Short-term folic acid supplementation induces variable and paradoxical changes in plasma homocyst(e)ine concentrations.

Folic acid is presently the mainstay of treatment for most subjects with elevated plasma homocyst(e)ine concentrations [Plasma or serum homocyst(e)ine, or total homocysteine, refers to the sum of the sulfhydryl amino acid homocysteine and the homocysteinyl moieties of the disulfides homocystine and homocystein-cysteine, whether free or bound to plasma proteins.] Changes in homocyst(e)ine in response to folic acid supplementation are characterized by considerable interindividual variation. The purpose of this study was to identify factors that contribute to heterogeneity in short-term responses to folic acid supplementation. The effects of folic acid supplementation (1 or 2 mg per day) for 3 wk on plasma homocyst(e)ine concentrations were assessed in 304 men and women. Overall, folic acid supplementation increased mean plasma folate 31.5 +/- 98.0 nmol/L and decreased mean plasma homocyst(e)ine concentrations 1.2 +/- 2.4 micromol/L. There was evidence of substantial interindividual variation in the homocyst(e)ine response from -18.5 to +7.1 micromol/L, including an increase in homocyst(e)ine in 20% of subjects (mean increase 1.5 +/- 1.4 micromol/L). Basal homocyst(e)ine, age, male gender, cigarette smoking, use of multivitamins, methylene tetrahydrofolate reductase, and cystathionine beta-synthase polymorphisms accounted for 47.6% of the interindividual variability in the change in homocyst(e)ine after folic acid supplementation, but about 50% of variability in response to folic acid was not explained by the variables we studied.

A proteomic approach for the discovery of early detection markers of hepatocellular carcinoma.

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post-translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to "cherry pick" or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.

Polymorphisms in the CBS gene associated with decreased risk of coronary artery disease and increased responsiveness to total homocysteine lowering by folic acid.

Elevated total plasma homocysteine (tHcy) is an established risk factor for the development of vascular disease and neural tube defects. Total homocysteine levels can be lowered by folic acid supplements but individual response is highly variable. In this case-control study, involving 142 coronary artery disease (CAD) patients and 102 controls, we have typed six genetic polymorphisms in three homocysteine metabolizing genes and examined their relationship to the incidence of CAD, tHcy levels, and lowering of tHcy levels in response to folic acid supplementation. We found that two single nucleotide polymorphisms in the cystathionine beta synthase (CBS) gene, 699C --> T and 1080T --> C, are associated with decreased risk of CAD and increased responsiveness to the tHcy lowering effects of folic acid. Individuals homozygous for 699T were significantly underrepresented in CAD patients as compared to controls (4.9% vs 17.3%, P = 0.0015), as were individuals homozygous for the 1080C (29.6% vs 44.2%, P = 0.018). Additionally, 699T and 1080C homozygous individuals were the most responsive to folate supplementation. 699T homozygotes lowered tHcy levels 13.6% on average, compared to 4.8% lowering in 699C homozygotes (P = 0.009), while 1080C homozygotes lowered 12.9% compared to just 2.7% for 1080T homozygotes (P = 0.005). The two polymorphisms in CBS are third codon changes and would not be predicted to affect the underlying protein. However, there is strong linkage disequilibrium between these two positions, suggesting that they may also be linked to other as yet unidentified polymorphisms within the CBS gene. These observations suggest that specific CBS alleles are a risk factor for the development of vascular disease and that genetic information could be predictive of individual response to folic acid supplementation.

Gender differences in genetic susceptibility for lung cancer.

