Methods. 883 patients under 25 years of age with T1D for at least 6 months were investigated. Anthropometric parameters, blood pressure, and microvascular complications were evaluated, and the lipid profile and HbA1c were determined for all patients. Results. Study subjects' mean HbA1c was 8.5 ± 2%; 19.5% were overweight and 3.6% obese. Hypertension and dyslipidemia were diagnosed in 29.8% and 62.6% of participants, respectively. HbA1c concentration was directly related to levels of total cholesterol , LDL , and triglycerides  and inversely associated with levels of HDL . Prevalence of dyslipidemia increased with duration of diabetes (p < 0.05). Hypertension was more prevalent in overweight and obese compared to normal-weight patients . Frequency of microvascular complications was higher among patients with dyslipidemia  and among those with hypertension . Conclusion. The frequency of cardiovascular risk factors is high in youth with T1D and associated with diabetes duration, obesity, and metabolic control.Cardiovascular risk and obesity are becoming major health issues among individuals with type 1 diabetes (T1D). The aim of this study was to evaluate cardiovascular risk factors and obesity in youth with T1D in Lithuania.  The main cause of death in European countries is cardiovascular diseases (CVD) . SeveralCVD tend to present at a younger age in patients with diabetes than in the general population . The SEAThe other growing issue in type 1 diabetes (T1D) patients is obesity, which aggravates the risk of hypertension and dyslipidemia .The aim of our study was to analyze the risk factors for CVD in children and young adults under the age of 25 years with T1D in Lithuania.The presented cohort included 883 subjects from the recent joint Lithuanian-Swiss project “Genetic Diabetes in Lithuania,” which had 1209 subjects overall, covering all children and 70% of young adult (under the age 25) patients with T1D in Lithuania, described previously [This analysis included patients with established diabetes longer than 6 months and treated with insulin: 590 of them (66.8%) were from 1- to 17-year-old children and adolescents, and 293 are young adults (33.2%) from 18 to 25 years old. Subjects with diabetes duration less than 6 months were not included in this analysis due to the weight fluctuation and metabolic instability that are usually seen at the onset and initial therapy of diabetes [At the time of involvement in the project, data about age, duration of T1D, insulin delivery method (pump/injection), and total daily insulin dose (U/kg/d) were collected and clinical and laboratory assessments done. The participants were consulted by a single ophthalmologist for evaluation of diabetic retinopathy and a pediatric or adult neurologist for assessment of diabetic neuropathy.The Lithuanian Bioethics Committee granted the approval for this biomedical research (No. BE-2-5). Subject information forms and informed consent forms were signed by each participant or official representative.Anthropometric parameters were measured by clinical nurses at the Endocrinology Department of the Hospital of Lithuanian University of Health Sciences. At this department, the Harpenden Stadiometer  is used for height measurement. Patients' height was measured to the nearest ±0.1 cm three times, then the average was estimated for analyses. seca 700 medical scales (seca GmbH & Co. KG) were used for weight in kg, with precision of 0.1 kg.2 was used.For body mass index (BMI), the equation weight in kg/recumbent length or standing height in mz-score was evaluated according to age and gender using the references of the World Health Organization (WHO); for participants aged 19 to 25 years, a BMI z-score was evaluated according to the references of WHO for 19-year-old individuals, assuming that their linear growth is over.For participants under 19 years, a BMI 2 at 19 years); overweight was defined as BMI less than +2 SD (which corresponds to BMI 30 kg/m2 at 19 years), obese as BMI>+2 SD, and underweight as BMI < ‐2 SD.For all participants, normal weight was defined as BMI ranging from -2 standard deviations (SD) to less or equal to +1 SD /hip circumference (cm)) and waist-to-height ratio (WtHR) (waist circumference (cm)/height (cm)) were used. The waist‐hip ratio ≥ 0.90 cm for men and ≥0.85 cm for women were considered significantly increased . WtHR < Arterial blood pressure for children and adults was measured after sitting in silence for 5 min using an oscillometric sphygmomanometer in the left arm with appropriate cuff size. For children, “The Fourth Report from the National High Blood Pressure Education Program (NHBPEP) Working Group on Children and Adolescents” guidelines was used to classify measurements of arterial blood pressure: “Normal BP was defined as systolic blood pressure (SBP) and diastolic blood pressure (DBP) less than the 90th percentile for sex, age, and height. Hypertension was defined as average SBP or DBP that was greater than or equal to the 95th percentile for sex, age, and height on at least three separate occasions” .Measurements of arterial blood pressure in adults were classified according to the American Heart Association (AHA) guidelines: “Normal blood pressure defined as SBP <120 mmHg and DBP <80 mmHg, hypertension beginning at 140/90 mmHg and higher” .Glycosylated hemoglobin (HbA1c) and lipid profiles were measured by the UniCel DxC 800 Synchron system . The normal cutoff values of HbA1c were 4-6% (20 mmol/mol-42 mmol/mol). International Society for Pediatric and Adolescent Diabetes (ISPAD) guidelines were used to define optimal metabolic control when HbA1c < 7.5% (58 mmol/mol) for children and adolescents and <7% for young adults with reference to American Diabetes Association guidelines , 15.Normal values for low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and triglycerides (Tg) were defined as <2.6 mmol/l, >1.1 mmol/l, and <1.7 mmol/l, respectively , 16. NorAll participants were screened for microvascular diabetes complications at the same Endocrinology Department. A single diabetes ophthalmologist and an adult/pediatric neurologist consulted with all diabetes patients