Introduction: Actually 0.7-1 percent of all deliveries can be followed by secondary uterine bleeding. There is a residual trophoblastic tissue diagnosed in most of these cases and it is often managed by repeat intrauterine interventions. These operations are connected with high risk of formation of intrauterine adhesions and their early diagnosis and management can be important for next fertility. Material and methods: There were generally 188 patients included into the study. All patients underwent ultrasound examination in 6 weeks after delivery and ambulant hysteroscopy after next 2 months without anesthesia. Described intrauterine pathologies (residual tissue and adhesions) were managed in one step. Results: In cases with suspect ultrasound finding, the retained trophoblastic tissue was diagnosed by hysteroscopy in 66 percent vs. in 96 percent with sensitivity 85 percent and specificity 85 percent. Patients with intrauterine adhesions had normal ultrasound finding in 74 percent and it did not recognized patients with severe adhesions in 94 percent. Clinical signs had generally 72 percent of patients with diagnosed left residual tissue. Number of severe residual tissue is increasing with delay of instrumental evacuation from delivery (10 vs. 30 percent). When is necessary to repeat the operation...

Mechanical interactions of cells with their immediately surrounding extracellular matrix (ECM) is now known to be critical in pathophysiology. For example, during cancer progression, while uncontrollable cell division leads to tumor formation, the subsequent metastatic migration of cells from the primary tumor site to distant parts of the body causes most cancer-related deaths. The metastatic journey requires cells to be able to adopt different shapes and move persistently through the highly fibrous native ECM, thereby requiring significant spatiotemporal reorganization of the cell cytoskeleton. While numerous studies performed on flat 2-dimensional culture platforms and physiological 3D gels have elucidated cytoskeletal reorganization, our understanding on how cells adapt to natural fibrous microenvironments and regulate their behavior in response to specific ECM biophysical cues including fiber size, spacing, alignment and stiffness remains in infancy. Here, we utilize the non -electrospinning Spinneret tunable engineered parameters (STEP) technique to manufacture ECM mimicking suspended fibrous matrices with precisely controlled fiber diameters, network architecture, inter-fiber spacing and structural stiffness to advance our fundamental understanding of how external cues affect cytoskeleton-based cellular forces in 3-distinct morphological processes of the cell cycle starting from division to spreading and migration. Mechanobiological insights from these studies are implemented to deliver intracellular cargo inside cells using electrical fields. Holistically, we conclude that fibrous environments elicit multiple new cell behaviors never before reported. Specifically, our new findings include (i) design of fiber networks regulates actin networks and cell forces to sculpt nuclei in varying shapes: compressed ovals, tear drop, and invaginations, and drive the nuclear translocation of transcription factors like YAP/TAZ. In all these shapes, nuclei remain rupture-free, thus demonstrating the unique adaptability of cells to fibers, (ii) dense crosshatch networks are fertile environments for persistent 1D migration in 3D shapes of rounded nuclei and low density of actin networks, while sparse fiber networks induce 2D random migration in flattened shapes and well-defined actin stress fibers, (iii) actin retraction fiber-based stability regulates mitotic errors. Cells undergoing mitosis on single fibers exhibit significant 3D movement, and those attached to two fibers can have rotated mitotic machinery, both conditions contributing to erroneous division, and (iv) a bi-phasic force response to electroporation that coincides with actin cytoskeleton remodeling. Cells on suspended fibers can withstand higher electric field abuse, which opens opportunities to deliver cargo of varying sizes inside the cell. Taken altogether, our findings provide new mechanobiological understanding of cell-fiber interactions at high spatiotemporal resolution impacting cell migration, division and nuclear mechanics-key behaviors in the study of pathophysiology. / Doctor of Philosophy / Cancer, one of the major pathophysiological conditions, progresses within the living body through spreading of malignant cells from the primary tumor to distant secondary sites, ultimately leading to life-ending outcomes. Such spreading of cancer also known as cancer metastasis requires mechanical interactions of cells with their immediately surrounding microenvironment or the extracellular matrix (ECM). Cells utilize their cytoskeleton, a dynamic internal network of filamentous proteins, to adopt various morphologies, exert mechanical forces and physically remodel their local environment as they navigate through the highly fibrous native ECM. While previous research has elucidated how biochemical factors and bulk matrix properties regulate such cytoskeletal organization and single cell behavior, our understanding of how cells adapt to fibrous environments and respond to local biophysical cues like fiber diameter, spacing, alignment