Prognosis of small bowel adenocarcinoma in Crohn's disease compares favourably with de novo small bowel adenocarcinoma.

AIM: Limited data exist on Crohn's disease (CD)-associated small bowel adenocarcinoma (SBA). A large-scale retrospective cohort study was conducted comparing the clinical features and outcome of CD-associated SBA and de novo SBA. METHOD: Data for patients with small bowel adenocarcinoma were gathered from the 1992-2010 United States Surveillance, Epidemiology and End Results cancer registry-Medicare linked database. We identified 2123 patients, of whom 179 had CD-associated and 1944 de novo SBA. The main outcome measures were overall survival (OS) and cancer-specific survival (CSS). RESULTS: CD-associated SBA was most commonly located in the ileum (62% vs 31%, P < 0.0001). CD patients were diagnosed at an earlier stage (I/II), compared with de novo SBA (55% vs 32%, P < 0.0001), and were more likely to undergo surgery (81% vs 72%, P = 0.0016). Chemotherapy use was similar (25% vs 21%, P = 0.1886). Patients with CD-associated SBA had better 5-year OS (43% vs 34%, P = 0.0121) but a similar CSS (65% vs 64%, P = 0.77). There was no difference in the OS between the cohorts when stratified by stage. On multivariate analysis, CD was not significantly related to OS [hazard ratio (HR) 0.97, 95% CI: 0.79-1.20, P = 0.7889]. Surgery and the extent of lymphadenectomy improved OS for all SBA patients (HR 0.73, 95% CI: 0.60-0.88, P = 0.001), whereas chemotherapy did not (HR 1.13, 95% CI: 0.99-1.28, P = 0.0665). CONCLUSION: Patients with CD-associated SBA present at an earlier stage than patients with de novo SBA, they receive more surgery but similar rates of chemotherapy, and have similar OS and CSS. The presence of CD does not worsen survival after treatment of SBA.

Long-term parental methamphetamine exposure of mice influences behavior and hippocampal DNA methylation of the offspring.

The high rate of methamphetamine (METH) abuse among young adults and women of childbearing age makes it imperative to determine the long-term effects of METH exposure on the offspring. We hypothesized that parental METH exposure modulates offspring behavior by disrupting epigenetic programming of gene expression in the brain. To simulate the human pattern of drug use, male and female C57Bl/6J mice were exposed to escalating doses of METH or saline from adolescence through adulthood; following mating, females continue to receive drug or saline through gestational day 17. F1 METH male offspring showed enhanced response to cocaine-conditioned reward and hyperlocomotion. Both F1 METH male and female offspring had reduced response to conditioned fear. Cross-fostering experiments have shown that certain behavioral phenotypes were modulated by maternal care of either METH or saline dams. Analysis of offspring hippocampal DNA methylation showed differentially methylated regions as a result of both METH in utero exposure and maternal care. Our results suggest that behavioral phenotypes and epigenotypes of offspring that were exposed to METH in utero are vulnerable to (a) METH exposure during embryonic development, a period when wide epigenetic reprogramming occurs, and (b) postnatal maternal care.

Chondro/osteoblastic and cardiovascular gene modulation in human artery smooth muscle cells that calcify in the presence of phosphate and calcitriol or paricalcitol.

Vitamin D sterol administration, a traditional treatment for secondary hyperparathyroidism, may increase serum calcium and phosphorus, and has been associated with increased vascular calcification (VC). In vitro studies suggest that in the presence of uremic concentrations of phosphorus, vitamin D sterols regulate gene expression associated with trans-differentiation of smooth muscle cells (SMCs) to a chondro/osteoblastic cell type. This study examined effects of vitamin D sterols on gene expression profiles associated with phosphate-enhanced human coronary artery SMC (CASMC) calcification. Cultured CASMCs were exposed to phosphate-containing differentiation medium (DM) with and without calcitriol, paricalcitol, or the calcimimetic R-568 (10(-11)-10(-7) M) for 7 days. Calcification of CASMCs, determined using colorimetry following acid extraction, was dose dependently increased (1.6- to 1.9-fold) by vitamin D sterols + DM. In contrast, R-568 did not increase calcification. Microarray analysis demonstrated that, compared with DM, calcitriol (10(-8) M) + DM or paricalcitol (10(-8) M) + DM similarly and significantly (P < 0.05) regulated genes of various pathways including: metabolism, CYP24A1; mineralization, ENPP1; apoptosis, GIP3; osteo/chondrogenesis, OPG, TGFB2, Dkk1, BMP4, BMP6; cardiovascular, HGF, DSP1, TNC; cell cycle, MAPK13; and ion channels, SLC22A3 KCNK3. R-568 had no effect on CASMC gene expression. Thus, SMC calcification observed in response to vitamin D sterol + DM may be partially mediated through targeting mineralization, apoptotic, osteo/chondrocytic, and cardiovascular pathway genes, although some gene changes may protect against calcification. Further studies to determine precise roles of these genes in development of, or protection against VC and cardiovascular disease are required.

