Expression of Lefty predicts Merkel cell carcinoma-specific death.

BACKGROUND: Lefty and Nodal are transforming growth factor ß-related proteins, which, beside their role in determination of laterality during embryogenesis, have also been linked with cancer progression. OBJECTIVES: Prompted by the observed significant left-sided laterality of Merkel cell carcinoma (MCC), we addressed whether Lefty and Nodal are expressed in MCC and correlated expression patterns with clinical parameters such as MCC laterality and patient outcome. METHODS: Expression of Lefty and Nodal in primary MCC was assessed in 29 patients by immunohistochemistry. The histology (H-)score was calculated and correlated with clinical parameters. RESULTS: The median (range) H-score of Lefty and Nodal was 17.6 (0-291) and 74.9 (0.7-272), respectively. There was a significant correlation between Lefty expression and Nodal expression (correlation coefficient of 0.60, P = 0.0006). There was no significant correlation between Lefty expression and Nodal expression with either tumour laterality, gender, age, Merkel cell polyomavirus status, disease stage, anatomical localization of primary tumours or disease relapse. On univariate analysis, low Lefty expression and Nodal expression were significantly associated with MCC-specific death (P = 0.010 and P = 0.019, respectively). On univariate analysis, low Lefty expression was the only significant independent predictor for MCC-specific death (P = 0.025) as indicated by an odds ratio of 14 (95% CI: 1.43-137.33). CONCLUSIONS: Lefty and Nodal are frequently expressed in MCC, but not correlated with tumour laterality. Importantly, our data suggest that a low level of Lefty expression in primary MCC is a strong predictor of MCC-specific death.

The impact of peppermint oil on the irritable bowel syndrome: a meta-analysis of the pooled clinical data.

BACKGROUND: Peppermint oil (PO) has intrinsic properties that may benefit patients with irritable bowel syndrome (IBS) symptoms. The study objective was to determine the effect of peppermint oil in the treatment of the IBS. METHODS: We systematically searched MEDLINE (PubMed), Cochrane Central Register of Controlled Trials (Cochrane CENTRAL), ClinicalTrials.gov, EMBASE (Ovid), and Web of Science for randomized controlled trials (RCTs) of PO for IBS. We appraised the eligible studies by the Cochrane risk of bias tool. We performed random-effects meta-analysis on primary outcomes including global improvement in IBS symptoms and abdominal pain. A PRISMA-compliant study protocol is registered in PROSPERO Register [2016, CRD42016050917]. RESULTS: Twelve randomized trials with 835 patients were included. For global symptom improvement, the risk ratio (RR) from seven RCTs for the effect of PO (n = 253) versus placebo (n = 254) on global symptoms was 2.39 [95% confidence interval (CI): 1.93, 2.97], I2 = 0%, z = 7.93 (p < 0.00001). Regarding abdominal pain, the RR from six RCTs for the effect of PO (n = 278) versus placebo (n = 278) was 1.78 [95% CI: 1.43, 2.20], I2 = 0%, z = 5.23 (p < 0.00001). Overall, there were no differences in the reported adverse effects: PO (32 events, 344 total, 9.3%) versus placebo (20 events, 327 total, 6.1%) for eight RCTs; RR 1.40 [95% CI: 0.87, 2.26] I2 = 0%, z = 1.39 (p = 0.16). The number needed to treat with PO to prevent one patient from having persistent symptoms was three for global symptoms and four for abdominal pain. CONCLUSIONS: In the most comprehensive meta-analysis to date, PO was shown to be a safe and effective therapy for pain and global symptoms in adults with IBS.

Histopathological, immunohistochemical criteria and confocal laser-scanning data of arthrofibrosis.

Arthrofibrosis (af) is defined as a fibrosing disease of the synovial membrane, after joint operations, with painful restricted range of motion. The aim of this paper was to describe the histopathological substrate of af, hitherto only defined by clinical criteria. Based on a group of 222 tissue samples, the characteristic changes to af were analyzed. The control group comprised 29 cases with neosynovialis of the indifferent type. Due to cytoplasmic SM-actin positivity and the absence of specific cytoplasmic reactivity in CD 68 representation, af fibroblasts were characterized as myofibroblasts. In confocal laser-scanning microscopy, ß-catenin-positive aggregates were detected in the cytoplasm. Over and above this, unequivocal colocalization of ß-catenin and the tight junction protein ZO-1 became manifest, particularly on the cell membrane and, partly, in the cytoplasm. A threshold value of 20 ß-catenin-positive cells/HPF was determined. This enables the histopathological diagnosis of an af to be made (sensitivity: 0.733, specificity: 0.867). Af is a fibrosing disease of the synovial membrane with variable grade of fibrotization (fibroblast cellularity). A threshold value of 20 ß-catenin-positive fibroblasts per HPF was defined, which enables the histopathological diagnosis of af.

