Rationale and design of the CV-PREVITAL study: an Italian multiple cohort randomised controlled trial investigating innovative digital strategies in primary cardiovascular prevention.

INTRODUCTION: Prevention of cardiovascular disease (CVD) is of key importance in reducing morbidity, disability and mortality worldwide. Observational studies suggest that digital health interventions can be an effective strategy to reduce cardiovascular (CV) risk. However, evidence from large randomised clinical trials is lacking. METHODS AND ANALYSIS: The CV-PREVITAL study is a multicentre, prospective, randomised, controlled, open-label interventional trial designed to compare the effectiveness of an educational and motivational mobile health (mHealth) intervention versus usual care in reducing CV risk. The intervention aims at improving diet, physical activity, sleep quality, psycho-behavioural aspects, as well as promoting smoking cessation and adherence to pharmacological treatment for CV risk factors. The trial aims to enrol approximately 80 000 subjects without overt CVDs referring to general practitioners' offices, community pharmacies or clinics of Scientific Institute for Research, Hospitalization and Health Care (Italian acronym IRCCS) affiliated with the Italian Cardiology Network. All participants are evaluated at baseline and after 12 months to assess the effectiveness of the intervention on short-term endpoints, namely improvement in CV risk score and reduction of major CV risk factors. Beyond the funded life of the study, a long-term (7 years) follow-up is also planned to assess the effectiveness of the intervention on the incidence of major adverse CV events. A series of ancillary studies designed to evaluate the effect of the mHealth intervention on additional risk biomarkers are also performed. ETHICS AND DISSEMINATION: This study received ethics approval from the ethics committee of the coordinating centre (Monzino Cardiology Center; R1256/20-CCM 1319) and from all other relevant IRBs and ethics committees. Findings are disseminated through scientific meetings and peer-reviewed journals and via social media. Partners are informed about the study's course and findings through regular meetings. TRIAL REGISTRATION NUMBER: NCT05339841.

Liraglutide preserves CD34+ stem cells from dysfunction Induced by high glucose exposure.

BACKGROUND: Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function. METHODS: In cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated. RESULTS: CD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation, CXCR4/SDF-1α axis activity and metabolic imbalance from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9-39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects. CONCLUSION: We provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.

The Long Telling Story of "Endothelial Progenitor Cells": Where Are We at Now?

Endothelial progenitor cells (EPCs): The name embodies years of research and clinical expectations, but where are we now? Do these cells really represent the El Dorado of regenerative medicine? Here, past and recent literature about this eclectic, still unknown and therefore fascinating cell population will be discussed. This review will take the reader through a temporal journey that, from the first discovery, will pass through years of research devoted to attempts at their definition and understanding their biology in health and disease, ending with the most recent evidence about their pathobiological role in cardiovascular disease and their recent applications in regenerative medicine.

The role of unhenanced CT in a ruptured parathyroid adenoma: A case report.

Acute bleeding is a rare and potentially life-threatening complication of a Parathyroid Adenoma described in just a few cases in literature. We describe the case of a healthy 53-years-old female patient without prior history of parathyroid pathology who presented with acute onset of neck and mediastinal hemorrhage. Ultrasound (US), Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) combined with laboratory tests led to the diagnosis of a bleeding Parathyroid adenoma. This case is presented to sensitize both Radiologists and Clinicians about this rare presentation that should be put into differential diagnosis of acute neck swelling and pain.

An ileal duplication cyst case report: From diagnosis to treatment.

Enteric duplication cysts (EDCs) are rare congenital malformations of the children and can develop everywhere along the gastrointestinal (GI) tract, being the ileum the most frequent localization. We herein present an unusual case of duplication cyst of ileal origin who show a tubular morphology and doesn't communicate with GI lumen. A 2-month-old boy was admitted to our hospital for investigation of an anechoic formation of the lower right abdomen for the surgical planning. The patient was asymptomatic. Ultrasound (US) and magnetic resonance imaging (MRI) showed features of a cystic lesion. Laparoscopic surgery was performed and the cyst excised. Macroscopic examination and histologic findings confirmed the diagnosis of a enteric duplication cyst arising from the ileum. In a patient with an abdominal cystic mass, although asymptomatic, it's worth assessing the nature of the lesion and planning a surgery in order to avoid future complications. A correct use of diagnostic it's fundamental to identify the etiology and the characteristics of a cystic mass.

