Expression of p21 has been shown to be up-regulated by the p53 tumor suppressor gene in vitro in response to DNA-damaging agents. However, p21 expression can be regulated independently of p53, and here we show that expression of p21 in various tissues during development and in the adult mouse occurs in the absence of p53 function. However, most tissues tested did require p53 for p21 induction following exposure of the whole animal to ~/irradiation. These results show that normal tissue expression of p21 to high levels is not dependent on p53 and confirm that induction of p21 by DNA-damaging agents does require p53. p21 is expressed upon differentiation of p53-deficient murine erythroleukemia (MEL1 cells, and the kinetics of induction of p21 in this system suggest that it may be involved in the growth arrest that precedes terminal differentiation. The gene is up-regulated in mouse fibroblasts in response to serum restimulation but the kinetics and levels of induction differ between wild-type and mutant cells. Expression of p21 message following serum restimulation is superinducible by cycloheximide in wild-type but not in p53-deficient cells. The increases in p21 mRNA are reflected in changes in p21 protein levels, p21 expression also appears to be regulated at the post-transcriptional level because moderate increases in mRNA expression, during differentiation of MEL cells and upon serum restimulation of fibroblasts, are followed by large increases in protein levels. Regulation of the mouse p21 promoter by p53 depends on two critical p53-binding sites located 1.95 and 2.85 kb upstream from the transcriptional initiation site. The sequences mediating serum responsiveness of the promoter map to a region containing the proximal p53 site. p53 appears to play a critical role in p21 induction following DNA damage. Moreover, p21 can be regulated independently of p53 in several situations including during normal tissue development, following serum stimulation, and during cellular differentiation.
Summary Background Findings of small studies have suggested that short treatments with anti-CD3 monoclonal antibodies that are mutated to reduce Fc receptor binding preserve β-cell function and decrease insulin needs in patients with recent-onset type 1 diabetes. In this phase 3 trial, we assessed the safety and efficacy of one such antibody, teplizumab. Methods In this 2-year trial, patients aged 8–35 years who had been diagnosed with type 1 diabetes for 12 weeks or fewer were enrolled and treated at 83 clinical centres in North America, Europe, Israel, and India. Participants were allocated (2:1:1:1 ratio) by an interactive telephone system, according to computer-generated block randomisation, to receive one of three regimens of teplizumab infusions (14-day full dose, 14-day low dose, or 6-day full dose) or placebo at baseline and at 26 weeks. The Protégé study is still underway, and patients and study staff remain masked through to study closure. The primary composite outcome was the percentage of patients with insulin use of less than 0.5 U/kg per day and glycated haemoglobin A1c (HbA1C) of less than 6.5% at 1 year. Analyses included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00385697. Findings 763 patients were screened, of whom 516 were randomised to receive 14-day full-dose teplizumab (n=209), 14-day low-dose teplizumab (n=102), 6-day full-dose teplizumab (n=106), or placebo (n=99). Two patients in the 14-day full-dose group and one patient in the placebo group did not start treatment, so 513 patients were eligible for efficacy analyses. The primary outcome did not differ between groups at 1 year: 19·8% (41/207) in the 14-day full-dose group; 13·7% (14/102) in the 14-day low-dose group; 20·8% (22/106) in the 6-day full-dose group; and 20·4% (20/98) in the placebo group. 5% (19/415) of patients in the teplizumab groups were not taking insulin at 1 year, compared with no patients in the placebo group at 1 year (p=0·03). Across the four study groups, similar proportions of patients had adverse events (414/417 [99%] in the teplizumab groups vs 98/99 [99%] in the placebo group) and serious adverse events (42/417 [10%] vs 9/99 [9%]). The most common clinical adverse event in the teplizumab groups was rash (220/417 [53%] vs 20/99 [20%] in the placebo group). Interpretation Findings of exploratory analyses suggest that future studies of immunotherapeutic intervention with teplizumab might have increased success in prevention of a decline in β-cell function (measured by C-peptide) and provision of glycaemic control at reduced doses of insulin if they target patients early after diagnosis of diabetes and children.
