Differential responses of VIPergic and nitrergic neurons in paediatric patients with Crohn's disease.

Inflammatory bowel disease is a recurrent intestinal inflammatory disorder that in adults has been associated with changes in enteric nervous system neuropeptide expression. The aim of the present study was to determine whether similar changes were observed in paediatric Crohn's disease. The distribution of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) was determined in colonic tissues from children with ileo-colonic (n=4) and colonic (n=3) Crohn's disease. The submucosal plexus of inflamed regions showed significant increase in density of VIP immunoreactive neurons (margin, 48% vs. inflamed tissue, 82% of HuC/D positive neurons). The density of submucosal plexus nNOS immunoreactive neurons was too low to be reliably quantified. Using the pan-neuronal marker HuC/D, no significant difference in numbers of HuC/D positive submucosal neurons was evident except where neurons were normalized to length of tissue (margins, 3.6+/-0.7 vs. inflamed tissue, 4.0+/-0.6 neurons/ganglia, p=0.33; margins, 2.7+/-0.4 vs. inflamed tissue, 5.7+/-1.2, neurons/mm, p=0.03). In the myenteric plexus, there was a significant increase in the percent of NOS neurons (38% vs. 82% of HuC/D positive neurons) while there was no significant difference in percent of VIP neurons (4% vs. 8%). No difference in number of HuC/D positive myenteric neurons among margin and inflamed tissues was observed (margin, 12.2+/-3.0 vs. inflamed tissue, 12.5+/-5.1 neurons/ganglia, p=0.50; margins 9.1+/-2.1 vs. inflamed tissue, 13.7+/-2.3 neurons/mm, p=0.11). These data demonstrate that inflammation is associated with a differential expression of VIP and nNOS neuronal subpopulations within the two major enteric plexi, likely due to phenotypic switch. Such changes might contribute to the pathogenesis of IBD and ongoing symptoms even in quiescent disease.

Myenteric plexus injury and apoptosis in experimental colitis.

Intestinal inflammatory conditions are associated with structural and functional alterations of the enteric nervous system (ENS). While injury to the enteric nervous system is well described, the mechanisms of neuronal injury and neuronal cell loss remain unclear. The aim of the present study was to examine the neural consequences of distal colitis and to assess the role of neutrophil granulocytes in mediating these changes. Colitis was induced in C3H/HEN female mice with dinitrobenzene sulfonic acid. The mice were then sacrificed at 0.5, 1, 1.5, 2, 3, 4, 6, 12, 24, 120 h post instillation of dinitrobenzene sulfonic acid. The inflammatory response was assessed by macroscopic damage score, myeloperoxidase activity and histology. HuC/D and PGP 9.5 immunostaining was used to examine myenteric plexus density and structure, neural cell body numbers and distribution in cross-section and whole mount preparations. Apoptosis was investigated in whole mount preparations double stained with HuC/D and activated caspase-3 or cleaved poly (ADP-ribose) polymerase (PARP). Dinitrobenzene sulfonic acid-induced colitis was associated with a rapid and significant loss of HuC/D immunoreactive myenteric plexus neuronal cell bodies (42% decrease relative to control) that remained unchanged between 6 and 120 h. No change in myenteric plexus density was observed with PGP 9.5 immunostaining. Neuronal apoptosis was evident between 0.5 and 3 h. PARP immunoreactive neurons ranged between 1% and 2.5%. Colitis was associated with significant impairment in colonic propulsive function. Pre-treatment of mice with anti-neutrophil serum attenuated the inflammatory response and partially reduced the extent of myenteric plexus neuronal cell loss. Taken together, these data suggest that acute colitis is associated with loss of myenteric plexus neurons that is partly mediated by neutrophil granulocyte infiltration and is accompanied by impairment of colonic motility.

Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication.