Research in Diseases, Disorders, and Health Conditions
Chronic Diseases and Organ Systems
Type 1 diabetes is an autoimmune disease that often strikes in infancy, childhood, or young adulthood. The NIDDK’s landmark Diabetes Control and Complications Trial (DCCT), conducted from 1983 to 1993, showed that intensive glucose control dramatically delays or prevents the eye, nerve, and kidney complications of type 1 diabetes. A paradigm shift in the way type 1 diabetes is controlled was based on this finding. The follow-on study (Epidemiology of Diabetes Interventions and Complications Study, EDIC) showed that tight glucose control also prevents or delays the cardiovascular complications of type 1 diabetes. Because of improvements in treatment of the disease and new technologies, the long-term survival of those with type 1 diabetes has dramatically improved in the last 30 years. However, disease management to reduce risk for complications places an enormous burden on patients. Thus, it is imperative to pursue research on new methods, such as artificial pancreas technology, to improve type 1 diabetes control and reduce the burden on patients. Major efforts to address type 1 diabetes—in particular those on diabetes complications and beta cell research, as well as hemoglobin A1c standardization—are also of great importance for reducing the burden of type 2 diabetes.
The incidence of type 1 diabetes is increasing at three percent per year, suggesting that one or more unknown environmental factor is involved in triggering the disease. Type 1 diabetes is one of the few polygenic diseases for which over 70 percent of the genetic basis of the disease has been identified. With the identification of additional risk genes and biomarkers, it is now possible to predict risk of developing type 1 diabetes. This has enabled the launch of prevention studies as well as studies to identify environmental trigger(s). Artificial pancreas technology—linking a continuous blood glucose sensor and an insulin delivery system—has high potential to have a positive impact on patients’ health and quality of life.
Current research on type 1 diabetes supports the following broad goals: 1) identify the genetic and environmental causes; 2) prevent or reverse the disease; 3) develop cell replacement therapy; 4) improve type 1 diabetes management and care (including development of the artificial pancreas); and 5) prevent or reduce the complications of the disease. Type 1 diabetes research at NIH is supported by regular appropriations and by the Special Statutory Funding Program for Type 1 Diabetes Research, a program administered by the NIDDK on behalf of the HHS Secretary and in collaboration with other NIH ICs.
Major research efforts include the Type 1 Diabetes TrialNet, an NIDDK-led international clinical trials network that screens large numbers of individuals and conducts trials of agents to prevent type 1 diabetes in at-risk people and to slow progression of the disease in people who are newly diagnosed. Blood tests can accurately identify relatives of people with type 1 diabetes who are at high or moderate risk of developing the disease within five years. This has enabled TrialNet to launch clinical trials of promising prevention strategies, two of which (oral insulin, anti-CD3) are currently ongoing.194 Recent results from three clinical trials testing agents targeting the immune system in people with new-onset type 1 diabetes reported that:
The drug abatacept slowed disease progression for 6–9 months compared to placebo.After that time, the effect of the drug diminished, and rate of loss of insulin production was similar in the abatacept and placebo groups. However, because of the initial beneficial effects, after two years, people in the abatacept group produced 59 percent more C-peptide, a marker of insulin production.195 Preservation of C-peptide production is associated with better glucose control, less hypoglycemia, and reduced risk of complications. Another study showed that a Glutamic Acid Decarboxylase (GAD) vaccine had no effect after one year.196 In type 1 diabetes, GAD is a major target of autoimmune response.
