Eradication of Helicobacter Pylori in Iran: A Review.

Helicobacter pylori (H. pylori) infection is one of the most common bacterial infections, affecting almost half of the world's population. It is associated with peptic ulcer disease, gastric adenocarcinoma, and lymphoma. In Iran, the prevalence of H. pylori infection has been reported to be between 36% and 90% in different geographic regions. Several studies have assessed the efficacy of different therapeutic options for firstline and second-line H. pylori eradication in Iran; however, the results are conflicting. Therefore, we conducted a review to evaluate different studies in order to select the best options and to provide recommendations for H. pylori eradication in Iran. Accordingly, we searched through PubMed to obtain relevant randomized clinical trials published in English language up to June 2017. According to our study, among first-line eradication regimens, bismuth-based furazolidone- or clarithromycin-containing quadruple therapies, hybrid regimen, and concomitant therapy seem to be appropriate options. Also, 10- or 14-day clarithromycin-containing triple therapy can be used if local H. pylori resistance to clarithromycin is known to be less than 15%. For second-line H. pylori eradication, bismuth-based quadruple therapies and 14-day levofloxacin-based triple therapy can be used, provided that antibiotics other than those used in the first-line regimen are used. Third-line H. pylori eradication regimens have not been addressed in Iranian studies. However, most guidelines recommend treatment according to the results of culture and susceptibility testing. Although we limited our investigation to H. pylori eradication regimens in Iran, the results are transferrable to any region as long as the patterns of antibiotic resistance are the same.

The trend of national and sub-national burden of gastrointestinal and liver diseases in Iran 1990 to 2013; study protocol.

BACKGROUND: It is expected that gastrointestinal (GI) and liver diseases inflict considerable burden on health systems in Iran; therefore, highlighting the significance of GI disorders across the other most burdensome diseases requires comprehensive assessment and regular updates of the statistics of such diseases in Iran. OBJECTIVE: To assess in-depth sub-national estimates and trends for the incidence and prevalence of selected GI and liver diseases by age, gender and province over the period 1990 - 2013 in Iran. METHODS: This is a national and sub-national burden of disease study on 21 GI diseases using all available data sources, including cancer registry, death registration system data, hospital data, and all available published data. Analyses will be performed separately by gender, age groups, year, and province. We will conduct 21 separated systematic reviews of the literature for 21 diseases categories through searching online international electronic databases (i.e. the Medline database of the National Library of Medicine, Web of Science, and Scopus), Iranian search engines (i.e., IranMedex, Scientific Information Database (SID), and IRANDOC), and gray literature. We will search the medical literature published between January 1985 and December 2013. We generated two models, Spatio-temporal and Multilevel Autoregressive models, to estimate mean and uncertainty interval for the parameters of interest by gender, age, year, and province. The models will be informed by data of gender, age, year, and province. Markov Chain Monte Carlo (MCMC) methods will be used to perform Bayesian inference in both modeling framework. All programs will be written in R statistical packages (version 3.0.1). RESULTS: We will calculate and present 1990 to 2013 trends in terms of prevalence, years of life lost due to premature mortality (YLLs), years lived with disability (YLDs), and disability-adjusted life years DALYs for the 21 selected GI diseases by gender, and province. We will also quantify the uncertainty interval for the estimates of interest. CONCLUSION: Results of the present study will have implications for policy making; as they allow for understanding geographic distributions of the selected GI diseases, and identifying health disparities across provinces.

Correlation of quality of life with gastroesophageal reflux disease amongst Qashqai nomads in Iran.

