areal cell density of decidual leukocytes in the tissue with exclusion of the trophoblast. The highest areal cell density of leukocytes was determined up to a distance of 20 µm from the trophoblast cells, whereas in more distant regions it was even lower than average, indicating a migration of these leukocytes towards the trophoblast invasion front. In the three-dimensional co-culture model, however, we found an enrichment of dMph (P ≤ 0.01) but not of dNK cells (P > 0,05) in the neighbourhood of the invasive trophoblast. LIMITATIONS, REASONS FOR CAUTION: The morphometric image analysis depends on intense immunohistochemical staining that is free of background and cross-reactivity. WIDER IMPLICATIONS OF THE FINDINGS: The presented method will be useful not only for the investigation of recurrent miscarriage but also in the fields of tumour immunology and inflammation. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the European Commission (Network of Excellence 'The Control of Embryo Implantation (EMBIC)', FP6-512040, lead researcher: P.S.), and by the Franz Lanyar Foundation of the Medical University of Graz, Austria (Grant #347). None of the authors declared a conflict of interests.

[Maternal-placental interactions and fetal programming]. / Mütterlich-plazentare Interaktionen und fetale Programmierung.

Pregnancy-related complications not only represent a risk for maternal and fetal morbidity and mortality, but are also a risk for several diseases later in life. Many epidemiological studies have shown clear associations between an adverse intrauterine environment and an increased risk of diabetes, hypertension, cardiovascular disease, depression, obesity, and other chronic diseases in the adult. Some of these syndromes could be prevented by avoiding adverse stimuli or insults including psychological stress during pregnancy, intake of drugs, insufficient diet and substandard working conditions. Hence, all of these stimuli have the potential to alter health later in life. The placenta plays a key role in regulating the nutrient supply to the fetus and producing hormones that control the fetal as well as the maternal metabolism. Thus, any factor or stimulus that alters the function of the hormone producing placental trophoblast will provoke critical alterations of placental function and hence could induce programming of the fetus. The factors that change placental development may interfere with nutrient and oxygen supply to the fetus. This may be achieved by a direct disturbance of the placental barrier or more indirectly by, e. g., disturbing trophoblast invasion. For both path-ways, the respective pathologies are known: while preeclampsia is caused by alterations of the villous trophoblast, intra-uterine growth restriction is caused by insufficient invasion of the extravillous trophoblast. In both cases the effect can be undernutrition and/or fetal hypoxia, both of which adversely affect organ development, especially of brain and heart. However, the mechanisms responsible for disturbances of trophoblast differentiation and function remain elusive.

[Recommendations on therapy for chronic constipation--working group for functional diagnosis and psychosomatics of the austrian society of gastroenterology and hepatology]. / Empfehlungen zur Therapie der chronischen Obstipation der Arbeitsgruppe für Funktionsdiagnostik und Psychosomatik der Österreichischen Gesellschaft für Gastroenterologie und Hepatologie.

Constipation is one of the most frequent gastroenterological problems, especially among elderly people. Chronic constipation is now defined by the new Rome III criteria. Life style counselling is usually only effective in early stages of disease and for mild types. In patients with constipation one should actively screen for possible causative, medically adverse events. Recently new effective opioid antagonists were introduced to act against constipation in patients on long-term opioid therapy. If these actions fail, medical therapy with polyethylene glycol or lactulose should be favoured in the long-term treatment. Psychodiagnostic queries concerning fear should be included in the diagnostic procedures. Biofeedback is an effective therapy in these cases and especially with pelvic floor dyssynergia. Surgical interventions are rarely indicated or successful with the exception of chronic outlet obstruction with severe anatomic changes.

Studies of the antibody-dependent killing of schistosomula of Schistosoma mansoni employing haptenic target antigens. I. Evidence that the loss in susceptibility to immune damage undergone by developing schistosomula involves a change unrelated to the masking of parasite antigens by host molecules.