In contrast to men, the incidence of lung cancer among women has increased over the past decade. The basis for this increase among female smokers remains unknown. Surgical patients with a diagnosis of lung cancer and control subjects without a history of malignancy completed a smoking questionnaire and donated a blood sample. DNA was extracted from peripheral mononuclear cells and genotyped for polymorphisms in cytochrome P450 1A1 (CYP1A1) (exon 7) and glutathione S-transferase M1 (GSTM1) (null). No gender differences in either age at diagnosis or histological subtype were observed among lung cancer patients. In both patients (n = 180) and controls (n = 163), females smoked significantly less than males. The pack-year history associated with adenocarcinoma was smaller than that for squamous cell carcinoma. No significant association was observed between the GSTM1 null genotype and cancer risk. However, women had a larger cancer risk than men (odds ratio 4.98 vs. 1.37) if they possessed the mutant CYP1A1 genotype. Female cancer patients were significantly more likely than female controls to have both the CYP1A1 mutation and GSTM1 null genotype. The combined variant genotypes conferred an odds ratio of 6.54 for lung cancer in women versus 2.36 for men, independent of age or smoking history. These data suggest that polymorphisms in CYP1A1 and GSTM1 contribute to the increased risk of females for lung cancer.

Geographic variation in viral load among hepatitis B carriers with differing risks of hepatocellular carcinoma.

The risk of hepatocellular carcinoma (HCC) varies significantly among hepatitis B virus (HBV) carriers from different geographic regions. We compared serological markers of HBV infection in adult male carriers from Haimen City, China and Senegal, West Africa, where the prevalence of chronic infection is similar. HCC mortality among HBV carriers is much higher in Haimen City than it is in Senegal (age-standardized rate, 878 versus 68 per l0(5) person-years). A dramatic difference was observed when HBV DNA levels in serum were assessed among carriers by Southern blot. In the Senegalese group (n = 289), 14.5% were HBV DNA positive by Southern blot in their 20s, and this percentage declined in each subsequent decade of age to 3.3, 2.9, and 0% thereafter. In the Chinese group (n = 285), a higher prevalence of HBV DNA positivity and a less consistent reduction were seen; 29.4% were positive in their 20s, and 30.2, 23.6, and 20.6%, respectively, were positive in each subsequent decade of age. Among 102 male Asian-American HBV carriers, the prevalence of HBV DNA positivity was intermediate between the Chinese and Senegalese populations (36.8, 10.7, 3.0, and 4.6% in each subsequent decade of age). Viral titers were similar among those who were HBV DNA positive in all three populations [median value, 10(7) virions/ml (range, 10(6)-10(9) virions/ml)]. The presence of HBV DNA in serum was positively associated with serum glutathione S-transferase, a marker of liver damage. These findings suggest that the more prolonged maintenance of productive virus infection in the Chinese carriers compared with the Senegalese carriers may explain their higher risk of HCC. This profound difference in the natural history of chronic infection may be due to earlier age of infection in China or to as yet unknown environmental or genetic factors.

Glutathione S-transferase expression in hepatitis B virus-associated human hepatocellular carcinogenesis.

Hepatitis B virus (HBV) and aflatoxin B1 represent the main risk factors for the development of hepatocellular carcinoma (HCC) in areas endemic for liver cancer. The glutathione S-transferases (GSTs) are a family of Phase II detoxification enzymes that catalyze the conjugation of a wide variety of endogenous and exogenous toxins, including aflatoxin B1, with glutathione. This study characterizes the GST isoenzyme composition (alpha, mu, and pi) of both HBV-infected normal hepatic tissues and HCCs. Analysis of matched pairs of hepatic tissue (normal and tumor) from 32 HCC patients indicated that total GST activity was significantly higher in normal tissues than in tumor tissues, although the percentage of samples expressing GST alpha and pi was equivalent. GST mu was detected by Western blot in the normal tissue from 87.5% of the subjects possessing the GST M1 gene but only 28.6% of the corresponding tumor tissues. The GST activity of normal tissue from GST M1 null patients was significantly decreased as compared to that of subjects possessing the GST M1 gene (264.6 and 422.2 nmol/min/mg, respectively; P = 0.005). GST pi appeared to be overexpressed in the normal tissue of GST M1 null patients, a potential compensatory effect. Patients positive for HBV DNA had significantly lower GST activity than those who were HBV negative (302.1 versus 450.0 nmol/min/mg, respectively; P = 0.02). These results suggest that cellular protection within the human liver is compromised by HBV infection and further decreased during hepatocellular tumorigenesis.