for the presence of retinopathy and neuropathy, respectively. Diabetic retinopathy was identified from stereoscopic fundal examination. Sensations for vibration, pressure, and temperature were evaluated for each patient, and all of them were surveyed with the Michigan Neuropathy Screening Questionnaire. If ≥2 of these tests were abnormal, peripheral neuropathy was diagnosed , 17, 18.For diabetic kidney damage, a 24-hour urine albumin excretion rate (AER) was evaluated. AER < 30 mg/24 h was defined as normal, 30-300 mg/24 h—microalbuminuria, and >300 mg/24 h—macroalbuminuria .t test, χ2 statistics, and parametric one-way ANOVA were used. For nonnormally distributed data, the Mann-Whitney U test was used, and for ordinal data, Kruskal-Wallis one-way ANOVA was used. For estimation of trends, linear regression models were used. For testing the hypothesis about relationships between dichotomous-dependent variables and continuous predictors, binary logistic regression analysis was carried out. p values <0.05 were considered as statistically significant. All p values were two-sided.The IBM SPSS Statistics Base version 22.0 was used for statistical analysis of the data. In case of normal data distribution, Student's 2-tailed n = 434) were males. The mean age of study subjects was 16.2 ± 5.6 yrs. The average diabetes duration was 6.7 ± 4.8 yrs . The distribution of patients by duration of T1D was as follows: 0.5-4 years 45.4% (n = 401), 5-9 years 30.9% (n = 273), 10-14 years 17.9% (n = 158), 15-19 years 4.3% (n = 38), and ≥20 years 1.5% (n = 13).Of the 883 subjects enrolled for the current analysis, 49.2% (z-score in the whole cohort was 0.29 ± 0.99. 75.8% of study subjects (n = 666) were of normal weight, 19.5% (n = 171) overweight, 3.6% (n = 32) obese, and 1.1% (n = 10) underweight. The distribution of weight status among different age groups is shown in p > 0.05). 20.5% of females and 18.3% of males were overweight (p > 0.05), and 3.3% of females and 3.9% of males were obese (p > 0.05).The mean BMI p < 0.05).Clinical characteristics according to weight group are shown in n = 192) of patients had WtHR higher than 0.5. As expected, overweight and obese individuals had higher WtHR than normal-weight subjects . However, no significant differences in the waist-hip ratio were found comparing different-weight-status participants.21.8% (n = 289) of patients had optimal glycemic control. The best glycemic control was recorded in the group of youngest patients: patients aged 1-4 yrs and 5-9 yrs had significantly lower HbA1c compared to patients aged 10-14 yrs, 15-19 yrs, and ≥20 yrs . In all age groups, females had significantly higher HbA1c than males, except in the youngest 1-4 yrs of age group (Study subjects' mean HbA1c was 8.5 ± 2% (69.2 ± 2 mmol/mol). 32.7% (ge group .p < 0.001). In the whole cohort, 30.2% of patients were on insulin pumps. HbA1c of patients treated with insulin pumps and multiple daily injections (MDI) was 8.5 ± 2% and 8.4 ± 1.8%, respectively, p > 0.05; also, there was a similar proportion of subjects with optimal glycemic control in insulin pump users or those on MDI .The average insulin dose was 0.83 ± 0.3 U/kg/d for the whole cohort. Adjusted for diabetes duration, patients with optimal glycemic control had a lower insulin dose compared to patients with suboptimal HbA1c  of the total cohort. Increased total cholesterol was found in 28.2% (n = 249) of patients, increased LDL in 54.9% (n = 484), increased Tg in 9.2% (n = 81), and decreased HDL in 13.5% (n = 119) of patients. The frequency of dyslipidemia was similar among all age groups: 50% (n = 8) among patients aged 1-4 years, 52% (n = 62)—5-9 years, 64.7% (n = 141)—10-14 years, 64.4% (n = 183)—15-19 years, and 64.7% (n = 156)—≥20 years.Dyslipidemia was found in 62.6% (p < 0.001). Also, the HbA1c level was significantly higher in patients with dyslipidemia compared to patients with a normal lipid profile . A significantly higher prevalence of dyslipidemia was found in patients with WtHR exceeding 0.5 compared to subjects whose WtHR was less than 0.5 .Dyslipidemia was more frequent in the group with poor glycemic control compared to that with optimal glycemic control  and directly related to LDL and Tg concentrations .In linear regression models, controlled for diabetes duration and glycemic control, BMI n = 263) of patients. It was more frequent among males compared to females . Hypertension was diagnosed more often in children (<18 yrs) than in adults (18-25 yrs) . Controlled for age and gender, the direct relationship between SBP, DBP, and BMI z-scores was found .Hypertension was diagnosed in 29.8% (n = 94), neuropathy in 11.5% (n = 97), and elevated AER in 9.4% (n = 83) of participants.In the whole cohort, 212 (24%) subjects were diagnosed with at least one microvascular complication. Retinopathy was diagnosed in 10.8% ; the same trend was found in patients with hypertension vs. patients with normal BP .Patients with an altered lipid profile had higher frequency of at least one microvascular complication compared to patients with normal levels of lipids , especially in children, compared to other authors , 23. Thiz-scores. Our results support findings from other studies that weight management is one of the principal strategies to lower the risk of CVD in T1D patients. In the future, it is expected that metformin would also bring some benefits for obese youth with T1D [We found a significant correlation between SBP, DBP, and BMI with T1D , 37.We report here similar frequency of microvascular complications among young T1D patients, compared to recently published data . Higher We discussed several limitations of our study. We did not perform BP monitoring at home, which would have elucidated the real prevalence of hypertensive and prehypertensive patients. Furthermore, in our cohort, we did not assess apolipoprotein B concentrations and carotid artery intima-media thickness, which are both significant predictors of CVD risk .Finally, the findings of our study highlight that the management of T1D should be multifaceted and most importantly include glycemic control, weight management, and dyslipidemia treatment.