When Good Guys Turn Bad: Bone Marrow's and Hematopoietic Stem Cells' Role in the Pathobiology of Diabetic Complications.

Diabetes strongly contributes to the development of cardiovascular disease, the leading cause of mortality and morbidity in these patients. It is widely accepted that hyperglycemia impairs hematopoietic stem/progenitor cell (HSPC) mobilization from the bone marrow (BM) by inducing stem cell niche dysfunction. Moreover, a recent study demonstrated that type 2 diabetic patients are characterized by significant depletion of circulating provascular progenitor cells and increased frequency of inflammatory cells. This unbalance, potentially responsible for the reduction of intrinsic vascular homeostatic capacity and for the establishment of a low-grade inflammatory status, suggests that bone BM-derived HSPCs are not only victims but also active perpetrators in diabetic complications. In this review, we will discuss the most recent literature on the molecular mechanisms underpinning hyperglycemia-mediated BM dysfunction and differentiation abnormality of HSPCs. Moreover, a section will be dedicated to the new glucose-lowering therapies that by specifically targeting the culprits may prevent or treat diabetic complications.

Abnormal DNA Methylation Induced by Hyperglycemia Reduces CXCR 4 Gene Expression in CD 34+ Stem Cells.

Background CD 34+ stem/progenitor cells are involved in vascular homeostasis and in neovascularization of ischemic tissues. The number of circulating CD 34+ stem cells is a predictive biomarker of adverse cardiovascular outcomes in diabetic patients. Here, we provide evidence that hyperglycemia can be "memorized" by the stem cells through epigenetic changes that contribute to onset and maintenance of their dysfunction in diabetes mellitus. Methods and Results Cord-blood-derived CD 34+ stem cells exposed to high glucose displayed increased reactive oxygen species production, overexpression of p66shc gene, and downregulation of antioxidant genes catalase and manganese superoxide dismutase when compared with normoglycemic cells. This altered oxidative state was associated with impaired migration ability toward stromal-cell-derived factor 1 alpha and reduced protein and mRNA expression of the C-X-C chemokine receptor type 4 ( CXCR 4) receptor. The methylation analysis by bisulfite Sanger sequencing of the CXCR 4 promoter revealed a significant increase in DNA methylation density in high-glucose CD 34+ stem cells that negatively correlated with mRNA expression (Pearson r=-0.76; P=0.004). Consistently, we found, by chromatin immunoprecipitation assay, a more transcriptionally inactive chromatin conformation and reduced RNA polymerase II engagement on the CXCR 4 promoter. Notably, alteration of CXCR 4 DNA methylation, as well as transcriptional and functional defects, persisted in high-glucose CD 34+ stem cells despite recovery in normoglycemic conditions. Importantly, such an epigenetic modification was thoroughly confirmed in bone marrow CD 34+ stem cells isolated from sternal biopsies of diabetic patients undergoing coronary bypass surgery. Conclusions CD 34+ stem cells "memorize" the hyperglycemic environment in the form of epigenetic modifications that collude to alter CXCR 4 receptor expression and migration.

A Review of the Long-Term Efficacy, Tolerability, and Safety of Exenatide Once Weekly for Type 2 Diabetes.