BACKGROUND: Gastroesophageal reflux disease (GERD) is one of the most common gastrointestinal diseases encountered in today's clinic practice. GERD symptoms are troublesome and disruptive to the physical, social and emotional well-being of many patients. This is a cross-sectional study performed on Qashqai nomads of Fars Province in southern Iran to determine the impact of GERD on quality of life. METHODS: We randomly selected 748 subjects over the age of 25 years. Subjects completed two questionnaires conducted by interviews. The first one consisted of questions on gastroesophageal reflux symptoms. The second questionnaire was the Short Form Health Survey (SF-36), a generic health-related quality of life instrument that consists of 36 items divided into eight dimensions. It has a 0- to 100-point scale where higher scores show better functioning and well-being. RESULTS: Of 748 Qashqai migrating nomads who participated in the study, 717 subjects) mean age: 43 ± 14.2 years) completed the GERD questionnaire and only 372 subjects completed the SF-36 questionnaire due to their busy lifestyles, and GERD was reported in 106 subjects (28.5%). For all dimensions of SF-36, the mean score was consistently lower in patients with GERD compared to non-GERD subjects (P < 0.001). The dimension most frequently impaired was role-physical (40.9 vs. 77.3) followed by role-emotional (44.7 vs. 77.5), physical functioning (66.9 vs. 84.6), and general health (46.8 vs. 63.8). An association existed between impairment in quality of life and frequency (P < 0.05), but not severity, of GERD symptoms.  CONCLUSION: In this group of Qashqai nomads, all dimensions of health-related quality of life as measured by the SF-36 questionnaire were meaningfully impaired in subjects with symptomatic GERD compared to non-GERD subjects. There was an association between impaired quality of life and frequency, but not severity, of GERD.

Construction and preparation of three recombinant adenoviruses expressing truncated NS3 and core genes of hepatitis C virus for vaccine purposes.

BACKGROUND: In spite of dozens of clinical trials to establish effective therapeutic and/or preventive vaccine to resolve HCV infection, no real vaccine has been proved to date. Genetic vaccines based on replication-defective adenoviruses have proved to elicit strong and long lasting T-cell responses against a number of viral antigens and are even currently being used for vaccine trials in humans. According to the controversy in the immune modulatory effects of both core and NS3 full length genes, it seemed more practical to employ some parts of these HCV proteins for vaccine design. OBJECTIVES: To generate recombinant Adenoviral vectors containing new overlapping-truncated region of NS3 gene or both the N- and C-terminal deleted parts of core gene, as well as a fusion fragment derived from both of them. MATERIALS AND METHODS: The corresponding transfer vectors expressing truncated fragments of core, NS3 or a fusion fragment of both genes were prepared. The integrity and sequence of the transfer vectors were confirmed, and followed by experiments involving homologous recombination between them and the adenovirus backbone plasmid in the bacterial host. Recombinant Ad-pNS3, Ad-pCore and Ad-pNS3pCore viruses were prepared by transfection of these new recombined constructs into 293 packaging cell lines. The virus titer was then calculated by an immunohistochemistry based method. The RT-PCR, Real-Time PCR and western blotting were used to evaluate gene expression by all recombinant constructs. The production of complete virion particles was evaluated by detailed electron microscopy in addition to the appearance of typical cytopathic effects (CPE) and GFP expression patterns in 293 cells. The RT-PCR and GFP detection were employed to monitor the integrity as well as infectivity potency of the viral particles in Hep-G2 cells. RESULTS: RT-PCR, Real-Time PCR or western blotting confirmed expression of truncated fragment of NS3, core or a fusion fragment of theirs by newly constructed Ad-pNS3, Ad-pCore, Ad- pNS3pCore particles. Electron microscopy, which revealed many adenovirus-like particles and characteristics of CPE in infected cells in addition to GFP detection, confirmed the infectivity, potency and integrity of recombinant adenoviral particles. CONCLUSIONS: These adenoviruses expressing novel fragments of NS3 and core genes may be suitable tools to overcome shortcomings associated with full gene expression in the setting of HCV vaccine therapy.

Irritable bowel syndrome in adults over 35 years in Shiraz, southern Iran: prevalence and associated factors.

BACKGROUND: The symptoms of irritable bowel syndrome (IBS) are common in the general population. The aim of this population-based study was to determine the prevalence of IBS and describe the associated factors including demographic, life style and health-seeking behaviors in Shiraz city, southern Iran. METHODS: From April to September 2004, 1978 subjects aged > 35 years old completed a validated and reliable questionnaire on IBS. RESULTS: The prevalence rate of IBS was 10.9%, higher in females, in 35-44 years old age group and among subjects eating fast food (14.1%) but was lower in those taking more fruits and vegetables (10.5%). The occurrence of anxiety, nightmare and restlessness was also significantly higher in subjects with IBS. It had an association with psychological distress and recurrent headaches but not with drinking tea/coffee, smoking or physical activity. CONCLUSIONS: In our area, IBS was correlated with gender, age, psychological distress, recurrent headaches and consumption of fast foods that necessitate health planning programs by health policy makers.

Distinct high-profile methylated genes in colorectal cancer.