Somatostatin receptor subtype mRNA expression in human colorectal cancer and normal colonic mucosae.

Somatostatin analogues may be useful novel agents in the systemic treatment of advanced colorectal cancer, as somatostatin inhibits proliferation in a wide variety of cell types. Here, we report the expression profiles of somatostatin receptor mRNAs in 32 pairs of malignant and normal colonic epithelia. Receptor subtype 2 (hSSTR2) mRNA was detected throughout nearly 90% of both malignant and normal tissue by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Subtype 5 (hSSTR5) mRNA was detected in 46% and 45% of tumour and mucosal samples respectively, but in 75% (9/12) of early-stage tumours (tubulovillous adenomas, Dukes' A and B) compared with 31% (5/16) of late-stage tumours (Dukes' C and 'D' tumours), 0.05>P>0.025 (chi2 with Yates' correction). There was also reduced expression of hSSTR5 in samples of metastatic tumour (11%, 1/9) compared with all tumour samples (56%, 18/32) 0.025>P>0.01 (chi2 with Yates' correction). Other hSSTRs (1, 3 and 4) were expressed infrequently. Thus, hSSTR2 expression is retained after malignant transformation in colonic epithelium and, although it may potentially be a target for antiproliferative therapy, its ubiquitous expression militates against this. hSSTR5 warrants investigation as a tumour suppressor.

Analysis of somatostatin receptor subtype mRNA expression in human breast cancer.

Somatostatin is a widely distributed inhibitory peptide with growth-inhibitory effects in several human tumours, including breast cancer, raising the possibility that it may have therapeutic potential. The effects of somatostatin are mediated via a family of cell-surface receptors that differ in their tissue distribution, pharmacological properties and intracellular response mediators, suggesting that they mediate different functions of the peptide. We have analysed the expression of somatostatin receptor subtype (SSTR1-5) mRNA in normal and malignant breast tissue. Receptor expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR) using receptor subtype-specific primers and by in situ hybridization (ISH) with riboprobes synthesized by in vitro transcription of cloned PCR products. A total of 51 breast carcinomas, 36 samples of matched normal tissue, two axillary node metastases and eight normal/benign breast tissue samples were analysed. SSTR2 expression was ubiquitous in both normal and malignant breast tissue. Expression of SSTR5 was detected in approximately one-third of tumour and normal tissue, but fewer than 13% of all tissues expressed SSTR1, 3 and 4. These data suggest that SSTR2 gene expression is ubiquitous in breast cancer. Although this is unlikely to have diagnostic or prognostic significance, SSTR2-specific somatostatin analogues may have therapeutic potential in breast cancer.

Gestational age and cell viability determine the effect of frozen storage on human fetal hematopoietic progenitor cell preparations.

We have assessed hematopoietic progenitor cell preparations (including CD34-positive stem cells) obtained from human fetal liver at 6-17 weeks of gestational age for total cell numbers, viability and the ability to tolerate (frozen) storage. Hematopoietic cell preparations obtained from the 16-to 17-week gestational age range had the highest total cell count and the greatest proportion of CD34-positive stem cells. However, these preparations were insufficiently robust to withstand the freeze/thaw protocol required. Cells obtained from the 13- to 15-week gestational age range showed optimal post-thaw viability and it is suggested that these cell preparations are the most applicable for in utero transplantation.

The epidemiology of hepatitis viruses B, C, and D.

