HIC1 tumour suppressor gene is suppressed in acute myeloid leukaemia and induced during granulocytic differentiation

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

Chondrogenic differentiation of bovine synovium: bone morphogenetic proteins 2 and 7 and transforming growth factor beta1 induce the formation of different types of cartilaginous tissue

OBJECTIVE: To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor beta1 (TGFbeta1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. METHODS: Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFbeta1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. RESULTS: BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFbeta1 differed in terms of both cell phenotype and gene expression profiles. CONCLUSION: The 3 tested members of the TGFbeta superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded alpha1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.

Cartilage T2 assessment at 3-T MR imaging: in vivo differentiation of normal hyaline cartilage from reparative tissue after two cartilage repair procedures--initial experience

PURPOSE: To prospectively compare cartilage T2 values after microfracture therapy (MFX) and matrix-associated autologous chondrocyte transplantation (MACT) repair procedures. MATERIALS AND METHODS: The study had institutional review board approval by the ethics committee of the Medical University of Vienna; informed consent was obtained. Twenty patients who underwent MFX or MACT (10 in each group) were enrolled. For comparability, patients of each group were matched by mean age (MFX, 40.0 years +/- 15.4 [standard deviation]; MACT, 41.0 years +/- 8.9) and postoperative interval (MFX, 28.6 months +/- 5.2; MACT, 27.4 months +/- 13.1). Magnetic resonance (MR) imaging was performed with a 3-T MR imager, and T2 maps were calculated from a multiecho spin-echo measurement. Global, as well as zonal, quantitative T2 values were calculated within the cartilage repair area and within cartilage sites determined to be morphologically normal articular cartilage. Additionally, with consideration of the zonal organization, global regions of interest were subdivided into deep and superficial areas. Differences between cartilage sites and groups were calculated by using a three-way analysis of variance. RESULTS: Quantitative T2 assessment of normal native hyaline cartilage showed similar results for all patients and a significant trend of increasing T2 values from deep to superficial zones (P < .05). In cartilage repair areas after MFX, global mean T2 was significantly reduced (P < .05), whereas after MACT, mean T2 was not reduced (P > or = .05). For zonal variation, repair tissue after MFX showed no significant trend between different depths (P > or = .05), in contrast to repair tissue after MACT, in which a significant increase from deep to superficial zones (P < .05) could be observed. CONCLUSION: Quantitative T2 mapping seems to reflect differences in repair tissues formed after two surgical cartilage repair procedures. (c) RSNA, 2008.

Pineal parenchymal tumor of intermediate differentiation: diagnostic pitfalls and discussion of treatment options of a rare tumor entity

Tumors of the pineal region are uncommon, comprising approximately 0.4-1% of all intracranial tumors in adults in European and American series. Histopathologically, they are a very heterogeneous group of tumors. Of genuine pineal tumors, pineal parenchymal tumors of intermediate differentiation (PPTIDs) are the least frequently found type. In this paper, we report on the case of a patient with an unexpected and difficult-to-diagnose PPTID. A 2.2 x 2.2-cm midline mass within the posterior part of the third ventricle with consecutive obstructive hydrocephalus was found in a 44-year-old man presenting with diplopia and gait disturbances. There was no clear connection of the tumor to the pineal gland. Differential diagnosis included all intraventricular and midline tumors, therefore a biopsy was taken. Preliminary histopathological diagnosis was germinoma or primitive neuroectodermal tumor, and the tissue sample was reexamined by a referential neuropathological institute. Final diagnosis was PPTID. The tumor was then resected through a transventricular/transchoroidal approach. Histopathological examination of tumor specimen confirmed the diagnosis of a PPTID. Postoperatively, the patient received gamma-knife radiosurgery. At 1-year follow-up, there are no signs of tumor regrowth. Diagnosis of pineal parenchymal tumors in general and PPTIDs in particular can be troublesome. Their histopathological features are still being defined, as is the biological behavior of the different tumor entities. Thus, treatment options including surgery, radiation therapy, and chemotherapy remain controversial. We recommend surgical removal of PPTID, preferably in toto whenever the size of the tumor permits that kind of excision.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.