The drug rituximab, which destroys immune cells called B lymphocytes, preserved insulin production in newly diagnosed patients for 1 year, but the effect dissipated at 2 years. As drugs such as rituximab broadly deplete B lymphocytes, they can increase risk of infection and therefore have significant side effects. Nonetheless, the finding is important because it is propelling research to find drugs targeting the specific B lymphocytes involved in type 1 diabetes without the associated side effects.197
Research is showing that people with type 1 diabetes are living longer, healthier lives than ever before, largely due to long-term NIH supported research. The Epidemiology of Diabetes Interventions and Complications (EDIC) study demonstrated that, compared to conventional therapy, near-normal control of blood glucose—beginning soon after diagnosis of type 1 diabetes and continuing an average of 6.5 years—reduced the long-term risk (average 22-year follow-up) of developing kidney disease by 50 percent. The Diabetes Control and Complications Trial (DCCT) showed reduced biomarkers of complications. A decade after DCCT ended, EDIC found reduced heart attack, stroke, and cardiovascular death. Now, two decades later, EDIC found that early control reduced development of chronic and end stage kidney disease. This finding shows that the benefits of early and intensive therapy can persist for decades. It also demonstrates the importance of long-term research, when the full benefit of treatment may not be seen for long time periods.198
The Environmental Determinants of Diabetes in the Young (TEDDY) is an NIDDK-led study to identify the infectious agents, dietary factors, or other environmental conditions that trigger type 1 diabetes in genetically susceptible individuals. The TEDDY study has completed enrollment of over 8,000 high-risk newborns and is collecting biosamples for analysis to identify potential triggers of type 1 diabetes. Children enrolled in the study are developing autoimmunity and type 1 diabetes at the predicted rates, indicating that those at risk can be accurately identified and that the study is on track to make a major contribution. Identification of an infectious agent that triggers autoimmunity could lead to a vaccine to protect against type 1 diabetes, or, if dietary factors are identified that protect from or contribute to the development of the disease, changes to infant feeding practices could be recommended. NIAID, NIEHS, and NICHD also participate in this study.199
194 For more information, see https://www.diabetestrialnet.org/ .
195Orban T, et al. Lancet. 2011;378(9789):412–9. PMID: 21719096.
196Wherrett DK, et al. Lancet. 2011;378(9788):319–27. PMID: 21714999.
197Pescovitz MD, et al. em>NEJM. 2009; 361:2143–52. PMID: 19940299.
198The DCCT/EDIC Research Group. N Engl J Med. 2011;365(25):2366–76. PMID: 22077236.
199For more information, see https://teddy.epi.usf.edu/ .
The Beta Cell Biology Consortium (BCBC), led by NIDDK, is a consortium of researchers studying pancreas and beta cell biology and development toward a cell-based treatment for type 1 diabetes. BCBC investigators are working to reconstruct components of human type 1 diabetes in the mouse to observe how human diabetes develops, and then pinpoint the molecules, genes, and cells responsible.200Consortiumresearchers recently discovered that pancreatic glucagon-producing alpha cells could convert to insulin-producing beta cells in a mouse model of diabetes. This insight suggests that it may be possible to develop therapies to promote conversion of alpha cells to beta cells to restore insulin production in people with diabetes, and opens up intriguing new avenues for research toward cell replacement therapy.201
The Clinical Islet Transplantation Consortium, co-led by NIDDK and NIAID, is conducting clinical and mechanistic studies in islet transplantation, with or without accompanying kidney transplantation, for the treatment of type 1 diabetes. The Consortium has completed enrollment of a pivotal (Phase III) islet transplantation trial (islet transplant alone), which is intended to support future FDA licensure of an islet product.202
To overcome the limitations of current insulin therapy, researchers have long sought to link glucose monitoring and insulin delivery by developing an artificial pancreas. There has been tremendous progress toward the development of an artificial pancreas that will mimic, as closely as possible, the way a healthy pancreas detects changes in blood glucose levels and responds automatically to secrete appropriate amounts of insulin. All of the current continuous glucose monitoring technology on the market benefitted from NIDDK support early in development. NICHD and NIBIB have joined NIDDK in supporting small-business initiatives aimed at further development of these and related technologies. Clinical studies on closed-loop technologies are now ongoing, including a TrialNet study of whether initiation of closed-loop glucose control at diagnosis of type 1 diabetes and subsequent continuous glucose monitoring can preserve insulin production in people with newly diagnosed type 1 diabetes.203 NIDDK is also supporting research training of engineers and behavioral scientists—fields that are critical for propelling progress in this area.
NICHD supports a diverse research portfolio related to type 1 diabetes in children and in pregnant women. For example, NICHD has funded research on the increased risk of pregnancy complications for women with type 1 diabetes; metabolic processes underlying the severity of type 1 diabetes in children; and behavioral research on the maintenance of glycemic control in children and adolescents and other areas of science. The Diabetes Research in Children Network,led by NICHD, is a research consortium investigating hypoglycemia and use of continuous glucose monitoring in children.204
200For more information, see https://www.betacell.org/ .
201Thorel F, et al. Nature. 2010; 464: 1149–54. PMID: 20364121.