BACKGROUND. Type 1 diabetes (T1D) results from destruction of pancreatic β cells by autoreactive effector T cells. We hypothesized that the immunomodulatory drug alefacept would result in targeted quantitative and qualitative changes in effector T cells and prolonged preservation of endogenous insulin secretion by the remaining β cells in patients with newly diagnosed T1D.METHODS. In a multicenter, randomized, double-blind, placebo-controlled trial, we compared alefacept (two 12-week courses of 15 mg/wk i.m., separated by a 12-week pause) with placebo in patients with recent onset of T1D. Endpoints were assessed at 24 months and included meal-stimulated C-peptide AUC, insulin use, hypoglycemic events, and immunologic responses.RESULTS. A total of 49 patients were enrolled. At 24 months, or 15 months after the last dose of alefacept, both the 4-hour and the 2-hour C-peptide AUCs were significantly greater in the treatment group than in the control group (P = 0.002 and 0.015, respectively). Exogenous insulin requirements were lower (P = 0.002) and rates of major hypoglycemic events were about 50% reduced (P < 0.001) in the alefacept group compared with placebo at 24 months. There was no apparent betweengroup difference in glycemic control or adverse events. Alefacept treatment depleted CD4 + and CD8 + central memory T cells (Tcm) and effector memory T cells (Tem) (P < 0.01), preserved Tregs, increased the ratios of Treg to Tem and Tcm (P < 0.01), and increased the percentage of PD-1 + CD4+ Tem and Tcm (P < 0.01).CONCLUSIONS. In patients with newly diagnosed T1D, two 12-week courses of alefacept preserved C-peptide secretion, reduced insulin use and hypoglycemic events, and induced favorable immunologic profiles at 24 months, well over 1 year after cessation of therapy.TRIAL REGISTRATION. https://clinicaltrials.gov/ NCT00965458.FUNDING. NIH and Astellas.
-Cell mass can expand in response to demand: during pregnancy, in the setting of insulin resistance, or after pancreatectomy. It is not known whether similar -cell hyperplasia occurs following immune therapy of autoimmune diabetes, but the clinical remission soon after diagnosis and the results of recent immune therapy studies suggest that -cell recovery is possible. We studied changes in -cell replication, mass, and apoptosis in NOD mice during progression to overt diabetes and following immune therapy with anti-CD3 monoclonal antibodies (mAbs) or immune regulatory T-cells (Tregs). -Cell replication increases in pre-diabetic mice, after adoptive transfer of diabetes with increasing islet inflammation but before an increase in blood glucose concentration or a significant decrease in -cell mass. The pathogenic cells are responsible for increasing -cell replication because replication was reduced during diabetes remission induced by anti-CD3 mAb or Tregs. -Cell replication stimulated by the initial inflammatory infiltrate results in increased production of new -cells after immune therapy and increased -cell area, but the majority of this increased -cell area represents regranulated -cells rather than newly produced cells. We conclude that -cell replication is closely linked to the islet inflammatory process. A significant proportion of degranulated -cells remain, at the time of diagnosis of diabetes, that can recover after metabolic correction of hyperglycemia. Correction of the -cell loss in type 1 diabetes will, therefore, require strategies that target both the immunologic and cellular mechanisms that destroy and maintain -cell mass. Diabetes 55:3238 -3245, 2006
Summary Background Type 1 diabetes (T1D) results from autoimmune targeting of the pancreatic beta cells, likely mediated by effector memory T cells (Tems). CD2, a T cell surface protein highly expressed on Tems, is targeted by the fusion protein alefacept, depleting Tems and central memory T cells (Tcms). We hypothesized that alefacept would arrest autoimmunity and preserve residual beta cells in newly diagnosed T1D. Methods The T1DAL study is a phase II, double-blind, placebo-controlled trial that randomised T1D patients 12-35 years old within 100 days of diagnosis, 33 to alefacept (two 12-week courses of 15 mg IM per week, separated by a 12-week pause) and 16 to placebo, at 14 US sites. The primary endpoint was the change from baseline in mean 2-hour C-peptide area under the curve (AUC) at 12 months. This