Long-term clinical and surrogate marker effects of subcutaneous intermittent interleukin-2 without antiretroviral therapy in HIV-infected patients.

OBJECTIVES: Subcutaneous administration of interleukin-2 (IL-2) has been shown to increase CD4 counts in HIV-infected patients. It remains unclear whether this effect is associated with a clinical benefit. PATIENTS AND METHODS: We conducted a long-term follow-up in the cohort of the UK-Vanguard study in which three groups of 12 antiretroviral-naive subjects with CD4 cell counts >350 cells/mm(3) received no treatment or IL-2 at either 4.5 or 7.5 MIU twice daily in 5 day cycles, respectively. RESULTS: Mean follow-up was 376 weeks. IL-2 therapy was associated with a higher area under the curve of CD4 cell count change from baseline at week 48 but not thereafter. HIV-RNA levels were unaffected. Highly active antiretroviral therapy (HAART) was initiated after a mean of 172, 175 and 152 weeks in the control group, low-dose and high-dose IL-2 treatment group, respectively, a statistically non-significant difference. There was a tendency to start HAART soon after discontinuation of IL-2 therapy which may have been triggered by the steep decay of CD4 counts. There were two serious adverse events in the control group, seven in the low-dose IL-2 group and eight in the high-dose IL-2 group. No pattern of disease was detected, making an association with IL-2 therapy unlikely. CONCLUSIONS: We could detect neither a benefit of IL-2 therapy after week 48 nor delayed initiation of HAART. This is currently the longest follow-up data comparing IL-2 therapy with no therapy in antiretroviral-naive HIV-infected patients and does not show a persistent benefit of the intervention.

Development of rebiana, a natural, non-caloric sweetener.

Rebiana is the common name for high-purity rebaudioside A, a natural non-calorie sweetener 200-300 times more potent than sucrose. It provides zero calories and has a clean, sweet taste with no significant undesirable taste characteristics. It is functional in a wide array of beverages and foods and can be blended with other non-calorie or carbohydrate sweeteners. It is stable under dry conditions, and has much better stability than aspartame or neotame in aqueous food systems. Studies undertaken for the development of a purification process and for the full characterization of the properties of rebiana are reported here.

Epoxide hydrolase Tyr113His polymorphism is associated with elevated risk of colorectal polyps in the presence of smoking and high meat intake.

Microsomal epoxide hydrolase (mEH) metabolizes polycyclic aromatic hydrocarbons, carcinogens found in cigarette smoke and cooked meat. Polymorphisms in exon 3 and exon 4 of the mEH gene have been found to alter mEH activity. We investigated the association between these polymorphisms and colorectal polyps within the Minnesota Cancer Prevention Research Unit case-control study. Cases were diagnosed with colonoscopically confirmed adenomas (n = 530) or hyperplastic polyps (n = 202); controls (n = 649) were polyp-free at colonoscopy. Smoking history and meat consumption were obtained from self-administered questionnaires before colonoscopy. mEH genotypes were determined by PCR/RFLP or oligonucleotide ligation assay. The overall risks associated with exon 3 or exon 4 polymorphisms for both adenomas and hyperplastic polyps were not statistically different from 1.0. Compared with exon 3 Tyr/Tyr, 0 pack-years, risk was highest among those with the exon 3 His/His genotype and >25 pack-years of smoking [adenoma, odds ratio (OR) = 4.9 (1.9-12.8); hyperplastic, OR = 7.7 (2.5-24.0)]. Risks were not elevated among exon 4 homozygous variants, even in the presence of heavy smoking. Fried, baked, or broiled meat intake of > or =two servings/week (high) compared with < or =one serving/week was associated with a 2-fold increase in risk of adenoma. The highest risks were seen for those with the exon 3 His/His genotype and high cooked meat intake [OR = 3.3 (1.4-7.9); reference group: Tyr/Tyr, < or = 1 serving/week). Although mEH polymorphisms are not associated with an increased risk of colorectal polyps overall, genotypes that produce a slow phenotype appear to be associated with an increased risk in the presence of smoking and high intakes of cooked meat.

CYP2C9 and UGT1A6 genotypes modulate the protective effect of aspirin on colon adenoma risk.

Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) has a protective effect on the incidence of colon neoplasia. However, polymorphisms in NSAID-metabolizing enzymes may alter this effect. NSAIDs, particularly aspirin, are glucuronidated by UGT1A6 and some classes of NSAIDs are also metabolized by cytochrome P450 (CYP) 2C9. Both of these enzymes have slow-metabolizing, variant forms. We tested the hypothesis that the slow alleles of these enzymes can modify the inverse association between NSAIDs and colon neoplasia in the Minnesota Cancer Prevention Research Unit (CPRU) adenomatous polyp case-control study. CYP2C9 and UGT1A6 genotypes were determined for 474 adenoma cases and 563 controls. NSAID use was inversely associated with adenoma risk [odds ratio (OR), 0.63; 95% confidence interval (CI), 0.44-0.90 for aspirin; and OR, 0.50; 95% CI, 0.31-0.82 for nonaspirin NSAID]. However, this association was absent in aspirin users who carried the CYP2C9 variant alleles (OR, 0.88; 95% CI, 0.51-1.53) or who were homozygous wild-type UGT1A6 (OR, 0.86; 95% CI, 0.50-1.50). Carriers of both of these alleles who use aspirin were also not at reduced risk of adenomatous polyps (OR, 1.59; 95% CI, 0.68-3.73). The variants of these enzymes did not influence the association between nonaspirin NSAIDs and adenoma risk. These data indicate that the effectiveness of chemopreventive drugs can be modulated by the genotype of metabolizing enzymes.

Closeout of four phase II Vanguard trials and patient rollover into a large international phase III HIV clinical endpoint trial.

Large phase III clinical trials typically require many years of planning and preparation. During this time, proposed study methods and overall trial feasibility can be assessed in smaller pilot studies. However, the patients enrolled in these pilot studies are not routinely included in the larger study. In preparation for a multinational randomized clinical end point trial of interleukin-2 in HIV-infected patients, four phase II "Vanguard" studies were initiated. These Vanguard trials served to increase safety and surrogate marker data in diverse patient cohorts, increase clinical experience with the study medication, and identify the optimal dose of medication for the phase III trial. These trials also served to assess patient recruitment potential and to develop international clinical trial coordination experience. The Vanguard trials were designed to allow continued follow-up of their patients as participants of the phase III trial once the feasibility of the phase III trial was confirmed. The purpose of this paper is to describe the steps taken in the closeout of these four phase II trials while reconsenting these patients to the phase III trial. Specifically, the reconsent process, the data collection transition plan, and the steps taken to minimize bias due to differential reconsent according to the assigned treatment arm in the phase II trial are described. The procedures employed are relevant to the reconsent of patients for long-term follow-up at the completion of clinical trials. Control Clin Trials 2001;22:42-48

Cutting edge: introduction of an endopeptidase cleavage motif into a determinant flanking region of hen egg lysozyme results in enhanced T cell determinant display.

The choice of which determinants of a whole Ag will be presented on cell surface MHC class II molecules after uptake and processing by APC is the result of the interplay between structural characteristics of the Ag and the processing machinery of the APC. In this study, we demonstrate that introduction of a dibasic motif adjacent to a subdominant determinant enhances the presentation of this determinant from the whole molecule. This is the first report showing that a single amino acid substitution in a whole Ag, designed to introduce an endopeptidase recognition site, enhances display of class II-restricted determinants, most likely by creating a peptide chain cleavage in the antigenic molecule. Our findings have important implications for the understanding of immunodominance and for vaccine design.

Lack of association between the C677T MTHFR polymorphism and colorectal hyperplastic polyps.