INTRODUCTION: Exenatide once weekly (ExeOW, Bydureon®, Astra Zeneca), a drug belonging to the class of glucagon-like peptide-1 (GLP-1) receptor agonists, is the first agent approved for treatment of type 2 diabetes (T2D) that can be administered on a weekly basis. METHODS: Data concerning treatment of T2D with ExeOW are reviewed with special reference to its long-term efficacy, tolerability, and safety. Relevant literature was identified through the PubMed database from inception to January 2015. RESULTS: In randomized clinical trials ExeOW, as add-on to oral antidiabetics, achieved significantly improved glycemic control compared to maximum recommended doses of exenatide twice daily, sitagliptin, pioglitazone, and insulin glargine, as measured by HbA1c. In drug-naïve patients ExeOW was superior to sitagliptin and non-inferior to metformin, whereas non-inferiority to pioglitazone and liraglutide was not proven. In different trials reductions in HbA1c ranged from -1.1% to -2.0%. ExeOW therapy over 6 months was also associated with a mean weight loss of -2 to -4 kg, improved systolic blood pressure and lipid profile, and no hypoglycemia unless associated to sulfonylurea. ExeOW long-term therapy up to 3-6 years allowed persistent glycemic control (HbA1c -1.6%), sustained decreases in blood pressure (-2 mmHg), and improvements of lipid profile. ExeOW tolerability was comparable to that of the other GLP-1 receptor agonists, with better gastrointestinal tolerability when direct comparison was done (namely liraglutide and exenatide BID), but higher incidence of injection site reactions and few treatment discontinuations mainly due to gastrointestinal events. CONCLUSION: ExeOW is a well-tolerated and convenient option for long-term treatment of T2D allowing significant and persistent glycemic control with moderate weight loss and low risk of hypoglycemia unless associated with sulfonylureas.

Epidemiology of diabetic kidney disease in adult patients with type 1 diabetes in Italy: The AMD-Annals initiative.

BACKGROUND: Patients with type 1 diabetes mellitus are at increased risk of death. This risk appears to be modulated by kidney dysfunction. The aim of this study was to evaluate the prevalence of diabetic kidney disease (DKD), its traits, and clinical correlates in a large sample of patients with type 1 diabetes. METHODS: Clinical data of 20 464 patients with type 1 diabetes were extracted from electronic medical records. Estimated glomerular filtration rate (eGFR) and increased urinary albumin excretion were considered. RESULTS: Mean age of the patients was 46 ± 16 years, 55.0% were males, and duration of diabetes 19 ± 13 years. The frequency of diabetic kidney disease, low eGFR, and albuminuria was 23.5%, 8.1%, and 19.5%, respectively. In the multivariate analysis the presence of diabetic kidney disease was associated with age (odds ratio [OR] = 1.14, 95% confidence interval [CI]: 1.10-1.18), duration of diabetes (OR = 1.05, 95% CI: 1.03-1.07), and worse glycemic control (OR = 1.24, 95% CI: 1.21-1.28, for every 1% glycated hemoglobin increase). Diabetic kidney disease was also independently associated with an atherogenic lipid profile and increased systolic blood pressure. Glucose control, systolic blood pressure, triglycerides, and high density lipoprotein cholesterol were associated with both low eGFR and albuminuria. Male gender, retinopathy and smoke were related to albuminuria, being female was related to low eGFR, while SUA levels were associated with DKD, low eGFR and albuminuria. CONCLUSIONS: In our sample of patients with type 1 diabetes, diabetic kidney disease entails an unsafe cardiovascular risk profile. Hyperglycemia, arterial hypertension, and atherogenic lipid profile affected both low eGFR and albuminuria. Retinopathy and smoking were related only to albuminuria while being female and elevated serum uric acid were associated only with low eGFR.

Glucagon and heart in type 2 diabetes: new perspectives.

Increased levels of glucagon in type 2 diabetes are well known and, until now, have been considered deleterious. However, glucagon has an important role in the maintenance of both heart and kidney function. Moreover, in the past, glucagon has been therapeutically used for heart failure treatment. The new antidiabetic drugs, dipeptidyl peptidase-4 inhibitors and sodium-glucose co-transporter-2 inhibitors, are able to decrease and to increase glucagon levels, respectively, while contrasting data have been reported regarding the glucagon like peptide 1 receptors agonists. The cardiovascular outcome trials, requested by the FDA, raised some concerns about the possibility that the dipeptidyl peptidase-4 inhibitors can precipitate the heart failure, while, at least for empagliflozin, a positive effect has been shown in decreasing both cardiovascular death and heart failure. The recent LEADER Trial, showed a significant reduction of cardiovascular death with liraglutide, but a neutral effect on heart failure. A possible explanation of the results with the DPPIV inhibitors and empagliflozin might be related to their divergent effect on glucagon levels. Due to unclear effects of glucagon like peptide 1 receptor agonists on glucagon, the possible role of this hormone in the Leader trial remains unclear.

