Liver steatosis in pre-transplant liver biopsies can be quantified rapidly and accurately by nuclear magnetic resonance analysis.

Donor livers marginally acceptable or acceptable according to extended criteria are more frequently transplanted due to the growing discrepancy between demand and availability of donor organs. One type of marginally acceptable graft is a steatotic donor liver, because it is more sensitive to ischemia-reperfusion injury. Thus, quantitative assessment of steatosis is crucial prior to liver transplantation. Extent of steatosis of 49 pre-reperfusion liver biopsies from patients who received orthotopic liver transplantation was assessed by three techniques: semi-quantitative histological evaluation, computerized histomorphometry, and NMR-based estimation of fat content. The findings were correlated to clinical data and to histological examination of corresponding post-reperfusion biopsies for quantification of ischemia-reperfusion injury. We found that values obtained through all three assessment methods were positively correlated. None of the values obtained by the three applied methods correlated with clinical outcome or extent of ischemia-reperfusion injury. Quantitative evaluation of steatosis by NMR yields results comparable to histological and morphometrical assessment. This technique is rapid (<5 min), accurately quantifies fat in donor livers, and provides results that can be used when evaluation by a pathologist is not available.

Acute short-term mental stress does not influence salivary flow rate dynamics.

BACKGROUND: Results of studies that address the influence of stress on salivary flow rate and composition are controversial. The aim of this study was to reveal the influence of stress vulnerability and different phases of stress reactivity on the unstimulated and stimulated salivary flow rate. We examined that acute mental stress does not change the salivary flow rate. In addition, we also examined the salivary cortisol and protein level in relation to acute mental stress stimuli. METHODS: Saliva of male subjects was collected for five minutes before, immediately, 10, 30 and 120 min after toothbrushing. Before toothbrushing, the subjects were exposed to acute stress in the form of a 2 min public speech. Salivary flow rate and total protein was measured. The physiological stress marker cortisol was analyzed using enzyme-linked immunosorbent assay. To determine the subjects' psychological stress reaction, the State-Trait-Anxiety Inventory State questionnaire (STAI) data were obtained. The subjects were divided into stress subgroup (S1) (psychological reactivity), stress subgroup (S2) (psychological and physiological reactivity) and a control group. The area under the curve for salivarycortisol concentration and STAI-State scores were calculated. All data underwent statistical analysis using one-way analysis of variance. RESULTS: Immediately after stress exposure, all participants exhibited a psychological stress reaction. Stress exposure did not change the salivary flow rate. Only 69% of the subjects continued to display a physiological stress reaction 20 minutes after the public talk. There was no significant change in the salivary flow rate during the psychological and the physiological stress reaction phases relative to the baseline. CONCLUSIONS: Acute stress has no impact on the salivary flow rate; however, there may be other responses through salivary proteins that are increased with the acute stress stimuli. Future studies are needed to examine specific proteins and their possible roles in acute stress responses.

Using human neural crest-derived progenitor cells to investigate osteogenesis: an in vitro study.

Human tooth contains a distinct population of neural crest-derived progenitor cells (dNC-PCs) which are known to give rise to specialized daughter cells of an osteogenic lineage. We hypothesised that dNC-PCs could develop into neural crest-derived bone in a self-propagating and extracorporal culture system. Thus, we examined the three-dimensional structure obtained from osteogenic-stimulated dNC-PCs by morphological, biochemical and spectroscopic methods. After the onset of stimulation, cells formed a multilayer with outer cells covering the surface and inner cells secreting a hyaline matrix. With prolonged culture, multilayers contracted and formed a three-dimensional construct which subsequently converted to a calcified mass. Differentiation of progenitor cells was associated with apoptosis. Cell types which survived were smooth muscle actin-positive cells and bone-like cells. The expression of osteoblastic markers and the secretion of a collagenous matrix indicate that the bone cells had acquired their functional phenotype. Furthermore, these cells produced and secreted membrane-bound vesicles into the newly forming matrix. Consequently, an early biomineralized extracellular matrix was found with calcium phosphate deposits being associated with the newly formed collagen matrix framework. The molar calcium-phosphorus-ratio of the mineralized collagen indicated that amorphous calcium phosphate was present within this matrix. The data suggest that stimulated cultures of dNC-PCs are able to recapitulate some processes of the early phase of osteogenesis.

The morphology of anisotropic 3D-printed hydroxyapatite scaffolds.