BACKGROUND: Mutations and promoters' methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes' promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining. METHODOLOGY/PRINCIPAL FINDINGS: The promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed. Seventy-seven and 34% of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was >65% for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was <50% for LGR6, RET, CD109, and RNF. The difference in methylation between the two populations was statistically significant for CHD5, ICAM5 and GPNMB. Thirty-one percent AA tumors showed MSI-H, compared to 28% in Iranians. CONCLUSIONS/SIGNIFICANCE: A significantly higher methylation rate was found for GPNMB, ICAM5, and CHD5 genes in AA patients compared to Iranians. These genes might play a role in the high incidence and aggressiveness of CRC in the AA population. The hypermethylation of the CAN genes can be considered as a marker of colon carcinogenesis.

Development of gastroenterology and hepatology in Iran: part I-training programs and the Iranian association of gastroenterology and hepatology.

During the first half of the 20th century, all subspecialties of internal medicine were managed by general internists and there was no gastroenterologist in Iran.1 The first group of Iranian gastroenterologists who had completed their training program in France started working in Tehran in 1951. Later on, other specialists trained in USA and the United Kingdom joined them. Some of these specialists and some internists with special interest in gastroenterology and hepatology were in charge of gastroenterology education in divisions of internal medicine in the Medical Schools of Tehran, Shiraz, Isfahan, and other universities, while most of them were also practicing gastroenterology in their private offices on a part-time basis. The establishment of subspecialty training program in gastroenterology and hepatology was firstly started in 1976 in Tehran University and later on and since 1984 in Shaheed Beheshti, and Shiraz Universities. This was a major step in the progress of this subspecialty, which is now one of the most active medical disciplines in Iran.2 In this paper, we aimed to describe the development and evolution of gastroenterology and hepatology in Iran during the last 60 years with emphasis on physicians and scientists, who played a major role in providing the first gastroenterology services in the country or had a major role in training, education, and research in this field. We also describe the attempts that led to the establishment of the Iranian Association of Gastroenterology and Hepatology, as the most important nongovernmental organization in this field.

Prevalence and risk factors of gastroesophageal reflux disease in Qashqai migrating nomads, southern Iran.

AIM: To investigate the prevalence and risk factors of gastroesophageal reflux disease (GERD) symptoms in Qashqai migrating nomads with a different life style in Fars province, southern Iran. METHODS: In summer 2006, 748 Qashqai migrating nomads aged 25 years or more were enrolled using a multiple-stage stratified cluster random sampling method. A questionnaire consisting of demographic characteristics, lifestyle and GERD symptoms (heartburn, regurgitation, chest pain, dysphagia, hoarseness and cough) as completed for each subject. RESULTS: The questionnaire was completed in 717 subjects. The prevalence rate of GERD, defined as reflux occurring at least one time per week in the preceding year, was 33% (237 subjects). The prevalence was higher in older individuals (36.0% vs 28.9%, P < 0.05) and in those with other gastrointestinal complaints (51.0% vs 27.8%, P < 0.001), but not different in obese and non-obese subjects. It was also higher in those consuming fruits and vegetables more than once a week (36.2% vs 17.3%, P < 0.001). GERD had a positive correlation with smoking (42.1% vs 27.8%, P < 0.001), but a negative relation with non-alcoholic beverages. The association between GERD and non-steroidal anti-inflammatory drugs (NSAIDs) consumption was also significant (40.2% vs 25.4%, P < 0.001). CONCLUSION: The prevalence of GERD (33%) is very high in Qashqai migrating nomads which may be due to a lower socioeconomic and educational level of these people and difference in the life style. Older age, frequent consumption of fruits and vegetables, smoking and NSAIDs are risk factors for GERD in this population.

Halothane hepatitis in Iran: a review of 59 cases.