We examined the within-person relationship between systolic blood pressure (SBP) and cognition and whether this association is moderated by hypertension status and stress level. Analysis was conducted on 50 (39 women and 11 men) community-dwelling African Americans ranging in age from 50 to 80 years . Participants’ blood pressure and cognition  was assessed on 8 different occasions over a 2-week time period. Stress was measured at baseline using the Elderly Life Stress Inventory. Findings show on days systolic blood pressure increased above an individual’s average level, their memory performance also tended to improve on that day. A significant 3-way interaction was further observed; such that on occasions when an individual’s systolic blood pressure was above his or her personal average, their executive functioning performance  was significantly better, particularly for participants who on average had a high SBP (or were hypertensive) and reported a history of high stress. These results suggest that higher levels of blood pressure are related to better cognitive performance among middle age to older African Americans. These counterintuitive findings may be attributed to the inclusion of a selective sample of resilient African Americans who have survived despite having adverse health conditions. This association may also arise from stress induced high effort coping, which is needed to perform well on cognitive tasks but also increases blood pressure.


Asthma-COPD overlap (ACO) has been under intensive focus; however, the levels of damage-associated molecular patterns (DAMPs) that can activate the innate and adaptive immune responses of ACO are unknown. The present study aimed to examine the levels of some DAMPs in asthma, COPD, and ACO and to identify the associations between clinical characteristics and DAMPs in ACO. Sputum from subjects with asthma (n = 87) or COPD (n = 73) and ACO (n = 68) or from smokers (n = 62) and never-smokers (n = 62) was analyzed for high mobility group protein B1 (HMGB1), heat shock protein 70 (HSP70), LL-37, S100A8, and galectin-3 . The concentration of HMGB1, HSP70, LL-37, and S100A8 proteins in sputum from ACO patients was significantly elevated, whereas that of Gal-3 was reduced, compared to that of smokers and never-smokers. The levels of HMGB1 and Gal-3 proteins in ACO patients were elevated compared to those in asthma patients. The sputum from ACO patients showed an increase in the levels of LL-37 and S100A8 proteins compared to that of asthma patients, whereas the levels decreased compared to those of COPD patients. The concentrations of HMGB1, HSP70, LL-37, and S100A8 proteins in the sputum of 352 participants were negatively correlated, whereas the levels of Gal-3 were positively correlated, with FEV1, FEV1%pred, and FEV1/FVC. Sputum HMGB1 had a high AUC of the ROC curve while distinguishing ACO patients from asthma patients. Meanwhile, sputum LL-37 had a high AUC of the ROC curve in differentiating asthma and COPD. The release of sputum DAMPs in ACO may be involved in chronic airway inflammation in ACO; the sputum HMGB1 level might serve as a valuable biomarker for distinguishing ACO from asthma, and the sputum LL-37 level might be a biomarker for differentiating asthma and COPD. Asthma and COPD differ from each other in their patterns of inflammation, immunological mechanisms, and the reversibility of their airflow limitations. However, some patients, especially elderly patients, may present several features associated with asthma and COPD, thereby complicating the diagnosis3. Recently, these patients have been termed asthma-COPD overlap (ACO) by international guidelines, which define ACO as a persistent airflow limitation along with high airflow reversibility2. Some studies demonstrated that these ACO patients might possess distinct clinical characteristics, such as cough or frequent and severe exacerbations, compared to patients with COPD or asthma4. Recently, great progress has been made in understanding the pathophysiology of asthma and COPD; however, the biological features of ACO are still poorly understood.Chronic obstructive pulmonary disease (COPD) and asthma are the most common chronic lung diseases worldwide, and obstructive airway diseases are characterized by airflow limitation and chronic airway inflammation2. Chronic airway inflammation in asthma and COPD is characterized by activation of the innate and adaptive immune systems5. However, the initiation of innate and adaptive immune responses in COPD and asthma has yet to be elucidated. Molecules derived from proteolytic allergens, such as viruses and bacteria, can activate pattern recognition receptors (PRRs) on structural and innate immune cells of the lung to induce the immune response5. Importantly, injured or dying cells release damage-associated molecular patterns (DAMPs) that might alarm the immune system by interacting with PRRs7. Therefore, Pouwels et al. postulated that DAMPs play a vital role in the initiation of chronic airway inflammation in COPD8.Airway inflammation plays an essential role in the development of both asthma and COPD. COPD is associated with an increased inflammatory reaction in the lungs, causing destruction of lung parenchyma. Asthma is associated with local chronic inflammation of the airway, resulting in airway remodeling and airway hyperresponsiveness7.DAMPs can be classified based on their origin. DAMPs include several nuclear proteins, such as high mobility group protein B1 (HMGB1), IL-1a, and IL-33, cellular organelles, such as mitochondria and histones, and other cellular components, such as adenosine triphosphate (ATP), uric acid, HSPs, heme, haptoglobin, defensins, LL-37, EDN, galectin-3 , heparan sulfate, hyaluronan, and S100 proteins11 that are implicated in the pathogenesis of asthma and COPD. Thus, HMGB1, HSPs, S100 proteins, and IL-33 have been under intensive focus in addition to other DAMPs, such as EDN, defensin, heme, and IL-1a; in particular, HMGB1 is widely considered the most classic DAMP6. Interestingly, accumulating evidence strongly supports the role of Gal-3 in the activation of allergic inflammation, such as eosinophil recruitment, airway remodeling, and development of a Th2 phenotype13. Similarly, some studies have shown that LL-37 is linked to airway inflammation in asthma and COPD14. To deduce the pathophysiology of ACO, we selected a panel of five DAMPs, including HMGB1, HSP70, LL-37, S100A8, and Gal-3, in the induced sputum.Increased levels of several DAMPs, including HMGB1, HSPs, and S100A8, have been observed in induced sputum and serum of asthma and COPD patients in our and other previous studiesIn this study, the levels of five DAMPs in the sputum of patients with asthma, COPD, and ACO were examined, followed by an analysis of the correlations between the concentrations of these DAMPs and different clinical variables.The induced sputum samples were collected from 352 participants . The participants were divided into five groups according to their medical history and pulmonary function tests: healthy never-smokers , healthy smokers , asthma patients , COPD patients , and ACO patients .1. The exclusion criteria included a respiratory tract infection characterized by purulent sputum and infiltration based on lung X-ray within the previous 1 month. Patients with stable COPD were diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines with incomplete reversible airflow obstruction. The exclusion criteria for all patients included respiratory tract infection, oral steroid treatment, or an exacerbation during the month before admission and a history of other lung diseases.The 62 healthy never-smokers were excluded if they had respiratory diseases, such as asthma, COPD, pneumonia, or failed to provide sputum samples. Additionally, all healthy smokers were excluded if they had respiratory diseases. The asthmatic patients were initially diagnosed in the two hospitals, as mentioned above, according to the Global Initiative for Asthma (GINA) guidelinesThe ACO patients were defined as smoking asthmatics with a smoking history ≥20 pack-years and COPD patients younger than an age of 40 years with a history of physician-diagnosed asthma or with reversible airway obstruction.The present study was approved by the Ethics Committee of Guangxi Medical University according to the 1964 Declaration of Helsinki and its later amendments, and all experiments on humans were performed in accordance with the relevant guidelines. All subjects provided informed consent for participation.Pulmonary function tests before sputum induction were performed using the Jaeger Masterscope spirometry system. Two reproducible measurements of each FEV1, FEV1/FVC, and FEV1%pred were obtained before and after inhalation of 400 mg salbutamol.9.Asthma and ACO patients were instructed to record their daytime and nocturnal symptoms for the preceding 24 h, as described previously9.COPD and ACO patients were required to record points by reading the 5-point Medical Research Council (MRC) dyspnea scale, as described previously9. The sputum induced by inhalation of hypertonic saline was weighed and diluted 4X in 0.1% dithiothreitol  in water. This suspension was mixed and incubated in a shaking water bath at 37 °C, followed by centrifugation at 750 × g for 10 min. The supernatant was stored at −80 °C, and the slides were stained with hematoxylin-eosin  for differential cell enumeration (n = 200 cells were counted).Sputum induction and processing has been described previouslyThe HMGB1, HSP70, LL-37, S100A8, and Gal-3 levels were quantified by ELISA kits [HMGB1 (Cat. No. HHE3533), HSP70 ; LL-37 ; S100A8 (Cat. No. DY4570-05), and Gal-3  curves of the DAMPs were drawn to assess the predictive capability for distinguishing ACO patients from those in other groups. Multiple stepwise regression analysis was performed to obtain the predictors of the DAMPs. P-values < 0.05 were considered statistically significant.The data were analyzed using SPSS 13.0 software and expressed as the mean ± SEM. The comparisons between groups were investigated using ANOVA, followed by the least significant difference (LSD) post hoc test. The frequency data  were analyzed by the χThe clinical characteristics of all participants are shown in Table 15. Two standard curves of each ELISA with normal reagent or 0.1% DTT were constructed to establish that DTT did not affect the estimations by ELISA in the present study (see Supplementary Table Dithiothreitol (DTT) is routinely selected to dissolve mucus proteins to obtain sputum supernatant. Although DTT has an effect on the analysis of some inflammatory markers in sputum supernatants such as IL-6, it had no effect on the assay standards for IL-1β or TNF-αThe sputum levels of HSP70 were elevated in the asthma, COPD, and ACO groups  compared to the NS and HS groups  but decreased in the asthma group compared to the HS group  indicated that S100A8 and S100A9 were elevated in the BALF of COPD patients compared to healthy smokers and never-smokers11. Furthermore, a recent computationally intensive analysis of induced sputum proteome demonstrated reduced levels of S100A8/9 in the induced sputum of asthmatic patients compared to healthy subjects25. To the best of our knowledge, this is the first report comparing sputum S100A8 in patients with asthma, COPD, and ACO. In the present study, the ACO group showed a higher sputum S100A8 level than asthmatic patients but a lower S100A8 level than COPD patients. Since S100A8 is constitutively expressed in neutrophils, the distinct sputum neutrophil counts may partially account for these differences in the levels of S100A8. However, the association between S100A8 and airway inflammation in these diseases remains unclear.S100A8 has been recognized as a DAMP molecule as extracellular S100 proteins can bind to receptors, such as RAGE and TLR4, and both lead to NF-κB activation26. LL-37 also displays a DAMP function as it can provoke a proinflammatory response by binding to TLR7, TLR9, and RAGE and induce necrosis in airway epithelial cells27. Consistent with the present study, some studies revealed that the increased LL-37 concentrations in COPD patients negatively correlated with lung function parameters29. Importantly, ROC curve analysis showed that sputum LL-37 had a high specificity and sensitivity and thus may be a useful and novel biomarker for the differentiation of asthma and COPD. Furthermore, we demonstrated that the sputum LL-37 levels in ACO were significantly higher compared to the non-smoking controls. Based on the previous literature and the current results, we speculated that LL-37 might participate in the inflammatory process underlying ACO.LL-37/hCAP-18 , primarily secreted by airway epithelial cells, possesses antimicrobial activity against bacteria, fungi, and viruses30. Only a few studies have investigated the role of Gal-3 in asthma and COPD. Gal-3 levels were significantly decreased in the BALF of patients with COPD and healthy smokers compared to controls31. Gao et al.32, for the first time, showed that the concentration of Gal-3 in sputum was significantly reduced in neutrophilic asthma compared to eosinophilic and paucigranulocytic asthma. However, the differences in levels of Gal-3 between asthmatics and healthy controls have not yet been determined. To the best of our knowledge, this is the first report to detect the sputum Gal-3 levels in COPD and ACO patients and to compare the level of Gal-3 in patients with asthma, COPD, and ACO. Interestingly, some studies33 showed that Gal-3 could remove apoptotic neutrophils, avoiding the amplification of inflammation, and that the decreased level of Gal-3 could be associated with defective efferocytosis of macrophages in COPD.Gal-3 is a β-galactoside-binding lectin with several physiological functions. Gal-3 is released into the extracellular space and can display a proinflammatory function, termed DAMPNotably, the relatively large sample size in the present study was beneficial for minimizing the statistical error. The asthmatics were first diagnosed without the use of medicine; the patients with asthma, COPD, and ACO neither suffered from nor had exacerbations during the month before admission, and this excluded the impact of infection. Furthermore, the participants were selected cautiously, especially the ACO patients, who were diagnosed according to the guidelines; the diagnosis criteria of ACO were based on both clinical history and objective parameters. Nonetheless, the present study has several limitations. Demographic differences were noted between the five groups with respect to age, smoking status, and use of drugs. In addition, comparisons between our results and those of other studies might be challenging due to the lack of consensus on the diagnostic criteria for ACO.In conclusion, the current data provide a novel approach to understanding the pathophysiological features of ACO. Interestingly, compared to asthma or COPD, ACO exhibited a distinct biomarker profile in sputum DAMP levels. Importantly, sputum HMGB1 is a novel biomarker for differentiating ACO from healthy controls, and sputum LL-37 may also serve as a valuable biomarker for differentiating asthma and COPD.Supplementary information