Short-term high glucose exposure impairs insulin signaling in endothelial cells.

BACKGROUND: Hyperglycemia is the hallmark of diabetes and its cardiovascular complications. Insulin plays an important role in the regulation of vascular homeostasis and maintenance of endothelial function. Insulin signaling occurs after binding to the insulin receptor, causing activation of two separate and parallel pathways: PI3K/AKT/eNOS and Ras/Raf/MAPK pathways. AKT phosphorylates eNOS at Ser1177, resulting in increased nitric oxide production and vasodilation. The MAPK pathway results in endothelin-1 production and vasoconstriction and mitogenic effects. METHODS: We studied the effects of physiological insulin treatment in human umbilical vein endothelial cells (HUVECs) on the two pathways under high glucose conditions, which mimic the in vivo condition of hyperglycemia. HUVECs were incubated with insulin at different physiological concentrations (from 10(-10) to 10(-8) M) for 30 min after 24 h of exposition to normal (5 mmol/L, NG) or high glucose (25 mmol/L, HG). Phosphorylated forms of AKT, eNOS, ERK1/2, p38, JNK and insulin receptor-ß subunit (IRß) were evaluated. RESULTS: In normal glucose, the active phosphorylated forms of AKT, eNOS, ERK1/2, p38 and JNK were increased in insulin treated cells, in a dose-dependent manner. In high glucose, insulin was not able to activate the PI3K/AKT/eNOS pathway, with the phosphorylated form of eNOS reduced with respect to the control. However, insulin was able to induce the up-regulation of phospho-ERK1/2, -p38 and -JNK. Moreover, we found reduced levels of IRß phosphorylated form in high glucose as compared to the control. Insulin was able to increase phospho-IRß in normal glucose but not in high glucose, in which the total protein levels remained reduced. CONCLUSIONS: Exposure to short-term high glucose negatively affects insulin signaling even when physiological insulin concentrations are added. The impairment of the PI3K/AKT/eNOS pathway after physiological insulin treatment could contribute to detrimental effects on cardiovascular homeostasis under high glucose conditions, and might shift toward the activation of certain mitogenic effectors, such as ERK1/2, p38 and JNK, the only ones that respond to physiological insulin treatment in high glucose.

Oscillating glucose and constant high glucose induce endoglin expression in endothelial cells: the role of oxidative stress.

AIM: High glucose-induced oxidative stress has been suggested as one of the mediators of endothelial damage in diabetes. The major endothelial protein, endoglin, has been found overexpressed in the vessels during pathological situations, but little is known about its relation to diabetic vascular complications. To clarify the role of endoglin in endothelial injury, we sought to determine the effects of high and oscillating glucose on its expression. MATERIALS: Furthermore, the activation of the Krüppel-like factor 6 (KLF-6) and the hypoxia-inducible factor-1α (HIF-1α) as possible regulators of endoglin expression has been evaluated. The possible role of the oxidative stress has been studied evaluating the effects of the antioxidant alpha-lipoic acid (ALA) and the cellular antioxidant response mediated by NAD(P)H: quinine-oxido-reductase-1 (NQO-1) and heme oxygenase-1 (HO-1). RESULTS: Primary HUVECs were cultured for 21 days in normal, high and oscillating glucose (5, 25 and 5/25 mmol/l every 24 h, respectively) with/without ALA. In oscillating and high glucose total endoglin, its soluble form (sEng), KLF-6 and HIF-1α were significantly increased. Simultaneously, the oxidative DNA stress markers 8-OHdG and H2A.X were elevated. Moreover, ENG gene transcriptional rate increased during glucose exposures concomitantly with increased KLF-6 nuclear translocations. ALA significantly reduced all these phenomena. Interestingly, during oscillating and chronic high glucose, NQO-1 and HO-1 did not increase, but ALA induced their overexpression. CONCLUSIONS: Together, these findings provide novel clue about endoglin in the regulation of high glucose-mediated vascular damage in HUVECs and the role of oxidative stress in this regulation.

