Fatigue-Crack Detection in a Multi-Riveted Strap-Joint Aluminium Aircraft Panel Using Amplitude Characteristics of Diffuse Lamb Wave Field.

Structural health monitoring of riveted aircraft panels is a real challenge for maintenance engineers. Here, a diffused Lamb wave field is used for fatigue-crack detection in a multi-riveted strap-joint aircraft panel. The panel is instrumented with a network of low-profile surface-bonded piezoceramic transducers. Various amplitude characteristics of Lamb waves are used to extract information on fatigue damage. A statistical outlier analysis based on these characteristics is also performed to detect damage. The experimental work is supported by simplified modelling of wave scattering from crack tips to explain complex response features. The Local Interaction Simulation Approach (LISA) is used for this modelling task. The results demonstrate the potential and limitations of the method for reliable fatigue-crack detection in complex aircraft components.

High-mobility group protein HMGA2-derived fragments stimulate the proliferation of chondrocytes and adipose tissue-derived stem cells.

In previous research, it was shown that recombinant HMGA2 protein enhances the proliferation of porcine chondrocytes grown in vitro, opening up promising applications of this embryonic architectural transcription factor for tissue engineering, such as in cartilage repair. In this paper, we describe the development and analyses of two synthetic fragments comprising the functional AT-hook motifs of the HMGA2 protein, as well as the nuclear transport domain. They can be synthesised up to large scales, while eliminating some of the problems of recombinant protein production, including unwanted modification or contamination by the expression hosts, or of gene therapy approaches such as uncontrolled viral integration and transgene expression even after therapy. Application of one of these peptides onto porcine hyaline cartilage chondrocytes, grown in in vitro monolayer cell culture, showed a growth-promoting effect similar to that of the wild type HMGA2 protein. Furthermore, it also promoted cell growth of adult adipose tissue derived stem cells. Due to its proliferation inducing function and vast availability, this peptide is thus suitable for further application and investigation in various fields such as tissue engineering and stem cell research.

Air contrast of the intervillous space enables non-disruptive Micro-CT analysis of paraffin-embedded archival placental tissue.

The morphometric parameters of the villous tree are a strong indicator of deviant placentas. Methods have been established to digitally reconstruct small peripheral branches by tracing with 3D Microscopy at subcellular resolution. Micro-CT can help scale up the scanning of villous trees with resolution in the range of a few micrometers. As placental tissue samples are routinely conserved and archived by fixation and paraffin embedding, the villous structures are inaccessible to Micro-CT imaging due to poor contrast between paraffin and paraffinized tissue. We present a novel procedure for contrast enhancement by selectively replacing wax by air in the intervillous space.

The volume of villi with γ-sm-actin positive perivascular cells correlates with placental weight and thickness.

INTRODUCTION: The classification of histologically stained villous cross sections in villous types (terminal, intermediate and stem villi) by stromal peculiarities is known to be observer predicated. Therefore, quantitative histology of villous trees has not become a routine endpoint of studies on the role of the placenta in prenatal programming, as opposed to the gross placental parameters weight and thickness. The classification of villous cross sections in central (stem) and peripheral (terminal) parts based on the presence or absence, respectively, of immunohistochemical detection of myofibroblasts in perivascular position is less observer dependent. We hypothesized that it will, possibly, identify microscopic correlates of placental weight and thickness within the villous tree. METHODS: 50 placentas from clinically normal pregnancies were processed for the present study. Thin villous cross sections, obtained in a systematic random manner, were stained immunohistochemically to detect γ-smooth muscle (sm) actin and to classify them subsequently as part of central or peripheral villous tree. The volume fractions of histological structures visible in villous cross sections (stroma, lumen, endothelium and syncytium) were estimated by design-based stereology. RESULTS: The present study reveals a significant correlation of placental weight and thickness with the volume estimate of stroma that have myofibroblasts in perivascular position. DISCUSSION: The positive linear correlation between the volume of central parts of villous trees and the placental weight and thickness is new. Surprisingly, the volume of more peripheral parts of villous trees, which is the main site of materno-fetal exchange does not correlate with placental weight and thickness.