Chronic viral hepatitis is caused mainly by chronic infection with hepatitis viruses B (HBV), C (HCV), or delta (HDV). Persons chronically infected with one or more of these viruses may develop chronic progressive hepatitis, cirrhosis, and liver failure. In addition, chronic HBV and HCV infections are major causal risk factors for hepatocellular carcinoma. Alcohol consumption accelerates the development of chronic liver disease among HCV-infected individuals and may have similar effects on persons chronically infected with HBV alone or HBV and HDV, but the reported studies are inconsistent.

Long-term storage of human fetal haematopoietic progenitor cells and their subsequent reconstitution. Implications for in utero transplantation.

Haematopoietic progenitor cells were isolated from human fetal liver, obtained between 6 and 15 weeks gestation. After preparation of a single cell suspension, the cells were stored using a stepwise freezing protocol; taking the cells from room temperature through -70 degrees C to liquid nitrogen. Viability (trypan blue exclusion), morphology (Leishman stain), identification of cell type (flow cytometry) and growth characteristics in semi-solid culture medium were assessed using the fresh cell suspension. We were able to confirm that the predominant cells in human fetal liver up to about 15 weeks gestation are those of the erythroid lineage. It was established that viability in excess of 75% was required to ensure adequate growth in culture after frozen storage and it was deemed important to ensure morphological integrity of the cell preparations. The colonies formed in culture were observed to be producing haemoglobin between 7 and 9 days after initial seeding. We have determined that cells can be stored in liquid nitrogen for up to 2 years without loss of (1) viability, (2) morphological features and (3) ability to form colonies and produce haemoglobin in culture. These findings offer encouragement for the implementation of a cell bank to support an in utero transplantation programme.

Molecular and genetic epidemiology of hepatocellular carcinoma: studies in China and Senegal.

To identify environmental, viral, and genetic factors that may influence the risk of developing hepatocellular carcinoma (HCC), large prospective studies are being conducted in Haimen City, China and Senegal, and a case-control study of genetic variation in the detoxification of aflatoxin-B1 was carried out in Shanghai, China. Analysis of 78 HCCs that have occurred among 51,020 men enrolled in a large prospective study in Haimen City, China showed a strong association of HCC with chronic hepatitis B virus (HBV) infection. There were also significant associations of HCC risk with occupation (farming), history of a clinical episode of hepatitis in adulthood, and a family history of HCC. Study of 52 HCC cases and 116 controls for genetic polymorphisms and HCC risk showed a significant association with epoxide hydrolase (EPHX) mutant alleles (1/2, 2/2) and a borderline association with homozygous deletion of the glutathione-S-transferase mu (GSTM1) gene. There was a multiplicative interaction of these polymorphisms with chronic HBV infection such that HBsAg-positive persons who were GSTM1 null and were EPHX 1/2 or 2/2 had 135 times the risk of HCC as HBsAg-negative persons with the wild type genotypes for GSTM1 and EPHX. The risk of HCC is not uniform among persons with chronic HBV or HCV infections. Studies of genetic, viral, and environmental interactions may permit identification of those individuals at highest risk within groups at increased risk of HCC. Prevention strategies could then be targeted at those individuals.

Viral, host and environmental risk factors for hepatocellular carcinoma: a prospective study in Haimen City, China.

To identify specific environmental, viral, and genetic risk factors for hepatocellular carcinoma (HCC) and the interaction of such factors, we are conducting a prospective study in a high-incidence area of China. Questionnaires were completed and biosamples collected by 60,984 men ages 30-64 years, at study entry. Within 2.5 years, 183 deaths from HCC had occurred. Each HCC case was matched with 5 controls and compared for items on the questionnaire. In addition to chronic hepatitis B virus (HBV) infection, the significant risk factors were: occupation (peasant), corn consumption (in the 1970s), family history of HCC, and history of an episode of acute hepatitis as an adult. HBV, consumption of aflatoxins, a genetic factor, and possibly a second hepatitis virus infection contribute to the risk of HCC.

Breast reconstruction at a district general hospital.

