High Frequency of Chromosome 14 Deletion in Early-Onset Colon Cancer

Purpose

Several genes have been recognized, when mutated in the germline, to highly predispose to colorectal cancer, impairing the DNA mismatch repair system in hereditary nonpolyposis colon cancer syndrome, or APC/MYH in adenomatous polyposis. However, 10 percent of microsatellite stable colorectal cancer is reported to develop in an unexplained context of genetic predisposition. This study was designed to depict the genetic mechanisms underlying early-onset microsatellite stable colon cancers.

Methods

Patients younger than aged 50 years undergoing primary surgical resection for colon carcinoma were collected prospectively between 1993 and 2003. A first series of 8 samples has been allelotyped using 361 poly–CA polymorphisms distributed on the 39 autosomal arms within a larger set of 166 sporadic tumors. Genotyping of 24 poly–CA polymorphisms distributed on the 8 chromosomes exhibiting allelic losses in more than 30 percent of the previous cases was then applied to an independent series of 40 tumors. A third series of 70 tumors has been genotyped on chromosome 14 only.

Results

Comparison of genomic profile from patients younger and older than aged 50 years at the 8 most frequently lost chromosomes allowed, identify chromosome 14 as showing a significant difference between the two groups. Dense chromosome 14 genotyping detected two partial deletions in a general background of 57 percent allelic loss, pointing at a region located between D14S63 and D14S292.

Conclusions

These observations suggest that a tumor-suppressor gene located on chromosome 14 might have an important role in microsatellite stable colon carcinogenesis. Because it seems to be more frequently involved in early-onset cases, it could be a good candidate in inherited conditions.

     

Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients

Proinsulin is a key autoantigen in type 1 diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8+ T cell responses to the insulin B chain and adjacent C peptide, we selected 8- to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type 1 diabetic patients produced IFN-gamma in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted peptides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-A1 and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients.     

Abnormal calcium transport into microsomes of grey platelet syndrome

Calcium uptake into isolated membrane vesicles from two patients with a grey platelet syndrome has been investigated. An increase in calcium transport appears in both patients when compared to controls. Determination of the kinetic parameters of the calcium transport system gave similar apparent affinity for calcium and an increase in the calcium uptake velocity. This increase in calcium transport is correlated with the increase of the associated Ca2+ activated ATPase activity. The results would suggest a new relationship between the ultrastructural and functional abnormalities of the grey platelet syndrome.     

Diminution of Circulating CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T Cells in Na?ve Crohn’s Disease

Crohn’s disease is considered to be caused either by an excess of T-cell effector functions and/or by a defective regulatory T-cell compartment. The aim of this study was to assess in Crohn’s disease the frequency of circulating CD4⁺CD25^high T cells that possess regulatory T-cell functions and CD4⁺CD25^low T cells that contain activated T cells. Flow cytometry of peripheral blood was used to assess CD4⁺CD25^high and CD4⁺CD25^low T-cell frequencies in a cohort of 66 patients with Crohn’s disease in comparison to 19 patients with ulcerative colitis and 31 healthy individuals enrolled as controls. The CD4⁺CD25^high T-cell frequency was significantly lowered in naïve Crohn’s disease (P = 0.013) and in ulcerative colitis (P = 0.001). CD4⁺CD25^low T-cell frequency was increased in Crohn’s disease (P = 0.0001) and in ulcerative colitis (P = 0.0002). Both CD4⁺CD25^high and CD4⁺CD25^low T-cell frequencies are altered in naïve Crohn’s disease resulting in an imbalance between both populations and a relative contraction of the CD4⁺CD25^high T-cell population.     

ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function

Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component β-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.     

Applying peptide antigens onto bare skin: induction of humoral and cellular immune responses and potential for vaccination.

The development of non-invasive immunisation procedures is a top priority for public health agencies when it is realised that the current immunisation practices are unsafe, particularly in developing countries due to the widespread reuse of non-sterile syringes. There is a risk of abscess formation resulting in impairment of meat quality or the value of the hide, and the risk of transmission of infectious diseases when vaccines are administered to food animals by injection. Recently, the skin has emerged as an alternative route for non-invasive delivery of vaccines. Topical application of various types of antigens (mainly proteins and toxoids) with an adjuvant resulted in the induction of systemic and mucosal immune responses. However, due to skin barrier constraints and the physicochemical properties of large molecular weight proteins, the immune responses are variable and require further optimisation. Small molecular size synthetic peptides when applied onto bare skin with an adjuvant are effective immunogens, inducing both humoral and cellular immune responses. Their use as vaccines offers considerable advantages over conventional preparations in terms of safety, purity, stability, availability and cost. Therefore, they could be the most suitable candidate immunogens for skin immunisation. This review describes our recent observations on the immunogenicity of synthetic peptides applied onto bare skin in relation to vaccination.     

Polyethylene glycol, unique among laxatives, suppresses aberrant crypt foci, by elimination of cells

OBJECTIVE: Polyethylene glycol (PEG), an osmotic laxative, is a potent inhibitor of colon cancer in rats. In a search for the underling mechanisms, the hypothesis that fecal bulking and moisture decrease colon carcinogenesis was tested. We also investigated the PEG effects on crypt cells in vivo. MATERIAL AND METHODS: Fischer 344 rats (n=272) were injected with the colon carcinogen, azoxymethane. They were then randomized to a standard AIN76 diet containing one of 19 laxative agents (5% w/w in most cases): PEG 8000 and other PEG-like compounds, carboxymethylcellulose, polyvinylpyrrolidone, sodium polyacrylate, calcium polycarbophil, karaya gum, psyllium, mannitol, sorbitol, lactulose, propylene glycol, magnesium hydroxide, sodium phosphate, bisacodyl, docusate, and paraffin oil. Aberrant crypt foci (ACF) and fecal values were measured blindly after a 30-day treatment regimen. Proliferation, apoptosis, and the removal of cells from crypts were studied in control and PEG-fed rats using various methods, including TUNEL and fluorescein dextran labeling. RESULTS: PEG 8000 reduced the number of ACF 9-fold in rats (p<0.001). The other PEGs and magnesium hydroxide modestly suppressed ACF, but not the other laxatives. ACF number did not correlate with fecal weight or moisture. PEG doubled the apoptotic bodies per crypt (p<0.05), increased proliferation by 25-50% (p<0.05) and strikingly increased (>40-fold) a fecal marker of epitheliolysis in the gut (p<0.001). PEG normalized the percentage of fluorescein dextran labeled cells on the top of ACF (p<0.001). CONCLUSIONS: Among laxatives, only PEG afforded potent chemoprevention. PEG protection was not due to increased fecal bulking, but in all likelihood to the elimination of cells from precancerous lesions.     

Long-term remodeling of a bilayered living human skin equivalent (Apligraf®) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix

Type I collagen is a clinically approved biomaterial largely used in tissue engineering. It acts as a regenerative template in which the implanted collagen is progressively degraded and replaced by new cell-synthesized tissue. Apligraf, a bioengineered living skin, is composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the extracellular matrix components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species-specific antibodies and electron microscopy techniques. We observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, i.e., type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species-specific immunohistochemistry tools is thus very useful for studying long-term tissue remodeling of bioengineered living tissues.