AIM: To study halothane hepatitis (HH) in Iran and its associated risk factors. METHODS: We retrospectively studied files of all cases diagnosed with HH referred to three referral hospitals and four private centers in Iran from April 1994 to September 2006. Information on age at surgery, gender, medications history, obesity, history of previous exposure, previous reaction to halothane, familial history, type of surgery, perioperative hypoxia or sepsis, morbidity and mortality were recorded and analyzed. RESULTS: A total of 59 cases were identified. Forty-eight (81%) were women. The median age at the time of surgery was 44 years (range, 18 to 80 years). Sixty percent of patients were above 40-year-old. Obesity was observed in 22.2%. Previous history of exposures to halothane was noted in 61% of which 50% had history of post-exposure reaction. Coronary artery bypass graft (CABG), cholecystectomy, and cosmetic surgeries (mainly weight reduction) were the most frequent surgeries. The mortality rate was 12.2%. In patients developing encephalopathy, it was as high as 50%. CONCLUSION: HH remains an important cause of morbidity and mortality in centers still using this anesthetic. However, a large percentage of these cases could have been avoided. To lessen occurrence of further cases of HH, the authors suggest that in female patients having a history of surgery (or delivery) with general anesthesia, the use of halothane should be absolutely avoided. Utilization of proper substitutes in adults' anesthesia is advocated.

Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study.

We have identified an alternative pathway of tumorigenesis in sporadic colon cancer, involving microsatellite instability due to mismatched repair methylation, which may be driven by mutations in the BRAF gene (V600E). Colorectal cancer (CRC) is the most common cancer in the world, and African Americans show a higher incidence than other populations in the United States. We analyzed sporadic CRCs in Omani (of African origin, N = 61), Iranian (of Caucasian origin, N = 53) and African American (N = 95) patients for microsatellite instability, expression status of mismatched repair genes (hMLH1, hMSH2) and presence of the BRAF (V600E) mutation. In the Omani group, all tumors with BRAF mutations were located in the left side of the colon, and for African Americans, 88% 7 of tumors with BRAF mutations were found in the right side of the colon. In African Americans, 31% of tumors displayed microsatellite instability at two or more markers (MSI-H), while this rate was 26% and 13% for tumors in the Iranian and Omani groups, respectively. A majority of these MSI-H tumors were located in the proximal colon (right side) in African American and Iranian subjects, whereas most were located in the distal colon (left side) in Omani subjects. Defects in hMLH1 gene expression were found in 77% of MSI-H tumors in both African Americans and Iranians and in 38% of tumors in Omanis. BRAF mutations were observed in all subjects: 10% of tumors in African Americans (8/82), 2% of tumors in Iranians (1/53), and 19% of tumors in Omanis (11/59). Our findings suggest that CRC occurs at a younger age in Omani and Iranian patients, and these groups showed a lower occurrence of MSI-H than did African American patients. Our multivariate model suggests an important and significant role of hMLH1 expression and BRAF mutation in MSI-H CRC in these populations. The high occurrence of MSI-H tumors in African Americans may have significant implications for treatment, since patients with MSI-H lesions display a different response to chemotherapeutic agents such as 5-fluorouracil.

Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status.

AIM: To evaluate joint effects of Methylentetra-hydrofolate reductase (MTHFR) C677T genotypes, and serum folate/vitamin B(12) concentrations on promoter methylation of tumor-associated genes among Iranian colorectal cancer patients. METHODS: We examined the associations between MTHFR C677T genotype, and promoter methylation of P16, hMLH1, and hMSH2 tumor-related genes among 151 sporadic colorectal cancer patients. The promoter methylation of tumor-related genes was determined by methylation-specific PCR. Eighty six patients from whom fresh tumor samples were obtained and 81 controls were also examined for serum folate and vitamin B(12) concentrations by a commercial radioimmunoassay kit. RESULTS: We found 29.1% of cases had tumors with at least one methylated gene promoter. In case-case comparison, we did not find a significant association between methylation in tumors and any single genotype. However, in comparison to controls with the CC genotype, an increased risk of tumor methylation was associated with the CT genotype (OR = 2.5; 95% CI, 1.1-5.6). In case-case comparisons, folate/vitamin B(12) levels were positively associated with tumor methylation. Adjusted odds ratios for tumor methylation in cases with high (above median) versus low (below median) serum folate/vitamin B(12) levels were 4.9 (95% CI, 1.4-17.7), and 3.9 (95% CI, 1.1-13.9), respectively. The frequency of methylated tumors was significantly higher in high methyl donor than low methyl donor group, especially in those with MTHFR CT (P = 0.01), and CT/TT (P = 0.002) genotypes, but not in those with the CC genotype (P = 1.0). CONCLUSION: We conclude that high concentrations of serum folate/vitamin B(12) levels are associated with the risk of promoter methylation in tumor-specific genes, and this relationship is modified by MTHFR C677T genotypes.

Dietary intake of benzo(a)pyrene and risk of esophageal cancer in north of Iran.