Simultaneous GLP-1 and insulin administration acutely enhances their vasodilatory, antiinflammatory, and antioxidant action in type 2 diabetes.

OBJECTIVE: To test the hypothesis that the simultaneous administration of GLP-1 and insulin may increase their vasodilatory, antiinflammatory, and antioxidant action in type 2 diabetes. RESEARCH DESIGN AND METHODS: In two groups of persons with type 2 diabetes, two sets of experiments were performed. The first group had two normoglycemic-normoinsulinemic clamps with or without GLP-1 and two normoglycemic-hyperinsulinemic clamps with or without GLP-1. The second group had two hyperglycemic-normoinsulinemic clamps and two hyperglycemic-hyperinsulinemic clamps with or without GLP-1. RESULTS: During the normoglycemic-hyperinsulinemic clamp, flow-mediated dilatation (FMD) increased, while soluble intercellular adhesion molecule (sICAM-1), plasma 8-iso-prostaglandin F2α (8-iso-PGF2α), nitrotyrosine, and interleukin (IL)-6 decreased compared with normoglycemic-normoinsulinemic clamp. Similar results were obtained with the infusion of GLP-1 during the normoglycemic-normoinsulinemic clamp. The combination of hyperinsulinemia and GLP-1 in normoglycemia was accompanied by a further FMD increase and sICAM-1, 8-iso-PGF2α, nitrotyrosine, and IL-6 decrease. During the hyperglycemic-normoinsulinemic clamp, FMD significantly decreased, while sICAM-1, 8-iso-PGF2α, nitrotyrosine, and IL-6 significantly increased. When hyperglycemia was accompanied by hyperinsulinemia or by the simultaneous infusion of GLP-1, these phenomena were attenuated. The simultaneous presence of hyperinsulinemia and GLP-1 had an increased beneficial effect. CONCLUSIONS: Our results show that the combination of insulin and GLP-1 is more effective than insulin or GLP-1 alone in improving endothelial dysfunction, inflammation, and oxidative stress in type 2 diabetes.

Type 1 diabetes and cardiovascular disease.

The presence of cardiovascular disease (CVD) in Type 1 diabetes largely impairs life expectancy. Hyperglycemia leading to an increase in oxidative stress is considered to be the key pathophysiological factor of both micro- and macrovascular complications. In Type 1 diabetes, the presence of coronary calcifications is also related to coronary artery disease. Cardiac autonomic neuropathy, which significantly impairs myocardial function and blood flow, also enhances cardiac abnormalities. Also hypoglycemic episodes are considered to adversely influence cardiac performance. Intensive insulin therapy has been demonstrated to reduce the occurrence and progression of both micro- and macrovascular complications. This has been evidenced by the Diabetes Control and Complications Trial (DCCT) / Epidemiology of Diabetes Interventions and Complications (EDIC) study. The concept of a metabolic memory emerged based on the results of the study, which established that intensified insulin therapy is the standard of treatment of Type 1 diabetes. Future therapies may also include glucagon-like peptide (GLP)-based treatment therapies. Pilot studies with GLP-1-analogues have been shown to reduce insulin requirements.

Vitamin C further improves the protective effect of GLP-1 on the ischemia-reperfusion-like effect induced by hyperglycemia post-hypoglycemia in type 1 diabetes.

