Mutagenesis studies have demonstrated the requirement for the CD28-responsive element (CD28RE) within the interleukin-2 (IL-2) promoter for transcriptional upregulation by CD28. Here, we demonstrate that CD28 responsiveness is conferred by a composite element containing both the CD28RE and the NF-IL-2B AP-1 sites (RE/AP). Mutations at either site within the RE/AP composite element abolish activity. The RE/AP composite element is a site for signal integration within the IL-2 promoter, since its activation is dependent on at least two separate signalling pathways being activated, through the T-cell receptor, CD28, and/or phorbol myristate acetate. Activation is maximal when all three signals occur simultaneously. By using a panel of CD28 cytoplasmic domain mutants, it was found that the transcriptional activation of the RE/AP composite element correlates exactly with the pattern of IL-2 secretion induced by these mutants upon stimulation. Similar to the upregulation of IL-2 secretion, the transcriptional upregulation of the RE/AP composite element by CD28 is FK506 insensitive. The pattern of activation of the RE/AP composite element is different from that observed for either an NFAT or consensus AP-1 site, implying that RE/AP represents a unique element. Using gel shift analysis, we demonstrate that stimulation by CD28 induces the association of the NF-B family member c-Rel to the CD28RE within the RE/AP composite element. The transcriptional upregulation of IL-2 by CD28 appears, therefore, to be mediated through the RE/AP composite element, involving the association of c-Rel with the CD28RE.Activation of T cells by recognition of antigens and major histocompatibility complex products on the surfaces of antigen-presenting cells initiates a series of biochemical events leading to T-cell proliferation and cytokine secretion (33). Stimulation through the T-cell receptor (TCR) alone is insufficient for T-cell proliferation or interleukin-2 (IL-2) production. In the absence of costimulation, a state of unresponsiveness, termed anergy, may develop (13, 18). The failures to produce and respond to IL-2 are the major determinants of anergy. CD28, a 44-kDa glycoprotein expressed as a homodimer on most T cells, can mediate costimulation. Induction of anergy in T-cell clones was found to be blocked by activation of CD28 by monoclonal antibodies (7).One consequence of T-cell activation is the expression of IL-2, due to both transcriptional upregulation and stabilization of IL-2 mRNA. Transcriptional upregulation is mediated by the IL-2 promoter, contained within approximately 300 bp upstream from the transcriptional start site (reviewed in references 11 and 22). Engagement of CD28 enhances activation of the IL-2 promoter resulting from either TCR stimulation or use of phorbol esters such as phorbol myristate acetate (PMA). Mutagenesis studies have demonstrated that a CD28-responsive element (CD28RE), located within the IL-2 promoter between Ϫ160 and Ϫ152 relative to the transcriptional start site, is required for CD28-induced ...

Background: Poor glycemic control is a risk factor for microvascular complications in patients with type 2 diabetes mellitus. Achieving glycemic control safely with insulin therapy can be challenging.Methods: A prospective, 16-week, multicenter, randomized, double-blind, placebo-controlled, parallelgroup study conducted at 50 sites in the United States and 1 site in Mexico between August 12, 2004, and December 28, 2005. Subjects had type 2 diabetes mellitus that was not adequately controlled (glycated hemoglobin level, 7.5%-9.5%, inclusive) receiving insulin therapy alone or in combination with oral antidiabetes agents. In total 287 subjects (52% men; mean age, 57 years; with a mean baseline glycated hemoglobin level of 8.3%) were randomized: 147 to receive colesevelam hydrochloride, 3.75 g/d, and 140 to receive placebo.Results: Using the least squares method, the mean (SE) change in glycated hemoglobin level from baseline to week 16 was −0.41% (0.07%) for the colesevelam-treated group and 0.09% (0.07%) for the placebo group (treatment difference, −0.50% [0.09%]; 95% confidence interval, −0.68% to −0.32%; P Ͻ .001). Consistent reductions in fasting plasma glucose and fructosamine levels, glycemic-control response rate, and lipid control measures were observed with colesevelam. As expected, the colesevelam-treated group had a 12.8% reduction in low-density lipoprotein cholesterol concentration relative to placebo (PϽ.001). Of recipients of colesevelam and placebo, respectively, 30 and 26 discontinued the study prematurely; 7 and 9 withdrew because of protocol-specified hyperglycemia, and 10 and 4 withdrew because of adverse events. Both treatments were generally well tolerated.Conclusions: Colesevelam treatment seems to be safe and effective for improving glycemic control and lipid management in patients with type 2 diabetes mellitus receiving insulin-based therapy, and it may provide a novel treatment for improving dual cardiovascular risk factors.