Based on non-clinical data, it is expected that azilsartan, an angiotensin II receptor blocker, will help improve insulin resistance in addition to its hypotensive action. The present study is aimed to explore the effect of azilsartan compared to telmisartan on insulin sensitivity in hypertensive patients in the clinical setting.This multicenter, randomized, open-label, parallel-group exploratory study was conducted in Japan. We randomized adult patients (≥20 years old) with grade I or II essential hypertension and coexisting type 2 diabetes (1:1) to receive either oral azilsartan  or telmisartan  for 12 weeks. The primary endpoint was the change in the homeostasis model assessment ratio of insulin resistance (HOMA-R) from the baseline at the end of the treatment period. We also evaluated its safety and efficacy on other diabetes-related variables and blood pressure.The mean changes in HOMA-R at the end of treatment were 0.22  in the azilsartan group and −0.23  in the telmisartan group. We found no clinically remarkable changes between the groups in diabetes-related variables such as fasting blood glucose, fasting insulin, HbA1c (NGSP), HOMA-β, or 1,5-anhydroglucitol. Reductions in clinic systolic and diastolic blood pressure were observed at week 4 and the reduced levels were maintained throughout the treatment period in both groups. No serious treatment-emergent adverse events (TEAEs) were observed. Only one drug-related TEAE (mild decrease in blood pressure) was reported in one patient in the azilsartan group.Neither azilsartan nor telmisartan had any clinically remarkable effects on insulin resistance parameters when administered for 12 weeks to patients with grade I or II essential hypertension and coexisting type 2 diabetes mellitus. Azilsartan (20 mg/day) and telmisartan (40 mg/day) exerted comparable antihypertensive effects.NCT02079805ClinicalTrials.gov  The Guidelines for the Management of Hypertension issued by the Japanese Society of Hypertension state that hypertension and type 2 diabetes mellitus are primary risk factors for major vascular disorders caused by endothelial dysfunction, atherothrombosis, and so on . Since tHypertension and type 2 diabetes mellitus represent components of metabolic syndrome, a disorder based on insulin resistance . TherefoAzilsartan is a relatively new ARB that has considerable clinical hypotensive action compared with other ARBs , 13. NonThus, we planned to explore the effects of azilsartan compared with telmisartan on insulin resistance by measuring the homeostasis model assessment ratio of insulin resistance (HOMA-R) and other diabetes-related variables in patients with concurrent grade I or II essential hypertension and type 2 diabetes mellitus.This multicenter, randomized, open-label, parallel-group exploratory study was conducted at 27 centers in Japan between June 2014 and April 2016. The study protocol was reviewed and approved by the Institutional Review Boards of all 27 centers, and the study was conducted in compliance with all Institutional Review Board regulations and in accordance with the principles of the Declaration of Helsinki and the Ethical Guidelines for Clinical Research . The trial was registered at ClinicalTrials.gov . Informed consent was obtained from all patients included in the study.2), markedly low bile secretion, or severe hepatic disorder; (l) having a history of hyper-sensitivity or allergy to azilsartan or telmisartan, or both; and (m) being pregnant, possibly pregnant, or breast-feeding.We assessed patients for eligibility after obtaining their informed consent. The inclusion criteria were as follows: (a) outpatients aged ≥20 years, (b) presence of grade I or II essential hypertension (sitting systolic blood pressure ≥130 mmHg and <180 mmHg or sitting diastolic blood pressure ≥80 mmHg and <110 mmHg at the start of the treatment period), (c) presence of coexisting type 2 diabetes with an HbA1c level of <8.4%  and a ≤0.3% change in HbA1c (peak minus nadir during three months before the start of the treatment period), and (d) fixed diet/exercise therapy, if applicable, for three months before the start of the treatment period. The exclusion criteria were as follows: (a) grade III essential hypertension , secondary hypertension, or malignant hypertension; (b) use of oral antihypertensive medication within two weeks before the start of the treatment period; (c) grade II essential hypertension treated with antihypertensive drugs (sitting systolic blood pressure ≥160 mmHg or sitting diastolic blood pressure ≥100 mmHg); (d) use of renin–angiotensin system (RAS) inhibitors or thiazolidines within three months before the start of the treatment period; (e) type 1 diabetes mellitus; (f) fasting blood glucose ≥180 mg/dL and HOMA-R ≤1.6 at the start of the treatment period (Week 0); (g) receiving or requiring treatment with insulin, glucagon-like peptide (GLP)-1 receptor agonists, or other parenteral hypoglycemic agents, or combination therapy with three or more oral hypoglycemic agents; (h) antidiabetic medication changes (including dosage and administration changes) within three months before the start of the treatment period; (i) having been diagnosed with myocardial infarction, cerebral infarction, cerebral hemorrhage, or transient ischemic attack, or having undergone a coronary revascularization procedure within three months before the start of the treatment period, or presenting with unstable conditions after the start of the treatment period; (j) having been diagnosed with or treated for advanced hypertensive retinopathy within three months before the start of treatment; (k) presenting with severe ketosis, diabetic coma (or precoma), severe infection, serious trauma, clinically evident renal disorder  and concurrent use of biguanides (yes or no) as stratification factors. The registration center performed the randomization, and the related information was not disclosed to the sponsors/investigators until the end of the study.The patients orally received azilsartan (20 mg/day) or telmisartan (40 mg/day) in the morning before or after breakfast. On days of scheduled site visits, we administered the study drugs after tests/examinations. Concomitant use of drugs affecting the HOMA-R or blood pressure, including thiazolidines, insulin, GLP-1 receptor agonists, and all antihypertensives, was prohibited during the study. We also prohibited the concomitant use of oral hypoglycemic agents other than those that had been taken at the time of obtaining informed consent. We allowed the continuous use of other drugs used to treat concurrent diseases. However, we did not allow changes in the dosage and administration of oral hypoglycemic agents from those used at the time of obtaining informed consent. We required participating patients using antihypertensives at the time of obtaining informed