Design and Rationale for the Veterans Affairs "Cooperative Study Program 594 Comparative Effectiveness in Gout: Allopurinol vs. Febuxostat" Trial.

BACKGROUND: Gout patients do not routinely achieve optimal outcomes related in part to suboptimal administration of urate lowering therapy (ULT) including first-line xanthine oxidase inhibitors allopurinol or febuxostat. Studies leading to the approval of febuxostat compared this agent to allopurinol in inappropriately low, fixed doses. We will compare allopurinol with febuxostat in gout using appropriately titrated doses of both agents and a "treat-to-target" strategy congruent with specialty guidelines. METHODS: We have planned and initiated the Veterans Affairs (VA) Cooperative Study Program (CSP) 594, Comparative Effectiveness in Gout: Allopurinol vs Febuxostat study. This large double-blind, non-inferiority trial will enroll 950 gout patients randomized to receive allopurinol or febuxostat. Patients will be followed for a total of 72 weeks encompassing 3 distinct 24-week study phases. During Phase I (0-24 weeks), participants will undergo gradual dose titration of ULT until achievement of serum uric acid (sUA) <6.0 mg/dL or <5.0 mg/dL if tophi are present. Dose escalation will not be allowed during final three study visits of Phase 2 (24-48 weeks) and during Phase 3 (48-72 weeks). The primary study outcome is the proportion of participants experiencing at least one gout flare during Phase 3. Subsequent to the 72-week study, participants will be followed passively for up to 10 years after the study to assess long-term health outcomes. CONCLUSION: With its completion, the VA Comparative Effectiveness in Gout: Allopurinol vs Febuxostat study will demonstrate the central role of gradual ULT dose escalation and a treat-to-target strategy in gout management.

Selective Inhibition of CBX6: A Methyllysine Reader Protein in the Polycomb Family.

The polycomb paralogs CBX2, CBX4, CBX6, CBX7, and CBX8 are epigenetic readers that rely on "aromatic cage" motifs to engage their partners' methyllysine side chains. Each CBX carries out distinct functions, yet each includes a highly similar methyllysine-reading chromodomain as a key element. CBX7 is the only chromodomain that has yet been targeted by chemical inhibition. We report a small set of peptidomimetic agents in which a simple chemical modification switches the ligands from one with promiscuity across all polycomb paralogs to one that provides selective inhibition of CBX6. The structural basis for this selectivity, which involves occupancy of a small hydrophobic pocket adjacent to the aromatic cage, was confirmed through molecular dynamics simulations. Our results demonstrate the increases in affinity and selectivity generated by ligands that engage extended regions of chromodomain binding surfaces.

Chromodomain antagonists that target the polycomb-group methyllysine reader protein chromobox homolog 7 (CBX7).

We report here a peptide-driven approach to create first inhibitors of the chromobox homolog 7 (CBX7), a methyllysine reader protein. CBX7 uses its chromodomain to bind histone 3, lysine 27 trimethylated (H3K27me3), and this recognition event is implicated in silencing multiple tumor suppressors. Small trimethyllysine containing peptides were used as the basic scaffold from which potent ligands for disruption of CBX7-H3K27me3 complex were developed. Potency of ligands was determined by fluorescence polarization and/or isothermal titration calorimetry. Binding of one ligand was characterized in detail using 2D NMR and X-ray crystallography, revealing a structural motif unique among human CBX proteins. Inhibitors with a ∼200 nM potency for CBX7 binding and 10-fold/400-fold selectivity over related CBX8/CBX1 proteins were identified. These are the first reported inhibitors of any chromodomain.

Synthetic trimethyllysine receptors that bind histone 3, trimethyllysine 27 (H3K27me3) and disrupt its interaction with the epigenetic reader protein CBX7.

Post-translational modifications act as 'on' or 'off' switches causing downstream changes in gene transcription. Modifications such as trimethylation of lysine 27 on histone H3 (H3K27me3) cause repression of transcription and stable gene silencing, and its presence is associated with aggressive cancers of many types. We report here macrocyclic host-type compounds that can bind H3K27me3 preferentially over unmethylated H3K27, and characterize their binding affinities and selectivities using a convenient dye-displacement method. We also show that they can disrupt the protein-protein interaction of H3K27me3 with the chromobox homolog 7 (CBX7), a methyllysine reader protein, using fluorescence polarization. These results show that sub-micromolar potencies are achievable with this family of host compounds, and suggest the possibility of their use as new tools to induce the disruption of methyllysine-mediated protein-protein interactions and to report on lysine methylation in vitro.