Colorectal hyperplastic polyps are benign lesions that share many risk factors with colorectal adenomas and cancers. Low folate intakes are associated with an increased risk of colon cancer. The enzyme 5,10-methylene-tetrahydrofolate reductase (MTHFR) may be linked to DNA methylation and nucleotide synthesis and thus play a role in the etiology of colorectal neoplasia. We investigated an association between the common MTHFR polymorphism (C677T) and colorectal hyperplastic polyps within the Minnesota Cancer Prevention Research Unit case-control study. Cases (n = 200) were diagnosed with colonoscopically confirmed hyperplastic polyps; controls (n = 645) were derived from the same gastroenterology practice and were polyp-free at colonoscopy. Dietary intakes were estimated from a self-administered food-frequency questionnaire prior to colonoscopy. Multivariate adjusted odds ratios (ORs) and 95% confidence intervals for MTHFR status were 0.8 (0.6-1.2; CT versus CC wild-type) and 0.9 (0.5-1.6; TT versus CC). In subgroup analyses stratified on dietary intakes of folate, vitamin B12, vitamin B6, or methionine, those with the TT genotype and either low intakes of folate or vitamin B6 were at increased risk relative to those with normal or high vitamin intake. However, most 95% confidence intervals included 1.0, and no consistent trends were observed. In contrast to our findings on colorectal adenomas, increasing alcohol consumption was associated with an elevated risk of colorectal hyperplastic polyps, regardless of genotype. The MTHFR (C677T) variant genotype does not appear to be related to risk of colorectal hyperplastic polyps, and there is no convincing evidence that MTHFR shows a different relation to risk, dependent on dietary intakes of nutrients related to its pathway.

Increasing vegetable and fruit intake: randomized intervention and monitoring in an at-risk population.

High vegetable and fruit (V&F) consumption has been associated with a lower risk of several cancers. However, little is known about the ability of individuals to increase their intakes markedly. In this 1-year randomized, controlled diet intervention study of men and women with a recent history of adenomas, the intervention group (n = 100) was asked to increase V&F intake to at least eight servings per day; the control group (n = 101) continued eating their usual diet. End-point measures included V&F intake assessed by 3-day diet records, plasma carotenoids, serum lipids, urinary sodium and potassium, and body weight. The intervention group increased their daily V&F intake an average of 5.5 servings over 1 year; the control group had an average decrease of 0.5 servings per day (P < 0.001). Plasma total carotenoids, alpha-carotene, beta-carotene, beta-cryptoxanthin, and lutein/zeaxanthin were each statistically significantly elevated over baseline (11-54%) in the intervention group compared with the control group over the duration of follow-up (P < 0.001). Urinary potassium excretion was elevated 14% over baseline in the intervention group compared with no change in the control group (P < 0.001). Modest decreases in the intervention but not the control group were observed for total and low-density lipoprotein cholesterol. Plasma lycopene, triglycerides, high-density lipoprotein cholesterol, body weight, and urinary sodium were not affected by the intervention. V&F intake was significantly increased in this motivated population at higher risk of colon cancer and maintained for at least 12 months, as assessed using diet records and an ensemble of biomarkers.

Effect of calcium supplementation on serum cholesterol and blood pressure. A randomized, double-blind, placebo-controlled, clinical trial.

OBJECTIVE: To test the effect of daily supplemental calcium on serum total and high-density lipoprotein cholesterol (HDL-C) levels and blood pressure in adults. DESIGN: Randomized, double-blind, placebo-controlled clinical trial; adjunct study to a trial of calcium and colon cell proliferation in patients with sporadic adenoma. SETTING: Outpatient clinic. PATIENTS: A total of 193 men and women, aged 30 to 74 years. INTERVENTION: Treatment with 1.0 and 2.0 g/d of elemental calcium vs placebo over a 4-month period for cholesterol determinations and 6 months for blood pressure. MAIN OUTCOME MEASURES: Serum total cholesterol and HDL-C levels, systolic and diastolic blood pressure. RESULTS: Because there were no apparent differences in responses between the 1.0-g and 2.0-g calcium groups, their data were combined and compared with those of the placebo group. Among all participants, the mean total cholesterol level dropped 0.07 mmol/L (2.9 mg/dL) (1.3%) (P = .43) more, and the mean HDL-C level dropped 0.01 mmol/L (0.4 mg/dL) (1.1%) (P = .71) less in the calcium group than in the placebo group. Among participants without a history of hypercholesterolemia, the mean total cholesterol level dropped 0.18 mmol/L (6.8 mg/dL) (3.3%) (P = .10) and the HDL-C level dropped 0.02 mmol/L (0.6 mg/dL) (1.5%) (P = .61) more in the calcium group than in the placebo group. Among all participants, there was no apparent change in blood pressure until 6 months, when the mean systolic blood pressure dropped 0.8 mm Hg (0.6%) (P = .85) and the mean diastolic blood pressure dropped 0.4 mm Hg (0.5%) (P = .80) more in the calcium group than in the placebo group. CONCLUSIONS: There were no substantial or statistically significant effects of calcium supplementation on total cholesterol or HDL-C levels or on blood pressure. There was a suggestion (not statistically significant) of a 0.07 to 0.18 mmol/L (3-7 mg/dL) or 2% to 4% drop in the total cholesterol level, a finding similar to that reported in other studies, which indicates the need for further study.