202For more information, see https://www.citisletstudy.org/ .
203For more information, see https://clinicaltrials.gov/ct2/show/NCT00891995?term=metabolic+control+in+new+onset+diabetes&rank=15.
The TRIGR clinical trial, led by NICHD, is examining whether hydrolyzed infant formula compared to standard cow’s milk-based formula decreases the risk of developing type 1 diabetes in at-risk children.205
The Diabetic Retinopathy Clinical Research Network is an NEI-led, collaborative, nationwide network of eye doctors and investigators conducting multi-center clinical trials of diabetes-induced eye disease, including comparative effectiveness research on new therapies. By providing infrastructure for conducting multiple concurrent studies, DRCR.net enables rapid development, initiation, and patient recruitment for new protocols and provides opportunities for industry collaborations while maintaining a rigorous academic environment.
The Hemoglobin A1C (HbA1C) Standardization Program, supported by CDC and NIDDK, is achieving international standardization and reliability in measurement of HbA1C, a blood test that measures glycosylated hemoglobin and is a good surrogate measure of long-term blood glucose control and, as such, reflects risk of diabetic complications.206
NIDDK-supported research uncovered a key factor necessary for making insulin-producing beta cells in both humans and mice. Mice lacking the newly identified protein—called Rfx6—can make islets, but these islets do not contain insulin-producing cells. Interestingly, a rare form of neonatal diabetes is associated with mutations in the human gene that produces the Rfx6 protein, suggesting that Rfx6 plays a critical role in beta cell development in humans as well as mice. Researchers now know they will have to ensure that Rfx6 is present in order to successfully generate beta cells from some other cell type for transplantation into people with diabetes.207
Researchers funded by NIDDK tested an implantable glucose sensor that monitors tissue glucose and reports data to an external wireless receiver.When implanted into pigs, the system functioned continuously for over a year. The implanted sensor also worked when tested for several months in diabetic pigs. These results are encouraging because an implantable device could potentially be used in the future as part of an artificial pancreas to automate glucose sensing and insulin delivery.208
NIDDK-supported scientists identified a novel genetic variant associated with type 1 diabetes risk that regulates a network of immune system genes.209Understanding genetic underpinnings of the disease could inform new targets for therapy.
NIDDK-supported scientists also discovered that a variant of an immune system molecule may contribute to type 1 diabetes by enabling an aberrant immune reaction against insulin. 210 This discovery could inform ways to intervene in the immune process to prevent type 1 diabetes or slow its progression.
204For more information, see https://www.nichd.nih.gov/research/supported/directnet.cfm.
205For more information, see https://www.nichd.nih.gov/research/supported/TRIGR.cfm.
206For more information, see http://www.ngsp.org/index.asp .
207Smith SB, et al. Nature. 2010. 463: 775–80. PMID: 20148032.
208Gough DA, et al. Sci Transl Med. 2010;2: 42ra53. PMID: 20668297.
209Heining M, et al. Nature. 2010; 467: 460–4. PMID: 20827270.
210Stadinski BD, et al. Proc Natl Acad Sci USA. 2010;107: 10978–83. PMID: 20534455.
Advances and Emerging Opportunities in Diabetes Research: A Strategic Planning Report of the Diabetes Mellitus Interagency Coordinating Committee, published in February 2011, identifies compelling opportunities for research over the next decade on diabetes and its complications.
The over 8,000 participants being followed in TEDDY provide an unparalleled resource to study the development of the human microbiome from birth through childhood. Planned studies will build on research in mice to identify how interaction between the immune system and bacteria in the gut may alter the risk of type 1 diabetes.
Research will build on the unprecedented recent discoveries of genes and gene regions contributing to type 1 diabetes to understand their function in health and disease. This research could illuminate new targets for therapy. Research will also develop, refine, and pilot test innovative strategies to improve adherence to medications and medical regimens in children, adolescents, and young adults with type 1 diabetes.
NIDDK is supporting a new study of the Joslin “medalists” – a population of people with type 1 diabetes who have survived with the disease for more than 50 years without serious development of eye, kidney, or nerve complications. The study seeks to identify factors that can protect against development of diabetes complications.