Analysis of anti-protective antigen IgG subclass distribution in recipients of anthrax vaccine adsorbed (AVA) and patients with cutaneous and inhalation anthrax.

The anti-PA IgG1, IgG2, IgG3, and IgG4 subclass responses to clinical anthrax and to different numbers of anthrax vaccine adsorbed (AVA, BioThrax) injections were determined in a cross-sectional study of sera from 63 vaccinees and 13 clinical anthrax patients. The data show that both vaccination with three AVA injections and clinical anthrax elicit anti-PA IgG1, IgG2, and IgG3 subclass responses. An anti-PA IgG4 response was detected in AVA recipients after the fourth injection. The anthrax lethal toxin (LTx) neutralization efficacy of sera from recipients who received 4 to > or =10 AVA injections did not vary significantly in relation to changes in distribution of anti-PA IgG1 and IgG4 subclasses.

Defective maturation of dendritic cells in common variable immunodeficiency.

Monocyte-derived dendritic cells (MdDCs) from many patients with common variable immunodeficiency (CVID) have been shown recently to have reduced expression of surface molecules associated with maturity. Using flow cytometry and confocal microscopy, we now show that this is due to a partial failure to fix Class II DR molecules on the surface during procedures that induce full maturation in vitro in cells from normal subjects. Major histocompatibility complex (MHC) class I, CD86 and CD83 expression were expressed normally, but CD40 was reduced. These abnormalities are unlikely to be due to prior in vivo exposure of monocytes to lipopolysaccharide (LPS), as addition of LPS to monocytes from normal subjects in vitro caused a different pattern of changes. CVID MdDCs retained Class II DR in the cytoplasm during maturation, showed increased internalization of cross-linked Class II DR surface molecules and were unable to polarize DR within a lipid raft at contact sites with autologous lymphocytes. These cells retained some features of monocytes, such as the ability to phagocytose large numbers of fixed yeast and fluorescent carboxylated microspheres and expression of surface CD14. These abnormalities, if reflected in vivo, could compromise antigen presentation and may be a fundamental defect in the mechanism of the antibody deficiency in a substantial subset of CVID patients.

Patterns of food allergen-specific cytokine production by T lymphocytes of children with multiple allergies.

BACKGROUND: The contribution of different T cell subsets to the overall measured cytokine response to food allergens is largely unexplored. METHOD: The patterns of cytokine production of peripheral blood-derived T cells after allergen stimulation were studied in 22 children with multiple food allergies and in 20 non-allergic children as controls, using flow cytometry. RESULTS: Proportions of T cells of food-sensitized children spontaneously secreting IFN-gamma and IL-10 (without antigen stimulation) were lower than non-atopic children and adult controls (P

Severe illness and death among injecting drug users in Scotland: a case-control study.

Between April and September 2000, 60 injecting drug users in Scotland died or were hospitalized with severe illness. Laboratory investigations suggested that Clostridium novyi and other bacteria were important aetiological agents. To determine associated environmental/behavioural factors a case-control study was undertaken with 19 'definite' and 32 'probable' cases in Glasgow, Scotland. For every deceased case (n=19), up to three proxy individuals were interviewed. Three controls were identified for each case. Multivariate logistic regression analyses compared (i) all cases and controls; (ii) definite cases and matched controls; (iii) probable cases and matched controls. In all three analyses injecting into muscle or skin and injecting most of the time with a filter used by someone else were the variables most strongly associated with illness. Comparing only muscle-injecting cases and controls, cases were significantly more likely to have injected larger amounts of heroin per average injection than were controls. The findings make an important epidemiological contribution to the understanding of the public health and clinical implications of the contamination of illicit drugs by histotoxic clostridia.

Monocyte derived dendritic cell responses in common variable immunodeficiency.

