Markers of Inflammation and Lineage on Exfoliated Colonic Cells In Pediatric Inflammatory Bowel Disease.

OBJECTIVES: The diagnosis (endoscopy, and biopsy) and continued clinical management of Inflammatory Bowel Disease (IBD), remain highly invasive, expensive, and inconvenient for the pediatric patient. The objective of this study was to see if colonocytes obtained from stools of subjects with IBD and normal controls would demonstrate higher levels of inflammatory markers (Cox 2 in CD45+ and CD45- cells) and if the inflammatory process and treatment effects would be reflected in an altered cytokine expression in the subjects compared to controls. SETTING: Outpatient hospital based pediatric gastroenterology clinic. METHODS AND MAIN OUTCOME MEASURES: Stool samples (~ 1 gm), were obtained from 18 children between the ages of 4 and 18 diagnosed with IBD, and from a normal first degree relative. Colonocytes were isolated using the Somatic Cell Sampling Recovery (SCSR) system and assessed for the expression of COX-2, CD-45, IgA, IgG, IL6, IL18, TGF ß, TNF, and IL16ß using flow cytometry. In addition, levels of COX-2 and cytokeratin 19 transcripts were measured by microwell plate hybridization assay. RESULTS: Expression of COX-2 and co-expression of IgA and IgG were significantly higher in the IBD cases compared to the controls. In ulcerative colitis, the expression of COX-2 and co-expression of COX-2 and CD45 were greater than that in patients with Crohn's disease. In contrast, cells expressing IgA and IgG were higher in Crohn's. Subjects on immunosuppressants and/or anti-inflammatory medications, expressed significantly lower levels of COX-2 and IL-18 compared to those who were not on treatment. CONCLUSIONS: This study indicates that the use of disease markers on exfoliated colonic cells can be used for non-invasive assessment of disease status, for follow-up of response to treatment and for forecasting flare-up of disease before its symptomatic manifestations.

Quasispecies of genotype 4 of hepatitis C virus genomes in Saudi patients managed with interferon alfa and ribavirin therapy.

BACKGROUND AND OBJECTIVES: Many patients with hepatitis C virus (HCV) infection do not respond to antiviral treatment, possibly due to viral quasispecies. We aimed to investigate whether the quasispecies population could be used as a predictor of response to therapy in our patients. METHODS: The quasispecies of HCV genotype 4 (HCV-4) were studied in 25 naïve Saudi patients at zero, three, and six months following interferon alfa and ribavirin combination therapy. Hypervariable region 1 within the E2/NS1 gene of the virus was analyzed by the single-strand conformation polymorphism (SSCP) technique after amplification. RESULTS: Pretreatment DNA bands by SSCP (2-7 bands) were detected in all patients. In those who achieved a complete virological response within six months (viral load P=.53). Two of the four patients with pretreatment high viral load and the same or decreased composition of quasispecies bands responded to the therapy. CONCLUSION: Quasispecies in our studied patients cannot be used to predict responsiveness to treatment, but may offer an explanation for failure of most HCV-4 patients to respond to interferon alfa and ribavirin therapy.

Human papilloma virus-16/18 cervical infection among women attending a family medical clinic in Riyadh.

BACKGROUND: Prevalence information is lacking on human papillomavirus types 16 and 18 (HPV-16/18) infection in cervical tissues of women residing in Riyadh, Saudi Arabia. In addition, there are no observations on progression to cervical intraepithelial neoplasia (CIN). SUBJECTS AND METHODS: Pap smear and HPV-16/18 DNA detection by PCR followed by Southern blotting was performed on 120 subjects (Saudi and other Arab nationals) during routine gynecological examination. Some HPV-positive subjects were followed for 4 years, by Pap smear every 6 months and by HPV DNA detection at the end of 4 years. RESULTS: Overall HPV-16/18 prevalence was 31.6%. HPV-16 prevalence alone was 13.3%, HPV-16 as a mixed infection with HPV-18 was 15%, and all HPV-16 was 28.3%. HPV-18 alone was 3.3%, HPV-18 as a mixed infection with HPV-16 was 15%, and all HPV-18 was 18.3%. Ten subjects had cervical abnormalities with the Pap smear test, six of whom were HPV-16/18 positive, 1 with HPV-16, 1 with HPV-18, and 4 with a mixed infection of HPV-16/18. Of all 23 HPV-16/18-positive subjects, either as individual or mixed infection, followed for 4 years, 7 showed abnormal cytology, 6 at initial examination and 1 during follow-up. Of these 7, 6 reverted to normal without treatment and 1 was treated and became normal after 3 years. None of the subjects progressed to CIN-III. CONCLUSION: A high prevalence of HPV-16/18 was found, but with a low rate of progression to CIN. A significant association with abnormal cytology was found only in patients with HPV-16/18 mixed infection.