A method was developed for coupling a hapten, trinitrophenyl (TNP), to the surface of schistosomula of Schistosoma mansoni which results in a minimal loss in their viability as judged by morphological examination in vitro and survival after injection in vivo. Skin-stage (3-h-old) and lung-stage (5-d-old) schistosomula surface labeled in this manner were then compared for their susceptibility to killing by anti-TNP antibody-dependent effector mechanisms both in vivo and in vitro. TNP skin-stage larvae were readily rejected in mice actively immunized against TNP bovine gamma globulin and were highly susceptible to anti-TNP-dependent killing mediated either by complement or purified human eosinophils in vitro. In contrast, TNP-lung-stage schistosomula, which were shown by microfluorimetry to bind anti-TNP antibody to approximately the same extent as skin-stage schistosomula, were found to be resistant to killing by the same in vivo and in vitro mechanisms. These findings suggest that the insusceptibility of postskin-stage schistosomula to antibody-dependent killing must result at least in part from an intrinsic structural change in the integument of the parasite and cannot be caused solely by the masking of parasite antigens by acquired host molecules, a mechanism of immune evasion previously proposed for schistosomes.

Endoglandular trophoblast, an alternative route of trophoblast invasion? Analysis with novel confrontation co-culture models.

BACKGROUND: Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands. METHODS: First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ. RESULTS: EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% +/- 7% (mean +/- SEM; n = 10, range 6-11 weeks) of glandular cross sections were associated with or infiltrated by EVTs. CONCLUSIONS: Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.

Factors involved in regulating trophoblast fusion: potential role in the development of preeclampsia.

In the human placenta, turnover of villous trophoblast involves proliferation, differentiation and fusion of mononucleated cytotrophoblasts with the overlying syncytiotrophoblast. In this way the syncytiotrophoblast is continuously supplied with compounds derived from the fusing cytotrophoblasts. Acquisition of fresh cellular components is balanced by a concomitant release of apoptotic material as syncytial knots from the syncytiotrophoblast to the maternal circulation. In the turnover of villous trophoblast, fusion is an essential step and has been shown to be regulated by multiple factors, such as cytokines, hormones, protein kinases, transcription factors, proteases and membrane proteins. Dysregulation of one or more of these fusion factors entails aberrant fusion of the cytotrophoblast with the syncytiotrophoblast, which adversely affects the maintenance and integrity of the placental barrier. Unbalanced trophoblast fusion and release of apoptotic material into the intervillous space may provoke a massive systemic inflammatory response by the mother and thus lead to preeclampsia.

Field theory of bicritical and tetracritical points. III. Relaxational dynamics including conservation of magnetization (model C).

We calculate the relaxational dynamical critical behavior of systems of O(n_{ parallel}) plus sign in circleO(n_{ perpendicular}) symmetry including conservation of magnetization by renormalization group theory within the minimal subtraction scheme in two-loop order. Within the stability region of the Heisenberg fixed point and the biconical fixed point, strong dynamical scaling holds, with the asymptotic dynamical critical exponent z=2varphinu-1 , where varphi is the crossover exponent and nu the exponent of the correlation length. The critical dynamics at n_{ parallel}=1 and n_{ perpendicular}=2 is governed by a small dynamical transient exponent leading to nonuniversal nonasymptotic dynamical behavior. This may be seen, e.g., in the temperature dependence of the magnetic transport coefficients.

Analysis of an in-house technique for temporary mandibulotomy and its impact on postoperative radiotherapy.

The purpose of this study was to describe a newly developed procedure for temporary mandibulotomy and subsequent osteosynthesis. Clinical outcomes were evaluated, including complications and the impact on postoperative treatment, particularly postoperative radiotherapy. Twenty-four patients underwent temporary mandibulotomies for the surgical resection of malignancies located in the posterior oral or oropharyngeal region. All were treated with postoperative radiotherapy. An angulated median mandibulotomy was followed by osteosynthesis with three anchor screws directed towards the inferior aspect of the mandible. Anchor screws are modified conventional lag screws that include an additional biconcave washer. This modification prevents the screw heads from cracking into the cancellous bone during tightening, improving their biomechanical qualities considerably. Insertion of screws at any angle to the bony surface therefore becomes possible, which is a precondition for this technique. Minor complications occurred in two patients in the early postoperative period. However, complications causing bony non-union, leading to postponed postoperative radiotherapy were not noted in this cohort.

Case series of seven women with uterine fibroids associated with venous thromboembolism and chronic thromboembolic disease.