The phenotype and function of monocyte derived dendritic cells (MdDC) were investigated in 25 patients with common variable immunodeficiency (CVID) to test for abnormalities that might help explain the failure of antibody production. Using MHC class II DR and CD86 as markers of maturation, DCs from the majority of CVID patients were normal. However 5 patients, the majority of whom had affected family members who had previously been shown to have a susceptibility genetic locus in the MHC region, expressed abnormally low levels of DR on repeated testing, in some cases associated with a reduced capacity to support antigen stimulated T cell proliferation; nevertheless costimulatory molecules for production of IL-13, IL-10 and IFN-gamma from T cells were intact. In contrast to DCs from healthy donors, DCs from many CVID patients had high spontaneous production of IL-8 and lipopolysaccharide stimulation often caused a reduction in DR expression. Expression of other cytokines (IL-1a, IL-6 and IL-12), either before or after LPS stimulation, was normal. The data suggests there is a fundamental defect in the maturation of MdDCs in a subset of CVID patients that may compromise antigen presentation and subsequent antibody production.

Mass value assignment of total and subclass immunoglobulin G in a human standard anthrax reference serum.

An anti-Anthrax Vaccine Adsorbed (anti-AVA) standard human reference serum pool, AVR414, has been prepared, and the total and protective antigen (PA)-specific immunoglobulin G (IgG) were quantified. AVR414 was prepared by plasmapheresis of healthy adults who had received a minimum of four subcutaneous injections of AVA. Mass values (in milligrams per milliliter) for total IgG and IgG subclasses 1 to 4 were determined by radial immunodiffusion. Anti-PA-specific IgG assignment (in micrograms per milliliter) was done by consensus of two complementary approaches: homologous enzyme-linked immunosorbent assay (ELISA) with affinity-purified anti-PA IgG as a calibrator and summation of mean PA-specific IgG subclass concentrations determined by IgG subclass-specific ELISA using the United States National Reference Preparation for Human Serum Proteins as a standard. The total IgG concentration assigned to AVR414 reference serum was 8.33 mg/ml. IgG subclass concentrations were the following: for IgG1, 4.48 mg/ml; for IgG2, 3.35 mg/ml; for IgG3, 0.37 mg/ml; and for IgG4, 0.30 mg/ml. The assigned mass value for total anti-PA-specific IgG was 141.2 microg/ml. Anti-PA-specific IgG subclass concentrations were the following: for IgG1, 79.6 microg/ml; for IgG2, 35.3 microg/ml; for IgG3, 3.2 microg/ml; and for IgG4, 25.3 microg/ml. Human reference serum pool AVR414 will have direct application in the standardization of anthrax serological assays, in reagent qualification, and as a standard for quantification of PA-specific IgG in humans who have been vaccinated with or otherwise exposed to Bacillus anthracis PA.

The shape of the dose-response curve for radiation-induced neoplastic transformation in vitro: evidence for an adaptive response against neoplastic transformation at low doses of low-LET radiation.

A dose-response curve for gamma-radiation-induced neoplastic transformation of HeLa x skin fibroblast human hybrid cells over the dose range 0.1 cGy to 1 Gy is presented. In the experimental protocol used, the spontaneous (background) frequency of neoplastic transformation of sham-irradiated cultures was compared to that of cultures which had been irradiated with (137)Cs gamma radiation and either plated immediately or held for 24 h at 37 degrees C prior to plating, for assay for neoplastic transformation. The pooled data from a minimum of three repeat large-scale experiments at each dose demonstrated a reduced transformation frequency for the irradiated compared to the sham-irradiated cells for doses of 0.1, 0.5, 1, 5 and 10 cGy for the delayed-plating arm. The probability of this happening by chance is given by 1/2(n), where n is the number of observations (5); i.e., 1/32 congruent with 0.031. This is indicative of an adaptive response against spontaneous neoplastic transformation at least up to a dose of 10 cGy of gamma radiation. The high-dose data obtained at 30 and 50 cGy and 1 Gy showed a good fit to a linear extrapolation through the sham-irradiated, zero-dose control. The delayed-plating data at 10 cGy and below showed a statistically significant divergence from this linear extrapolation.

Racial and ethnic colorectal cancer patterns affect the cost-effectiveness of colorectal cancer screening in the United States.

