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.

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.

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.

The formation of somite compartments in the avian embryo.

The somites develop from the unsegmented paraxial mesoderm that flanks the neural tube. They form in an intrinsic process which lays down the primary segmental pattern of the vertebrate body. We review the processes of somitogenesis and somite differentiation as well as the mechanisms involved in these developmental events. Long before overt differentiation occurs, different compartments of the still epithelial somites give rise to special cell lines and to particular derivatives. By means of isotypic grafting between quail and chick embryos, it is possible to follow the fate of groups of somitic cells. In this way, the development of the myotome and the back dermis from the dorsomedial quadrant and of the hypaxial body wall and limb musculature from the dorsolateral quadrant was established. The two ventral quadrants and the somitocoele give rise to the chondrogenic/fibroblastic lineage of the sclerotome and form the vertebral column. Somite compartments can first be visualized by the expression pattern of Pax genes. Pax-3 is expressed in the dorsal part of the epithelial somite, while the ventral two thirds express Pax-1, a marker of sclerotome development. Pax-3 expression is retained also in the premitotic myogenic cells that migrate into the limb buds. In differentiating myoblasts, Pax-3 expression is turned down and taken over by the activation of MDF's. This initial event in myogenesis occurs in the absence of local signals, whereas the expression of Pax-1 in the sclerotome can be shown to be induced by signals from the notochord and floor-plate of the neural tube. Epaxial myotome differentiation is supported by the neural tube, after the neural tube has received patterning signals from the notochord. The hypaxial musculature and limb musculature differentiate independently of the axial structures. The myogenic cells migrating within the limb buds respond to signals of the lateral plate mesoderm which guide their distalward migration and pattern the muscle.

Collagen type VI gene expression in the skin of trisomy 21 fetuses.

OBJECTIVE: To determine whether the mechanism for the retention of interstitial fluid in trisomy 21 fetuses presenting with nuchal translucency at 10-14 weeks' gestation is an alteration in the composition of collagen type VI, which is normally a triple helix formed of three single chains, alpha1, alpha2, and alpha3. The genes responsible for the alpha1 and alpha2 chains, COL6A1 and COL6A2, are located on chromosome 21 and therefore may be overexpressed in trisomy 21, whereas COL6A3 is located in chromosome 2. METHODS: Skin tissue was obtained after termination of pregnancy at 11-16 weeks' gestation in five fetuses with trisomy 21 and five normal controls. Total RNA was extracted and the steady-state levels of COL6A1 and COL6A3 mRNA expression of the gene transcripts were determined. Additionally, the distribution of collagen type VI in the skin of trisomy 21 and normal fetuses was analyzed using an immunohistochemical method. RESULTS: The ratio of the normalized densitometric scores for the mRNA expression of COL6A1 to COL6A3 in the skin of trisomy 21 fetuses was twice as high as in normal fetuses. Immunohistochemistry demonstrated that in trisomy 21 fetuses collagen type VI formed a dense network extending from the epidermal basement membrane to the subcutis, whereas in normal fetuses dense staining was confined to the upper region of the dermis. CONCLUSION: The distribution for collagen type VI is different from normal in the skin of trisomy 21 fetuses, and there is overexpression of COL6A1 compared with COL6A3.

Increased expression of platelet-derived growth factor receptor alpha and beta and vascular endothelial growth factor in the skin of patients with chronic venous insufficiency.