Breast reconstruction is normally carried out by plastic surgeons, but the advent of permanent tissue expanders places post-mastectomy reconstruction within easy reach of the general surgeon. Nineteen patients underwent breast reconstruction between 1989 and 1991 using a subpectoral silicone-based, double lumen tissue expander. Assessment of results was by: (a) patient completed questionnaire; and (b) third party evaluation of standardized photographs. The mean operating time was 58 min (40-80 min) and mean hospital stay 3 days (2-7 days). Complications included one flap necrosis and one leaking injection port. Outpatient tissue expansion required an average of seven visits (4-11) and was completed in an average of 12 months (7-19). The injection port was subsequently removed under local anaesthetic as a day case. The fully dressed appearance following reconstruction was graded good or excellent by 100% of patients and in over 80% of third-party assessments. Equivalent figures for the appearance when wearing a bra were 93% and 60% and undressed 57% and 47%, respectively. All patients recommended the procedure but 25% found inflation uncomfortable. Subpectoral tissue expansion is a safe, cosmetically acceptable method of breast reconstruction which is associated with a high level of patient satisfaction.

Role of hepatitis C virus in non-B chronic liver disease.

To assess the contribution of the recently identified hepatitis C virus to chronic liver diseases of unknown cause and chronic hepatitis attributed by exclusion to non-A, non-B hepatitis, we tested for antibody to hepatitis C in hepatitis B surface antigen-negative patients with a spectrum of chronic liver diseases. Antibody to hepatitis C virus, a marker of hepatitis C infection, was detected with a first-generation radioimmunoassay at the following frequencies in the following patient groups: 69% of transfusion-associated non-A, non-B hepatitis; 53% of non-transfusion-associated non-A, non-B hepatitis; 26% of hepatitis B surface antigen-negative hepatocellular carcinoma; 8% of cryptogenic cirrhosis; 5% to 7% of autoimmune chronic liver diseases; 19% of patients with miscellaneous types of chronic liver disease; and 0.67% of healthy controls. Among non-transfusion-associated cases, 81% with a history of intravenous drug use but only 18% with occupational exposure as health workers had antibody to hepatitis C virus. Among cases of hepatocellular carcinoma, 63% of Japanese patients but only 11% of American patients had evidence of hepatitis C infection. Comparison in a subgroup of 79 serum samples of a second-generation radioimmunoassay with the first-generation assay demonstrated a 12% increase in antibody frequency from 30% to 42%. We conclude that hepatitis C plays a substantial role in transfusion-associated and non-transfusion-associated non-A, non-B hepatitis as well as in hepatocellular carcinoma, especially in Japan, a limited role in cryptogenic cirrhosis, and essentially no role in autoimmune chronic liver diseases. Application of more sensitive immunoassays will increase the frequency of antibody seropositivity in all subgroups, but relative distinctions among risk groups are likely to remain.

Preoperative total parenteral nutrition for bowel resection in Crohn's disease.

To examine the effect of preoperative total parenteral nutrition (TPN) on patients with Crohn's disease undergoing bowel resection, an historical cohort was assembled of 103 patients resected between 1982 and 1984 by a single surgical team. Preoperative, perioperative, and postoperative variables were compared between patients receiving TPN and patients not receiving TPN. Analysis was stratified for three surgical procedures: segmental small bowel resection, ileocectomy, and segmental or total colectomy The effect of TPN was most pronounced in patients having small bowel surgery. For segmental small bowel resection, 12 of 17 patients had TPN, and these patients had 20.4 +/- 14.3 cm less bowel resected than did those in the non-TPN group, an effect not dependent on duration of TPN. For ileocectomy patients, 31 of 62 patients received TPN, and these patients had 11.2 +/- 4.2 cm less small bowel resected than the non-TPN group, an effect not dependent on the duration of TPN. For large bowel resection patients, 6 of 24 patients had TPN, and there was no difference in length of bowel resection, preoperative and perioperative variables, or recurrence. The total hospital stay was 13.5 +/- 2.6 days longer for those having TPN; 3.5 +/- 1.9 days of the longer stay was postoperative. In conclusion, TPN was associated with reduced length of small bowel resection at the expense of longer hospital stay.

