CD271 is expressed in melanomas with more aggressive behaviour, with correlation of characteristic morphology by in vivo reflectance confocal microscopy.

BACKGROUND: Melanoma is the most highly aggressive type of skin cancer. Its resistance to existing treatments and the rapid rise in incidence underscore the importance of acquiring a better understanding of melanomagenesis. OBJECTIVES: To assess the impact of reflectance confocal microscopy (RCM) on the description of cell morphology, which may influence the growth pattern and changes with increasing tumour severity, correlating with biological aspects. METHODS: A retrospective analysis of 30 primary melanomas in vivo, evaluated by RCM, to correlate cell morphology and cellular arrangement with a marker of melanoma progression (CD271) using immunohistochemical evaluations. RESULTS: Typical cells organized in dermal nests with peculiar in vivo confocal morphology result in melanoma with high malignancy and positivity to CD271. This architecture might be due to the presence of a type of cells, intrinsically predisposed to invasion, as a result of dedifferentiation programming, revealed by expression of the neural crest marker CD271. CONCLUSIONS: With the hypothesis that dedifferentiated cells would be strongly responsible for initiation of tumour development and progression, we propose that CD271 detection could be associated with RCM evaluation in order to detect more aggressive melanoma subtypes.

MATER protein as substrate of PKCepsilon in human cumulus cells.

High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKCepsilon) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER(+)HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKCepsilon. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKCepsilon. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKCepsilon is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.

Pathology databanking and biobanking in The Netherlands, a central role for PALGA, the nationwide histopathology and cytopathology data network and archive.

Since 1991, a nationwide histopathology and cytopathology network and archive is in operation in The Netherlands under the name PALGA, encompassing all sixty-four pathology laboratories in The Netherlands. The overall system comprises decentralized systems at the participating laboratories, a central databank, and a dedicated communication and information exchange tool. Excerpts of all histopathology and cytopathology reports are generated automatically at the participating laboratories and transferred to the central databank. Both the decentralized systems and the central system perform checks on the quality and completeness of excerpts. Currently, about 42 million records on almost 10 million patients are stored in the central databank. Each excerpt contains patient identifiers, including demographic data and the so-called PALGA diagnosis. The latter is structured along five classification axes: topography, morphology, function, procedure, and diseases. All data transfer and communication occurs electronically with encryption of patient and laboratory identifiers. All excerpts are continuously available to all participating pathology laboratories, thus contributing to the quality of daily patient care. In addition, external parties may obtain permission to use data from the PALGA system, either on an ongoing basis or on the basis of a specific permission. Annually, 40 to 60 applications for permission to use PALGA data are submitted. Among external users are the Dutch cancer registry, population-based screening programs for cancer of the uterine cervix and breast cancer in The Netherlands, and individual investigators addressing a range of research questions. Many scientific papers and theses incorporating PALGA data have been published already. In conclusion, the PALGA system is a unique system that requires a minimal effort on the part of the participating laboratories, while providing them a powerful tool in their daily practices.

Microsatellite instability and mismatch-repair protein expression in hereditary and sporadic colorectal carcinogenesis.

Aberrant crypt foci (ACF) are microscopic clusters of altered colonic crypts considered premalignant lesions in the large bowel. Genomic instability at short tandem repeats in the DNA, referred to as microsatellite instability (MSI) is the hallmark of hereditary nonpolyposis colorectal carcinoma (HNPCC) caused by mutations in DNA mismatch-repair genes, mostly hMLH1 and hMSH2. In this study, we evaluated for MSI ACF (n = 16), adenomas (n = 18), carcinomas (n =22), and lymph node metastases (n = 3) from 17 patients with colorectal cancer positive for MSI. Ten patients were members of HNPCC families; 7 patients had no family history of cancer. MSI was found in 7 of 7 (100%) ACF and 11 of 12 (91%) adenomas from patients with HNPCC. MSI was not related to histology and size of ACF. A progressive increase in instability as estimated by the number of shifted bands was observed along the ACF-adenoma-carcinoma sequence. In contrast, two of nine (22%) ACF and none of six adenomas from patients with MSI sporadic carcinoma were unstable at microsatellite loci. hMLH1 or hMSH2 protein expression was altered only in MSI-positive premalignant lesions (ACF and/or adenomas), but not in all MSI-positive lesions in patients with HNPCC. These observations provide evidence of the premalignant nature of ACF in HNPCC and suggest that MSI is a very early event both in HNPCC and in sporadic colorectal carcinogenesis, although in the latter it seems infrequent.

Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells.