One etiologic factor for high incidence of esophageal squamous cell carcinoma (ESCC) in Golestan (Northeastern Iran) might be exposure to polycyclic aromatic hydrocarbons. We examined whether food and water are major sources of benzo(a)pyrene (BaP) exposure in this population. We used a dietary questionnaire to assess the daily intake of staple food (rice and bread) and water in 3 groups: 40 ESCC Golestan cases, 40 healthy subjects from the same area, and 40 healthy subjects from a low-risk area in Southern Iran. We measured, by high-performance liquid chromatography combined with fluorescence detection, the BaP concentration of bread, rice, and water in samples obtained from these 3 groups and calculated the daily intake of BaP. Mean BaP concentration of staple foods and water was similar and within standard levels in both areas, but the daily intake of BaP was higher in controls from the high-risk area than in controls from the low-risk area (91.4 vs. 70.6 ng/day, P < 0.01). In the multivariate regression analysis, having ESCC had no independent effect on BaP, whereas residence in the low-risk area was associated with a significant decrease in total BaP intake. Polycyclic aromatic hydrocarbons might, along with other risk factors, contribute to the high risk of ESCC in Golestan.

Risk factors of gastroesophageal reflux disease in Shiraz, southern Iran.

AIM: To determine the prevalence and symptoms of gastroesophageal reflux disease (GERD) in a healthy general population in relation to demographic, lifestyle and health-seeking behaviors in Shiraz, southern Iran. METHODS: A total of 1978 subjects aged > 35 years who referred to Gastroenterohepatology Research Center and who completed a questionnaire consisting of 27 questions for GERD in relation to demographic, lifestyle and health-seeking behaviors were included in this study for a period of five months. The validity and reliability of the questionnaire were determined. RESULTS: The prevalence of GERD was 15.4%, which was higher in females (17.3%), in rural areas (19.8%), and in illiterate subjects (21.5%) and those with a mean age of 50.25 years. The prevalence was significantly lower in subjects having fried food (14.8%), and fruit and vegetables (14.6%). More symptoms were noticed in subjects consuming pickles (22.1%), taking aspirin (21%) and in subjects with psychological distresses (27.2%) and headaches (22%). The correlation was statistically significant between GERD and halitosis (18.3%), dyspepsia (30.6%), anxiety (19.5%), nightmares (23.9%) and restlessness (18.5%). Their health seeking behavior showed that there was a significant restriction of diet (20%), consumption of herbal medicine (19%), using over-the-counter drugs (29.9%) and consulting with physicians (24.8%). Presence of GERD symptoms was also significantly related to a previous family history of the disease (22.3%). CONCLUSION: GERD is more common in females, rural and illiterate subjects and correlated with consumption of pickles, occurrence of headache, psychological distress, dyspepsia, halitosis, anxiety, nightmare and restlessness, and a family history of GERD and aspirin intake, but the correlation was negative with consumption of fat and fiber intake.

Correlation of gastroesophageal reflux disease with positive family history and headache in Shiraz city, southern Iran.

BACKGROUND/AIMS: To analyze the potential correlation of a positive family history of gastroesophageal reflux disease (GERD) and the history of headaches as a risk factor for and complication of the disease. MATERIALS AND METHODS: Three thousand and six hundred subjects were selected by cluster random sampling from all seven districts of Shiraz city, who were invited for interview. In five months, 1956 subjects participated in this study. A questionnaire organized into three sections of demographic, signs and symptoms of GERD, headache and family history of GERD was completed for each patient. Social and demographic variables were also recorded. RESULTS: The presence of GERD symptoms (72%) had a significant correlation with a positive family history of the disease ( P = 0.000). Patients showed a variable frequency of headache, ranging from once daily (16.7%), three to five times a week (5.6%), once-twice a week (26.7%), once to three times a month (15.0%) and less than once a month (8.3%). There was a significant correlation between the headaches and the GERD symptoms ( P = 0.000). CONCLUSION: A positive family history of GERD can be considered as a risk factor for the disease and the presence of headache at the time of diagnosis as a complication of this disease. Therefore, in the management of GERD, attention should be given to these factors.

Analysis of cytotoxic T lymphocyte associated antigen 4 gene polymorphisms in patients with ulcerative colitis.