Trophoblastic invasion in vitro and in vivo: similarities and differences.

BACKGROUND: The basic mechanisms of trophoblast invasion are not completely understood. This may be due to the lack of suitable in vitro models which enable experimental modulation of this complex process. In the present study, a three-dimensional co-culture model is used for comparing two factors considered to be implicated in the regulation of trophoblast invasion, the expression of HLA-G and apoptosis, in vitro and in vivo. METHODS: Tissue fragments from human first trimester decidua parietalis were put in close contact with spheroids of AC-1M59 trophoblast/choriocarcinoma hybrid cells as a model of the invasive trophoblast. Cryostat sections from these co-cultures were immunohistochemically stained and compared with first trimester placentation sites in vivo. RESULTS: Only the invasive trophoblast-derived cells showed an intensive staining for HLA-G, whereas the cells on the periphery of the confrontation culture exhibited only a weak staining. A similar staining pattern was found in vivo. Both in vitro and in vivo CD45(+) apoptotic leukocytes were frequently detected in close proximity to the invasive trophoblastic cells. CONCLUSIONS: In this co-culture system, key factors considered to be implicated in trophoblast invasion in vivo can also be demonstrated in vitro. Therefore, it may help in finding strategies for the management of diseases associated with deficient trophoblast invasion.

Syncytial nuclei accumulate at the villous surface in IUGR while proliferation is unchanged.

INTRODUCTION: Placental syncytiotrophoblast is responsible for feto-maternal nutrient exchange during pregnancy. It is assumed that in IUGR, placental dysfunction is crucially bound to compromised stability and function of syncytiotrophoblast, the latter being related to altered proliferation of villous trophoblast. Cell cycle data obtained on conventional thin sections has produced inconsistent results. In the present study we investigated cell cycle markers found in the villous trophoblast using a novel 3D histological quantification method. METHODS AND FINDINGS: We analyzed 40 placentas from IUGR pregnancies and 42 placentas from clinically normal pregnancies by immunohistochemical detection of the cell cycle marker PCNA. Nuclei immuno-positive for PCNA were quantified using 3D microscopy, and the results were compared to corresponding results obtained on conventional thin histological sections. These data did not show any evidence of altered trophoblast proliferation in IUGR, while the density of post-proliferative (i.e. PCNA-negative) trophoblast nuclei was statistically significantly increased in IUGR. The latter could be revealed by 3D topological microscopy, but not by conventional histology of thin sections. DISCUSSION: The data of the present study indicate a previously unknown type of regulation of syncytial stability and function, independent of proliferation. We hypothesize that in IUGR, post-proliferative trophoblast nuclei accumulate at the villous surface of peripheral villous branches. This could possibly reflect the presence of an unknown mechanism controlling syncytial function and stability by modulation of syncytial passage time rather than by modulation of proliferative supply.

Video games and seizures.

Two teenagers had seizures while playing video games. These cases may well have been a variety of photoconvulsive epilepsy. Some patients with seizures may be at risk of precipitating a seizure while playing these games.

Mechanisms and products of azo coupling in histochemical protease procedures based on primary aromatic amines as unspecific moieties.