A randomized, controlled 24-week study of intermittent subcutaneous interleukin-2 in HIV-1 infected patients in Thailand.

OBJECTIVES: To assess the immunological and virological effects, safety profile and feasibility of subcutaneous interleukin-2 (scIL-2) therapy in an HIV-infected Thai population. DESIGN: Seventy-two patients with baseline CD4 cell count of > or = 350 x 10(6)/l and no history of opportunistic infection were randomized to receive antiretroviral therapy plus scIL-2 (scIL-2 group) or antiretroviral therapy alone (control group). scIL-2 was administered at one of three doses for at least 24 weeks. The main measure of treatment efficacy was change in CD4 cell count. RESULTS: The time-weighted mean change in CD4 cell count from baseline to week 24 was + 252 x 10(6)/l for the scIL-2 group compared with + 42 x 10(6)/l for the control group (P< 0.0001). Changes in plasma HIV RNA were not significantly different between the groups over the same time period: there was a 0.83 log10 copies/ml decrease for the scIL-2 group and a 0.70 log copies/ml decrease for the control group (P= 0.362). CONCLUSIONS: This study provides the most extensive experience of scIL-2 therapy in HIV-1 infected women and Asians, and demonstrates the immunological efficacy, tolerability and feasability of scIL-2 therapy in this population. Data from this study were instrumental in guiding the selection of the scIL-2 dosing regimen for ongoing phase III trials.

Colorectal adenomatous and hyperplastic polyps: smoking and N-acetyltransferase 2 polymorphisms.

Arylamine N-acetyltransferase 2 (NAT2) is involved in both the detoxification and bioactivation of carcinogenic arylamines and other mutagens. This enzyme is polymorphic, and the fast and slow phenotypes are thought to be risk factors for colon and bladder cancer, respectively. Here, we report on a case-control study of adenomatous and hyperplastic polyps, with particular attention to tobacco smoking, a known risk factor for adenomas, and polymorphisms of NAT2. All participants underwent complete colonoscopy and were subsequently divided into case and control groups on the basis of pathology. Cases were diagnosed with confirmed adenomas (n = 527) or hyperplastic polyps (n = 200); controls (n = 633) had no history of colonic neoplasia and no polyps at colonoscopy. NAT2 genotype was determined using an oligonucleotide ligation assay and fast, intermediate, or slow phenotype imputed. Multivariate-adjusted odds ratios (ORs) and 95% confidence intervals were computed using logistic regression adjusting for age, sex, nonsteroidal anti-inflammatory drug use, and hormone replacement therapy use. Smoking was associated with an increased risk of adenomas [current versus never smoking OR = 2.0 (95% confidence interval, 1.4-2.9)] and hyperplastic polyps [current versus never smoking OR = 4.1 (2.6-6.5)]. NAT2 status among adenomatous polyp patients and hyperplastic polyp patients, respectively, showed ORs of 1.1 (0.8-1.4) and 1.2 (0.8-1.6; intermediate versus slow) and 1.1 (0.6-1.9) and 0.9 (0.4-1.9; fast versus slow). There were no differences in risk when adenoma patients were stratified on multiplicity, size, or histopathological subtype of polyps. Never-smokers showed no variation in risk across acetylator status for either species of polyp, whereas current smokers showed ORs of 2.0 (1.2-3.2) and 2.3 (1.4-3.9) for adenomas and 3.9 (2.1-7.1) and 4.9 (2.6-9.4) for hyperplastic polyps for slow and intermediate/fast NAT2, respectively, compared with slow-NAT2 never-smokers. Risks of both multiple [OR = 4.3 (2.1-8.8)] and large [OR = 3.8 (1.9-7.5)] adenomas were somewhat elevated in current smokers with an intermediate/fast phenotype compared with smokers with a slow NAT2 phenotype, but the interaction was not statistically significant. Risk of hyperplastic polyps and adenomatous polyps is strongly related to smoking. There is little suggestion of interaction between NAT2 status and smoking and no relationship with NAT2 genotype alone.