Molecular epidemiology and genotyping of TT virus isolated from Saudi blood donors and hepatitis patients.

BACKGROUND: In Saudi Arabia, the epidemiology and clinical significance of Torque Teno virus (TTV) infection alone and in patients with hepatitis virus infections have not been determined in a single study. In this paper, we molecularly investigated the rate and genotypes of TTV infection among Saudi Arabian blood donors and patients with viral hepatitis. The effect of TTV coinfection on viral hepatitis was also examined. SUBJECTS AND METHODS: DNA was extracted from the sera of 200 healthy blood volunteers, 45 hepatitis B virus patients, 100 hepatitis C virus patients, 19 hepatitis G virus patients, and 56 non-A-G hepatitis patients. TTV DNA was amplified using primers derived from the ORF1 and 5'UTR regions. The alanine aminotransferase (ALT) level was determined for each specimen. Sequencing of ORF1 amplicons was carried out to investigate TTV genotypes. RESULTS: Using primers derived from ORF1 and 5'UTR, TTV DNA was detected in 5.5% and 50.5%, respectively, of healthy blood donors, in 2.2% and 88.8% in hepatitis B patients, in 2.0% and 70% of hepatitis C patients, in 15.8% and 100% of hepatitis G patients, in 5.4% and 12.5% of non-A-G hepatitis patients and in 4.8% and 56.4% overall. No detrimental effect of TTV coinfection in viral hepatitis patients was noted. An overall prevalence of 4.8% and 56.4% was established. Phylogenetic analysis indicated that the most common genotype of TTV among Saudis is 2c. CONCLUSION: The rate of TTV infection among Saudi Arabians seems to be lower than that stated in previous reports on Saudi Arabia and in some other countries. The virus does not seem to worsen the status of those who are suffering from viral hepatitis infection.

Exfoliated colonic epithelial cells: surrogate targets for evaluation of bioactive food components in cancer prevention.

There is significant evidence supporting the hypotheses that lifestyle, diet, and bioactive components in foods are important modifiers of cancer risk. However, our ability to assess host response noninvasively is limited. To overcome this, we have developed a technology to isolate several million viable exfoliated somatic colonic cells from a small sample of stool (0.5-1.0 g) by a procedure known as somatic cell sampling and recovery (SCSR). Orally administered carotenoids appear in these cells several days after consuming the supplement, usually showing a peak concentration between 5-7 d after their ingestion. The time lag observed for the appearance of orally administered carotenoids in SCSR cells corresponds to the turnover rate of the colonic mucosa. This is an example of how changes in cell turnover rates can be carefully assessed using the SCSR system. The specific mechanisms by which individual constituents of diet affect the cancer process are not fully understood. However, host response to dietary constituents may be investigated, noninvasively, by following the modulation of tumor-associated molecular markers in these exfoliated SCSR cells. We have demonstrated the feasibility of using SCSR cells to detect the expression of carcinoembryonic antigen, CD44, and its splice variants, c-myc, c-erbb2, and mutations in the p53 gene. In this regard, SCSR cells are a readily available surrogate cellular target that may serve to monitor changes in cell turnover, differentiation, and expression of cancer-associated biomarkers that are likely to be modulated by bioactive food components.

Gene therapy of anaplastic thyroid carcinoma with a single-chain interleukin-12 fusion protein.