DNA methyltransferase inhibition in normal human fibroblasts induces a p21-dependent cell cycle withdrawal.

Maintenance of methylation patterns in the mammalian genome by DNA (cytosine-5) methyltransferases (DNAMeTase) is required for normal cell and tissue function. Inhibition of DNAMeTase in cultured cells induces the expression of p21, a cyclin-dependent kinase (Cdk) inhibitor critical for cells to enter replicative senescence. We investigated the effects of DNAMeTase inhibition in normal human fibroblasts and found that it induces an irreversible growth arrest. Cells arrested by DNAMeTase inhibition became enlarged and had a flat morphology, exhibited an increased expression of collagenase and p21, and the DNA synthesis block could be overcome by the introduction of the SV40 large T antigen, all characteristics of senescent cells. In contrast, normal human fibroblasts lacking a functional p21 gene fail to undergo cell cycle arrest following DNAMeTase inhibition, indicating that p21 is an essential component of this arrest. Furthermore, DNAMeTase activity was reduced as cells approached the end of their proliferative potential. These data suggest that DNAMeTase could be an integral part of the mechanisms by which cells count the number of cell divisions completed and initiate a signaling cascade that ultimately results in the senescent phenotype.

Authentic cell-specific and developmentally regulated expression of pro-opiomelanocortin genomic fragments in hypothalamic and hindbrain neurons of transgenic mice.

The pro-opiomelanocortin (POMC) gene is expressed in a subset of hypothalamic and hindbrain neurons and in pituitary melanotrophs and corticotrophs. POMC neurons release the potent opioid beta-endorphin and several active melanocortins that control homeostasis and feeding behavior. POMC gene expression in the CNS is believed to be controlled by distinct cis-acting regulatory sequences. To analyze the transcriptional regulation of POMC in neuronal and endocrine cells, we produced transgenic mice carrying POMC27*, a transgene containing the entire 6 kb of the POMC transcriptional unit together with 13 kb of 5' flanking regions and 8 kb of 3' flanking regions. POMC27* was tagged with a heterologous 30 bp oligonucleotide in the third exon. In situ hybridization studies showed an accurate cell-specific pattern of expression of POMC27* in the arcuate nucleus and the pituitary. Hypothalamic mRNA-positive neurons colocalized entirely with beta-endorphin immunoreactivity. No ectopic transgenic expression was detected in the brain. Deletional analyses demonstrated that neuron-specific expression of POMC transgenes required distal 5' sequences localized upstream of the pituitary-responsive proximal cis-acting elements that were identified previously. POMC27* exhibited a spatial and temporal pattern of expression throughout development that exactly paralleled endogenous POMC. RNase protection assays revealed that POMC27* expression mimicked that of POMC in different areas of the CNS and most peripheral organs with no detectable ectopic expression. Hormonal regulation of POMC27* and POMC was identical in the hypothalamus and pituitary. These results show that distal 5' sequences of the POMC gene located between -13 and -2 kb target expression into the CNS of transgenic mice in a precise neuron-specific, developmentally and hormonally regulated manner.

Accurate spatial and temporal transgene expression driven by a 3.8-kilobase promoter of the bovine beta-casein gene in the lactating mouse mammary gland.