consent to undergo a two-week washout period before the start of treatment.We chose the change in HOMA-R from baseline at the end of the treatment period as the primary efficacy endpoint. HOMA-R was calculated according to the formula: fasting insulin (μU/mL) × fasting glucose (mg/dL) / 405. The secondary efficacy endpoints were changes in the following variables: fasting blood glucose, fasting insulin, HbA1c (NGSP), HOMA-β, and 1, 5-anhydroglucitol (AG). HOMA-β was calculated according to the formula: fasting insulin (μU/mL) × 360 / (fasting glucose [mg/dL] − 63). We also assessed changes in clinic and home blood pressure levels.We monitored adverse events (AEs) during the study and evaluated their severity and causal relationship with the study drugs; we used the system organ class and preferred term to report AEs coded according to the Medical Dictionary for Regulatory Activities (MedDRA) version 19.0.We analyzed the efficacy endpoints using the full analysis set (FAS), which comprised patients who were randomized and received at least one dose of the tested drugs during the study period. For the primary endpoint, we calculated descriptive statistics of HOMA-R for each treatment group on the FAS population as the primary analysis. As a secondary analysis of the primary endpoint, we also examined the change in HOMA-R for subgroups stratified using the baseline HOMA-R values (<2.5 or ≥2.5) and concurrent use of biguanides (Yes or No). We analyzed other efficacy endpoints using the FAS population. We performed safety analysis using the safety analysis set (SAS), which comprised patients who received at least one dose of the test medication during the study period.Since this was an exploratory study, we did not perform statistical significance tests on each variable.We had originally planned to include 50 patients in each treatment group in consideration of feasibility, not based on a statistical rationale. However, we reduced that number to 20 in each group due to the slow enrollment of patients meeting the stringent eligibility criteria and the limited study period.Of the 78 patients who provided informed consent, we excluded 45 who did not meet the inclusion criteria or who met the exclusion criteria (n = 42) or who withdrew voluntarily (n = 3). Thus, we randomly assigned 33 patients to either one of the two treatment groups  and included them in the FAS and SAS populations . Of thesThe baseline characteristics in the FAS were comparable between the two groups .The mean (SD) of baseline HOMA-R was 4.24 (±1.843) in the azilsartan group and 3.31 (±1.366) in the telmisartan group. The mean [95% confidence interval (CI)] changes in HOMA-R from the baseline at the end of treatment were 0.22 (−1.09–1.52) in the azilsartan group and −0.23 (−0.72–0.27) in the telmisartan group . The meaPatient populations were stratified by subgroups based on the baseline HOMA-R values of <2.5 or ≥2.5 and with or without concurrent use of biguanides. No clinically remarkable differences were observed in the changes in HOMA-R from baseline in the subgroups with HOMA-R values of ≥2.5 and without concurrent use of biguanides. The number of the patients in the subgroups with HOMA-R values <2.5 and concurrent use of biguanides was too small to be evaluated .We also found no clinically remarkable effects of azilsartan or telmisartan on the other diabetes-related variables .The clinic systolic and diastolic blood pressure values in the azilsartan and telmisartan groups decreased at comparative levels by week 4 and then remained stable at similar low levels until the end of the treatment .The changes from the baseline values in blood pressure at the end of the treatment period are summarized in We observed no apparent changes from the baseline values in home systolic and diastolic blood pressure in terms of day-by-day and circadian blood pressure variability at the end of treatment in either group –S8 Figs.The incidences of treatment-emergent AEs (TEAEs) during the treatment period were 35.3% (6/17) in the azilsartan group and 50.0% (8/16) in the telmisartan group as summarized in It is important to strictly control blood pressure levels to prevent microvascular and macrovascular diseases in patients with hypertension coexisting with type 2 diabetes mellitus . VariousHere, we compared the effects of azilsartan and telmisartan on the insulin resistance index (HOMA-R) and other diabetes-related variables in patients with grade I or II essential hypertension complicated with type 2 diabetes mellitus. The mean (95% CI) changes from the baseline values in HOMA-R at the end of the treatment period were 0.22 (−1.09–1.52) in the azilsartan group and −0.23 (−0.72–0.27) in the telmisartan group, thus indicating that azilsartan and telmisartan did not clinically remarkably improve insulin resistance, although telmisartan showed a trend of slightly decreasing insulin sensitivity. The mean difference in the changes from the baseline values in HOMA-R between the azilsartan and telmisartan groups was 0.44 (−0.89–1.78). The stratifying factors of the baseline HOMA-R values (<2.5 vs. ≥2.5) and the concurrent use of biguanides (yes vs. no) did not impact the changes in HOMA-R in either group. Moreover, we found no clinically remarkable changes from the baseline values concerning other diabetes-related variables, such as fasting blood glucose, fasting insulin, HbA1c (NGSP), HOMA-β, or 1, 5-AG, in either group.Despite the results from meta-analysis of randomized studies demonstrating significant improvements in insulin resistance with telmisartan compared with active controls in patients with hypertension , the effBoth azilsartan and telmisartan resulted in reductions in the clinic diastolic and systolic blood pressure levels, and some patients achieved normal blood pressure levels at the end of the treatment period. Azilsartan (20 mg/day) appeared as effective as telmisartan (40 mg/day) in controlling blood pressure in this patient population. Although the reductions in clinic systolic and diastolic pressure levels in the azilsartan group were numerically higher than those in the telmisartan group, our findings cannot be considered conclusive because the direct comparison of the antihypertensive efficacy of the two treatments was not a pre-specified objective of the study.Azilsartan and telmisartan both demonstrated favorable safety and tolerability profiles in our patient population, and we did not identify any new safety signals.The main limitations of this study were the open-label design, small number of enrolled patients, and relatively short treatment period. Double-blind studies utilizing larger patient populations and longer treatment periods are required to conclusively determine the effects of azilsartan and telmisartan on insulin resistance and glycemic control in patients with hypertension and type 2 diabetes mellitus.In conclusion, we did not observe any clinically remarkable effects of 12-week treatment with azilsartan and telmisartan on the variables of insulin resistance and the disease state of diabetes mellitus, including HOMA-R, fasting blood glucose, fasting insulin, HbA1c (NGSP), HOMA-β, and 1, 5-AG in hypertensive patients with type 2 diabetes mellitus enrolled in this study.S1 Table(DOCX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S2 Protocol(PDF)Click here for additional data file.


