Soy diet induces intestinal inflammation in adult Zebrafish: Role of OTX and P53 family.

Inflammatory bowel diseases (IBDs) are a group of inflammatory conditions of the colon and small intestine, including Crohn's disease and ulcerative colitis. Since Danio rerio is a promising animal model to study gut function, we developed a soy-dependent model of intestinal inflammation in adult zebrafish. The soya bean meal diet was given for 4 weeks and induced an inflammatory process, as demonstrated by morphological changes together with an increased percentage of neutrophils infiltrating the intestinal wall, which developed between the second and fourth week of treatment. Pro-inflammatory genes such as interleukin-1beta, interleukin-8 and tumour necrosis factor alpha were upregulated in the second week and anti-inflammatory genes such as transforming growth factor beta and interleukin-10. Interestingly, an additional expression peak was found for interleukin-8 at the fourth week. Neuronal genes, OTX1 and OTX2, were significantly upregulated in the first two  weeks, compatible with the development of the changes in the gut wall. As for the genes of the p53 family such as p53, DNp63 and p73, a statistically significant increase was observed after two weeks of treatment compared with controls. Interestingly, DNp63 and p73 were shown an additional peak after four weeks. Our data demonstrate that soya bean meal diet negatively influences intestinal morphology and immunological function in adult zebrafish showing the features of acute inflammation. Data observed at the fourth week of treatment may suggest initiation of chronic inflammation. Adult zebrafish may represent a promising model to better understand the mechanisms of food-dependent intestinal inflammation.

Homeoprotein OTX1 and OTX2 involvement in rat myenteric neuron adaptation after DNBS-induced colitis.

BACKGROUND: Inflammatory bowel diseases are associated with remodeling of neuronal circuitries within the enteric nervous system, occurring also at sites distant from the acute site of inflammation and underlying disturbed intestinal functions. Homeoproteins orthodenticle OTX1 and OTX2 are neuronal transcription factors participating to adaptation during inflammation and underlying tumor growth both in the central nervous system and in the periphery. In this study, we evaluated OTX1 and OTX2 expression in the rat small intestine and distal colon myenteric plexus after intrarectal dinitro-benzene sulfonic (DNBS) acid-induced colitis. METHODS: OTX1 and OTX2 distribution was immunohistochemically investigated in longitudinal muscle myenteric plexus (LMMP)-whole mount preparations. mRNAs and protein levels of both OTX1 and OTX2 were evaluated by qRT-PCR and Western blotting in LMMPs. RESULTS: DNBS-treatment induced major gross morphology and histological alterations in the distal colon, while the number of myenteric neurons was significantly reduced both in the small intestine and colon. mRNA levels of the inflammatory markers, TNFα, pro-IL1ß, IL6, HIF1α and VEGFα and myeloperoxidase activity raised in both regions. In both small intestine and colon, an anti-OTX1 antibody labeled a small percentage of myenteric neurons, and prevalently enteric glial cells, as evidenced by co-staining with the glial marker S100ß. OTX2 immunoreactivity was present only in myenteric neurons and was highly co-localized with neuronal nitric oxide synthase. Both in the small intestine and distal colon, the number of OTX1- and OTX2-immunoreactive myenteric neurons significantly increased after DNBS treatment. In these conditions, OTX1 immunostaining was highly superimposable with inducible nitric oxide synthase in both regions. OTX1 and OTX2 mRNA and protein levels significantly enhanced in LMMP preparations of both regions after DNBS treatment. CONCLUSIONS: These data suggest that colitis up-regulates OTX1 and OTX2 in myenteric plexus both on site and distantly from the injury, potentially participating to inflammatory-related myenteric ganglia remodeling processes involving nitrergic transmission.

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas.

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

Shwachman-Diamond syndrome with clonal interstitial deletion of the long arm of chromosome 20 in bone marrow: haematological features, prognosis and genomic instability.

In Shwachman-Diamond syndrome (SDS), deletion of the long arm of chromosome 20, del(20)(q), often acquired in bone marrow (BM), may imply a lower risk of developing myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), due to the loss of the EIF6 gene. The genes L3MBTL1 and SGK2, also on chromosome 20, are in a cluster of imprinted genes, and their loss implies dysregulation of BM function. We report here the results of array comparative genomic hybridization (a-CGH) performed on BM DNA of six patients which confirmed the consistent loss of EIF6 gene. Interestingly, array single nucleotide polymorphisms (SNPs) showed copy neutral loss of heterozygosity for EIF6 region in cases without del(20)(q). No preferential parental origin of the deleted chromosome 20 was detected by microsatellite analysis in six SDS patients. Our patients showed a very mild haematological condition, and none evolved into BM aplasia or MDS/AML. We extend the benign prognostic significance of del(20)(q) and loss of EIF6 to the haematological features of these patients, consistently characterized by mild hypoplastic BM, no or mild neutropenia, anaemia and thrombocytopenia. Some odd results obtained in microsatellite and SNP-array analysis demonstrate a peculiar genomic instability, in an attempt to improve BM function through the acquisition of the del(20)(q).

gDNA qPCR is statistically more reliable than mRNA analysis in detecting leukemic cells to monitor CML.

Chronic Myeloid Leukemia (CML) is a stem cell cancer that arises when t(9;22) translocation occurs in a hematopoietic stem cells. This event results in the expression of the BCR-ABL1 fusion gene, which codes for a constitutively active tyrosine kinase that is responsible for the transformation of a HSC into a CML stem cell, which then gives rise to a clonal myeloproliferative disease. The introduction of Tyrosine Kinase Inhibitors (TKIs) has revolutionized the management of the disease. However, these drugs do not seem to be able to eradicate the malignancy. Indeed, discontinuation trials (STIM; TWISER; DADI) for those patients who achieved a profound molecular response showed 50% relapsing within 12 months. We performed a comparative analysis on 15 CML patients and one B-ALL patient, between the standard quantitative reverse-transcriptase PCR (qRT-PCR) and our genomic DNA patient-specific quantitative PCR assay (gDNA qPCR). Here we demonstrate that gDNA qPCR is better than standard qRT-PCR in disease monitoring after an average follow-up period of 200 days. Specifically, we statistically demonstrated that DNA negativity is more reliable than RNA negativity in indicating when TKIs therapy can be safely stopped.