The spatial, temporal, and hormonal pattern of expression of the beta-casein gene is highly regulated and confined to the epithelial cells of the lactating mammary gland. Previous studies have shown that 1.7 kb of the bovine beta-casein promoter were able to drive cell-specific and hormone-dependent expression to a mouse mammary cell line but failed to induce accurate expression to the mammary gland of transgenic mice. We investigated here the ability of 3.8 kb of the bovine beta-casein gene promoter to drive the expression of the human growth hormone (hGH) gene in transgenic mice. A Northern blot analysis using total RNA obtained from different tissues of lactating and nonlactating females revealed the presence of hGH mRNA only in the mammary gland of lactating females. hGH mRNA was not detectable in the mammary gland of virgin females or males. A developmental analysis showed that hGH mRNA only peaked on parturition, resembling more closely the bovine beta-casein temporal expression pattern rather than the murine. In situ hibridization studies performed on mammary gland sections showed that the cellular pattern of hGH expression was homogeneous in all lobules from heterozygous and homozygous transgenic mice. Silver grain counts on the tissue sections highly correlated with the hGH contents in the milk determined by radioimmunoassay (r = 0.996). Thus 3.8 kb of the bovine beta-casein promoter direct a high-level expression of a reporter gene to the lactating mammary gland of transgenic mice in a tissue-specific and developmentally regulated manner.

Activation of mechanical responses in leukocytes.

Different kinds of leukocytes undergo cytoskeleton-dependent mechanical responses associated with their specific physiological functions. We have investigated cellular stiffening of several types of leukocytes using a method which measures the force resisting cellular indentation. We have found that lymphocytes stiffen in response to crosslinking cell surface antigens in a process associated with the much studied capping and patching processes. Further studies of myosin-deficient mutants of the ameba Dictyostelium discoideum suggest that this stiffening process results from a myosin dependent contractile process. Rat basophilic leukemia cells and pancreatic islet cells stiffen when triggered to secrete. The function of these cytoskeleton dependent processes is now unknown, but, at least in the islet cells, may be related to a regulation of the rate of secretion. Primary neutrophils stiffen in response to the chemotactic agent, fMet-Leu-Phe. This stiffening may be responsible for retention of these cells in the pulmonary microcirculation during response to inflammation. These observations pose the challenge of determining the structural basis, mechanism, and physiological function of each of these cellular responses.

Rat basophilic leukemia cells stiffen when they secrete.

RBL cells provide a useful model of the IgE and antigen-dependent stimulus-secretion coupling of mast cells and basophils. We have measured cellular deformability to investigate the participation of cytoskeletal mechanical changes. Cross-linking cell-surface IgE-receptor complexes with multivalent ligands not only triggered secretion but also caused the cells to stiffen, i.e., to become more resistant to deformation. This mechanical response required receptor cross-linking, had a time course similar to that of secretion, and was reversed by DNP-L-lysine, a competitive inhibitor of antigen binding. Hence the same stimulus seems to elicit both stiffening and secretion. Cytochalasin D, which inhibits actin filament assembly, prevented or reversed stiffening, thereby implicating the cytoskeleton in the mechanical response. Increasing intracellular calcium ion concentration with the ionophore A23187 stiffened cells and stimulated secretion. Activation of protein kinase C with a phorbol ester also stiffened cells and enhanced both the stiffening and secretion caused by the ionophore. Yet cytochalasin D enhances secretion whereas activation of protein kinase c alone is insufficient for secretion. Therefore stiffening is neither necessary nor sufficient for secretion. These results characterize a cytoskeletal mechanical response triggered by the same receptor-dependent stimulus that elicits secretion and by second messengers that are thought to mediate between the receptor signal and secretion. The function of the mechanical response, however, remains to be determined.

Cross-linking surface immunoglobulin increases the stiffness of lymphocytes.

Cross-linking surface immunoglobulin (sIg) on B-lymphocytes causes a substantial increase in the mechanical stiffness of the cells. This has been demonstrated using a new method for measuring cellular deformability. The method is based on a device, the "Cell Poker", which we use to determine the force required slightly to indent or compress a cell adherent to a rigid substrate in culture. Cross-linking of sIg by bivalent anti-sIg antibodies is necessary to elicit the increase in stiffness; binding of monovalent Fab fragments is insufficient. The increase in stiffness is partially reversed by cytochalasin D and by completion of the capping of the cross-linked sIg. The modulation of cellular deformability and the induction of cellular dynamic processes such as capping are similar in their requirements for cross-linking sIg and in their sensitivity to cytochalasins. This suggests that both kinds of responses stem from similar cellular processes and structures. These results emphasize the mechanical capability of lymphocytes and suggest that the physiological functions of these cells are likely to employ this capability.