Natural history of acute upper GI bleeding due to tumours: short-term success and long-term recurrence with or without endoscopic therapy.

BACKGROUND: Scant information is available regarding patients with upper gastrointestinal bleeding (UGIB) from tumours. AIM: To determine the presentation, endoscopic findings, treatment and outcomes in patients with UGIB from malignant tumours and identify risk factors associated with rebleeding. METHODS: Consecutive patients who were hospitalised with haematemesis, melena or haematochezia and underwent upper endoscopy were identified retrospectively by reviewing an endoscopy database. Patients with UGIB due to biopsy-proven malignant tumours were studied. RESULTS: Tumours were the source of bleeding in 106 (5%) of 2,166 patients with UGIB. Tumours were oesophageal in 17 (16%), gastric in 77 (73%) and duodenal in 12 (11%). At presentation, 84 (79%) did not have known cancer previously, and 79 (75%) had metastatic disease. Seventy-seven (73%) received transfusions at index hospitalisation. At endoscopy, 32 (30%) had active bleeding (31 oozing, 1 spurting). Among actively bleeding patients, haemostasis was achieved in 12 (86%) of 14 receiving endoscopic therapy and all 18 not receiving endoscopic treatment. Hospitalisation for rebleeding occurred in 50 (49%) of 103 at a median of 30 days (3-885). On multivariate analysis, age ≤60 years (OR = 2.49, 95% CI 1.06-5.81) and haemodynamic instability (OR = 2.42, 95% CI 1.08-5.46) were associated with rebleeding. CONCLUSIONS: Patients presenting with tumour-associated UGIB have substantial blood loss, with three-quarters requiring transfusion at presentation. Initial haemostasis occurs in almost all patients, with or without endoscopic therapy, but rebleeding requiring repeat hospitalisation occurs in approximately half the patients and is more common in patients who are ≤60 years of age and have haemodynamic instability at presentation.

Apert syndrome: report of a case with emphasis on oral manifestations.

To report the oral findings, including dental anomalies, ectopic eruption of the maxillary permanent first molars and periodontal disease and soft tissue alterations, in a subject with Apert syndrome. Clinical and radiographic examination of a patient with Apert syndrome, aged 21 years old, not previously submitted for orthodontic or orthognathic treatment.Dental anomalies were present in a patient. Intraoral evaluation revealed poor oral hygiene with varying degrees of periodontal involvement, an arched swelling (pseudo cleft configuration), class III malocclusion, anterior open bite, posterior crossbite, supernumerary teeth, ectopic eruption and creamy white enamel opacities, an excessively large appearing tongue and a v-shaped maxillary arch. The occurrence of typical lateral palatal swellings agrees with the literature. The high prevalence of dental anomalies and ectopic eruption may suggest a possible etiologic relationship with the syndrome.

Lymphatic capillary hypoplasia in the skin of fetuses with increased nuchal translucency and Turner's syndrome: comparison with trisomies and controls.

Fetuses with Turner's syndrome or trisomies 21, 18 and 13 show excess of skin, which can be visualized by ultrasonography as increased nuchal translucency at 11-13(+6) weeks' gestation. The objective of this study was to gain insight in the development and distribution of blood vessels, lymphatic capillaries of the cutis and lymphatic collectors of the cutis and subcutis and to study developmental changes with increasing gestation. Immunofluorescence of cryosections with 10 specific antibodies was used to investigate the nuchal skin of three fetuses with Turner syndrome's and to differentiate lymphatics, lymph capillaries (FLT4, PTN 63, LYVE1, PROX1), blood vessels (KDR, CD 31, PDPN), blood clotting activity (von Willebrand factor), basement membranes and big vessels (Laminin, Collagen Type IV). The findings were compared with those in seven fetuses with trisomy 21 and two fetuses each with trisomies 18 or 13, respectively, as well as six normal controls. Immunoreactive receptors for vascular endothelial growth factors (FLT4) were decreased in lymphatic capillaries of the skin of Turner fetuses. Accordingly, LYVE1 was scarce and PROX1 staining was less intense in the dermis of Turner fetuses. Lymphatic collectors were, however, evenly stained. In normal fetuses and in those with trisomies, lymphatic capillaries were evenly distributed. We conclude that lymphatic capillary hypoplasia might be responsible for nuchal cystic hygroma in Turner syndrome. The biological basis for increased nuchal translucency in trisomies may however be different.