In order to effectively develop personalized medicine for kidney diseases we urgently need to develop highly accurate biomarkers for use in the clinic, since current biomarkers of kidney damage  apply to a later stage of disease, lack accuracy, and are not connected with molecular pathophysiology. Analysis of urine peptide content (urinary peptidomics) has emerged as one of the most attractive areas in disease biomarker discovery. Urinary peptidome analysis allows the detection of short and long-term physiological or pathological changes occurring within the kidney. Urinary peptidomics has been applied extensively for several years now in renal patients, and may greatly improve kidney disease management by supporting earlier and more accurate detection, prognostic assessment, and prediction of response to treatment. It also promises better understanding of kidney disease pathophysiology, and has been proposed as a “liquid biopsy” to discriminate various types of renal disorders. Furthermore, proteins being the major drug targets, peptidome analysis may allow one to evaluate the effects of therapies at the protein signaling pathway level. We here review the most recent findings on urinary peptidomics in the setting of the most common kidney diseases. Kidney diseases can be caused by a variety of insults such as hypertension, genetic or metabolic disorders, infections, toxins, ischemia, immunological disorders or allograft rejection. These renal insults may promote the development of chronic kidney disease (CKD), a major health problem affecting ~ 10% of the general population and causing a huge economic and personal burden [In clinical practice, kidney damage is generally detected by changes in serum creatinine and a creatinine-based estimate of the glomerular filtration rate (eGFR), and/or urinary albumin/protein excretion. However, both methods have major limitations, including nonspecificity, substantial variability, and lack of accuracy . A signiUrinary proteomics has progressively emerged as one of the most attractive topics in disease biomarker discovery . The proBoth proteins and peptides (short aminoacid chains) are contained in urine but peptides are mainly the results of complex molecular post-translational modifications of larger polypeptide molecules. In fact, their specific structures are not only representative of a single gene product, but effectively represent the action of multiple biochemical steps. Peptidomics focuses on the discovery and quantitation of such endogenous peptides, given their wide range of encoded biological functions and their associated diagnostic and therapeutic potential . EndogenIn this article, after a brief section on available technologies, we review the most recent findings on urinary peptidomics in the setting of the most common kidney diseases, covering the publication years 2005–2019.In contrast to proteomics, that pursues the identification of proteins through the analysis of proteolityc peptides obtained upon enzymatic digestion (bottom up) or entire proteins (top down), peptidomics analysis implies the characterization of the complete collection of naturally occurring peptides present in the sample without any preventive protein digestion. Pros and cons can be defined for both these approaches .For peptides analysis, technologies that can assure limited pre-analytical manipulation, low dynamic range of detection, high mass accuracy, high reproducibility and rapid analysis times are required. The complexity of the chemical properties of the peptidome has been only partially addressed mainly due to the large chemical space in which this class of molecules falls. Multiple analytical separation techniques have been employed including nano- and microscale ultra performance liquid chromatography (UPLC) and capillary electrophoresis (CE) in combination with mass spectrometry (MS).Mass spectrometry is an analytical technique that allows the characterization of mass, structure and concentration of unknown molecules, provided that molecules are in their ionized form. Ionized molecules in gas phase pass through a mass analyzer and their mass over charge ratio is measured as a function of their relative abundance. Peptides ionization step prior to MS scan can be performed by means of Electrospray Ionization (ESI) or Matrix Assisted Laser Desorption Ionization (MALDI).CE-MS has been widely used for urine peptidome analysis. The separation of sample in CE is based on the assumption that the electrophoretic mobility of the molecule is directly proportional to its charge and inversely proportional to its size. The application of a high voltage to a capillary filled with an electrolyte solution, generates both an electrosmotic flow and an electrophoretic migration, allowing to separate the analytes with a high resolution. Before CE peptide separation, sample needs to pass through some pre-analytical manipulation, to reduce its complexity and to eliminate contaminant compounds. Several methods have been developed to prepare native peptides from urine samples .CE, which is commonly coupled with an ESI ion source, in comparison with LC, shows some advantages: it allows the separation of small multicharged peptides, which are poorly retained by the stationary phases commonly used in the reverse phase column used in LC, or can separate large molecules which are not efficiently eluted from the LC column . MoreoveDirect analysis by MALDI TOF MS has also been used for urinary peptide profiling. The advantage is that it does not require preventive purification step and that all the peptides are simultaneously evidenced in the detection window. It represents an attractive high throughput platform for urine biomarker discovery, though particular care is required in sample manipulation to reduce pre-analytical and analytical variability . In MALDLastly, though MALDI reproducibility may be affected by different sources of measurement error, the application of biostatistical tools during the data analysis can improve the reliability of