In type 2 diabetes, cells in muscle, fat, and liver tissue do not properly respond to insulin. At first, the pancreas produces more insulin to compensate. Gradually, however, blood glucose levels rise as the pancreatic beta cells lose their capacity to secrete insulin, and the timing of insulin secretion becomes abnormal. Treatment approaches for controlling glucose levels include diet, exercise, and oral and injected medications; insulin often is required as the disease progresses. Risk factors include being overweight, age, racial or ethnic background, history of gestational diabetes (GDM), and family history of type 2 diabetes.
NIH supports research to better understand metabolism and the mechanisms that lead to the development and progression of diabetes; such research will ultimately spur the design of potential new intervention strategies. In parallel, based on knowledge from past scientific research investments, NIH is vigorously pursuing studies of prevention and treatment approaches. Research on type 2 diabetes and its complications is spearheaded by NIDDK, but encompasses efforts by and collaborations with many other NIH Institutes and Centers and Offices, including NHLBI, NEI, NINR, NIEHS, NINDS, and ORWH.
Critical programs in type 2 diabetes include a new NIDDK-led consortium Optimizing Recovery and Preservation of Endogenous Insulin Secretion that is exploring approaches to slow beta cell loss in pre-diabetes and early in type 2 diabetes.211 Another NIDDK-led consortium, the Multiethnic Study of Type 2 Diabetes Genes Consortium is working to identify genes or gene regions conferring type 2 diabetes risk in multiple ethnic groups.212
NIDDK’s Translational Research for the Prevention and Control of Diabetes213 and NIDDK Centers for Diabetes Translation Research fund type II translation research (e.g., bedside to practice and the community) based on past successful diabetes clinical trials. Among the projects funded by these programs will be those that lower the cost and increase the availability of lifestyle interventions to prevent diabetes based on the intervention found highly effective in NIDDK’s landmark Diabetes Prevention Program clinical trial. 214
Major NIH clinical trials in people with type 2 diabetes include the Look AHEAD (Action for Health in Diabetes) trial. Led by NIDDK, with additional support from NHLBI, NINR, NIEHS, and ORWH, Look AHEAD is a multicenter randomized clinical trial to examine the effects of a lifestyle intervention designed to achieve and maintain weight loss over the long term through exercise and decreased caloric intake in overweight or obese adults with type 2 diabetes. The primary Look AHEAD outcome measure is cardiovascular disease events. The study is also examining effects on other diabetes complications, as well as co-morbid conditions such as depression.
This NIDDK leads TODAY (Treatment Options for Type 2 Diabetes in Adolescents and Youth), aclinical trial that compared safety, efficacy, and cost-effectiveness of three different treatments for type 2 diabetes in youth. NIDDK also led the Diabetes Prevention Program Outcomes Study with additional support from NHLBI, ORWH, NIA, NICHD, and NEI. The Diabetes Prevention Program Outcomes Study is following participants in the landmark Diabetes Prevention Program to determine long term outcomes and durability of the DPP interventions. The Diabetes Prevention Program Outcomes Study has found that the lifestyle intervention continues to be effective for at least 10 years, and the study has also made numerous other important findings including the pharmacogenomic characterization of a gene influencing the transport and effectiveness of metformin, currently the most important medication in the treatment of type 2 diabetes. The study is also examining intervention effects on other health-related outcomes, including co-morbid conditions such as depression.
211For more information, see https://grants.nih.gov/grants/guide/rfa-files/RFA-DK-10-013.html.
212For more information, see https://grants.nih.gov/grants/guide/rfa-files/RFA-DK-09-004.html.
213For more information, see https://grants.nih.gov/grants/guide/pa-files/PAR-06-532.html.
214For more information, see https://grants.nih.gov/grants/guide/rfa-files/RFA-DK-10-009.html.
NHLBI leads an observational follow-up study (ACCORDIAN) of over 8,000 participants who were treated and followed in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) Trial is designed to further elucidate the long-term effects of the ACCORD interventions on cardiovascular disease (CVD) and type 2 diabetes outcomes. ACCORD was a large, randomized clinical trial in people with type 2 diabetes who were treated and followed for an average of approximately five years, through mid-2009. ACCORD tested the effects of three treatment strategies for control of glucose, blood pressure, and lipids on the rate of CVD events. The original trial found a modest decrease in the rate of non-fatal heart attacks and some indicators of eye and kidney disease, but there was a statistically significant increase in mortality in the intensive glycemia treatment group (targeting an Hba1c below 6.5 percent). The blood pressure and lipid interventions had no effect on CVD, but both the intensive blood sugar control and the lipid therapy reduced the progression of diabetic eye disease.