Growth factors produced by a variety of cells act as signalling peptides through specific cell surface receptor pathways. Functions such as cell proliferation, migration and differentiation have been assigned to each of them. Here, we report alterations of platelet-derived growth factor receptor alpha (PDGFR-alpha) and beta (PDGFR-beta) and vascular endothelial growth factor (VEGF) expression patterns in the progressive clinical stages of chronic venous insufficiency (CVI). A total of 30 punch biopsies were taken from patients with CVI, and VEGF and PDGFR were detected by indirect immunofluorescence and immunoperoxidase techniques. PDGFR-alpha and PDGFR-beta expression was strongly increased in endothelial cells of capillaries, pericapillary cells and connective tissue cells in the stroma of the skin of venous eczema and venous leg ulcer patients, and to a smaller extend in the dermis of those with lipodermatosclerosis. VEGF staining showed a similar expression pattern in the progressive CVI stages. However, staining of vessels in particular might simply reflect binding of VEGF, secreted by keratinocytes or fibroblasts, to its receptors. Growth factor and receptor expression in specimens from telangiectases and reticular veins, and from pigmented areas, resembled that of normal skin. We conclude that PDGFR-alpha, PDGFR-beta and VEGF play an important role in mediating inflammation and epithelial hyperproliferation in venous eczema, inducing connective tissue sclerosis in lipodermatosclerosis, and causing the reduced reepithelialization tendency in venous ulcers. We speculate that endothelial proliferation with chronic venous hypertension might be mediated by these growth factors.

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.

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.

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.

Defect repair after somite removal in avian embryos is not true regeneration.

The question of regeneration after experimental somite extirpation has been controversial in the literature. While all workers agree that repair of the defects occurs, results concerning the extent and mechanism of this process, as well as the origin of the cells filling the defect, show great discrepancies. Our approach towards a re-examination of this question involved microsurgical removal of individual somites in 2-day-chick embryos in combination with grafting of quail somites and lateral plate. We show that the defect in the paraxial mesoderm is filled within a day after extirpation and that the reconstituting cells are derived only from the cranial and caudal somites, but not from the contralateral somites or from the lateral plate. There are no indications of an increase of proliferation in the neighbouring somites. In order to examine the differentiation capacities of the cells that fill the defect, we used immunohistochemistry and in situ-hybridization. We show that the cells in the defect are mesenchymal in morphology and express Pax-1 and Twist. There are a few desmin-positive cells in the defect that can be shown to derive from adjacent somites. An epithelial dermomyotome and myotome are absent at the operation site. Neural crest cells do not participate in the reconstitution of the defect. We conclude that cells in the defect either already have or adopt a ventral somitic (sclerotomal) identity, whereas derivatives with dorsal identity are absent from the defect except for a few individual cells.

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.

The control of directed myogenic cell migration in the avian limb bud.

Interspecific grafting experiments between chick and quail embryos were carried out in order to investigate the mechanism controlling myogenic cell migration in the avian limb bud. In six series, various experimental set-ups were prepared involving different age combinations of donor and host. The migration of the myogenic cells contained in the quail donor could be traced due to the prominent perinucleolar heterochromatin of the quail nucleus. Irrespectively of the presence or absence of the apical ectodermal ridge (AER), myogenic cells were found to migrate distally when implanted at a more distal site or into a younger host. They were even found to migrate in the reverse direction when younger host tissue was located proximal to the graft. From these findings, we conclude that the state of differentiation ("juvenility") of the limb bud mesenchyme controls the directed migration of myogenic cells.

Local signalling in dermomyotomal cell type specification.

The development and differentiation of the avian myotome was studied after removal of the neural tube, including neural crest, and after replacement of dorsal half-somites by ventral half-somites. Results show that in the absence of neural tissue myoblast differentiation within the somites does not take place. Ventral half-somites are able to undergo muscle differentiation if they were grafted in place of dorsal half somites. It is suggested that local signals must be responsible for the dorsalisation of the newly formed somite including myoblast differentiation. Neural crest cells are discussed as possible sources of these signals.

The ventralizing effect of the notochord on somite differentiation in chick embryos.

The dorso-ventral pattern formation of the somites becomes manifest by the formation of the epithelially organized dorsal dermomyotome and the mesenchymal ventrally situated sclerotome. While the dermomyotome gives rise to dermis and muscle, the sclerotome differentiates into cartilage and bone of the axial skeleton. The onset of muscle differentiation can be visualized by immunohistochemistry for proteins associated with muscle contractility, e.g. desmin. The sclerotome cells and the epithelial ventral half of the somite express Pax-1, a member of a gene family with a sequence similarity to Drosophila paired-box-containing genes. In the present study, changes of Pax-1 expression were studied after grafting an additional notochord into the paraxial mesoderm region. The influence of the notochord and the floor-plate on dermomyotome formation and myotome differentiation has also been investigated. The notochord is found to exert a ventralizing effect on the establishment of the dorso-ventral pattern in the somites. Notochord grafts lead to a suppression of the formation and differentiation of the dorsal somitic derivatives. Simultaneously, a widening of the Pax-1-expressing domain in the sclerotome can be observed. In contrast, grafted roof-plate and aorta do not interfere with dorso-ventral patterning of the somitic derivatives.