Colorectal adenomas and the C677T MTHFR polymorphism: evidence for gene-environment interaction?

5,10-Methylene-tetrahydrofolate reductase (MTHFR), an enzyme in folate metabolism, may play a role in the etiology of colorectal adenomas via effects on DNA methylation and nucleotide synthesis. We investigated the association between a common polymorphism (C677T, reduced MTHFR activity) and colorectal adenomas within the Minnesota CPRU case-control study. Cases (n = 527) were diagnosed with colonoscopically confirmed adenomas; controls (n = 645) were derived from the same gastroenterology practice and were polyp free at colonoscopy. Dietary intakes were obtained from a self-administered food-frequency questionnaire prior to colonoscopy. Age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals for the MTHFR genotype were 0.9 (0.7-1.2; CT versus CC wild-type) and 0.8 (0.6-1.3; TT versus CC). The associations between dietary intakes of folate, vitamin B12, vitamin B6, or methionine and risk of adenomas showed consistent patterns dependent upon MTHFR genotype. Individuals with the TT genotype and intakes of any of these nutrients in the lowest tertile were at elevated risk for adenomas (about 2-3-fold when compared with TT genotype with high intakes). These trends were more pronounced among individuals over age 60, resulting in a 3-6-fold increase for low intakes of folate, B12, and B6. An increased risk with increasing alcohol consumption was observed only among those with the CC genotype (P-trend = 0.005); among those with the TT genotype, those with moderate alcohol consumption were at lowest risk (P for interaction P = 0.02). In conclusion, nutrients involved in the MTHFR metabolic pathway may modify the relationship between the MTHFR C677T polymorphism and colorectal adenomas. Low intakes of folate, vitamin B12, and vitamin B6 increase risk among those (particularly the elderly) with the MTHFR TT genotype.

Methodological findings and considerations in measuring colorectal epithelial cell proliferation in humans.

The methodological issues for measuring colorectal epithelial cell proliferation, an intermediate end point for studies of colon neoplasia, in epidemiological studies are deceptively numerous and complex, with few methodological data available. Accordingly, during our experience with measuring colorectal epithelial cell proliferation from nearly 500 participants attending over 1300 study visits over a 6-year period, we recorded data on a variety of measurement variations. Methods investigated included rectal biopsy technique, general histological and labeling procedures [including the tritiated thymidine, 5-bromodeoxyuridine (BrdUrd), and the proliferating cell nuclear antigen (PCNA) immunohistochemical techniques used to label S-phase cells in colonic crypts in rectal biopsy specimens], biopsy scoring procedures, and summary scoring methods. Findings include that the PCNA technique was the simplest, most economical, and least time-consuming. The BrdUrd labeling failure rate was 15% versus < 1% for PCNA. The percentage of labeled cells (labeling index) was highest using PCNA in biopsies processed without prior incubation, intermediate using PCNA in biopsies processed with prior incubation as for BrdUrd, and lowest using BrdUrd. The percentage of labeled cells that were in the upper 40% of the crypt (phi h) was higher using BrdUrd than PCNA; visit-to-visit correlations were higher using PCNA (r = 0.51 versus 0.35), and visit-to-visit variability was lower and between-person variability was higher using PCNA. Intra- and inter-rater reliabilities for the techniques were comparable (PCNA intra-rater r = 0.93, inter-rater r = 0.92). The PCNA technique, compared to the BrdUrd technique, is more feasible and reliable, provides a more accurate estimate of the labeling index, and cell proliferation measures determined with PCNA have statistical properties that are generally more favorable for detecting differences in clinical trials. Thus, the PCNA technique may be preferable to techniques requiring incubation of biopsies. Other methodological findings lead us to recommend that, for larger studies measuring colorectal epithelial cell proliferation on outpatient rectal biopsies, biopsies should be taken 10 cm above the anus using a flexible, preferably jumbo cup, endoscopic forceps through a rigid sigmoidoscope, and histological sections should be 3 microns thick taken 50 microns apart.