trial is registered with ClinicalTrials.gov, number NCT00965458. Findings The mean 2-hour C-peptide AUC at 12 months increased by 0.015 nmol/L (95% CI -0.080 to 0.110 nmol/L) in the alefacept group and decreased by 0.115 nmol/L (95% CI -0.278 to 0.047) in the placebo group, which was not significant (p=0.065). However, key secondary endpoints were met: the mean 4-hour C-peptide AUC was significantly higher (p=0.019), and daily insulin use and the rate of hypoglycemic events were significantly lower (p=0.02 and p<0.001, respectively) at 12 months in the alefacept vs. placebo groups. Safety and tolerability were comparable between groups. There was targeted depletion of Tems and Tcms, with sparing of naïve and regulatory T cells (Tregs). Interpretation At 12 months, alefacept preserved the 4-hour C-peptide AUC, lowered insulin use, and reduced hypoglycemic events, suggesting a signal of efficacy. Depletion of memory T cells with sparing of Tregs may be a useful strategy to preserve beta cell function in new-onset T1D.
Protégé was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC >0.2 nmol/L, those randomized ≤6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <0.4 units/kg/day, and 8–17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose.
Despite extensive and ongoing investigations of the immune mechanisms of autoimmune diabetes in humans and animal models, there is much less information about the natural history of insulin secretion before and after the clinical presentation of type 1 diabetes and the factors that may affect its course. Studies of insulin production previously published and from the Diabetes Prevention Trial (DPT)-1 suggest that there is progressive impairment in insulin secretory responses but the reserve in response to physiological stimuli may be significant at the time of diagnosis, although maximal responses are more significantly impaired. Other factors, including insulin resistance, may play a role in the timing of clinical presentation along this continuum. The factors that predict the occurrence and rapidity of decline in -cell function are still largely unknown, but most studies have identified islet cell autoantibodies as predictors of future decline and age as a determinant of residual insulin production at diagnosis. Historical as well as recent clinical experience has emphasized the importance of residual insulin production for glycemic control and prevention of end-organ complications. Understanding the modifiers and predictors of -cell function would allow targeting immunological approaches to those individuals most likely to benefit from therapy. Diabetes 54 (Suppl. 2):S32-S39, 2005 R efinements in screening approaches have greatly improved the ability to identify individuals who will develop type 1 diabetes among first-degree relatives of affected individuals and possibly in the general population (1,2). In the recently completed Diabetes Prevention Trial (DPT)-1, a prospective study of first-degree (aged 3-35 years) and second-degree (aged 3-20 years) relatives of subjects with type 1 diabetes, ϳ3.7% of relatives of type 1 diabetic patients have islet cell autoantibodies (ICAs) (1). Among those ICA ϩ individuals, a high-risk cohort could be identified on the basis of insulin autoantibodies and diminished first-phase insulin response (FPIR) to intravenous glucose. These studies identified a group of individuals with a 60% risk of developing type 1 diabetes within a 5-year period, and the incidence may be higher with more observational time-a similar experience was seen in the European Nicotinamide Diabetes Intervention Trial (ENDIT) (3). Adding other autoantibodies to screening may improve prediction of disease because the risk of diabetes appears to increase as the autoimmune response becomes diversified, characterized by multiple autoantibodies, which appear sequentially over time (4,5).Although immunological markers have been extensively studied for their ability to predict diabetes, the metabolic course of pre-diabetes has not been as well characterized. Data from the NOD mouse have been conflicting. Sreenan et al. (6) reported an extended course of -cell failure that begins with the onset of insulitis. There were increased rates of -cell proliferation before the onset of hyperglycemia, and there was e...
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