A planning grant has been funded by NIDDK to develop a major new trial Glycemic Reduction Approaches for Treating Diabetes: An Effectiveness Study, which will be designed to compare effectiveness of commonly used diabetes medications to determine the most effective treatment strategies for patients early in the course of their type 2 diabetes, with the aim of achieving and maintaining glycemic levels known to reduce long-term complications.
Other significant efforts include the NIEHS National Toxicology Program in which researchers are examining how to incorporate information on diabetes-related signaling pathways into the Tox21 framework for chemical screening. These efforts create an opportunity to use Tox21 approaches in a targeted testing framework to identify substances of concern, exposure to which may increase diabetes risk.
NIMH is supporting a randomized control trial enrolling persons with serious mental illness and comorbid diabetes from primary care safety net clinics. The psychosocial intervention, Targeted Training in Illness Management, involves nurses and peer educators—persons who have both a serious mental illness and diabetes and have been trained in illness self-management. These interventionists meet with patients in groups and model positive behavior change and the patient’s active self-management to address both the mental disorder and diabetes in an integrated fashion. The intervention targets mental health and diabetes outcomes as well as overall functioning and quality of life.
Adverse Metabolic Side Effects of Second Generation Psychotropic Medications Leading to Obesity and Increased Diabetes Risk was a funding opportunity announcement (PA) issued jointly by NIMH and NIDDK to address the metabolic syndrome resulting from medications used by people with serious mental illness. Several innovative studies are underway. In addition, the first new therapy for diabetic retinopathy in 25 years was established in a trial demonstrating VEGF-inhibitor Lucentis is effective for slowing or reversing vision loss caused by diabetic retinopathy. Nearly 50 percent of patients who received the combination of Lucentis and laser treatment experienced substantial visual improvement after one year, compared with only 28 percent who received laser treatment alone.215 The trial was conducted as part of the NEI and NIDDK co-funded Diabetic Retinopathy Clinical Trials Network.
Metabolomics (the study of chemical processes involving metabolites) revealed that plasma concentrations of five specific amino acids were strong predictors of future diabetes in a 12 year prospective study of 2,422 people with normal glucose levels. Testing for three of them—Ile, Phe, and Tyr—revealed a five- to seven-fold increase in type 2 diabetes likelihood between the top and bottom quartiles.216
Diabetes Prevention Program (DPP) participants homozygous for the most common allele of an SLC47A1 SNP (roughly a third of the population) received no diabetes-prevention benefit from metformin, although the metformin arm of the study developed diabetes 31 percent less often than those taking placebo. This could represent a major new advance for personalized medicine, as a test could identify which of millions of Americans with prediabetes would likely benefit from metformin.217
The peptide hormone FGF19, produced by cells of the distal small intestine in response to the presence of bile salts, signals to the liver to complete the task of storing glucose as glycogen. It is therefore an important regulator of blood glucose levels, and because it acts through a different pathway than insulin, stimulation of its effectors may be a valuable new approach to type 2 diabetes treatment.218 Other studies have shown that in response to glucagon, the class IIa histone deacetylases (HDACs) activate FOXO to stimulate liver glucose production. Experimentally limiting HDAC activity in the livers of mice with type 2 diabetes helps normalize blood glucose, suggesting another potential route for type 2 diabetes therapy.219
A high-fat diet can interfere with the post-translational modification of the glucose transporter in pancreatic beta cells of mice, so an insufficient supply of the protein reaches the cell surface to detect increases in glucose levels. Mature, membrane-embedded transporter was also reduced in beta cells from type 2 diabetes patients. This suggests insufficient beta cell insulin production could precede or occur in tandem with peripheral insulin resistance, leading to type 2 diabetes.220
215Elman M, et al. Ophthalmology. 2010;117 (6):1064–77. PMID: 20427088.
216Wang TJ, et al. Nat Med. 2011;17(4):448–53. PMID: 21423183.
217Jablonski KA. Diabetes. 2010;59(10):2672–81. PMID: 20682687.