AIMS: Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects. MAIN METHODS: hAFSCs were cultured in a standard medium or in an osteoblastic medium for 14 or 21days and ferutinin was added at 10-8M. Immunofluorescence techniques and Western-blot 21analysis were used to study estrogen receptors and signaling pathways. KEY FINDINGS: In both undifferentiated and differentiated hAFSCs we identified ERα and GPR30 with a nuclear or cytoplasmatic localization, respectively. The presence of ferutinin in the osteoblastic medium leads to an increase in ERα expression. To dissect the role of estrogen receptors, MPP and G15 were used to selectively block ERα and GPR30, respectively. Notably, ferutinin enhanced osteoblastic differentiation in cells challenged with G15. Ferutinin was able to increase ERK and Akt phosphorylations with a different timing activation. These phosphorylations were antagonized by PD0325901, a MEK inhibitor, and wortmannin, a PI3K inhibitor. Both MPP and G15 inhibited the ferutinin-induced MEK/ERK and PI3K/Akt pathway activations. In the osteoblastic condition, PD0325901, but not wortmannin, reduced the expression of OPN and RUNX-2, whereas ferutinin abrogated the down-modulation triggered by PD0325901. SIGNIFICANCE: PI3K/Akt pathways seems to mediate the enhancement of hAFSCs osteoblastic differentiation triggered by ferutinin through ERα.

Enrichment in c-Kit improved differentiation potential of amniotic membrane progenitor/stem cells.

INTRODUCTION: Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. METHODS: After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. RESULTS: Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. DISCUSSION: AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC.

Aberrant crypt foci in colorectal carcinogenesis. Cell and crypt dynamics.

Aberrant crypt foci (ACF) have been identified on the colonic mucosal surface of rodents treated with colon carcinogens and of humans after methylene-blue staining and observation under a light microscope. Several lines of evidence strongly suggest that ACF with certain morphological, histological, cell kinetics, and genetic features are precursor lesions of colon cancer both in rodents and in humans. Thus, ACF represent the earliest step in colorectal carcinogenesis. This paper has the main purpose of reviewing the evidence supporting this view, with particular emphasis on cell and crypt dynamics in ACF. ACF have been used as intermediate biomarkers of cancer development in animal studies aimed at the identification of colon carcinogens and chemopreventive agents. Recently, evidence has also shown that ACF can be effectively employed in chemopreventive studies also in humans.

Insulin selectively stimulates nuclear phosphoinositide-specific phospholipase C (PI-PLC) beta1 activity through a mitogen-activated protein (MAP) kinase-dependent serine phosphorylation.

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the beta family of PI-PLC (i.e. beta1, beta2, beta3, and beta4), insulin only activated the beta1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [(32)P]orthophosphate, we ascertained that nuclear PI-PLC-beta1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-beta1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-beta1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.

Increased activity and nuclear localisation of inositol lipid signal transduction enzymes in rat hepatoma cells.

To elucidate the relationship between inositol lipid signal transduction and oncogenic transformation, the activity and subcellular distribution of phospholipase C isoforms and of phosphatidylinositol 3-kinase were analysed in Morris hepatoma cells, MH(1)C(1), with respect to normal rat liver cells. The results provide evidence of a gain of function of the enzymes involved in inositide signal transduction, the amount of which increased mainly at the nuclear level. Phospholipase C and phosphatidylinositol 3-kinase activities are significantly higher in rat hepatoma than in rat liver cells. Moreover, some phospholipase C isoforms are expressed at higher levels at the nuclear level; this is particularly evident in the case of the delta 1 isoform which is not expressed at the nuclear level in rat liver cells. Therefore, the autonomous nuclear signal transduction system, formerly reported as involved in the modulation of cell proliferation and differentiation, appears also affected in oncogenic transformation.

Apoptosis in different stages of human oogenesis.

Apoptosis is an active form of cell death characterized by a series of morphological changes that become particularly evident at the ultrastructural level. The majority of ovarian germ cells undergo degeneration during prenatal and reproductive life and only in recent studies has it been demonstred that this drop is due to an apoptotic process. We evaluated this process during human oogenesis in prenatal life and we studied the ultrastructural changes that occur in apoptosis in various phases of the meiotic process. From our observations it is clear that apoptosis involves two main phases of the meiotic process: an earlier one concerning the oogonia and oocytes in the preleptotene stage, and a later one that mainly concerns the oocytes in the pachytene stage.

Scanning electron microscopy of aberrant crypt foci in human colorectal mucosa.