Congenital myopathy with arrest of myogenesis prior to formation of myotubes.

We report a novel type of congenital myopathy, which is characterized by an early arrest of muscle formation prior to formation of myotubes. A female infant born prematurely at 32 weeks of gestational age died after six weeks of continuous ventilatory support. Various muscle specimens including quadriceps, deltoid, pectoral, neck, psoas, tongue, and diaphragm musculature were studied. Light and electron microscopy revealed well-demarcated fascicular structures interspersed with undifferentiated, mononuclear myogenic cells. Multinucleated myotubes and muscle fibres were not detectable, pointing towards a defect prior to the generation of myotubes during myogenesis. Immunohistochemistry identified the absence of dystrophin, N-CAM, MyoD and myogenin expression in these myogenic cells, compatible with a block of the complex transcriptional network necessary for correct embryonic muscle formation at an early stage of muscle development. These myopathological findings were absent in cardiac muscle, indicating that the defect exclusively affects skeletal muscle formation.

The scoliosis (sco) mouse: a new allele of Pax1.

We describe the spontaneous mutant mouse scoliosis (sco) that carries a new allele of Pax1 (un-i, undulated intermediate). The Pax1(un-i) allele is lacking the 5'-flanking region and exon 1 to 4 which is mapped to nt -2636 to -640 and -272 to 4271 of the Pax1 gene. Homozygous mice show a mild form of the known phenotypes of other Pax1 mutants. Adult mice have a lumbar scoliosis and kinky tails. In homozygous embryos the skeleton ossifies early, ossification centers of the vertebral bodies are fused with the ossification centers of the pedicles. Neural arches and spinous processes are underdeveloped but the pedicles and transverse processes are overdeveloped which is in contrast to other Pax1 mutants. In the scapula, the acromion is missing and the deltoid tuberosity of the proximal humerus is shortened and thickened. Among the inner organs the thymus development is affected. In late embryos, the thymus is small and thymocyte numbers are reduced. T-cell development from CD4- and CD8- double negative (DN) to CD4+ and CD8+ double positive (DP) is decelerated. The percentage of CD90+ cells is also reduced but in contrast to other Pax1 mutants no alteration of the expression level of the CD90 (Thy-1) could be found.

Characterization of migration behavior of myogenic precursor cells in the limb bud with respect to Lmx1b expression.

Limb buds develop from lateral plate-derived stationary mesenchyme and are invaded by cells from extrinsic regions. The largest populations of these cells are myogenic precursor cells that originate from the lateral dermomyotomes. After detachment under the influence of SF/HGF, myogenic precursor cells migrate in a proximo-distal direction and populate a dorsal and ventral zone. The patterning mechanism leading to the segregation of dorsal and ventral myogenic cells is at present not understood. Lmx1b, a LIM homeodomain transcription factor expressed in the dorsal mesenchyme of the developing limb bud, forms a sharp dorso-ventral boundary of expression within the limb. We have investigated the mechanisms of dorso-ventral patterning of muscle precursor cells in the limb buds with respect to Lmx1b expression using quail-chick chimeras and transgenic mice. Although cells appeared to be capable of migrating either ventrally or dorsally, their migration was restricted to the position they had attained during normal development or in the experimental situation. They were never found to cross the dorso-ventral boundary. Immunohistochemistry and histological analysis of mice carrying a LacZ reporter gene under the control of the endogenous Lmx1b locus confirmed that myogenic precursors in the limb bud were devoid of Lmx1b expression. In addition, it was shown that Lmx1b is not only expressed at early stages of limb development but maintains its pattern, at least until after birth. The present study provides new insights into migratory pathways of myogenic precursor cells and reveals details of Lmx1b expression on a cellular basis within the limb.

Expression of kinesin kif5c during chick development.

