Emilia Saraga

Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease

BACKGROUND/AIMS Because Helicobacter pylori is a potentially dangerous human pathogen, the protective potential of oral immunization with H. pylori urease and its subunits was evaluated in an animal model. METHODS Mice were orally immunized with H. pylori sonicate, urease, or recombinant enzymatically inactive urease subunits and then challenged with Helicobacter felis. Control mice were sham-immunized. RESULTS H. felis colonization was present 5 days after challenge in 9 of 10 sham-immunized, 6 of 9 sonicate-immunized, and 3 of 10 urease-immunized animals (P = 0.031 vs. sham-immunized). Twelve days after challenge, urease B-immunized mice had a weaker colonization than sham-immunized controls, whereas urease A had no effect. After 70 days, most urease A- and urease B-immunized mice had cleared the colonization (10/17: P = 0.0019; 16/20: P = 0.00002 vs. sham-immunized). In urease B-immunized animals, protection was often associated with corpus gastritis. CONCLUSIONS Oral immunization with H. pylori urease protects mice against H. felis infection. Enzymatically inactive urease A and B subunits contain protective epitopes. It is unclear whether protection depends on the development of a mononuclear inflammatory response in the gastric corpus. Our observations should encourage the development of a human vaccine.

Oral immunization with Helicobacter pylori urease B subunit as a treatment against Helicobacter infection in mice

BACKGROUND & AIMS Eradication of Helicobacter pylori infections in humans results in the healing of gastritis and gastric ulcers. This study used a mouse model to test whether oral vaccination can cure Helicobacter infection and gastritis. METHODS Mice were infected with Helicobacter felis. Three weeks after infection, the mice were orally immunized with H. pylori urease B subunit. Control mice were simultaneously infected but sham immunized. RESULTS Three to 8 weeks after oral immunization of H. felis-infected mice with recombinant H. pylori urease B subunit, the infection cleared and there was no evidence of gastritis. Vaccinated mice remained protected against two consecutive H. felis challenges. CONCLUSIONS These results show that the lack of natural immunity against Helicobacter can be overcome by oral immunization and that vaccination offers a novel therapeutic approach to Helicobacter-induced gastritis.

Intratumor genetic heterogeneity in advanced human colorectal adenocarcinoma.

Colorectal carcinogenesis is widely accepted as one of the best‐characterized examples of stepwise progression. The existing colorectal carcinogenesis model assumes genetic homogeneity of individual tumors for the main known genetic alterations: K‐ras and p53 genes point mutations and loss of heterozygosity (LOH) of chromosome 5q and 18q. The object of the present study was to demonstrate the existence of an intratumor genetic heterogeneity in advanced sporadic colorectal carcinoma for these genetic alterations. Using improved tissue microdissection and DNA extraction, for each tumor, amplifiable DNA was obtained from 15 to 20 areas, of which 1 to 2 concerned lymph node metastases (LNM). This study revealed that 10 of 15 (67%) analyzed tumors were heterogeneous for at least 1 genetic alteration, with between 2 and 6 genotypically different clones detected per tumor. No correlation was observed between the genotype of these subclones and histological differentiation or invasive propensity. Intratumor heterogeneity was more frequently observed for LOH than for point mutations, 67% and 58% for LOH at APC and DCC locus, and 20% for mutation of either the K‐ras or p53 gene. In 5 of the 9 (56%) heterogeneous cases with available LNM, the genotype observed in the LNM was different from that of the main clone in the primary tumor, and moreover, 2 of the LNM displayed a genotype undetected in the primary tumor. In conclusion, intratumor genetic heterogeneity was demonstrated in advanced sporadic colorectal carcinoma and was represented as topographically distinct genotypic subclones. Taking into account such a significant genetic heterogeneity of colorectal tumors, the use of genetic markers for prognosis management should be reconsidered.

The Type of K-ras Mutation Determines Prognosis in Colorectal Cancer

BACKGROUND Mutations involving the oncogene K-ras in colorectal cancer may be related to tumor aggressiveness. However, the value of K-ras gene determination as a prognostic marker has not been clearly established. PATIENTS AND METHODS The results from 98 patients recruited in a prospective study analyzing the effect of a K-ras mutation as a prognostic factor in colorectal cancer are reported. RESULTS Disease-free (P = 0.02) and overall survival (P = 0.03) were significantly reduced for patients harboring a K-ras mutation. Two specific mutations demonstrated a significantly increased risk of disease recurrence, namely, 12-TGT (P = 0.04) and 13-GAC substitutions (P = 0.002). Patients with either of these substitutions had a 2-year disease-free survival rate of 37% compared with that of 67% for the group of patients harboring any other mutation type or a wild-type status (P = 0.01). CONCLUSIONS The results herein presented suggest that K-ras acts as a prognostic factor in colorectal cancer and that this effect is probably related to a limited number of defined mutations.