It is presumed that the azo dyes generated by histochemical protease reactions are formed by substitution of a reactive aromatic carbon. They are referred to as dyes of the C-azo series. To confirm this assumption, the absorption spectra between 330 and 630 nm of azo dyes resulting from coupling between various aromatic amines of the aniline and naphthylamine series and the diazonium salts Fast Blue B and Fast Garnet GBC were studied in test tube experiments. Some of the amines were blocked by methylation to prevent coupling either at the amino group (N-methylated) or at the aromatic nucleus (C-methylated). Coupling was performed in buffered aqueous solutions of the diazonium salts. For analysis the azo dyes were dissolved in dimethylformamide. For acid rearrangement these solutions were acidified and incubated at elevated temperatures. After detection of dipeptidyl peptidase IV in tissue sections using Gly-Pro-4-methoxy-2-naphthylamine as substrate, the resulting dye was extracted and compared with the test tube compounds. All aromatic amines yielded azo dyes. Dyes extracted from sections and those test tube compounds derived from unmethylated or C-methylated amines showed almost identical spectral maxima, whereas dyes formed by N-methylated amines yielded different spectra. Acid rearrangement did not influence the spectral maxima of the N-methylated amine-derived dyes. Dyes resulting from C-methylated amines were destroyed. The results indicate that under histochemical conditions diazonium salts react primarily with the liberated free amino group but not with the aromatic nucleus of the unspecific moiety. Therefore, it is proposed that the formula of the final reaction product in naphthylamine-based protease histochemistry should be given as an N-azo dye, e.g., as a triazene.

Characterization of trophoblast cell isolations by a modified flow cytometry assay.

The purity of isolated trophoblast cells is an important checkpoint in in vitro studies on the human placenta. Maintaining viability of primary cells for a prolonged period, or achieving cell proliferation in primary cell cultures, is often a matter of concern. In this paper we present a method for characterizing the purity of isolated trophoblast cells based on the expression of cytoskeletal proteins, as well as for assessing their cell cycle status, by flow cytometry. We show that after a simple permeabilization and fixation step in 70 per cent methanol, staining for cytokeratin 7 and vimentin could discriminate between trophoblast cells and contaminating populations. The method was applicable to trophoblast cells both from villous and extravillous origin. By staining the proliferation-related antigen Ki-67 and DNA, information was gained about the cell cycle status and viability of freshly isolated and cultured villous trophoblast cells. This method may help to quickly and quantitatively characterize preparations of isolated trophoblast, as well as to search for culture conditions favouring long-term survival and proliferation.

Phage display: a molecular tool for the generation of antibodies--a review.

Phage display comprises an array of methods, which can be used to display proteins on bacteriophages. We present in this article a summary of techniques, which can be used to express antibody libraries on bacteriophages. Since many researchers are more familiar with the conventional hybridoma technique for production of monoclonal antibodies we describe analogies and differences between these two techniques, both of which are used to reach comparable scientific objectives. We focus on the features which are specific to phage display techniques rather than for hybridoma techniques. These comprise the freedom to choose other animals than the mouse for immunization, the enormously large sample (up to 10(9) clones) which can be drawn and immortalized from a single immunized animal and the opportunity to enhance affinity of isolated antibodies by in vitro affinity maturation. The panning techniques, which are used to enrich specifically binding phage antibodies from the huge libraries are briefly summarized.

The human placenta is encircled by a ring of smooth muscle cells.

The marginal zone of the human term placenta was studied by transmission electron microscopy and immunohistochemistry using antibodies against cytoskeletal filaments, extracellular matrix molecules and endothelial markers. The marginal sinus of the intervillous space is separated from the chorionic and basal plates by a layer of cells expressing vimentin, desmin, alpha- and gamma-smooth muscle actins, and smooth muscle myosin. Also ultrastructurally, these cells share all features with smooth muscle cells. This muscular ring is continuous with the media of uteroplacental veins entering the marginal sinus. In the basal plate the muscle cells may extend far into the central parts of the placenta. The muscular ring is separated from the intervillous space by a layer of endothelial cells. They are continuous with the maternal endothelium of the marginal uteroplacental veins. Moreover this endothelium covers neighbouring parts of the chorionic and basal plates, locally extending to the surfaces of large stem villi. The data suggest (1) that the marginal zone of the intervillous space ('marginal sinus') represents the dilated and merged parts of uteroplacental veins and (2) that lateral growth of the human placenta partly takes place by expansion into the uteroplacental veins. The functional importance of this muscular ring remains unknown.

Pregnancy-induced de-differentiation of media smooth muscle cells in uteroplacental arteries of the guinea pig is reversible after delivery.