Kinesins are molecular motors associated with microtubules. They act mainly as intracellular transport proteins carrying different cargos like organelles along the microtubules. We cloned the avian homologue of the mammalian kif5c gene, a member of the khc family coding for the heavy chain of conventional kinesin. Its murine homologue has been described to be specific for neuronal tissue. Here we present the expression pattern of kif5c in chick embryos. We found a highly dynamic expression pattern for kif5c in a variety of developing tissues including neuronal and mesodermal tissues. In young embryos the expression pattern around Hensen's node is asymmetric with stronger expression on the right side, implying that kif5c is involved in the formation of the left-right body axis. A connection with intracellular transport linked to early asymmetric morphogenesis in the node is likely. Vesicles containing signaling molecules could be possible cargos. At later stages, kif5c expression is found in the paraxial, intermediate and somatic mesoderm and in the tail bud. The expression in the paraxial mesoderm occurs first during segmentation and continues in the epithelial somites and the dermomyotome. During neurulation kif5c is expressed in ectodermal and neural-plate cells. In older embryos, the expression is restricted to the dorsal root and cranial ganglia, neural tube and olfactory tract. Taken together, our results demonstrate that in the chick embryo, kif5c plays a role during different morphogenetic processes.

Expression of thymosin beta4 during chick development.

We cloned the chick homologue of Homo sapiens thymosin beta4, encoding a G-actin sequestering factor which plays an important role in angiogenesis, cell motility and tumorigenesis. The thymosin beta4 gene is highly conserved between chick and human. Its expression was analyzed during different stages of development. At early stages thymosin beta4 is expressed in the mesoderm and endoderm and in Hensen's node. Later, thymosin beta4 transcripts are found in the head mesenchyme, somites, dorsal root ganglia, neural tube, brain, blood vessels and feather buds. The pattern of thymosin beta4 expression in blood vessels indicates a function mainly in development of the blood circulatory system which closely parallels findings in vitro. The observed expression pattern shows a high similarity to expression data published for mice, mainly in the heart and in the nervous system. Important new aspects are the early onset of expression, the expression in the mesoderm preceding heart formation and the involvement in feather development.

Glycosaminoglycans and proteoglycans in the skin of aneuploid fetuses with increased nuchal translucency.

BACKGROUND: First trimester increased fetal nuchal translucency is associated with fetal aneuploidies. One of the mechanisms of pathophysiology could be an abnormal extracellular matrix facilitating the formation of an interstitial edema. A previous study investigating interstitial edema in first trimester fetuses found large amounts of hyaluronan in the skin of fetuses with trisomy 21. The aim of this study was to establish distribution patterns for a number of other glycosaminoglycans-dermatan, heparan and keratan sulphate, chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan-in the nuchal skin of normal and chromosomally abnormal fetuses at 11-14 weeks. We also investigated whether biglycan (BGN), which is located on chromosome X, is underexpressed in fetuses with Turner syndrome. Decorin (DCN), a similar-sized proteoglycan located on chromosome 12, was taken as a control. METHODS: We studied the distribution and concentration of various extacellular matrix components using immunohistochemistry, a double staining technique, in-situ hybridization, Northern and Western blot analysis. RESULTS: Chondroitin-6-sulphate and chondroitin-4-sulphate proteoglycan were increased in Turner syndrome fetuses and BGN seemed to be underexpressed compared with normal controls, while DCN was not. Dermatan, heparan and keratan sulphate showed no significant abnormal distribution in trisomies 21, 18, 13, or in Turner syndrome, compared with normal. Western and immunohistochemical analysis revealed that absence of a second X chromosome, as is the case in Turner syndrome, affects BGN protein pattern. CONCLUSIONS: An abnormal amount of glycosaminoglycans and proteoglycans presumably contributes to increased nuchal translucency.

Regulation of Epha4 expression in paraxial and lateral plate mesoderm by ectoderm-derived signals.

Somitogenesis in all vertebrates involves a mesenchymal to epithelial transition of segmental plate cells. Such a transition involves cells altering their morphology and their adhesive properties. The Eph family of receptor tyrosine kinases has been postulated to regulate cytoskeletal organization. In this study, we show that a receptor belonging to this family, EphA4, is expressed in the segmental plate in a region where cells are undergoing changes in cell shape as a prelude to epithelialization. We have identified the ectoderm covering the somites and the midline ectoderm as sources of signals capable of inducing EphA4. Loss of EphA4 results in cells of irregular morphology and somites fail to form. We also show that when somites fail to develop, expression of EphA4 in the lateral plate is also lost. We suggest that signaling occurs between the somites and the lateral plate mesoderm and provide evidence that retinoic acid is involved in this communication.

cDermo-1 expression indicates a role in avian skin development.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be important regulatory proteins for tissue determination and differentiation. We cloned the chicken homologue of the gene of the murine Twist-related bHLH protein Dermo-1, which we named cDermo-1, and analyzed its sequence and embryonic expression. Our sequence data suggest a decisive role of Dermo-1 proteins in the evolution of amniote skin. We present a detailed analysis of cDermo-1 expression during avian embryonic development. cDermo-1 is first expressed in a variety of mesodermal tissues of the chick embryo including the limb buds, but later becomes restricted to the subectodermal mesenchyme of the integument and the developing feather buds, indicating a role of cDermo-1 during avian skin and feather development.