Three-dimensional (3D) scaffolds with tailored pores ranging from the nanometer to millimeter scale can support the reconstruction of centimeter-sized osseous defects. Three-dimensional-printing processes permit the voxel-wise fabrication of scaffolds. The present study rests upon 3D-printing with nano-porous hydroxyapatite granulates. The cylindrical design refers to a hollow bone with higher density at the periphery. The millimeter-wide central channel follows the symmetry axis and connects the perpendicularly arranged micro-pores. Synchrotron radiation-based micro computed tomography has served for the non-destructive characterization of the scaffolds. The 3D data treatment is essential, since, for example, the two-dimensional distance maps overestimate the mean distances to the material by 33-50% with respect to the 3D analysis. The scaffolds contain 70% micrometer-wide pores that are interconnected. Using virtual spheres, which might be related to the cells migrating along the pores, the central channel remains accessible through the micro-pores for spheres with a diameter of up to (350+/-35)mum. Registering the tomograms with their 3D-printing matrices has yielded the almost isotropic shrinking of (27+/-2)% owing to the sintering process. This registration also allows comparing the design and tomographic data in a quantitative manner to extract the quality of the fabricated scaffolds. Histological analysis of the scaffolds seeded with osteogenic-stimulated progenitor cells has confirmed the suitability of the 3D-printed scaffolds for potential clinical applications.

Defining properties of neural crest-derived progenitor cells from the apex of human developing tooth.

The connective tissue of the human tooth arises from cells that are derived from the cranial neural crest and, thus, are termed as "ectomesenchymal cells." Here, cells being located in a pad-like tissue adjacent to the apex of the developing tooth, which we designated the third molar pad, were separated by the microexplant technique. When outgrowing from the explant, dental neural crest-derived progenitor cells (dNC-PCs) adhered to plastic, proliferated steadily, and displayed a fibroblast-like morphology. At the mRNA level, dNC-PCs expressed neural crest marker genes like Sox9, Snail1, Snail2, Twist1, Msx2, and Dlx6. Cytofluorometric analysis indicated that cells were positive for CD49d (alpha4 integrin), CD56 (NCAM), and PDGFRalpha, while negative for CD31, CD34, CD45, and STRO-1. dNC-PCs could be differentiated into neurogenic, chondrogenic, and osteogenic lineages and were shown to produce bone matrix in athymic mice. These results demonstrate that human third molar pad possesses neural crest-derived cells that represent multipotent stem/progenitor cells. As a rather large amount of dNC-PCs could be obtained from each single third molar, cells may be used to regenerate a wide range of tissues within the craniofacial region of humans.

Epithelial-mesenchymal transition in Rhesus monkey embryonic stem cell colonies: a model for processes involved in gastrulation?

A characteristic feature of embryonic stem (ES) cells is their ability to give rise to differentiated cell types that are derived from all three primary germ layers. In the embryo of higher vertebrates, formation of mesoderm and definitive endoderm (gastrulation) occurs at the primitive streak through a spatially highly ordered process of cell ingression, combined with epithelial-mesenchymal transition (EMT). With respect to ES cell differentiation in vitro, however, germ layer derivative formation has not been studied in much detail, and data on any degree of spatial order that may be attained here are lacking. In the investigations to be reviewed here, rhesus monkey ES cells (line R366.4) were grown on mouse embryonic fibroblast feeder layers for up to 10 days during which time they formed multilayered disc-like colonies with an upper epithelial and a lower mesenchymal cell layer. Processes of epithelialization as well as EMT were studied by transmission electron microscopy, immunohistochemistry combined with confocal laser scanning microscopy, and marker mRNA expression (in situ hybridization, RT-PCR). It was found that under the culture conditions used most of the ES cell colonies developed transitorily a central pit where the epithelial upper layer cells underwent an EMT-like process and appeared to ingress to form the lower, mesenchymal layer, accompanied by appropriate changes of morphology and molecular markers. Similarities and differences in comparison with gastrulation/primitive streak formation in vivo are briefly discussed, as are ethical implications with respect to human ES cells. It is concluded that this rhesus ES cell colony system may be an interesting in vitro model for studies on some basic processes involved in early embryogenesis such as EMT/gastrulation and may open new ways to study the regulation of these processes experimentally in vitro in nonhuman primates.

Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells: a model for processes involved in gastrulation.

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial-mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells.

BACKGROUND: Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells. METHODS: Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting. RESULTS: Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction. CONCLUSION: Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo.

Human uterine epithelial RL95-2 cells reorganize their cytoplasmic architecture with respect to Rho protein and F-actin in response to trophoblast binding.