and stiffness remains in infancy. Here we use the non -electrospinning Spinneret tunable engineered parameters (STEP) to generate suspended nanofiber networks of tunable geometric and mechanical properties to mimic the native cellular environment. We discover that cells elongated within these ECM-mimicking environments utilize a unique cytoskeletal caging structure to regulate the shape and response of their nuclei in a fiber -diameter and organization-dependent manner. Additionally, we demonstrate that these elongated cell morphologies often observed during metastatic cancer cell movements, is achievable not only in aligned fibers but can also be induced by dense networks of fibers in a crossing organization. Specifically, such dense crosshatch networks allow cells to migrate persistently at high speeds while cells on sparsely spaced networks demonstrate slower and random movements. As cells elongated during interphase rounded up to undergo division, we find that the underlying fiber-geometry modulates mitotic dynamics through differential levels of actin retraction fiber-mediated stability, leading to significant alterations in orientation of mitotic machinery and mitotic spindle defects. Finally, we utilize these mechanobiological insights on cytoskeletal organization and cell shape control to optimize intracellular delivery of cargo using high-voltage electric fields. We demonstrate suspended cells are capable of withstanding higher electric fields and identify multistage cell contractility recovery dynamics, which correlate with cytoskeletal disruption and reassembly. Taken altogether, our findings provide a comprehensive understanding of the fibrous ECM-mediated regulation of the cytoskeletal organization and its impact in cell migration, division and nuclear mechanics. Knowledge obtained from this study will improve our understanding of cancer metastasis and provide predictive data for in vivo cellular response, essential for cytoskeleton-targeting cancer therapies.

No / We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Peritoneale Verwachsungen sind die häufigste Komplikation nach operativen Eingriffen. Sie haben abdominelle Beschwerden, chronische Ileuszustände und den akuten Ileus zur Folge. Diese peritonealen Reaktionen sind bei Neugeborenen und Säuglingen besonders ausgeprägt. Seit es operative abdominelle Eingriffe gibt, stellt das Vermeiden von peritonealen Adhäsionen ein zentrales Problem dar. Eine Vielzahl von Substanzen, lokal oder systemisch angewendet, wurde getestet, um Adhäsionen zu vermeiden. Bis heute hat sich kein gesicherter Standard für die Sekundärprophylaxe von Adhäsionen etabliert. Eine nicht ausreichende Wirksamkeit oder klinisch nicht akzeptable Nebenwirkungen sind dafür die Ursache. Das primäre Vermeiden von Verwachsungen steht nach wie vor im Mittelpunkt chirurgischer Bemühungen. Das organschonende Operieren und das Verbannen von Talkum an den Handschuhen der Chirurgen sind weltweit anerkannte Maßnahmen der Primärprophylaxe von Adhäsionen. Dass man auch den Abrieb von Bauchtüchern, die üblicherweise aus Baumwolle gefertigt sind, für die Entstehung von Adhäsionen verantwortlich machen muss, ist seit langem bekannt, wurde aber bisher in der operativen Praxis weitestgehend ignoriert. Die vorliegende tierexperimentelle Arbeit am Rattenmodell zeigt, dass die Verwendung alternativer Werkstoffe (Polyestergewebe) anstelle der üblichen Baumwolltücher zu einer deutlichen Verminderung der Adhäsionszahl und -stärke führt. Im zweiten Teil der Arbeit wird gezeigt, dass neben diesen primärprophylaktischen Maßnahmen auch sekundär Adhäsionen reduziert werden können. Wir untersuchten hierzu die Wirkung von PF 5080, einem Perfluorcarbon, auf das Peritoneum. Eine deutliche Reduktion der postoperativen Verwachsungen am Rattenmodell ließ sich nachweisen. Die lokale und systemische Wirkungsweise dieser Substanz ist noch weitgehend unbekannt und bedarf weiterer Forschung. / Peritoneal adhesions are the most common complication after surgical procedures. They often cause abdominal pain and bowel obstruction. Those peritoneal reactions are very intense in newborn and infants. Since the first days of open abdominal surgery the avoidance of adhesions has been a key problem. A countless array of drugs for local or systemic use to reduce adhesions has been tested. Until now no proven standard of secondary prophylaxis against adhesions has been established. The reasons are inefficiency or unacceptable side effects. The primary avoidance of adhesions is still the main focus for surgeons. Surgical techniques which keeps the organ carefully and the banishment of talcum from surgical gloves are widely acknowledged standards in the primary prophylaxis of adhesions. The fact that cotton particles from surgical swabs also contribute to postoperative adhesions has been known for along time but until now ignored in surgical practice. This study shows that the use of alternative material like polyester instead of cotton during operation does strongly reduce the formation of adhesions in rat. A further finding of this study is that the instillation of PF 5080, a Perfluorocarbon, in the late phase of the operation in the abdominal cavity does also reduce postoperative adhesions in rat. The local and systemic mode of function remains unknown and will be the subject to further research.