Reliability and comparability of three dietary assessment methods for estimating fruit and vegetable intakes.

Although fruits and vegetables have been evaluated in numerous epidemiologic studies, few validation studies have examined fruits and vegetables. We examined the reproducibility and comparability of fruit and vegetable intakes estimated by diet records, food frequency questionnaires, and modules (brief food frequency questionnaires) in 101 control participants of a 1-year diet intervention trial. For each method, mean intakes at baseline and 3 months were generally within 0.3 serving per day for juice, fruits, vegetables, and total fruits and vegetables. In addition, Pearson correlations for the two time periods generally exceeded 0.55 for these four groups for each method. We evaluated comparability of intakes for 15 days of diet records, 1-year food frequency questionnaires, and modules, respectively. Mean total fruit and vegetable intakes were 6.3, 6.5, and 3.8 servings per day for diet records, food frequency questionnaires, and modules. For each pair-wise combination of methods, Pearson correlations exceeded 0.45 for juice, fruits, and total fruits and vegetables; correlations were lower for vegetables. Exact agreement in quintile assignment was less than 45%, however. These results indicate that estimates of fruit and vegetable intakes and disease associations may differ depending on the method used to assess fruit and vegetable intake.

Colorectal epithelial cell proliferative kinetics and risk factors for colon cancer in sporadic adenoma patients.

Colorectal epithelial cell proliferative kinetics are altered in patients at increased risk for colon cancer: proliferation rates [labeling index (LI)] are higher and there is a shift of the proliferative zone from one confined to the lower 60% of the colonic crypt to one that includes the entire crypt (higher phi(h)). To assess factors associated with LI and phi(h), we performed a cross-sectional analysis using baseline rectal mucosal biopsies from sporadic adenoma patients participating in a chemoprevention trial. Biopsies (taken without preparatory cleansing) were taken 10 cm above the level of the anus, and proliferation was assessed by detection of endogenous S-phase-associated proliferating cell nuclear antigen by immunohistochemical methods. High-quality, scorable biopsies were obtained for 115 patients, and using analysis of covariance and multiple linear regression, the LI and phi(h) were evaluated in relation to diet and other lifestyle factors, demographics, anthropometrics, family history of colon cancer, and polyp history. Statistically significant findings included the following: (a) The LI for those in the upper versus the lowest tertile of vegetable and fruit consumption was, proportionately, 35% lower (3.4% versus 5.3%; P < 0.001); for vitamin supplement users versus nonusers, it was 36% lower (3.3 versus 5.2%; P < 0.001); for recurrent versus incident polyp patients, it was 36% higher (6.2 versus 4.0%; P < 0.001); and for those with rectal polyps only versus those with colon polyps only, it was 28% higher (6.0 versus 4.3%; P = 0.05); and (b) the phi(h) for those in the upper versus the lowest tertile of sucrose consumption was, proportionately, 48% higher (7.1% versus 3.7%; P = 0.01). These results indicate that (a) colorectal epithelial cell proliferation rates are higher in recurrent adenoma patients than in incident adenoma patients and in patients with rectal adenomas only versus those with colon adenomas only, but they are lower in patients with higher intakes of vegetables and fruit and in those who take vitamin/mineral supplements, and (b) the distribution of proliferating cells is shifted toward more inclusion of the upper 40% of the crypt in patients with higher intakes of sucrose. The pattern of positive, negative, and null associations of potential risk factors with cell proliferation is similar to that commonly found with colonic neoplasms.

Hormone replacement therapy is associated with lower risk of adenomatous polyps of the large bowel: the Minnesota Cancer Prevention Research Unit Case-Control Study.