BACKGROUND & AIMS: Colorectal cancer screening beginning at age 50 is recommended for all Americans considered at "average" risk for the development of colorectal cancer. METHODS: We used 1988-1995 California Cancer Registry data to compare the cost-effectiveness of two 35-year colorectal cancer screening interventions among Asians, blacks, Latinos, and Whites. RESULTS: Average annual age-specific colorectal cancer incidence rates were highest in blacks and lowest in Latinos. Screening beginning at age 50 was most cost-effective in blacks and least cost-effective in Latinos (measured as dollars spent per year of life saved), using annual fecal occult blood testing (FOBT) combined with flexible sigmoidoscopy every 5 years and using colonoscopy every 10 years. A 35-year screening program beginning in blacks at age 42, whites at age 44, or Asians at age 46 was more cost-effective than screening Latinos beginning at age 50. CONCLUSIONS: Colorectal cancer screening programs beginning at age 50, using either FOBT and flexible sigmoidoscopy or colonoscopy in each racial or ethnic group, are within the $40,000-$60,000 per year of life saved upper cost limit considered acceptable for preventive strategies. Screening is most cost-effective in blacks because of high age-specific colorectal cancer incidence rates.

The topography of colorectal cancer varies by race/ethnicity and affects the utility of flexible sigmoidoscopy.

Colorectal cancer screening beginning at age 50 is recommended for all Americans considered at "average" risk for the development of colorectal cancer either with flexible sigmoidoscopy and fecal occult blood testing (FOBT) or with colonoscopy. Patients who elect flexible sigmoidoscopy and FOBT undergo full colonoscopy only if left-sided neoplasia is detected or if the FOBT is positive. Unfortunately in blacks and whites most right-sided colorectal lesions are unaccompanied by left-sided sentinel lesions, which leads some to prefer colonoscopic screening in these patients. The topography of colorectal cancer in Asians and Latinos is unavailable. We used 1988-1995 California Cancer Registry data to determine the topography of 105,906 consecutive colorectal cancers among Asian, black, Latino, and white patients. We found that the proportion of colorectal cancer distal to the splenic flexure and therefore detectable by flexible sigmoidoscopy varied by ethnicity: Asian (71%) > Latino (63%) > white (57%) > black (55%); P < 0.001. These differences were significant after adjusting for age and sex. The risk of distal disease relative to whites was 1.61 in Asians, 1.15 in Latinos, and 0.82 in blacks (P < 0.001). Flexible sigmoidoscopy detects a higher proportion of colorectal cancers in Asians and Latinos than in whites or blacks. Further study is needed to assess whether the topography of benign colorectal neoplasia parallels that of malignant disease. Colorectal screening recommendations may need to incorporate racial and ethnic differences in colorectal neoplasia topography.

DNA vaccines.

The premise that DNA coding for antigens produces proteins to stimulate the immune system when inoculated directly into muscle tissues, has the immense attractions of simplicity, versatility and economy. When other vaccination approaches are experiencing practical problems, meeting such challenges as AIDS and malaria, considerable attention has focused on DNA vaccines with entire conferences and a flood of commercial companies devoted to exploring the possibilities. A number of clinical trials for both infectious diseases and cancer have already commenced, even though a number of major issues have to be resolved.

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines.

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.

Cancer risk estimates for family members of a population-based family registry for breast and ovarian cancer.

Population-based breast and ovarian cancer family registries can facilitate studies to evaluate genetic and environmental factors in the etiology of these malignancies. The purpose of this study is to describe what is, as far as we know, the first population-based breast and ovarian cancer family registry and to estimate breast and ovarian cancer risk in relatives of breast and ovarian cancer probands. Population-based consecutive incident cases of breast and ovarian cancer were invited to participate in the University of California, Irvine breast and ovarian family registry. In this study, we report data on 1567 breast cancer and 328 ovarian cancer probands. The operational components of this family registry include enrollment of probands, family history interviewing, confidentiality, pathology, verification and review, biospecimen bank, statistical/genetic analysis, and special studies on positional cloning of known genes. All of the components are tracked through the University of California, Irvine Genetic Research Information System. In non-Hispanic-white breast cancer probands, relative risk (RR) of breast cancer in mothers and sisters is significantly elevated [RR = 1.7 and 95% confidence interval (CI) = 1.4-2.0 and RR = 2.8 and 95% CI = 2.3-3.3, respectively]. In families of ovarian cancer probands, mothers are at increased risk of ovarian cancer (RR = 4.6; 95% CI, 2.1-8.7). RR of breast cancer in mothers of Hispanic breast cancer probands is significantly elevated (RR = 4.9; 95% CI, 2.6-8.5). No elevation of breast or ovarian cancer risk was observed among relatives of Asian probands. In general, there is a decrease in RR among mothers and sisters with increase in age of onset of probands. In second-degree relatives and first cousins, the breast cancer hazards ratios increase with increase in the number of affected first-degree relatives and decrease with increase in age at onset of the proband.