Cigarette smoking in Crohn's disease.

Crohn's disease is a chronic disease of unknown etiology. Previous reports have suggested that cigarette smoking may be associated with the development of Crohn's disease. To examine this association, we conducted a case-control study of patients referred to a single practice over a 7-month period. The cigarette-smoking habits of 115 patients with Crohn's disease were compared with the cigarette-smoking exposure of 109 patients with the irritable bowel syndrome. Patients with Crohn's disease were more likely to smoke at the time of symptom onset than were irritable bowel syndrome controls (age and sex adjusted odds ratio 3.71, 95% confidence interval 1.93-7.13). After the diagnosis of Crohn's disease, patients were less likely to quit smoking (odds ratio 0.35, 95% confidence interval 0.18-0.69) than controls. This study demonstrates an association and a temporal relationship between cigarette smoking and Crohn's disease. For the exposure to be considered an etiologic factor for disease, biologic plausibility and pathophysiologic mechanisms require elucidation.

Chronic hepatitis: disease factors at diagnosis predictive of mortality.

PURPOSE: Chronic hepatitis is known to be a disease with substantial mortality. The purpose of this study was to identify prognostic factors in a large group of patients with chronic hepatitis. We also wanted to determine whether the aminopyrine breath test (ABT) is of additional prognostic value in evaluation of this disease. PATIENTS AND METHODS: We studied 94 patients who had had a biopsy-proven diagnosis and an ABT between June 1, 1977, and June 30, 1981. Clinical features and biochemical test results at the time of diagnosis were retrieved from medical records, and histologic severity was assessed by reviewing all liver biopsy specimens under code. Survival was determined at a mean of 60 months. Data were studied with a Cox proportional hazards model to identify predictors of mortality and to control for confounding variables. RESULTS: Cumulative mortality as of December 31, 1985, was 5 percent in chronic persistent hepatitis, 6 percent in chronic active hepatitis, 29 percent in chronic active hepatitis with bridging necrosis, and 53 percent in chronic active hepatitis with cirrhosis. Histologic severity was a predictor of death (p less than 0.005). Other predictors of mortality were disease caused by hepatitis B virus (p less than 0.005), a high alkaline phosphatase level (p less than 0.025), a low alanine aminotransaminase level (p less than 0.001), and a depressed ABT result (p less than 0.005). CONCLUSION: The results suggest that patients with chronic hepatitis with one or more of these risk factors have an increased mortality and should be followed closely for liver failure, which may necessitate medical therapy or surgical intervention.

Prevalence and incidence of inflammatory bowel disease in family members.

To determine the risk of having or developing inflammatory bowel disease (IBD) in a family member of an IBD patient, a population of 245 IBD probands was randomly selected from the University of Chicago IBD Registry and their family history was elucidated by questionnaire and follow-up telephone call. One hundred seventy-nine (73%) probands responded to the questionnaire. There were no demographic distinctions between those eligible for the study, those who were complete responders, those who were nonresponders, and those with a positive family history of IBD. Fifty-four family members from 40 proband families (22%) had confirmed IBD. Prevalence of IBD in family members at the time of diagnosis of the proband was highest for parents (4.6%), siblings (2.6%), and children (1.9%). Grandparents, aunts and uncles, and first cousins had prevalence of IBD of less than 1%. Incident case frequency was determined by dividing the number of cases incident after the diagnosis of the proband by all those ever at risk. The incident case frequency was highest for siblings (1.9%), parents (1.0%), and children (1.0%). There was concordance noted for type of disease in the proband and the relative. No association could be discerned between the familial risk of IBD and gender, race, or religion of the proband. Despite a high occurrence rate of proband families with IBD, the specific risk to first, second, or third degree family members is low.