Chronic pelvic pain (CPP) is defined as constant or intermittent lower, cyclical or non-cyclical abdominal pain of at least six months’ duration. In the United Kingdom, over 1 million women suffer from CPP, with an estimated annual healthcare cost above £150 million. The aetiology of CPP is unknown in up to 50% of women, and in the remainder, the symptoms of CPP is associated with endometriosis, pelvic adhesions, irritable bowel syndrome or painful bladder syndrome. CPP is often accompanied by painful periods, pain during sexual intercourse and defaecation. Fatigue, sleep disturbances and depression are also common among this group of women. CPP asserts a heavy emotional, social and economic burden. Standard treatments such as hormonal and analgesic regimens are often associated with unacceptable side effects, even if helpful for the pain, underlining an urgent need for a satisfactory treatment. The meridian balanced method (BM) electro-acupuncture (EA) treatment (acupuncture needling + traditional Chinese medicine health consultation [TCM HC]) may be effective in managing CPP symptoms. Thus, I have completed a pilot study comprising of a three-armed randomised controlled trial (RCT), using a mixed methods research (MMR) approach, to assess the feasibility of a future large-scale RCT to determine the effectiveness of the meridian BMEA treatment on CPP in women. My hypothesis is that it is feasible to conduct such a large-scale RCT for CPP in women. The primary objectives were to determine recruitment and retention rates. The secondary objectives were to evaluate the, acceptability of the methods of recruitment, randomisation, interventions and assessment tools and any signals of effectiveness of the interventions. Thirty (30) women with CPP were randomised into three groups: BMEA treatment, TCM HC, or National Health Service standard care (NHS SC) group. The effects of my interventions were assessed by validated pain, physical and emotional functioning questionnaires, completed at weeks 0, 4, 8 and 12 of the study. Semi-structured telephone interviews and focus group discussions to explore participants’ experience of the study were conducted. Of the 59 women who were referred to the study, 30 women (51%) were randomised. There was a statistically significant difference in retention rates between the three groups. The retention rates were 80% (95% CI 74-96), in the BMEA treatment group, 53 % (95% CI 36- 70) in the TCM HC group and 87% (95% CI 63-90) in the NHS SC group. (Chi-square test, p=0.08) The attendance rates of the BMEA treatment group were 90% compared to 56% in the TCM HC group. There was a statistically significant difference (Mann-Whitney test, p=0.023) in attendance between the two intervention groups. Telephone interviews regarding the acceptability of the methods of recruitment, randomisation, assessment tools and interventions were positive. No adverse effects that were directly related to BMEA treatments were reported or observed. A higher proportion of the BMEA treatment group achieved clinical significance in the VAS-pain, BPI-pain severity, interference, and sleep scores, when compared to the other two groups. Due to small sample sizes, there was insufficient power to show statistically significant difference. (Fishers Exact Test, p=1.0) Analyses of the questionnaire data per group showed statistically significant differences in the following: the BMEA treatment group experienced less in pain at weeks 4 (p=0.01) and 8 (p=0.005); less helplessness (p=0.03) and their anxiety and depression scores declined at week 4 (p=0.04). The NHS SC group also reported less pain at week 4 (p=0.04). However, this group scored higher in anxiety and depression at weeks 8 and 12 (p=0.04). No statistically significant differences were achieved between the three groups at baseline, weeks 4, 8 and 12 in all scores. The therapeutic benefits gained by the TCM HC group were less compared to those of the BMEA treatment group, but better when compared to the NHS SC group. The BMEA treatment and TCM HC groups showed lower scores in anxiety and depression while the NHS SC group showed higher scores in anxiety and depression. The NHS SC group also tended to ruminate and magnify their problems as well as feeling more helpless than the other two groups. The three key themes that emerged from thematic analysis of focus group discussions were the “whole person effects” where participants reported an improvement in pain, sleep and a general sense of wellbeing in the two intervention groups; the “experience of standard care” and “impact of living with CPP”. In conclusion, the results of my pilot study are supportive of the feasibility of a future large-scale study. There were signals of effectiveness of interventions but the sample size was too small to make a definitive conclusion.