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancy with a mean survival time of less than 8 months. No effective therapeutic approach is currently available, making the development of novel treatments necessary. Interleukin (IL)-12 is a proinflammatory heterodimeric cytokine with strong antitumor activity. In the present study, we investigated the potential of IL-12 gene therapy for anaplastic thyroid carcinoma in BALB/c (nu/nu) nude mice. A single-chain IL-12 fusion protein construct was created to assure equal expression of its p35 and p40 subunits. Human anaplastic thyroid carcinoma cell line ARO was stably transfected with an IL-12 expression plasmid under the control of cytomegalovirus (CMV) promoter (scIL-12/CMVpDNA). High levels of functional IL-12 (26.78 +/- 4.11 ng/ml per 10(6) cells per 48 hr) were produced by scIL-12-transfected ARO cells (ARO/IL-12). Tumorigenicity in nude mice was completely lost in scIL-12-transfected ARO cells, as demonstrated by the lack of tumor formation after subcutaneous injection of 2 x 10(6) ARO/IL-12 cells, even though there was no difference in cell proliferation between ARO and ARO/IL-12 cells. Tumor growth was observed after challenge with ARO tumor cells, indicating that protective immunity had not developed against the parental cells. Furthermore, the growth rate of established subcutaneous ARO tumors was significantly reduced by either subcutaneous injection of 2 x 10(6) ARO/IL-12 cells weekly or intramuscular injection of 50 microg scIL-12/CMVpDNA twice weekly. The antineoplastic activity of ARO/IL-12 cells was, however, abrogated by intraperitoneal injection of anti-natural killer (NK) cell antibody. Moreover, significantly higher number of ARO/IL-12 cells and ARO cells were killed by splenocytes from nude mice previously treated with ARO/IL-12 compared to those treated with ARO cells (32% vs. 9% when ARO were used as target cells, 43% vs. 17% when ARO/IL12 were used as target cells; p < 0.01) in an in vitro cytotoxicity assay. Again, tumor cell killing was neutralized by the addition of anti-NK cell antibody in the assay. In conclusion, we have demonstrated successful gene therapy with a scIL-12 fusion protein against anaplastic thyroid carcinoma in an in vivo model. The immune response against ARO/IL-12 cells is mediated by NK cells. These results may set the stage for clinical application of IL-12 gene therapy for poorly differentiated thyroid carcinoma.

A comparative study on the application of 3 molecular methods in epidemiological typing of bacterial isolates using MRSA as a prototype.

OBJECTIVE: To evaluate and compare 3 widely used molecular techniques, namely, restriction endonuclease analysis of plasmid deoxyribonucleic acid (REAP), randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for their suitability and usefulness in the typing and fingerprinting of bacterial isolates. METHODS: Twenty-four epidemiologically unrelated methicillin-resistant Staphylococcus aureus (MRSA) isolates were used to evaluate the molecular typing methods (REAP, RAPD and PFGE). The study was conducted at the Research and Diagnostic Laboratories of King Faisal Specialist Hospital and Research Center from January 2002 through January 2003. RESULTS: Only 20.8% of all isolates studied were of the same genotypes by all 3 methods. Two major clusters of strains each representing 33% of the total number of isolates were identified by REAP analysis. Each of RAPD and PFGE however, identified one major cluster represented by 54% and 83% of the total number of isolates, all 3 typing methods, therefore, showed the clonal genetic relatedness among distant MRSA isolates. However inter-strain comparison of fingerprint data generated from each method revealed differences in clonal representation of the MRSA isolates. CONCLUSION: Although a variety of molecular assays are available for typing of bacterial species, there is no single standardized protocol for routine analysis. Reproducibility and interpretation of genotypic data are therefore, highly dependent on methodologies employed by the individual laboratory. Our findings illustrate the importance of using a combination of methods in typing schemes of bacterial isolates. In terms of reproducibility and typeability we found that PFGE is superior to REAP and RAPD and, therefore, more suitable for routine, standardized tracing of nosocomial bacterial isolates.

Hepatitis C virus detection and genotyping in liver tissues and sera of Saudi patients using PCR and a line probe assay.

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