The majority media of smooth muscle cells of uteroplacental arteries of the guinea pig is not destroyed during trophoblast invasion. Rather, most of these cells de-differentiate during pregnancy-induced arterial dilatation, forming a population of mesenchyme-like myoblasts ready to reconstitute the media after birth. We have studied the re-differentiation of these cells after delivery by means of transmission electron microscopy and immunohistochemistry using antibodies against a panel of cytoskeletal proteins. The data reveal that post partum re-differentiation of the media myoblasts starts immediately after birth where some of the invasive trophoblast cells are still present. The process of re-differentiation is completed at day 8 after parturition. Post partum re-differentiation can be subdivided into two steps: until day 5 after parturition, the central parts of the media are reconstituted out of the reservoir of vimentin-positive myoblasts by stepwise acquisition of desmin, alpha-smooth muscle actin, gamma-smooth muscle actin and smooth muscle myosin. Only thereafter the same re-differentiation takes place in the peripheral parts of the media. On the ultrastructural level immunohistochemical re-expression of cytoskeletal proteins is accompanied by reconstitution of the intracellular contractile apparatus. The data support our earlier notion that the majority of media smooth muscle cells in the guinea pig uterus does not degenerate during trophoblast-invasion but rather de-differentiate temporarily.

Mechanisms of syncytial fusion: a review.

Syncytial fusion of trophoblast is a key process in placental morphogenesis and physiology. Disturbed syncytial fusion may lead to a number of pregnancy-associated pathologies. The mechanisms regulating syncytial fusion are only partly understood. This review tries to summarize the available knowledge on trophoblast fusion, originating from different scientific disciplines. Among the themes addressed in this paper are: morphogenesis and functions of syncytiotrophoblast; early apoptotic events and changes in plasmalemmal phospholipid orientation; proteins involved in membrane fusion: ADAMs and retrovirally-derived proteins and short-lived proteolipid intermediates in membrane fusion. Deeper understanding of syncytiotrophoblast fusion in future studies is only to be anticipated from collaborative studies focusing in parallel on physicochemical events in the participating plasmalemmas, early apoptotic/differentiation events preceding the fusion and role of the fusogenic membrane proteins.

Human trophoblast contains an intracellular protein reactive with an antibody against CD133--a novel marker for trophoblast.

CD133 is a protein expressed on the cell membrane of a subfraction of haematopoietic stem and progenitor cells, as well as on some epithelial cells. Previously available antibodies against CD133 recognized only the glycosylated protein, localized to membrane protrusions or microvilli. Due to this, immature intracellular stages of the CD133 protein could not be visualized using these antibodies. We describe reactivity of a commercially available antibody against CD133, called AC133-2, with an intracellular protein in trophoblast. Both villous and extravillous cytotrophoblast, as well as syncytiotrophoblast were stained by AC133-2 in cryostat sections of first trimester and term placenta. Villous stroma was not stained. AC133-2 reactivity was seen in methanol-fixed primary trophoblast cells and trophoblast-derived cell lines, and was coexpressed with cytokeratin-7. CD133 messenger RNA was present in trophoblast and trophoblast-derived cell lines, but also in cells not displaying any reactivity with CD133 antibodies. AC133-2 recognized a 55-60 kDa protein on Western blots of cell extracts including trophoblast. The exact nature of this protein is not yet understood. However, AC133-2 is applicable as a positive marker for the characterization of all subtypes of trophoblast and for trophoblast cell lines.

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity.

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.

A two-colour fluorescence assay for the measurement of syncytial fusion between trophoblast-derived cell lines.