Células de músculo liso vascular são capazes de perceber estímulos mecânicos do sistema cardiovascular, coordenando pressão sanguínea e perfusão tecidual por meio da modulação do tônus e do diâmetro vascular via resposta contrátil. O gatilho inicial à contração é um aumento na concentração intracelular de cálcio e diversas vias de sinalização têm sido descritas na sustentação deste sinal inicial. Evidências atuais indicam que adesões focais desempenham papel crucial na contração através da organização do citoesqueleto de actina e engajamento com o aparato contrátil. Nosso grupo demonstrou que a proteína rica em cisteína e glicina 3 (CRP3) interage com a quinase de adesão focal (FAK) em resposta a um aumento do estiramento mecânico e existem evidências de que CRP3 modula a dinâmica do citoesqueleto de actina. Neste trabalho testamos a hipótese de que a proteína CRP3 atua como uma proteína de adesão focal que regula a contração de células musculares lisas aórticas. Por meio de ensaios de imunoprecipitação e colocalização, verificou-se a presença de CRP3 nas adesões focais de células selvagens. Evidenciou-se que a ausência de CRP3 está associada a aumento no tamanho médio de adesões focais em células musculares lisas aórticas de forma independente do substrato. Entretanto, em resposta à angiotensina II, células nocaute para CRP3 apresentam incapacidade de maturação das adesões focais, um evento que está associado ao reduzido conteúdo proteico de FAK, paxilina e MLC2 (plataformas moleculares envolvidas na maturação de adesões focais) observada em células nocaute. Consistente com o maior tamanho médio das adesões focais, células nocaute são mais rígidas e, portanto, menos elásticas que células selvagens, sendo que a rigidez avaliada por citometria magnético-óptica de oscilação se reflete na reduzida capacidade contrátil, seja em condições basais, em resposta à angiotensina II ou ao inibidor de ROCK, como evidenciado no ensaio de contração em gel de colágeno. Em síntese, os dados deste trabalho mostram que CRP3 está presente nas adesões focais, regulando tamanho e sinalização, com reflexos na rigidez (viscoelasticidade) e capacidade contrátil, variáveis fundamentais ao correto funcionamento de células musculares lisas aórticas. Em conjunto, as evidências deste trabalho suportam a hipótese de que CRP3 é um modulador de contratilidade e mecanotransdução em células musculares lisas aórticas / Smooth muscle cells act also as mecanosensors of the cardiovascular system, coordinating blood pressure and tissue perfusion by means of vascular tone and diameter modulation via the contractile response. The trigger for contraction is a rise in the intracellular calcium concentration and several signaling pathways have been described to sustain the initial calcium signal. Recent evidences highlight the crucial role of focal adhesions to the contractile response, given its role in actin cytoskeleton assembly and engagement with the actomyosin contractile apparatus. We have demonstrated that the cysteine and glycine-rich protein-3 (CRP3) interacts with focal adhesion kinase (FAK) in response to increased hemodynamic stress. Additionally, it has also been shown that CRP3 modulates actin cytoskeleton dynamics. Here, we tested the hypothesis that CRP3 acts as a focal adhesion protein that regulates the contraction of aortic smooth muscle cells. Through colocalization and immunoprecipitation studies we found that CRP3 is a focal adhesion protein in aortic smooth muscle cells. Focal adhesion mean size evaluation showed that in the baseline, CRP3 KO smooth muscle cells display greater focal adhesion size. However, upon angiotensin II (a contraction-triggering molecule) stimulation, CRP3 KO cells fail to maturate focal adhesions, an event that might be related to the reduced protein levels of FAK, paxillin, and MLC2 (key signaling molecules involved in focal adhesion maturation) observed in KO cells. Consistent with the greater mean focal adhesion size, CRP3 KO cells exhibited increased stiffness and therefore, reduced viscoelasticity when compared to wild type cells. The reduced viscoelasticity of KO cells seems to influence cell contractility, as CRP3 KO cells also displayed reduced contractile response in the baseline and in response to angiotensin II. In summary, these data showed that CRP3 is present at focal adhesions, regulating their size and signaling. Thus, CRP3 at focal adhesions influences cell stiffness and contractile capacity, which are key features of smooth muscle cell physiology. Altogether, our findings support the idea that CRP3 is a key modifier of contractility and mechanotransduction in aortic smooth muscle cells