Uterine fibroids have been described as an associate to acute venous thromboembolism (VTE), with case reports showing an association between large uterine fibroids, acute deep venous thrombosis (DVT), and acute pulmonary embolism (PE). However, there is little known about the association or causation between uterine fibroids, chronic thromboembolic disease (CTED), and chronic thromboembolic pulmonary hypertension (CTEPH). We report on six women with uterine fibroids and CTEPH, as well as one woman with CTED, all of whom presented with exertional dyspnea, lower extremity swelling, and in the cases of CTEPH, clinical, echocardiographic, and hemodynamic evidence of pulmonary hypertension and right heart failure. Compression of the pelvic veins by fibroids was directly observed with invasive venography or contrast-enhanced computed tomography in five cases. All seven women underwent pulmonary thromboendarterectomy (PTE) followed by marked improvement in functional, clinical, and hemodynamic status.

Field theory of bicritical and tetracritical points. I. Statics.

We calculate the static critical behavior of systems of O(n_||)(plus sign in circle)O(n_perpendicular) symmetry by the renormalization group method within the minimal subtraction scheme in two-loop order. Summation methods lead to fixed points describing multicritical behavior. Their stability border lines in the space of the order parameter components n_|| and n_perpendicular and spatial dimension d are calculated. The essential features obtained already in two-loop order for the interesting case of an antiferromagnet in a magnetic field ( n_|| =1, n_perpendicular =2 ) are the stability of the biconical fixed point and the neighborhood of the stability border lines to the other fixed points, leading to very small transient exponents. We are also able to calculate the flow of static couplings, which allows us to consider the attraction region. Depending on the nonuniversal background parameters, the existence of different multicritical behavior (bicritical or tetracritical) is possible, including a triple point.

Field theory of bicritical and tetracritical points. II. Relaxational dynamics.

We calculate the relaxational dynamical critical behavior of systems of O(n_||)(plus sign in circle)O(n_perpendicular) symmetry by renormalization group method within the minimal subtraction scheme in two-loop order. The three different bicritical static universality classes previously found for such systems correspond to three different dynamical universality classes within the static borderlines. The Heisenberg and the biconical fixed point lead to strong dynamic scaling whereas in the region of stability of the decoupled fixed point weak dynamic scaling holds. Due to the neighborhood of the stability border between the strong and the weak scaling dynamic fixed point to the dynamical stable fixed point a very small dynamic transient exponent of omega(Beta)_(v) =0.0044 is present in the dynamics for the physically important case n_|| =1 and n_perpendicular =2 in d=3 .

Predicting genetic merit for mastitis and fertility in dairy cattle using genome wide selection and high density SNP screens.

Two novel methods for genome wide selection (GWS) were examined for predicting the genetic merit of animals using SNP information alone. A panel of 1,546 dairy bulls with reliable EBVs was genotyped for 15,380 SNPs that spanned the whole bovine genome. Two complexity reduction methods were used, partial least squares (PLS) and regression using a genetic algorithm (GAR), to find optimal solutions of EBVs against SNP information. Extensive internal cross-validation was used tofind the best predictive models followed by external validation (without direct use of the pedigree or SNP location). Both PLS and GAR provided both accurate fit to the training data set for somatic cell count (SCC) (max r = 0.83) and fertility (max r = 0.88) and showed an accuracy of prediction of r = 0.47 for SCC, and r = 0.72 for fertility. This is the first empirical demonstration that genome wide selection can account for a very high proportion of additive genetic variation in fitness traits whilst exploiting only a small percentage of available SNP information, without use of pedigree or QTL mapping. PLS was computationally more efficient than GAR.

Right Axillary Artery Cannulation for Endovascular Repair of an Acute Type A Aortic Dissection.

We present the case of a 48-year-old woman with an acute type A aortic dissection that was treated with thoracic endovascular aortic repair at our institution. The patient was found to have a focal type A dissection with pericardial effusion but no tamponade physiology and no involvement of the aortic valve or root. We elected to treat the patient's type A aortic dissection with an endovascular stent because of the patient's favorable anatomy and no evidence of neurologic deficits or signs of distal malperfusion. The patient was successfully treated with an abdominal aortic cuff deployed through the axillary artery. An axillary approach was necessary because of the short length of the delivery sheath preventing a transfemoral delivery. At 2-year follow-up, the patient remains free of complications with computed tomography scan revealing complete false lumen thrombosis and a stable endovascular repair. This report demonstrates a case of acute type A aortic dissection successfully treated using thoracic endovascular aortic repair and illustrates the utility of axillary cannulation for precise deployment of stent grafts in the ascending aorta.