Evidence of a role for steroid hormones and reproduction in colon neoplasia remains tantalizing but unclear. Hormone replacement therapy (HRT) has been reported in a number of recent studies to be associated with a reduced risk of colon cancer. A case-control study was undertaken to establish whether HRT is associated with lower risk of adenomatous polyps. This case-control study was undertaken as a project of the Minnesota Cancer Prevention Research Unit. Cases (n = 219) were women, ages 30-74 years with colonoscopy-proven, pathology-confirmed, adenomatous polyps of colon and rectum recruited at Digestive Healthcare PA (Minneapolis, MN). Two control groups were selected: women without polyps at colonoscopy (n = 438) at Digestive Healthcare and age- and zip code-matched women selected from the general community (n = 247). Response rates were 68% among those colonoscoped and 65% among community controls. Parity, age at first live birth, and oral contraceptive use did not distinguish cases from either control group. Multivariate adjusted odds ratios and 95% confidence limits for use of HRT for less than 5 years (compared with never use) among postmenopausal women were 0.52 (0.32-0.85) versus colonoscopy-negative controls and 0.74 (0.44-1.26) versus community controls. For 5 years of use or greater, the corresponding figures were 0.39 (0.23-0.67) and 0.61 (0.34-1.07). These results were not materially different when stratified on body mass index, oophorectomy, hysterectomy, aspirin use, or family history. There is no marked increase in risk even 5 years after cessation of HRT use. HRT appears to lower risk of colorectal adenomatous polyps, suggesting that it acts quite early in the neoplastic process. Mechanisms remain unclear. Reduction of risk of colorectal neoplasia is an additional benefit of postmenopausal HRT.

Reproducibility of the measurement of colonic proliferation using bromodeoxyuridine across laboratories.

Although measures of colonic cell proliferation are being used as potential intermediate markers in chemoprevention studies, measurement standardization is still ongoing. This study was designed to assess the reproducibility of the labeling index quantification, as measured by bromodeoxyuridine, across four laboratories experienced in its use. Each institution submitted 10 slides, with one circled area of each slide to be scored. Each site followed its standard procedures for scoring colonic crypts; no attempts to standardize these procedures were made. There was high concordance among the laboratories on whether scorable crypts were present on a particular slide, but only two pairs of laboratories demonstrated agreement statistically greater than that predicted by chance. The overall difference among the sites on the number of scorable crypts was marginally significant (P = 0.083), and there was a highly significant overall difference in the magnitude of the labeling index (P < 0.0001). Sites 1 and 2 tended to have similar results, as did sites 3 and 4, most likely due to common training. Even with these discrepancies, high correlation (r > 0.75) was observed among the reported labeling index values for each pair of laboratories. Without standardized training, these laboratories may differ in the crypts considered appropriate for counting and in whether cells are counted as labeled or unlabeled. These results suggest that standardized training in scoring across all sites performing labeling index determinations is required to assure reproducibility across sites or studies. These results may also help explain discrepancies in the average values of the labeling index reported in the literature.

Quantification of proliferating cell nuclear antigen in large intestinal crypt by computer-assisted image analysis.

Measurements of proliferating cell nuclear antigen (PCNA) labeling of the large intestinal crypts scored by experienced observers were compared with those generated by computer-assisted image analysis (CAIA). (CAIA was performed at Pathology Expertise, Inc., Newton, MA, by A. V. Sotnikov.) Serial sections (3 microns) of the rectal biopsy specimens from 32 patients were immunostained for PCNA and then counterstained with hematoxylin. The same set of slides and rules for the location/acquisition of complete crypts was used to assess a minimum of ten complete crypts/patient. Each crypt was subdivided longitudinally into five equal compartments. With CAIA, the images were stored digitally, and once the color references were set, the areas occupied by labeled and counterstained nuclei were quantified automatically. The labeling index (LI) was calculated from the PCNA-labeled nuclei area/total nuclei area in CAIA and from the number of labeled cells/total number of cells in visual scoring. The LI of whole crypts averaged 1.04 +/- 0.18 by CAIA and 3.91 +/- 0.46 by the visual method, and the Spearman correlation (rs) between the two methods was 0.89. The different modes of evaluation and color reference selection are likely to have contributed to the differences in the LI ranges observed in the two methods. The high correlation between PCNA quantification by CAIA and visual scoring by experienced technicians indicates that CAIA can reliably rank individual subjects. Thus, measurement of PCNA labeling by CAIA is a practical alternative for evaluating colorectal epithelial cell proliferation.