218Kri S, et al. Science. 2011;331(6024):1621–4. PMID: 21436455. Potthoff MJ, et al. Cell Metab. 2011;13(6):729–38. PMID: 21641554.
219Wang B, et al. Cell. 2011;145(4):596–606. PMID: 21565616. Mihaylova MM, et al. Cell. 2011;145(4):607–21. PMID: 21565617.
220Ohtsubo K, et al. Nat Med. 2011;17(9):1067–75. PMID: 21841783.
A compound was found that binds PPAR-γ221 and inhibits its phosphorylation without broadly simulating it. In mice, a new compound was found that improved insulin sensitivity without causing the side effects observed in treatment with currently approved medicines that work via the same mechanism.222A Phase I clinical trial with the compound has begun.223
Vitamin D supplementation was found to improve insulin sensitivity in a 16-week clinical trial of people with prediabetes.224 A planning grant has been funded for a larger, longer-term randomized clinical trial, the Vitamin D for Type 2 Diabetes (D2D) trial. Although diabetes is a known risk factor for periodontal disease, new findings show that people with prediabetes are also at significantly elevated risk for periodontal disease.225 An observational study of over 65,000 women aged 50 to 75 found that not only does diabetes increase the risk for depression, but also depression increases the risk for type 2 diabetes.226p> NIMH and NIDDK recently co-funded a landmark study to improve care for patients with both depression and diabetes. The researchers examined a primary care approach called TEAMCare in which nurses worked with patients and their physicians to manage care for depression and poorly controlled diabetes in an integrated fashion, using evidence-based care guidelines. The study found that TEAMCare patients experienced less depression, better control of blood sugar, improved quality of life, and higher satisfaction with care, as compared to patients receiving usual care.227
In addition to research efforts, the National Diabetes Education Program, a joint effort of NIDDK and the Centers for Disease Control and Prevention, translates the latest science and spreads the word that diabetes is serious, common, and costly, yet controllable and, for type 2 diabetes, preventable. The ORWH contributes funding for the NDEP’s GDM awareness campaign, “It’s Never Too Early to Prevent Diabetes," an effort to inform women with a history of GDM, their families, and health care providers of the increased risk for future type 2 diabetes in both mothers and babies affected by GDM, and steps they can take to reduce this risk.
The February 2011 report titled Advances and Emerging Opportunities in Diabetes Research: A Strategic Planning Report of the Diabetes Mellitus Interagency Coordinating Committee, spearheaded by NIDDK, identifies compelling opportunities for NIH-supported research over the next decade on diabetes and its complications. Key type 2 diabetes research priorities include:
Following the landmark two-year diabetic macular edema (DME) trial228 that demonstrated that intravitreal injection of VEGF-inhibitor Lucentis (ranibizumab) with prompt or deferred laser treatment was superior to the standard therapy of laser alone, the NIH-funded Diabetic Retinopathy Clinical Research Network is launching a comparative effectiveness trial of Lucentis versus Avastin. Avastin, a similar yet significantly less expensive drug also produced by Genentech/Roche, has been used off-label for DME, but data on its effectiveness for this indication is not available, although a recent head-to-head trial (Comparison of AMD Treatment Trial) in a different eye disease, macular degeneration found the drugs to be equally effective for visual acuity.229
221Peroxisome proliferator-activated receptor gamma (PPAR-γ) regulates glucose metabolism and fatty acid storage.
222Choi JH, et al. Nature. 2011;477(7365):477–81. PMID: 21892191.
223Lee JM, et al. Nature. 2011;474(7352):506–10. PMID: 21614002.
224Mitri J, et al. Am J Clin Nutr. 2011;94(2):486–94. PMID: 21715514.
225Choi YH, et al. Diabetes Care. 2011;34(2):381–6. PMID: 21216848.
226Pan A, et al. Arch Intern Med. 2010;170(21):1884–91. PMID: 21098346.
227Katon WJ, et al. N Engl J Med. 2010;363(27):2611–20. PMID: 21190455. See also: https://www.nimh.nih.gov/science-news/2010/case-managed-care-improves-outcomes-for-depressed-patients-with-multiple-medical-conditions.shtml.
228Elman MJ, et al. Ophthalmology. 2011;118(4):609–14. PMID: 21459214.
229Martin D, et al. Ophthalmology. 2012;119(7):1388–98. PMID: 22555112.