BACKGROUND: Aberrant crypt foci (ACF) are clusters of morphologically altered crypts which can be observed by light or stereomicroscopy on the mucosal surface of the colon after staining with methylene-blue. They probably represent one of the earliest events in human colorectal carcinogenesis. The main purpose of the present study was to observe the surface features of aberrant and normal colonic crypts in humans using scanning electron microscopy (SEM) in order to find and measure differences between aberrant and normal. MATERIALS AND METHODS: Fifteen mucosal specimens containing ACF and 8 with normal mucosa taken from patients operated on for colon cancer were observed under a scanning electron microscope. RESULTS: By SEM ACF were easily observed on the mucosal surface, because they showed a well defined border and were elevated on the mucosal surface. Under higher magnification luminal openings of aberrant crypts had a larger overall average diameter than normal (37.6 microns +/- 13.5, mean +/- SD, vs 15.9 microns +/- 4.9, P = 0.001), though when crypt multiplicity of ACF (number of crypts per ACF) was higher, the diameter of luminal openings tended to be smaller and similar to those of normal crypts, with weak negative correlation between crypt multiplicity of ACF and mean diameter of aberrant luminal openings (r = 0.27). Finally, the mucosal surface among aberrant crypts was flattened because of a loss of microvilli. in conclusion, scanning electron microscopy allows a better definition of the topological features of aberrant crypt foci than light or stereomicroscopy.

Developmental pathways of vertebral centra and neural arches in human embryos and fetuses.

The ossification pathways of both vertebral centra (i.e., vertebral bodies) and neural arches were studied in human embryos and fetuses (CR-length between 38 and 116 mm). A clearing and double-staining method for whole embryo or fetus, using alcian blue and alizarin red S, allowed an easy and precise detection of the morphology of the whole vertebral column and every single vertebra. Both cartilaginous and bony components were clearly visible. Different temporal and topographical patterns of ossification were shown for the centra and arches; the latter were respectively proximal-distal (i.e., bidirectional from a defined starting tract in T10-L1) and cranial-caudal (i.e., monodirectional). The patterns could be related to the morphogenetic processes of other structures (i.e., muscles and nerves). Moreover, the numerical survey of ossification centers provided a possible parameter for the determination of the fetal developmental age. This could be useful in the study of pathological conditions.

Effects of estrogens and oxytocin on the development of neonatal mammalian ovary.

The preservation and death of germ cells in the neonatal mammalian ovary are linked with the presence of hormones. Estrogens and oxytocin are present at birth in all mammalian vertebrates. The aim of this study was to examine their role in the development of the neonatal ovary and also in the preservation and death of germ cells in the neonatal period: apoptotic phenomena play a fundamental role in the control of their number. Female neonatal mice were treated at birth with estradiol monobenzoate or oxytocin and sacrificed after 5 days. The ovaries were sectioned in toto into semi-thin sections, in order to calculate their volume. Thin sections were also carried out to verify, under the transmission electron microscope (T.E.M.), the cells in apoptosis. The ovaries treated with the greater concentration of estradiol monobenzoate showed a volume that was significantly greater than that of the controls and a reduction of germ cells in apoptosis. The ovaries treated with oxytocin at all degrees of concentration had a volume significantly less than the controls and they also had a higher number of germ cells in apoptosis.

Perinatal mortality in breech presentations as compared to vertex presentations in singleton pregnancies: an analysis based upon 57819 computer-registered pregnancies in The Netherlands.

In 1982, nationwide registration of obstetric data was instituted in The Netherlands with about 70% of all Dutch hospitals participating. The resultant data from 57819 singleton pregnancies in vertex or breech presentation at delivery was studied. The vertex and breech groups were compared. The proportion of breech presentations relative to vertex presentations was greater in low gestational age infants and those of low birthweight. After correction for gestational age and birthweight, the perinatal mortality was higher in the breech groups than in the vertex groups. Congenital malformations occurred more frequently in the breech group but, even after exclusion of infants with congenital malformations, perinatal mortality remained higher in the breech group at any gestational age. Caesarean section was more frequently performed in the breech group than in the vertex group but did not appear to improve the outcome of breech presentation. It is possible that breech presentation is not coincidental but is a consequence of poor fetal quality, in which case medical intervention is unlikely to reduce the perinatal mortality associated with breech presentation to the level associated with vertex presentation.

Human neonatal ovary: proposal of a three-dimensional model.

A model of human neonatal ovary is presented, derived from morphometric, evaluations carried out on left ovaries removed from five full-term neonates with a 46, XX karyotype, free from malformations of the genital apparatus. According to this model, the gonad can be represented by a triaxial ellipsoid with a central medullary core surrounded by a cortical stratum of constant thickness. The germinal population, consisting of follicles and primitive cortical tissue, occupies the cortex, intermingled with the interstitium or stroma. In the cortex it is then possible to describe an outer layer formed by primitive cortical tissue, and an inner portion occupied by follicles. The primary and secondary follicles fill the portion near the medulla and the primordial ones are contained in the middle and outer zones. Since the variability observed among ovaries is slight, we can propose a mean model of neonatal ovary in which the spatial relationships among the different components, the total number of follicles and their position in the cortex can be calculated.