Embryo implantation is initiated by interaction of trophoblast with uterine epithelium via the apical cell poles of both partners. Using spheroids of human trophoblastoid JAR cells and monolayers of human uterine epithelial RL95-2 cells to simulate this initial interaction, we previously demonstrated that formation of stable cell-to-cell bonds depends on actin cytoskeleton (F-actin) and small GTPases of the Rho family, most likely RhoA. In this study, we determined the apical as well as the basal distribution of these proteins by fluorescence confocal microscopy before and after binding of JAR spheroids. We focussed on changes in cytoplasmic organization with respect to apicobasal polarity of RL95-2 cells. Before binding of spheroids, significantly higher fluorescence signals of RhoA [37 +/- 6 grey scale values (gsv)] and of F-actin (41 +/- 3 gsv) were found in the basal region of RL95-2 cells as compared to the apical pole (RhoA: 24 +/- 3 gsv, F-actin: 28 +/- 2 gsv). After binding of JAR spheroids, this apicobasal asymmetry was inverted (RhoA: 55 +/- 10 gsv apical vs. 25 +/- 3 gsv basal; F-actin: 108 +/- 17 gsv apical vs. 57 +/- 7 gsv basal). Inactivation of Rho GTPases in RL95-2 cells by Clostridium difficile toxin A leads to a loss of their apical adhesion competence, as previously published. Here, we observed a uniform distribution of RhoA and F-actin between apical and basal region rather than an asymmetric one in toxin A-treated cells. These data suggest that activation of Rho GTPases and coordinated rearrangement of F-actin within uterine epithelial cells in response to trophoblast binding are part of a generalized structural and functional reorganization of the cytoplasm. This involves not only the immediate contact zone (apical) but also the opposite (basal) cell pole and may be a critical element of uterine epithelial reactions during transition between trophoblast adhesion and transmigration.

In vitro studies on endometrial adhesiveness for trophoblast: cellular dynamics in uterine epithelial cells.

Initiation of embryo implantation involves adhesion of trophoblast cells to the epithelial lining of the endometrium. The mechanisms regulating the adhesive properties of the uterine epithelium for trophoblast during initiation of human embryo implantation, however, are still incompletely understood. We report here on model studies that we have performed in our laboratory, and in particular on certain methodological approaches that seem to yield new insight into basic mechanisms involved. Of central interest is the ability of the uterine epithelium to develop an adhesion competence at its apical cell pole. This confronts us with a cell biological paradox in that adhesion must be established at the pole which in simple epithelia is typically specialized to resist adhesion. Gain of apical adhesion competence by uterine epithelial cells should be related to cellular rearrangements, i.e. a modulation of their apicobasal cell polarity. Here, we used monolayer-cultured uterine epithelial RL95-2 cells as an in vitro model for the human receptive uterine epithelium. We demonstrated that formation of stable cell-to-cell bonds between the free (apical) pole of these cells and attaching trophoblast (modelled by JAr cells) depends on a number of structural and functional peculiarities that RL95-2 cells have in contrast to other uterine epithelial cells (HEC-1-A cells) which resist attachment via this cell pole. RL95-2 cells were shown to lack tight junctions and to exhibit only rudimentary adherens junctions and a non-polar organization of the actin cytoskeleton. Using the atomic force microscope in a force spectroscopy mode, we exactly defined the time dependence of adhesive interactions between RL95-2 cells and trophoblast, measured the pressure force needed to initiate this process, and screened the buildup of the adhesive forces between the binding partners. A dynamic interaction between the actin cytoskeleton and integrins (a prerequisite for functional activity of integrins) was shown to be an important aspect of the adhesive properties of RL95-2 cells. In addition, at least two types of calcium channels in the plasma membrane of RL95-2 cells seem to play a role in activation of a variety of calcium-sensitive response mechanisms including adhesiveness for trophoblast, i.e. diltiazem-sensitive channels seem to contribute to the initiation of JAr cell binding and SKF-96365-sensitive channels to participate in a feedback loop that controls the balance of bonds. By extrapolation, these data suggest an active role of the uterine epithelium in the process of embryo implantation which we are just beginning to understand in terms of its cell biology.

Adhesiveness of human uterine epithelial RL95-2 cells to trophoblast: rho protein regulation.

Embryo implantation involves adhesion of trophoblast cells to the epithelial lining of the endometrium. Using an in-vitro model to simulate this initial interaction, we previously reported that attachment of human trophoblast-like JAR spheroids to human uterine epithelial RL95-2 cells provokes a Ca(2+) influx in RL95-2 cells depending on apically localized integrin receptors. Here, we demonstrate that adhesiveness of RL95-2 cells for JAR spheroids, measured by a centrifugal force-based adhesion assay, is dependent on Rho GTPases, most likely RhoA. Cellular expression and distribution of RhoA were studied by fluorescence confocal microscopy, focusing on the localization of RhoA and F-actin within the adhesion sites between JAR and RL95-2 cells. Contact areas contained high amounts of RhoA and F-actin fibres near the plasma membrane. To determine whether Rho GTPases may influence JAR cell binding, we treated RL95-2 cells with Clostridium difficile toxin A, which specifically inactivates Rho GTPases. Toxin A treatment changed the subcellular distribution of endogenous RhoA in RL95-2 cells and altered RhoA and F-actin colocalization. Adhesion of JAR spheroids to RL95-2 cells treated with toxin A was largely suppressed. These data indicate that Rho GTPases, most likely RhoA, play an important role in uterine epithelial RL95-2 cells for trophoblast binding, and suggest that RhoA may be involved in local signalling cascades during early embryo implantation in vivo.