Effectiveness of educational and lifestyle interventions to prevent paediatric obesity: systematic review and meta-analyses of randomized and non-randomized controlled trials.

OBJECTIVE: This review and meta-analysis aim at updating a previous meta-analysis carried out by Waters et al. on the efficacy of interventions aimed at preventing childhood obesity and at identifying predictors of outcome. METHODS: Using an ad-hoc search string, PubMed database was searched for studies assessing body mass index reduction associated with programmes lasting ≥12 weeks in overweight and obese children aged 2-18 years. Studies designed for children with eating disorders or relevant comorbidities were excluded. Studies meeting inclusion criteria were classified according to patient age (<6, 6-12 and 13-18 years), and intervention type (physical activity, diet or both), setting (educational, family or both) and duration (≤1 or >1 year). The search was also extended to other databases. Hand-searching techniques were also applied. The Cochrane 'risk of bias' was applied for quality assessment. RESULTS: Seventy-two studies were meta-analysed. Overall, the best results were achieved by programmes combining diet and physical activity (n = 39). With regard to the setting, programmes involving both school and family and lasting ≤1 year were the most efficacious for 6- to 12-year-old children (n = 26); family-based-only interventions were also effective in children <6 years old (n = 2), although results have to be interpreted cautiously because of the small number of patients enrolled and the high study heterogeneity. In 13- to 18-year-old patients, interventions delivered at school (n = 8) were substantially unsuccessful. CONCLUSIONS: Interventions for childhood obesity prevention should include both diet and physical activity, be preferentially targeted towards school age children and involve both the school and family setting. However, because of the important methodological limitations associated with currently available literature, additional studies are needed to draw definite conclusions.

Anterior Leaflet Augmentation With CorMatrix Porcine Extracellular Matrix in Twenty-Five Patients: Unexpected Patch Failures and Histologic Analysis.

BACKGROUND: This study reported on the treatment of Carpentier type IIIa and type IIIb mitral regurgitation (MR) with a large patch anterior mitral valve leaflet augmentation technique using CorMatrix (CorMatrix Cardiovascular Inc, Alpharetta, GA) extracellular matrix (ECM). METHODS: A single-site chart review was conducted on patients who underwent anterior leaflet augmentation performed with the Da Vinci surgical robot (Intuitive Surgical, Sunnyvale, CA) or through a median sternotomy. Only patients who had anterior leaflet augmentation with porcine intestine ECM or autologous pericardium were included. Follow-up echocardiography was performed on all patients. Histologic specimens were available on ECM patches from a subset of patients who required reoperation. RESULTS: Between August 2011 and April 2014, 44 patients (mean age, 62.6 ± 12.2 years) underwent anterior leaflet augmentation with either porcine intestinal ECM or autologous pericardium at the Temple University Hospital in Philadelphia. Two (4%) late deaths occurred, one in each group. One patient who underwent ECM augmentation died of non-cardiac-related causes 7 months after discharge as a result of complications of chronic renal failure. The second late death occurred 5 months after discharge because of complications of a stroke in a patient in the pericardial augmentation group. Eight (32%) of the patients with ECM had recurrence of severe MR on echocardiography at an average time of 201 ± 98 days. Seven (28%) patients required reoperation because of failure of the ECM patch including perforation (4%), excessive patch dilation (20%), and suture line dehiscence (4%). In contrast, none of the patients with pericardial augmentation developed severe MR or required operation. Statistical analysis of the patients with ECM augmentation demonstrated no correlation of the following: age; sex; comorbid conditions such as diabetes, chronic obstructive pulmonary disease, and hypertension; left ventricular ejection fraction; surgical approach; annuloplasty size; or type of MR with patch failure. A lower body mass index was the only factor associated with recurrent severe regurgitation on univariate analysis (p = 0.039). Histologic study of the four available explants demonstrated intense inflammation without evidence of host integration. The remaining 15 (60%) patients had normal mitral leaflet structure and function on follow-up echocardiograms out to 12 months. CONCLUSIONS: For type III MR, a large anterior leaflet patch technique with porcine ECM was associated with a 32% recurrence rate of severe MR related directly to patch failure. Further research and development should be performed on the use of ECM materials with a goal to decrease the failure rate experienced in this study.