Intestinal macrophages: differentiation and involvement in intestinal immunopathologies

Intestinal macrophages, preferentially located in the subepithelial lamina propria, represent the largest pool of tissue macrophages in humans. As an adaptation to the local antigen- and bacteria-rich environment, intestinal macrophages exhibit several distinct phenotypic and functional characteristics. Notably, microbe-associated molecular pattern receptors, including the lipopolysaccharide (LPS) receptors CD14 and TLR4, and also the Fc receptors for IgA and IgG are absent on most intestinal macrophages under homeostatic conditions. Moreover, while macrophages in the intestinal mucosa are refractory to the induction of proinflammatory cytokine secretion, they still display potent phagocytic activity. These adaptations allow intestinal macrophages to comply with their main task, i.e., the efficient removal of microbes while maintaining local tissue homeostasis. In this paper, we review recent findings on the functional differentiation of monocyte subsets into distinct macrophage populations and on the phenotypic and functional adaptations that have evolved in intestinal macrophages in response to their antigen-rich environment. Furthermore, the involvement of intestinal macrophages in the pathogenesis of celiac disease and inflammatory bowel diseases is discussed.

Pollinator responses to variation in floral display and flower size in dioecious Sagittaria latifolia (Alismataceae)

In animal-pollinated plants with unisexual flowers, sexual dimorphism in floral traits may be the consequence of pollinator-mediated selection. Experimental investigations of the effects of variation in flower size and floral display on pollinator visitation can provide insights into the evolution of floral dimorphism in dioecious plants. Here, we investigated pollinator responses to experimental arrays of dioecious Sagittaria latifolia in which we manipulated floral display and flower size. We also examined whether there were changes in pollinator visitation with increasing dimorphism in flower size. In S. latifolia, males have larger flowers and smaller floral displays than females. Visitation by pollinators, mainly flies and bees, was more frequent for male than for female inflorescences and increased with increasing flower size, regardless of sex. The number of insect visits per flower decreased with increasing floral display in males but remained constant in females. Greater sexual dimorphism in flower size increased visits to male inflorescences but had no influence on the number of visits to female inflorescences. These results suggest that larger flower sizes would be advantageous to both females and males, and no evidence was found that females suffer from increased flower-size dimorphism. Small daily floral displays may benefit males by allowing extended flowering periods and greater opportunities for effective pollen dispersal.

Spatial isolation and genetic differentiation in naturally fragmented plant populations of the Swiss Alps

Aims The effect Of anthropogenic landscape fragmentation on the genetic diversity and adaptive potential of plant populations is a major issue in conservation biology. However, little is known about the partitioning of genetic diversity in alpine species, which occur in naturally fragmented habitats. Here, we, investigate molecular patterns of three alpine plants (Epilobium fleischeri, Geum reptans and Campanula thyrsoides) across Switzerland and ask whether Spatial isolation has led to high levels of populations differentiation, increasing over distance, and a decrease of within-population variability. We further hypothesize that file contrasting potential for long-distance dispersal (LDD) of Seed in these Species will considerably influence and explain diversity partitioning. Methods For each study species, we Sampled 20-23 individuals from each of 20-32 populations across entire Switzerland. We applied Random Amplified Polymorphic Dimorphism markers to assess genetic diversity within (Nei's expected heterozygosity, H-e; percentage of polymorphic hands, P-P) and among (analysis of molecular variance, Phi(st)) populations and correlated population size and altitude with within-populalion diversity. Spatial patterns of genetic relatedness were investigated using Mantel tests and standardized major axis regression as well as unweighted pair group method with arithmetic mean cluster analyses and Monmonier's algorithm. To avoid known biases, We standardized the numbers of populations, individuals and markers using multiple random reductions. We modelled LDD with a high alpine wind data set using the terminal velocity and height of seed release as key parameters. Additionally, we assessed a number of important life-history traits and factors that potentially influence genetic diversity partitioning (e.g. breeding system, longevity and population size). Important findings For all three species, We found a significant isolation-by-distance relationship but only a moderately high differentiation among populations (Phi(st): 22.7, 48 and 16.8%, for E. fleischeri, G. reptans and C. thyrsoides, respectively). Within-population diversity (H-c: 0.19-0.21, P-p: 62-75%) was not reduced in comparison to known results from lowland species and even small populations with < 50 reproductive individuals contained high levels of genetic diversity. We further found no indication that a high long-distance seed dispersal potential enhances genetic connectivity among populations. Gene flow seems to have a strong stochastic component causing large dissimilarity between population pairs irrespective of the spatial distance. Our results suggest that other life-history traits, especially the breeding System, may play an important role in genetic diversity partitioning. We conclude that spatial isolation in the alpine environment has a strong influence on population relatedness but that a number of factors can considerably influence the strength of this relationship.