[In vitro study of periodontal ligament cells]. / Studio in vitro delle cellule del legamento periodontale.

BACKGROUND: The aim of this research is to outline a procedure able to promote specific cellular differentiation and proliferation with consequent periodontal regeneration. To achieve this goal, use was made of various compounds supposed to have the capacity of aiding periodontal regeneration. METHODS: The cells utilised for this study were obtained from explants of human periodontal ligaments. Their proliferation and differentiation capacity was examined in the presence of: coral granules (350, 500 mu), collagene type 1, growth factors (Platelet derived growth factor, PDGF and Transforming growth factor beta 1, TGF beta 1), both on their own and in different combination with one another. The differentiation activity was evaluated by ultrastructural morphological method (Transmission electron microscope-TEM) and by spectrophotometric investigation of the alkaline phosphatasis (ALP). RESULTS: The data show that the coral granules and among the growth factors used only TGF beta 1 stimulate the differentiation activity of the periodontal ligament cells valued on the basis of their capacity of producing ALP. These data are supported by the observation with TEM. CONCLUSIONS: From these results it is suggested that there may be therapeutic efficiency in the periodontal field of substances promoting cellular proliferation and differentiation.

Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells.

AIMS: The phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs). MAIN METHODS: AFSCs and DPSCs were cultured in a differentiation medium for 14 or 21days with or without the addition of Ferutinin at a concentration ranging from 10(-11) to 10(-4)M. 17ß-Estradiol was used as a positive drug at 10(-8)M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed. KEY FINDINGS: MTT assay revealed that Ferutinin, at concentrations of 10(-8) and 10(-9)M, enhanced proliferation of both AFSCs and DPSCs after 72h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14days of culture in both populations. SIGNIFICANCE: The enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.

Enrichment in c-Kit+ enhances mesodermal and neural differentiation of human chorionic placental cells.

OBJECTIVE: Human term placenta (HTP) has attracted increasing attention as an alternative source of stem cells for regenerative medicine since the amniochorionic membrane harbors stem cells populations that are easily accessible, abundantly available without ethical objections. In the chorionic side of HTP we found a progenitor perivascular "niche" in which rare cells co-express Oct-4 and c-Kit. We investigated the stem cell characteristics and differentiation potential of a chorionic derived population enriched in c-Kit(+) cells and compared this to the unenriched population. STUDY DESIGN: Cells, isolated from the chorion of HTP, were expanded and enriched in c-Kit(+) cells (Chorionic Stem Cells-CSC). Histological staining, immunofluorescence, Western blot and flow cytometry were used to verify the stem cells characteristics of the populations and to compare the differentiation capability towards mesodermal and neural lineages in vitro. RESULTS: The expression of the pluripotent marker Oct-4 was greater in the CSCs compared to the unselected cells (Chorionic Cell-CC) but both Oct-4 and c-Kit expression decreased during passages. After differentiation, CSC displayed stronger chondrogenic and osteogenic potential and a greater adipogenic forming capacity compared to unselected ones. CSC differentiated better into immature oligodendrocytes while CC showed a neuronal progenitor differentiation potential. Moreover, both populations were able to differentiate in hepatogenic lineage. CONCLUSION: CSC display improved Oct-4 expression and a high differentiation potential into mesodermal lineages and oligodendrocytes.

Human dental pulp stem cells produce mineralized matrix in 2D and 3D cultures.

The aim of this study was to characterize the in vitro osteogenic differentiation of dental pulp stem cells (DPSCs) in 2D cultures and 3D biomaterials. DPSCs, separated from dental pulp by enzymatic digestion, and isolated by magnetic cell sorting were differentiated toward osteogenic lineage on 2D surface by using an osteogenic medium. During the differentiation process, DPSCs express specific bone proteins like Runx-2, Osx, OPN and OCN with a sequential expression, analogous to those occurring during osteoblast differentiation, and produce extracellular calcium deposits. In order to differentiate cells in a 3D space that mimes the physiological environment, DPSCs were cultured in two distinct bioscaffolds, Matrigel™ and Collagen sponge. With the addition of a third dimension, osteogenic differentiation and mineralized extracellular matrix production significantly improved. In particular, in Matrigel™ DPSCs differentiated with osteoblast/osteocyte characteristics and connected by gap junction, and therefore formed calcified nodules with a 3D intercellular network. Furthermore, DPSCs differentiated in collagen sponge actively secrete human type I collagen micro-fibrils and form calcified matrix containing trabecular-like structures. These neo-formed DPSCs-scaffold devices may be used in regenerative surgical applications in order to resolve pathologies and traumas characterized by critical size bone defects.