Syncytial fusion is a key event in implantation and placentation. Its regulation is only poorly understood. We present a cell-cell fusion assay based on staining of cells in two portions with a green and a red fluorescent cytoplasmic dye that become intracellularly mixed only after syncytial fusion. We quantified cell-cell fusion by fluorescence microscopy in choriocarcinoma cell lines BeWo, JAR and JEG3 and in some non-trophoblastic cell lines and found clear differences in fusion behaviour. Only BeWo cells fused with each other, while the other cell lines tested did not. BeWo cells also fused with all other cell lines tested. The efficiency of cell-cell fusion of BeWo cells was stimulated by forskolin. We tried to correlate messenger levels of syncytin and its receptor RDR with the fusion index of choriocarcinoma cells. BeWo and JAR cells contained readily detectable and forskolin-inducible levels of syncytin mRNA, whereas this messenger was barely detectable in JEG3 cells. RDR transcript levels were similar in all cell lines tested and were unaffected by forskolin treatment. The data suggests that the expression of syncytin and RDR messengers alone does not guarantee successful fusion. The fusion assay presented in this paper is a useful tool to study syncytial fusion in an accurate and quantitative way.

The distribution of macrophages in spiral arteries of the placental bed in pre-eclampsia differs from that in healthy patients.

Placental bed biopsies taken during caesarean section from 10 patients with pre-eclampsia and six healthy pregnancies were studied. We applied antibodies against cytokeratin and different macrophage markers to analyse the distribution of invasive extravillous trophoblast cells as compared to that of macrophages in myometrial segments of uteroplacental arteries. The data were evaluated quantitatively. We found a clear inverse relationship between local infiltration with macrophages and trophoblast invasion. In pre-eclampsia, vessel cross-sections prevailed which were characterized by large numbers of macrophages but a low degree of trophoblast invasion. In contrast, in normal third trimester pregnancies the respective arterial segments had a high degree of trophoblast invasion but were largely void of macrophages. These data suggest causal links between macrophages and inhibition of intra-arterial trophoblast invasion in pre-eclampsia.

Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid.

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.

Placental toxicity in rats after administration of synthetic glucocorticoids. A morphological, histochemical and immunohistochemical investigation.

Administration of the synthetic glucocorticoids dexamethasone and triamcinolone to pregnant rats between gestational day (GD) 16 and 20 caused dose-dependent placental lesions on GD 21 and 22 which were detected by morphological, histochemical and immunohistochemical means. Maternal blood spaces, trophoblast layer and fetal blood vessels were altered primarily in the centre of the placental labyrinth. Less severe changes were found in the junctional zone, chorionic plate and intraplacental yolk sac. On GD 21, low doses increased the amount of glycogen, while high doses induced a loss of glycogen. gamma-glutamyl transpeptidase activity was increased in the spongiotrophoblast and the labyrinthic trophoblast and dipeptidyl peptidase IV activity in fetal capillary endothelium, whereas alpha-glutamyl aminopeptidase and microsomal alanyl amino-peptidase were not affected. Additionally, in the fetal capillary endothelium an increase of immunoreactivity for the von Willebrand factor occurred. These data suggest that synthetic glucocorticoids affect placental tissues at different and rather specific levels, which may in turn disturb placental function and contribute to fetal maldevelopment.

The apoptosis cascade--morphological and immunohistochemical methods for its visualization.

Apoptosis is involved in morphogenesis of embryonic tissues as well as in homeostasis of adult organs and tissues. It is the main process by which organs maintain cell mass and at the same time eliminate excess and aged cells that have lost their functional importance. The typical morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events, some of which are inextricably linked to the process of differentiation. Studies that analyze all stages of this cascade, rather than the final morphological stages of apoptotic death, are essential in order that specific link(s) between differentiation and apoptosis are appreciated. This review outlines the main stages of the apoptosis cascade together with current methods for their morphological visualization. Starting with (a) receptors and ligands known to induce apoptosis, we continue with (b) early initiator stages of apoptosis, and (c) proteins regulating and potentially inhibiting further progression of the cascade, into (d) irreversible execution stages of the cascade, and finally (d) the morphological events of apoptotic death. For each stage we present those aspects of the biochemical background that are morphologically relevant, together with proven methods for their visualization. We offer technical advice at each stage based upon our experience of studying differentiation and apoptosis in human placental trophoblast.