Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Evacetrapib and Cardiovascular Outcomes in High-Risk Vascular Disease

BACKGROUND: The cholesteryl ester transfer protein inhibitor evacetrapib substantially raises the high-density lipoprotein (HDL) cholesterol level, reduces the low-density lipoprotein (LDL) cholesterol level, and enhances cellular cholesterol efflux capacity. We sought to determine the effect of evacetrapib on major adverse cardiovascular outcomes in patients with high-risk vascular disease. METHODS: In a multicenter, randomized, double-blind, placebo-controlled phase 3 trial, we enrolled 12,092 patients who had at least one of the following conditions: an acute coronary syndrome within the previous 30 to 365 days, cerebrovascular atherosclerotic disease, peripheral vascular arterial disease, or diabetes mellitus with coronary artery disease. Patients were randomly assigned to receive either evacetrapib at a dose of 130 mg or matching placebo, administered daily, in addition to standard medical therapy. The primary efficacy end point was the first occurrence of any component of the composite of death from cardiovascular causes, myocardial infarction, stroke, coronary revascularization, or hospitalization for unstable angina. RESULTS: At 3 months, a 31.1% decrease in the mean LDL cholesterol level was observed with evacetrapib versus a 6.0% increase with placebo, and a 133.2% increase in the mean HDL cholesterol level was seen with evacetrapib versus a 1.6% increase with placebo. After 1363 of the planned 1670 primary end-point events had occurred, the data and safety monitoring board recommended that the trial be terminated early because of a lack of efficacy. After a median of 26 months of evacetrapib or placebo, a primary end-point event occurred in 12.9% of the patients in the evacetrapib group and in 12.8% of those in the placebo group (hazard ratio, 1.01; 95% confidence interval, 0.91 to 1.11; P=0.91). CONCLUSIONS: Although the cholesteryl ester transfer protein inhibitor evacetrapib had favorable effects on established lipid biomarkers, treatment with evacetrapib did not result in a lower rate of cardiovascular events than placebo among patients with high-risk vascular disease. (Funded by Eli Lilly; ACCELERATE ClinicalTrials.gov number, NCT01687998 .)

The receptor for advanced glycation endproducts and S100A11 modulate pathologic chondrocyte differentiation and dysregulated cartilage matrix catabolism in osteoarthritis

Changes within the synovium and cartilage associated with low-grade inflammation modulate the pathogenesis of OA, and many studies have shown that the inflammatory cytokines/chemokines IL-1[beta], TNF[alpha], CXCL1 and CXCL8 significantly contribute to the disease progression. The Receptor for Advanced Glycation Endproducts (RAGE) and S100/calgranulins have been implicated in the pathogenesis of chronic arterial, renal, and neurological degenerative states associated with low-grade tissue inflammation. Therefore, the studies in this dissertation proposed that the association of RAGE and its ligands (specifically S100A11) are important in the pathogenesis of OA. RAGE and S100A11 expression were upregulated in OA cartilage compared to normal cartilage. CXCL8-, TNF[alpha]-, and S100A11-induced chondrocyte hypertrophy were suppressed by treatment with soluble RAGE (sRAGE) or RAGE-specific blocking antibodies. Finally, it was determined that S100A11 induced MKK3 and p38 MAPK activation.S100/ calgranulins normally exist as homo/heterodimers and the type of bond formed and subsequent conformation affect their activities. As it was previously determined that S100A11 was a substrate for Transglutaminase 2 (TG2), an S100A11 transamidation mutant was created (S100A11 K3R/ Q102N), which formed only monomers. In mouse cartilage explants and chondrocytes, S100A11 K3R/Q102N mutant lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans (GAG) release. S100A11 failed to induce hypertrophy and GAG release in RAGE-/- and TG2-/- cartilages. The role of RAGE in the pathogenesis of OA was examined in vivo. Instability was induced surgically on RAGE-/- and congenic wild-type controls with an anterior cruciate ligament tear (ACL-T) through a blind "stab" incision. Cartilage degeneration, osteophyte formation and type X collagen expression significantly increased as instability increased, but there was no significant difference between RAGE-/- and congenic wild-type controls. Though RAGE gene deletion does not protect cartilage from degeneration in the ACL-T model of OA, the function of RAGE has not been assessed in another model of surgically-induced OA. In addition, the role of RAGE ligands, involvement of other receptors that recognize RAGE ligands and sRAGE have not been addressed. Taken together, the studies in this dissertation demonstrate critical roles for RAGE and S100A11 in modulating pathologic chondrocyte hypertrophic differentiation and dysregulated matrix catabolis