New experimental evidence for somite resegmentation.

According to the concept of resegmentation, the boundaries of vertebrae are shifted one half a segment compared with somite boundaries. This theory has been experimentally confirmed by interspecific transplantations of single somites. Due to the difficulty of exactly orientating individual somites in the host embryo, the outcome and interpretations of these experiments have occasionally been questioned. This is especially true for the formation of neural arches, their processes, and the ribs. We reinvestigated the formation of vertebrae in the avian embryo by grafting one and one half somites from quail to chick embryos. This method eliminates the possibility of a wrong somite orientation in the host embryo. Results show that the vertebral body, the neural arch and its processes are made up of material of two adjacent somites. This is also true for the rib, with the exception of the costal head, which is formed by only one somite. Whereas in the proximal part of the costal body the chick and quail cell regions border on each other in the middle of the rib, in its distal part quail cells gradually begin to mix with chick cells. The intersegmental muscles and their skeletal attachments sites are formed from the same somite. These results support and complete the data of previous studies and confirm the resegmentation concept.

Hyaluronan in the nuchal skin of chromosomally abnormal fetuses.

Nuchal skin oedema at 10-14 weeks gestation, observed by ultrasonography as increased nuchal translucency (NT), is found in approximately 70% of fetuses with trisomies 21, 18 and 13 as well as those with Turner's syndrome. This study investigates the possibility that one mechanism for increased translucency is an altered composition of the skin with a higher concentration of hyaluronan; large amounts of hyaluronan can lead to excessive hydration of the extracellular matrix. We isolated the hyaluronic acid binding region (HABR) from aggrecan in the extracellular matrix of hyaline cartilage and used it in a biotinylated form in combination with a fluorescent probe as a marker for hyaluronan. Immunohistochemistry was then used to examine the nuchal skin of chromosomally abnormal and normal fetuses, obtained after termination of pregnancy. In fetuses with trisomy 21 there was a substantial increase in hyaluronan, whereas in trisomies 18 and 13 and Turner's syndrome the amount was similar to that in chromosomally normal controls. This finding suggests that hyaluronan may be implicated in the pathogenesis of increased NT in fetuses with trisomy 21, but the common phenotypic expression of increased translucency in different chromosomal abnormalities may be the consequence of other mechanisms.

Evolution and development of distinct cell lineages derived from somites.

In the vertebrate embryo, the somites arise from the paraxial mesoderm as paired mesodermal units in a craniocaudal sequence. Segmentation is also the underlying principle of the body plan in annelids and arthropods. Genes controlling segmentation have been identified that are highly conserved in organisms belonging to different phyla. Segmentation facilitates movement and regionalization of the vertebrate body. Its traces in humans are, for example, vertebral bodies, intervertebral disks, ribs, and spinal nerves. Somite research has a history of at least three centuries. Detailed morphological data have accumulated on the development of the avian somite. Especially in connection with the quailchick interspecific marker system, progress was made toward an understanding of underlying mechanisms. At first each somite consists of an outer epithelium and a mesenchymal core. Later, the ventral portion of the somite undergoes de-epithelialization and gives rise to the sclerotome, whereas the dorsal portion forms the dermomyotome. The dermomyotome is the source of myotomal muscle cells and the dermis of the back. It also yields the hypaxial muscle buds at flank level and the myogenic cells invading the limb buds. The dorsal and ventral somitic domains express different sets of developmental control genes, for example, those of the Pax family. During later stages of development, the sclerotomes undergo a new arrangement called "resegmentation" leading to the fusion of the caudal half of one sclerotome with the cranial half of the following sclerotome. Further somitic derivatives include fibroblasts, smooth muscle, and endothelial cells. While sclerotome formation is controlled by the notochord, signals from the dorsal neural tube and ectoderm support the development of the dermomyotome. Myogenic precursor cells for the limb bud are recruited from the dermomyotome by the interaction of c-met with its ligand scatter factor (SF/HGF). In the evolution of metamerism in vertebrates, the first skeletal elements were primitive parts of neural arches, while axial elements developed only later in teleosts as pleurocentra and hypocentra.