<p>Epithelial damage and shedding of the epithelium are common observations in many airway diseases such as asthma, Sjögren’s syndrome, chronic obstructive pulmonary disease and cystic fibrosis. The ability of the cells to attach to each other and/or to the matrix seems to be altered. In the present study, cultured normal human bronchial epithelial cells were used as a model system. The desmosomes and also the focal adhesions were investigated to see if changes in these structural components as well as metabolic alterations could explain the observed shedding of the epithelium.</p><p>Inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), hypochlorous acid (HOCl) and nitric oxide (NO) are present in increased amounts in inflammation. The Th-1 cytokines, IFN-γ and TNF-α, as well as HOCl and NO affected the number of desmosomes and their ability to attach to each other. Interestingly, the Th-2 cytokines IL-4, IL-5 and IL-13 did not affect the cell-cell adhesion. HOCl and NO also affected the focal adhesions of the cells. </p><p>Both morphological and functional studies indicated that TNF-α, IFN-γ, HOCl and NO affect the mitochondria. A decreased glucose oxidation rate could result in a decreased production of ATP, which in turn could lead to inhibition of many cellular activities including an impaired ability of the ciliary activity in bronchial epithelial cells and mucus transport. The antioxidant N-acetyl-L-cysteine and the nitric oxide synthase inhibitor N-propyl-L-arginine inhibited these effects of HOCl. This indicates that HOCl can induce damage both by induction of free radicals and also through an increased production of NO. TNF-α and IFN-γ also induced an increased production of NO. N^ω-monomethyl-L-arginine reduced the cytokine-induced production of NO. The NO donor DETA NONOate reduced the total protein biosynthesis as well as the DNA content. NO can react with superoxide anions generated by inflammatory cells in the airways to form peroxynitrite ions, which in turn could generate hydroxyl radicals. These toxic ions may contribute to damage of the airway epithelial cells. </p><p>In conclusion, pro-inflammatory cytokines such as TNF-α, IFN-γ and also the reactive oxygen species HOCl and NO could contribute to airway epithelial shedding by affecting the adhesion properties of the epithelial cells. More generalized morphological and metabolic changes could be other contributing factors, together with the increased production of NO.</p>

Epithelial damage and shedding of the epithelium are common observations in many airway diseases such as asthma, Sjögren’s syndrome, chronic obstructive pulmonary disease and cystic fibrosis. The ability of the cells to attach to each other and/or to the matrix seems to be altered. In the present study, cultured normal human bronchial epithelial cells were used as a model system. The desmosomes and also the focal adhesions were investigated to see if changes in these structural components as well as metabolic alterations could explain the observed shedding of the epithelium. Inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), hypochlorous acid (HOCl) and nitric oxide (NO) are present in increased amounts in inflammation. The Th-1 cytokines, IFN-γ and TNF-α, as well as HOCl and NO affected the number of desmosomes and their ability to attach to each other. Interestingly, the Th-2 cytokines IL-4, IL-5 and IL-13 did not affect the cell-cell adhesion. HOCl and NO also affected the focal adhesions of the cells. Both morphological and functional studies indicated that TNF-α, IFN-γ, HOCl and NO affect the mitochondria. A decreased glucose oxidation rate could result in a decreased production of ATP, which in turn could lead to inhibition of many cellular activities including an impaired ability of the ciliary activity in bronchial epithelial cells and mucus transport. The antioxidant N-acetyl-L-cysteine and the nitric oxide synthase inhibitor N-propyl-L-arginine inhibited these effects of HOCl. This indicates that HOCl can induce damage both by induction of free radicals and also through an increased production of NO. TNF-α and IFN-γ also induced an increased production of NO. Nω-monomethyl-L-arginine reduced the cytokine-induced production of NO. The NO donor DETA NONOate reduced the total protein biosynthesis as well as the DNA content. NO can react with superoxide anions generated by inflammatory cells in the airways to form peroxynitrite ions, which in turn could generate hydroxyl radicals. These toxic ions may contribute to damage of the airway epithelial cells. In conclusion, pro-inflammatory cytokines such as TNF-α, IFN-γ and also the reactive oxygen species HOCl and NO could contribute to airway epithelial shedding by affecting the adhesion properties of the epithelial cells. More generalized morphological and metabolic changes could be other contributing factors, together with the increased production of NO.

No description available.

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.