Differences in the fibronectin-dependence of migrating cell populations.

In avian embryos, the migration behaviour of several cell populations, melanoblasts, Schwann cells, myogenic cells and axons after application of antibodies directed against the cell-attachment fragment of fibronectin (alpha-CAF) was investigated. The migration of the different cell types was influenced in different ways. 1. Epidermal melanoblasts did not colonize areas into which the antibody had been injected, i.e. distal to the grafting site. They frequently spread proximally to the back and neck, sometimes even as far as to the ipsilateral leg. When grafted to the dorsal side of the wing bud, melanoblasts never spread to the ventral side after injection of the antibody. Non-epidermal melanoblasts continued to migrate distally. 2. Grafted Schwann cells and host axons were not noticeably affected by the antibody injections. Both were found proximally and far distally to the grafting site, i.e. also within the injected area. 3. Myogenic cells were immobilized near the grafting site, where they differentiated biochemically, but sometimes only partially underwent fusion into myotubes. They participated in the formation of host muscle blastemas only immediately adjacent to the non-migratory cell population of the graft such as fibroblasts and cartilage. 4. The injected antibody could be localized up to 5 h after the application in the distal third of the limb bud. We conclude that migrating cell populations show differences in their fibronectin-dependence which probably reflect their use of fibronectin during migration.

Participation of individual brachial somites in skeletal muscles of the avian distal wing.

In this paper we investigate the somitic origin of the individual muscles of the forearm and hand using quail-chick chimeras. Our results show that only somites 16-21 give rise to wing muscle, but they take part in muscle formation to different extents. Somite 21 does not always participate in the formation of muscle of the forearm and hand. The most cranial somite (16) takes part in the radial muscles and the most caudal somites (20, 21) in the ulnar muscles, reflecting their position with respect to the limb bud. The centrally located somites (17, 18, 19) are involved in all (18) or most (17, 19) muscle primordia. This pattern of distribution is clearest in the forearm, whereas the participation of somites in particular muscle groups is not so distinct in the hand. Hand muscles are mainly made up of cells from somites 18-20. All brachial somites participate in dorsal (extensor) as well as ventral (flexor) muscles of the forearm and hand. Each somite takes part in more than three muscle primordia in a reproducible fashion, and every muscle primordium is derived from at least three somites. Especially the M. ulnimetacarpalis ventralis takes origin from all somites involved in limb muscle formation (16-21). Apart from muscle cells, endothelial cells also and a few fibroblasts of quail origin are found in the limb bud after somite grafting.

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.

Segmentation of the vertebrate body.

The segmental character of the vertebrate body wall is reflected by metamerically arranged tissues that are patterned during embryonic life as a consequence of somite formation, compartmentalization and differentiation. The somites bud off the paraxial mesoderm in a cranio-caudal sequence and are compartmentalized by local signals from adjacent structures. These signals may be mediated by diffusible substances such as Sonic hedgehog (Shh), Wnts and Bone morphogenetic protein (BMPs) or by cell-cell interactions via membrane-bound receptors and ligands such as Delta and Notch. Compartmentalization of the somites and their derivatives is reflected by the differential expression of developmental regulatory genes such as Pax-1, 3, 7 and 9, MyoD, paraxis, twist and others. Secondary segmentation is imposed upon other tissues, such as blood vessels and nerves, by the rearrangement and regionalization of the somitic derivatives, especially the sclerotome. Early cranio-caudal identity is determined by the expression of different Hox genes. Finally, fusion of segmental anlagen occurs to form segment-overbridging skeletal elements and muscles. The expression of homologous genes indicates that the process of segmentation in vertebrates and invertebrates is homologous, derived by descent from a common ancestor.