Aberrant crypt foci in patients with neoplastic and nonneoplastic colonic disease

Aberrant crypt foci (ACF) are putative preneoplastic lesions that might represent the earliest morphological lesion visible in colonic carcinogenesis. However, findings concerning the growth and morphological features of these lesions in human studies suggest that ACF are highly heterogeneous in nature. In this study, we evaluated the morphological features of a large number of ACF in colon mucosa of 26 patients with colorectal carcinoma (CRC), four patients with adenoma as well as seven patients with nonneoplastic colonic diseases. By dissecting microscope, 508 ACF were identified, and of these, 378 were sampled for histological examination. The median ACF density (number of ACF/cm2) was significantly higher in the left colon than in the right colon (0.047 v 0.014 ACF/cm2). Unexpectedly, in our series, the overall ACF density was higher in the nonneoplastic colonic diseases than in CRC (0.13 v 0.032 ACF/cm2, P=.0087), cases of nonneoplastic diseases, however, being limited to 7 patients. ACF were significantly larger in colons with CRC or adenoma than in colons with nonneoplastic disease (P < .03). On histological examination, we observed 133 ACF with normal epithelium, 189 ACF with hyperplasia, 27 ACF with atypical hyperplasia, and 29 ACF with dysplasia. We noted a progressive increase of median ACF size from normal mucosa to hyperplasia, atypical hyperplasia, and dysplasia. Dysplastic ACF were more frequently observed in patients with CRC or adenoma and showed predominantly elongated crypt orifices (P < .0001). We conclude that ACF are histologically heterogeneous, encompass a spectrum of lesions of which only a subset are associated with dysplasia and then represent an early step in colorectal carcinogenesis. ACF with dysplasia are characterized by larger size, elongated crypt orifices, and an association with CRC.

Genetic heterogeneity in sporadic colorectal adenomas

The majority of colorectal cancers develop from adenomatous polyps under the influence of factors that are still poorly understood. Tumourigenesis is generally considered a multistep process in which multiple genetic alterations occur, eventually reflected in abnormalities of the cellular DNA content. Macroscopical features such as tumour size and tumour architecture (tubular, tubulovillous, or villous) are correlated wit the chance of malignancy in the lesion. Grade of dysplasia can be considered an indicator for the level of progression of the adenoma towards invasive carcinoma. These characteristics were correlated with the presence or absence of K‐ras mutations and the DNA ploidy in a prospective study performed on 46 large sporadic colorectal adenomas resected by endoscopy. DNA ploidy and K‐ras mutations were analysed in two samples taken at distant sites in the adenomas. Aneuploidy was present in 12 adenomas (26 per cent) and K‐ras mutations occurred in 26 (57 per cent). A highly significant correlation was found between aneuploidy and adenoma size, architecture, and grade of dysplasia. The presence of K‐ras mutations was significantly correlated only with the size of the adenomas. The proportion of adenomas with aneuploidy and/or a K‐ras mutation increased when two samples were analysed instead of one. This observation suggests that the prevalence of genetic mutations and of aneuploidy is probably underestimated, as generally only one sample is investigated. No correlation was observed between K‐ras mutations and ploidy. This study demonstrates the presence of genetic heterogeneity in colorectal adenomas and supports the notion that K‐ras mutation is an early event, while aneuploidy is a late event in the adenoma–carcinoma sequence.

Fas and fas ligand expression in tumor cells and in vascular smooth-muscle cells of colonic and renal carcinomas

CD95/APO‐1 ligand (FasL) is implicated in the maintenance of immune privileged sites by inducing apoptosis of activated infiltrating T lymphocytes. Therefore, progressive tumors might express high levels of FasL and develop as immune privileged sites. In this study, we investigated the expression of FasL and CD95/APO‐1 (Fas, the FasL‐receptor) in vitro in rat adenocarcinoma cell lines and the localization in situ in normal human kidney and colon and in their adenocarcinomas. The rat cell line PROb (a progressive tumor in vivo) expressed a higher level of FasL than the sister cell line REGb (a regressive tumor in vivo), as detected by flow cytometry. The 2 cell lines expressed the same level of Fas, but were resistant to FasL‐induced apoptosis. In human tissue, both kidney and colon extracts expressed FasL by Western blot. Further investigations, using immunohistochemical staining of paraffin sections, showed that normal colon mucosa expressed Fas and FasL in crypt epithelial cells in the sub‐nuclear compartment. Normal kidney showed Fas and FasL labeling mostly restricted to epithelial cells of proximal tubules and Henlés loop, showing that this expression is not uniform throughout the organ. Smooth‐muscle cells of muscularis propria and blood vessels in and around the tumors were also intensly but more uniformly labeled. In colon‐cancer cells, FasL expression remained strong, whereas Fas expression was significantly reduced. A similar reduction in Fas expression was noted in renal‐cancer cells. Tumor‐infiltrating immune cells of the macrophage lineage do not express FasL. Our results show that smooth‐muscle cells of muscularis propria and blood vessels are able to express FasL and to a slight extent Fas. In normal epithelial cells of colon and kidney, Fas and FasL are often co‐expressed. The reduced expression of Fas in corresponding cancer cells in combination with the ability to express FasL might facilitate immune escape. Int. J. Cancer 81:772–778, 1999.