BACKGROUND: It has been reported that hyperglycemia following hypoglycemia produces an ischemia-reperfusion-like effect in type 1 diabetes. In this study the possibility that GLP-1 has a protective effect on this phenomenon has been tested. METHODS: 15 type 1 diabetic patients underwent to five experiments: a period of two hours of hypoglycemia followed by two hours of normo-glycemia or hyperglycemia with the concomitant infusion of GLP-1 or vitamin C or both. At baseline, after 2 and 4 hours, glycemia, plasma nitrotyrosine, plasma 8-iso prostaglandin F2alpha, sCAM-1a, IL-6 and flow mediated vasodilation were measured. RESULTS: After 2 h of hypoglycemia, flow mediated vasodilation significantly decreased, while sICAM-1, 8-iso-PGF2a, nitrotyrosine and IL-6 significantly increased. While recovering with normoglycemia was accompanied by a significant improvement of endothelial dysfunction, oxidative stress and inflammation, a period of hyperglycemia after hypoglycemia worsens all these parameters. These effects were counterbalanced by GLP-1 and better by vitamin C, while the simultaneous infusion of both almost completely abolished the effect of hyperglycemia post hypoglycemia. CONCLUSIONS: This study shows that GLP-1 infusion, during induced hyperglycemia post hypoglycemia, reduces the generation of oxidative stress and inflammation, improving the endothelial dysfunction, in type 1 diabetes. Furthermore, the data support that vitamin C and GLP-1 may have an additive protective effect in such condition.

Glucagon-like peptide 1 reduces endothelial dysfunction, inflammation, and oxidative stress induced by both hyperglycemia and hypoglycemia in type 1 diabetes.

OBJECTIVE: Hyperglycemia and hypoglycemia currently are considered risk factors for cardiovascular disease in type 1 diabetes. Both acute hyperglycemia and hypoglycemia induce endothelial dysfunction and inflammation, raising the oxidative stress. Glucagon-like peptide 1 (GLP-1) has antioxidant properties, and evidence suggests that it protects endothelial function. RESEARCH DESIGN AND METHODS: The effect of both acute hyperglycemia and acute hypoglycemia in type 1 diabetes, with or without the simultaneous infusion of GLP-1, on oxidative stress (plasma nitrotyrosine and plasma 8-iso prostaglandin F2alpha), inflammation (soluble intercellular adhesion molecule-1 and interleukin-6), and endothelial dysfunction has been evaluated. RESULTS: Both hyperglycemia and hypoglycemia acutely induced oxidative stress, inflammation, and endothelial dysfunction. GLP-1 significantly counterbalanced these effects. CONCLUSIONS: These results suggest a protective effect of GLP-1 during both hypoglycemia and hyperglycemia in type 1 diabetes.

[Gender differences of at risk patients with overt heart failure in the real world of general practice. Data from the GIPSI (Gestione Integrata Progetto Scompenso in Italia) registry]. / Differenze tra la popolazione maschile e femminile a rischio ed affetta da insufficienza cardiaca nel mondo reale della medicina generale. I dati del registro GIPSI (Gestione Integrata Progetto Scompenso in Italia).

BACKGROUND: Controlled clinical trials have defined the characteristics of specialized world populations, different from the real world population. On this basis, the GIPSI registry was created, aiming to collect data from heart failure populations managed by general practitioners, focusing on gender differences. METHODS: The registry was based on family history, clinical and laboratory data collection from general practitioners. Patients were considered as being at risk for heart failure if data applied to stage A/B, or presenting overt heart failure if data applied to stage C/D of the American College of Cardiology/American Heart Association classification. RESULTS: From June 2006 to October 2007, 757 consecutive patients (475 male, 62.7%) were enrolled from 260 general practitioner's practices; 227 patients (143 male, 63.0%) had overt heart failure. In the female population at risk, higher systolic and diastolic blood pressure values were observed, whereas males showed more frequently ischemic heart disease, hypercholesterolemia and hypertriglyceridemia, and were more often prescribed with statins and antiplatelet drugs. There were more heart failure females with diabetes and of advanced age. Moreover, females showed a higher pulse pressure and a significantly lower estimated glomerular filtration rate (by simplified MDRD equation) than males. CONCLUSIONS: The data collected in a real world population show that heart failure has significantly different gender characteristics, especially for risk factors, age, blood pressure and renal function. This kind of investigation should be extended to larger patient populations for a better understanding of the disease.