Clinical modulation of oral leukoplakia and protease activity by Bowman-Birk inhibitor concentrate in a phase IIa chemoprevention trial.

Bowman-Birk inhibitor is a protease inhibitor derived from soybeans that has demonstrated chemopreventive activity in a number of in vitro and animal systems. We conducted a 1-month phase IIa clinical trial of Bowman-Birk inhibitor concentrate (BBIC) in patients with oral leukoplakia. BBIC was administered to 32 subjects with oral leukoplakia for 1 month. We assessed toxicity and clinical and histological response of the lesions, and oral mucosal cell protease activity (PA) and serum micronutrient levels were measured. Clinical response was determined by measurement of pre- and posttreatment individual and total lesion areas and analysis of blinded clinical judgments of photographs. On the basis of prespecified response criteria, 31% of patients achieved a clinical response (two with complete and eight with partial responses). BBIC was nontoxic in doses up to 1066 chymotrypsin inhibitory units. The mean pretreatment total lesion area decreased from 615 to 438 mm2 after BBIC treatment (P < 0.004). A linear fit of the dose-response relationship between dose of BBIC and decrease in total lesion area was suggested (P < 0.08), and analysis of blinded clinical impression from lesion photographs confirmed this relationship (P < 0.01). Overall, at all doses tested, a 24.2% decrease in total lesion area was observed following treatment (sign rank = -142; P < 0.004). High pretreatment PA was associated with greater decreases in PA after BBIC administration (P < 0.02). BBIC demonstrated clinical activity after oral administration to patients with oral leukoplakia. These results indicate that BBIC should be investigated for chemopreventive activity in a randomized clinical trial.

Survival differences in breast cancer among racial/ethnic groups: a population-based study.

In women, breast cancer is the most frequent solid tumor and the second leading cause of cancer death. Differences in survival of breast cancer have been noted among racial/ethnic groups, but the reasons are unclear. This report presents the characteristics and the survival experience of four racial/ethnic groups and evaluates the effects of stage, age, histology, and treatment on survival time. The distributions of prognostic factors and treatment among racial/ethnic groups are compared using female breast cancer patients from two population-based registries in Southern California. The main end points are observed survival time and survival by cause of death. The Cox model is used to estimate the relative risk of death in three minority groups compared with non-Hispanic whites, while controlling for several covariates. Breast cancer cases included in this study were 10,937 non-Hispanic whites, 185 blacks, 875 Hispanics, and 412 Asians. The median follow-up period was 76 months (range: 48-132). The median age at diagnosis was 64 years among non-Hispanic whites, 55 years among Hispanics (p = 0.001), 52 years among blacks (p = 0.001), and 50 years among Asians (p = 0. 001). There was more localized disease among non-Hispanic whites (61. 4%) than among blacks (50.8%) and Hispanics (52.2%), but not compared to Asians (59.7%). After controlling for stage, age, histology, treatment, and registry, overall survival significantly differed between non-Hispanic whites and blacks [relative risk (RR) = 2.27, 95% confidence interval (95% CI) 1.82-2.84) and between non-Hispanic whites and Hispanics (RR = 1.18, 95% CI 1.04-1.34). The same results were found for breast cancer death in blacks (RR = 2.32, 95% CI 1.76-3.07) and Hispanics (RR = 1.28, 95% CI 1.10-1.50). We found no difference between Asians and non-Hispanic whites in overall and cancer-related survival. These results show that stage of disease, age at diagnosis, histologic features and treatment for breast cancer differed among racial/ethnic groups. Moreover, black women, in particular, and Hispanic women with breast cancer had a higher risk of death compared to non-Hispanic white women, even after controlling for prognostic factors. These findings underline the necessity of improved screening and access to appropriate treatment among minority women for breast cancer.