Oxygen radical production and thiol depletion are required for Ca(2+)-mediated endogenous endonuclease activation in apoptotic thymocytes.

Glucocorticoid hormones stimulate apoptosis in thymocytes via a mechanism that involves changes in intracellular Ca2+, and exogenous Ca2+ can also directly promote the nuclear alterations of apoptosis (lamin degradation and chromatin cleavage) in isolated nuclei. Here we report that glucocorticoid treatment resulted in the production of reactive oxygen species and the depletion of reduced glutathione. Separation of apoptotic cells on Percoll gradients demonstrated that both effects selectively occurred in thymocytes undergoing apoptosis. Moreover, glucocorticoid-induced endonuclease activation was partially blocked by the antioxidant N-acetyl-L-cysteine. Although abrogation of methylprednisolone-induced Ca2+ increases using the intracellular Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid resulted in inhibition of endonuclease activation, it failed to prevent GSH depletion. However, N-acetyl-L-cysteine almost completely blocked methylprednisolone-induced elevations in cytosolic calcium levels, indicating that oxidative stress was playing a role in the Ca2+ response. Our results support the idea that oxidative stress is a key component of the apoptotic effector pathway in thymocytes, and that it interacts, at least in part, with the Ca2+ response.

Calcium and colorectal epithelial cell proliferation in sporadic adenoma patients: a randomized, double-blinded, placebo-controlled clinical trial.

BACKGROUND: The kinetics of colorectal epithelial cell proliferation is altered in patients at increased risk for colon cancer. Calcium administration ameliorates such proliferative changes in rodents. Findings in preliminary clinical trials have suggested similar effects in humans. PURPOSE: A randomized, double-blind, placebo-controlled, clinical trial was designed to determine whether calcium supplementation will reduce the colorectal epithelial cell proliferation rate and normalize the distribution of proliferating cells within colorectal crypts (i.e., shift the zone of proliferation from the entire crypt to the lower 60% of the crypt, which is thought to be the normal proliferative zone of the crypt) in patients with sporadic adenomas. METHODS: Sporadic adenoma patients (n = 193) were treated with placebo (n = 66), 1.0 g calcium (n = 64), or 2.0 g calcium (n = 63) daily for 6 months. Rectal mucosa biopsy specimens were obtained at base line and at 1-, 2-, and 6-month follow-up. Cell proliferation was measured by detection of S-phase-associated proliferating cell nuclear antigen by immunohistochemical methods. The cell proliferation rate, called labeling index (LI), was calculated as the proportion of labeled cells in the crypts. The deviation of the proliferative zone from the normal location in the lower 60% of the crypt was calculated as the proportion of labeled cells in the upper 40% of the crypt, called distributional index (phi h). The effects of calcium treatment on the LI and phi h were expressed as relative effects--(calcium follow-up/calcium base line)/(placebo follow-up/placebo base line). Calculations and inference testing of the relative effects were accomplished using a repeated-measures mixed model on log-transformed LI and phi h values. All statistical tests were two-sided. RESULTS: Scorable biopsy specimens were obtained on 170 patients at base line, 164 at 1 month, 161 at 2 months, and 163 at 6 months. The difference in the change in the LI between the combined calcium groups and the placebo group was insignificant, with a relative effect of calcium versus placebo of 0.97 (P = .87). However, for the phi h, the relative effect of calcium versus placebo was 0.50 (P = .05) in the combined calcium groups, 0.56 (P = .16) in the 1.0-g calcium group, and 0.44 (P = .05) in the 2.0-g calcium group. CONCLUSIONS: Calcium supplementation normalizes the distribution of proliferating cells without affecting the proliferation rate in the colorectal mucosa of sporadic adenoma patients. IMPLICATIONS: These results support further study of whether alterations in colon cell proliferative kinetics represent true intermediate steps in colon carcinogenesis that can be used to investigate the etiology and prevention of, and whether a higher calcium consumption can reduce the risk of, colon cancer.