Niche differentiation between diploid and hexaploid Aster amellus

The maintenance of separated diploid and polyploid populations within a contact zone is possible due to both prezygotic and postzygotic isolation mechanisms. Niche differentiation between two cytotypes may be an important prezygotic isolating mechanism and can be studied using reciprocal transplant experiments. We investigated niche differentiation between diploid and hexaploid Aster amellus in their contact zone in the Czech Republic. Diploid populations are confined to habitats with low productivity, whereas hexaploid populations occur in habitats with both low and high productivity. Thus, we chose three diploid populations and six hexaploid populations, three in each of the two different habitat types. We analyzed habitat characteristics and carried out reciprocal transplant experiments in the field using both seeds and adult plants. Sites of diploid and hexaploid populations differed significantly in vegetation and soil properties. The mean number of juveniles was higher at sites of home ploidy level than at sites of foreign ploidy level, suggesting niche differentiation between the two cytotypes. On the other hand, transplanted adult plants survived at all sites and juvenile plants were able to establish at some sites of the foreign cytotype. Furthermore, the mean number of juveniles, survival, and flowering percentages were higher at home sites than at foreign sites, indicating local adaptation. We conclude that niche differentiation between the two cytotypes and local adaptation within each cytotype may contribute to the maintenance of diploid and hexaploid populations of A. amellus in their contact zone. Moreover, further factors, such as differences in flowering phenology and exclusion of minority cytotypes, should also be considered.

Adaptive rather than non-adaptive evolution of Mimulus guttatus in its invasive range

Adaptive and non-adaptive evolutionary processes are likely to play important roles in biological invasions but their relative importance has hardly ever been quantified. Moreover, although genetic differences between populations in their native versus invasive ranges may simply reflect different positions along a genetic latitudinal cline, this has rarely been controlled for. To study non-adaptive evolutionary processes in invasion of Mimulus guttatus, we used allozyme analyses on offspring of seven native populations from western North America, and three and four invasive populations from Scotland and New Zealand, respectively. To study quantitative genetic differentiation, we grew 2474 plants representing 17 native populations and the seven invasive populations in a common greenhouse environment under temporarily and permanently wet soil conditions. The absence of allozyme differentiation between the invasive and native range indicates that multiple genotypes had been introduced to Scotland and New Zealand, and suggests that founder effects and genetic drift played small, if any, roles in shaping genetic structure of invasive M. guttatus populations. Plants from the invasive and native range did not differ in phenology, floral traits and sexual and vegetative reproduction, and also not in plastic responses to the watering treatments. However, plants from the invasive range produced twice as many flower-bearing upright side branches than the ones from the native populations. Further, with increasing latitude of collection, vegetative reproduction of our experimental plants increased while sexual reproduction decreased. Plants from the invasive and native range shared these latitudinal clines. Because allozymes showed that the relatedness between native and invasive populations did not depend on latitude, this suggests that plants in the invasive regions have adapted to the local latitude. Overall, our study indicates that quantitative genetic variation of M. guttatus in its two invasive regions is shaped by adaptive evolutionary processes rather than by non-adaptive ones. (C) 2007 Gesellschaft fur Okologie. Published by Elsevier GmbH. All rights reserved.