Abstract 1571: Developing antigen-specific Th1 epitope based vaccines by excluding immunosuppressive peptide sequences

Abstract We have determined that IGFBP-2 is immunogenic in ovarian cancer. Our aim is to develop a multi-epitope vaccine that will elicit Th1 immunity to IGFBP-2. Antigen specific Th1 can modulate the tumor microenvironment to enhance cross priming, supporting the proliferation of cytotoxic T cells which are capable of eradicating ovarian cancer cells. Although some clinical benefit has been demonstrated with this strategy, recent ex vivo analyses of human PBMC have described the presence of antigen-specific CD4+ T regulatory cell (Tregs) in cancer patients that were not detected in healthy individuals. Thus, we questioned if we could optimize our vaccine to include only Th1-stimulating epitopes. Using a combined scoring system from five algorithms for predicting class II binding to determine Th epitopes, we identified 14 IGFBP-2 peptides. Th1 immunogenicity (IFN-gamma) and potential immunosuppression (IL-10) was evaluated by ELISPOT for 40 different donors. Twenty-two percent of the donors only responded with IL-10 secretion to any peptide, 22% only responded with IFN-gamma and 53% of the patients had a mixed IFN-gamma and IL-10 response. To determine which peptides would induce a predominantly Th1 response in the greatest number of people, we used a ratio of IFN-gamma to IL-10 and analyzed both the magnitude and frequency of ELISPOT responses for each of peptides using the following algorithm: (corrected mean SPW) x (percent of responding donors). The peptides were then ranked from highest IL-10 response to highest IFN-gamma response. Interestingly, 6 of the 14 peptides demonstrated a preference to secrete IFN-gamma over IL-10 and are only located in the N-terminus (amino acids 1-163) of IGFBP-2; the remaining potentially immunosuppressive peptides are located in the C-terminus (amino acids 164-328). Vaccination with p1-163 in MMTV-neu mice demonstrated a robust Th1 response (p=0.03) and concomitant inhibition of tumor growth by 70% compared to adjuvant only control animals, p164-328-vaccinated or full length protein-vaccinated mice (p&amp;lt;0.001 for all). Vaccination with p164-328 or full length protein did not inhibit tumor growth. These data suggest that more effective vaccines can be designed when both Th1 epitopes and immunosuppressive epitopes are screened simultaneously and epitopes that are most likely to induce robust Th1 responses in the majority of individuals can be identified and included as vaccine components. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1571. doi:1538-7445.AM2012-1571</jats:p

Elimination of IL-10–Inducing T-Helper Epitopes from an IGFBP-2 Vaccine Ensures Potent Antitumor Activity

Immunization against self-tumor antigens can induce T-regulatory cells which inhibit proliferation of Type I CD4(+) T-helper (Th1) and CD8(+) cytotoxic T-cells. Type I T-cells are required for potent anti-tumor immunity. We questioned whether immunosuppressive epitopes could be identified and deleted from a cancer vaccine targeting IGFBP-2 and enhance vaccine efficacy. Screening breast cancer patient lymphocytes with IFN-γ and IL-10 ELISPOT, we found epitopes in the N-terminus of IGFBP-2 that elicited predominantly Th1 while the C-terminus stimulated Th2 and mixed Th1/Th2 responses. Epitope-specific Th2 demonstrated a higher functional avidity for antigen than epitopes which induced IFN-γ (p=0.014). We immunized TgMMTV-neu mice with DNA constructs encoding IGFBP-2 N-and C-termini. T-cell lines expanded from the C-terminus vaccinated animals secreted significantly more Type II cytokines than those vaccinated with the N-terminus and could not control tumor growth when infused into tumor-bearing animals. In contrast, N-terminus epitope-specific T-cells secreted Th1 cytokines and significantly inhibited tumor growth, as compared with naïve T-cells, when adoptively transferred (p=0.005). To determine whether removal of Th2 inducing epitopes had any effect on the vaccinated anti-tumor response, we immunized mice with the N-terminus, C-terminus and a mix of equivalent concentrations of both vaccines. The N-terminus vaccine significantly inhibited tumor growth (p<0.001) as compared to the C-terminus vaccine which had no anti-tumor effect. Mixing the C-terminus with the N-terminus vaccine abrogated the anti-tumor response of the N-terminus vaccine alone. The clinical efficacy of cancer vaccines targeting self-tumor antigens may be greatly improved by identification and removal of immunosuppressive epitopes