BACKGROUND AND AIM: Ulcerative colitis (UC) is a multifactorial disease associated with dysregulated immunity. Recently, cytotoxic T lymphocyte associated antigen 4 (CTLA-4) gene polymorphisms have been reported in association with several autoimmune diseases in several populations. In the present study, the possible implication of the CTLA-4 gene as a risk factor for UC in the Iranian population was investigated. METHODS: One hundred UC patients and 100 healthy subjects were studied. CTLA-4 exon 1 position 49 (A/G: codon 17: Thr/Ala) polymorphisms were investigated by polymerase chain reaction single strand confirmation polymorphism method. Four of the patients and one of the healthy controls were excluded from the study because of incomplete DNA extraction. RESULTS: The allele frequencies of A and G in 96 patients (A: 66.1%; G: 33.9%) were not significantly different from the 99 control subjects (A: 63.1%; G: 36.9%, P > 0.05). No significant differences in the distribution of genotype frequencies were observed between A + 49G gene polymorphisms and UC in the Iranian population (P > 0.05). CONCLUSION: CTLA-4 polymorphism is not associated with UC in the Iranian population.

Effects of psycho-educational intervention on health-related quality of life (QOL) of patients with chronic liver disease referring to Shiraz University of Medical Sciences.

BACKGROUND: Chronic liver diseases (CLDs) are progressive disorder which has a significant impact on the well-being of patients and leads to significant morbidity. CLDs are characterized by disturbances in physical, psychological and social aspects of well-being. It causes significant health-related quality of life (QOL) impairment. Psycho-educational interventions targeting to functional factors could be beneficial for patients with CLDs. METHODS: An interventional study was conducted on 110 patients with CLDs in Shiraz Liver Transplantation Center (SLTC). Subjects with the required CLDs criteria were selected and randomly divided into experimental (55) and control (55) groups. A two part questionnaire with 25 items concerning demographic and general information and 29 items regarding QOL was used. The psycho-educational needs of the experimental group were assessed in a session before the intervention, then the experimental group took part in 3 sessions individually and one session in groups. The questionnaires were filled in again for both groups but the control group did not receive the intervention program. The questionnaires were filled in again for both groups one day and three months after the intervention. RESULTS: Findings revealed no significant differences between the two groups from the view point of demographic characteristics such as marital status, gender, etc... (p > 0.05) and from the point of clinical variables no statistically significant differences were found (p > 0.05). Findings revealed statistically significant differences in all domain of QOL in the experimental group three months after the intervention (p = 0.001), while there was no statistically significant differences in the control group. CONCLUSION: The results of this study revealed that psycho-educational intervention had a significant effect on QOL of patients with CLDs.

Effective production of carcinoembryonic antigen by conversion of the membrane-bound into a recombinant secretory protein by site-specific mutagenesis.

Carcinoembryonic antigen (CEA), the most widely used human tumor marker, is a heavily glycosylated protein over-expressed by a wide range of tumors. It has been indicated that CEA might be a useful target for human anti-tumor immunotherapy. CEA assay for research as well as clinical trials demands a continuous source of CEA protein preparations. In a multi-purpose research program to provide a reliable source for large production of CEA, we converted the membrane-bound carcinoembryonic antigen into a secretory protein by site-specific mutagenesis. We made the secretory CEA protein by introducing a new stop codon at 99 bp upstream of the original stop codon in CEA cDNA by PCR-based mutagenesis. The glycosylation of recombinant CEA proteins, especially those destined for administration to human trials is crucially important. To produce CEA with the same glycosylation pattern and immunogenicity as the native CEA expressed by human tumors in vivo, the truncated CEA cDNA which does not encode the last C-terminal 33-amino acid hydrophobic domain was transfected into HT29, a human colon carcinoma cell line by the calcium phosphate method. Stable transfectants were selected and pooled. CEA secretion from the cells was verified by analysis of the transfectant culture supernatant for CEA protein. As determined by ELISA, 16 microg/L of recombinant CEA was secreted per 106 transfectants within 48 hrs, an increase over 40 times relative to the untransfected cells. The size of the recombinant CEA secreted by HT29 transfectants in our experiment is identical to that of reference CEA secreted from tumors and is fully antigenic. It seems that the C-terminal truncation does not affect CEA glycosylation in HT29 cells. It is predicted that human cancer immunotherapy using recombinant CEA expressed in this system would be more effective than the commercial protein which is usually prepared from bacterial or other heterologous expression systems.