Budesonide in the Treatment of Collagenous Colitis

Collagenous colitis is characterized by watery diarrhea and inflammatory infiltration associated with a subepithelial collagen deposit on colonic biopsies despite a normal or subnormal endoscopic appearance. We here describe 5 patients treated with the locally active steroid budesonide. Complete and partial response was observed in 3 and 2 patients, respectively. Budesonide thus seems to be of therapeutic benefit in collagenous colitis. Prospective randomized long-term studies are needed to support this hypothesis.

Telomerase activation in colorectal carcinogenesis.

Telomerase activity has been detected in germ cells as well as in the developing embryo. Activity is no longer detectable in most somatic cells of the neonate, although low levels of activity persist in regenerative tissues. Telomerase has been found to be reactivated or up‐regulated in the majority of cancers. The colorectal adenoma–carcinoma sequence is one of the best‐characterized models of multistep tumourigenesis and is thus suitable for determining at which stage telomerase is activated. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in 96 cases of colorectal tissues, including 50 carcinomas, 31 adenomas, and 15 normal colonic tissues. For each case, histological diagnosis and telomerase activity were determined on consecutive frozen sections. In order to reduce the chance of a false‐negative TRAP assay due to RNA degradation, the integrity of rRNA in the tissues was verified in each case. Twenty‐five carcinomas, 30 adenomas, and all of the 15 normal colorectal mucosal samples showed no or only partial rRNA degradation and only in these cases was the TRAP assay interpreted. None of the normal tissues exhibited telomerase activity. In contrast, all of the 25 cancers and 47 per cent (14/30) of the adenomas were positive. In adenomas, telomerase activation was highly significantly related to the grade of dysplasia ( p< 0·0001). All adenomas which contained high‐grade dysplasia revealed telomerase activity, whereas telomerase activity was detectable in only 20 per cent (4/20) of cases with exclusively low‐grade dysplasia. These results indicate that telomerase activation, which may be an obligatory step in colorectal carcinogenesis, occurs in the progression from low‐grade to high‐grade dysplasia in adenomas. Furthermore, in the adenoma–carcinoma sequence, telomerase activation seems to occur later than K‐ ras mutation but earlier than p53 mutation. Copyright

Expression of telomerase genes correlates with telomerase activity in human colorectal carcinogenesis

The human telomerase enzyme is composed of two essential components, hTR, which acts as a template for reverse transcription, and hTERT, which is the putative catalytic subunit for the enzyme. Recent studies have demonstrated a good correlation between hTERT expression and telomerase activation, whereas RT‐PCR results seemed to reveal that hTR is ubiquitously expressed in all cells. These observations left unclear the role of hTR, and to a lesser extent hTERT, in the regulation of telomerase activation. In the present study, the correlation of telomerase activity and the expression of these genes was examined in a total of 70 colorectal tissues (25 adenocarcinomas, 30 adenomas, and 15 samples of normal colorectal mucosa). Total RNA for RT‐PCR analysis and cell extracts for TRAP assay were obtained from consecutive sections and histological control was simultaneously performed. To avoid false‐positive results, due to the fact that hTR cDNA and genomic hTR DNA are identical (the gene has no introns), extensive DNase digestion was performed before cDNA synthesis. RT‐PCR analysis revealed that hTERT mRNA was expressed in all cancers and in 13 of 14 telomerase‐positive adenomas, but never in telomerase‐negative colorectal tissues. hTR transcripts were observed in all telomerase‐positive samples but also in three telomerase‐negative samples, two adenomas, and one normal colonic mucosa. It is concluded that hTERT and hTR expression is strongly correlated with telomerase activity. hTR transcripts, however, also occur in some telomerase‐negative tissues and these results are in keeping with the concept that hTERT expression is a major regulator of telomerase activity. Copyright