the instrumental measurement .The analytical technologies so far applied are still in a framework of research application only. Validation of these mass spectrometer platforms, as observed in other applications , is stil2 for at least three months [CKD is defined as structural renal damage, or as a glomerular filtration rate (GFR) < 60 mL/min/1.73 mOne large study involved 379 healthy subjects and 230 patients suffering from various kidney diseases identified by CE-MS 273 peptides that significantly distinguished between CKD and healthy controls . These 22), the CKD273 analyzer outperformed albuminuria in predicting eGFR loss, whereas albuminuria performed better in CKD subjects with late-stage disease (eGFR < 50 mL/min/1.73 m2). In a longitudinal study including 2087 patients with baseline eGFR > 60 mL/min/1.73 m2, over a median 4.6 years, CKD273 improved prediction of decline in glomerular filtration and progression to CKD stage 3 (eGFR < 60 mL/min/1.73 m2), while accounting for baseline eGFR, albuminuria and covariables [n = 9; death, n = 6) displayed a much higher CKD273 score than subjects alive and not dialyzed [Beside detection of CKD, application of the CKD273 classifier was also found to enable prediction of progression of kidney disease in patients with CKD of various etiologies ,19,20. Tariables . Note thariables  proposedIn the CKD273 classifier, the sequenced markers for the diagnosis of CKD  were divn = 16; type 2, n = 19), application of the CKD273 classifier led to earlier detection (1.5 years) of progression to macroalbuminuria (AUC 0.93) than did routinely used urinary albumin (AUC 0.67); the prominent biomarkers before onset of macroalbuminuria were decreased collagen fragments [post hoc analysis of 737 samples from the DIRECT  2 study cohort showed the better predictive role of CKD273 over albuminuria in the development of microalbuminuria from a baseline normoalbuminuric state [p = 0.004), and retained significance after adjustment for sex, age, blood pressure, coronary artery calcium score, and N-terminal pro-brain natriuretic peptide [Several studies have demonstrated the validity of urinary peptidome analysis in the setting of diabetes mellitus, the leading cause of CKD worldwide. In a 5-year follow-up study in 35 normoalbuminuric diabetic patients  or spironolactone  as an adjunct to inhibition of the renin-angiotensin system. The percentage change in the urine albumin to creatinine ratio (UACR) represented the primary end-point of the study. The authors found that higher values of CKD273 at baseline were associated with a larger decrease in UACR in patients treated with spironolactone (p = 0.049), but not in the placebo group (p = 0.25). After stratification into tertiles of baseline CKD273 score, a significant reduction in UACR during treatment  was found in the highest tertile only; the reduction there was significantly higher (p = 0.013) than in the other two tertiles combined. These results suggest that the urinary proteomics classifier may represent a valuable tool for tailoring therapy, distinguishing between those diabetic patients at greatest risk of progression and those with the best albuminuria-lowering response to treatment with spironolactone [Apart from diagnostic and prognostic value, analysis of urinary polypeptide patterns in diabetics has also shown its potential in assessing therapeutic intervention. Eighteen type 2 diabetic patients with diabetic retinopathy and macroalbuminuria (≥300 mg/die) were studied in a randomized double-blinded, cross-over trial including four treatment periods each lasting two months . Each paal trial . Patientolactone .2 and normoalbuminuria, funded by the European Union [n = 102) or placebo (n = 107) on top of standard care for 2.5 years. Standard care was continued in the low-risk group. The aims of the PRIORITY study were (I) to test CKD273 prospectively as predictive of development of microalbuminuria (primary end-point), and (II) to evaluate in subjects with a high-risk score whether intervention with spironolactone can reduce the risk of microalbuminuria. Traditional diabetic disease risk factors differed slightly between participants at high risk and those at low-risk of disease progression based on the CKD273 score, suggesting that the classifier may provide additional prognostic information over and above the clinical data [p < 0.0001). However, there was no difference in the occurrence of new kidney disease between patients treated with spironolactone or placebo , which might be related to the study design or to spironolactone only having an effect at more advanced stages of kidney damage [The combination of risk assessment and treatment benefit with spironolactone was tested in the PRIORITY trial. The Proteomic prediction and Renin angiotensin aldosterone system Inhibition prevention of early diabetic nephRopathy in TYpe 2 diabetic patients with normoalbuminuria (PRIORITY) trial NCT02040441) was a randomized, double-blind, placebo-controlled multicenter clinical trial and observational study in diabetics with eGFR > 45 mL/min/1.73 man Union . Particical data . Results441 was apost-hoc analysis in 40 uncomplicated type 1 diabetics treated for 8 weeks with the sodium glucose cotransporter 2 inhibitor, empagliflozin, showed that treatment significantly changed the amount of 107 peptides in the urine, the direction being toward “CKD absent” [Stratification of CKD patients by CKD273 classifier was also recently investigated in urine samples derived from the completed MARLINA-T2D clinical trial . The Eff absent” . These o absent” ,43 sugge2, and even predicted a rapid CKD progression in individuals with eGFR > 60 mL/min/1.73 m2 and albuminuria < 30 mg/day. The association between CKD273 subclassifiers and rapid CKD progression remained significant after adjustment for sex, age, diabetes, albuminuria, baseline eGFR, and systolic blood pressure. In rapid progressors with eGFR > 60 mL/min/1.73 m2, down-regulated collagen fragments were the predominant differentially expressed peptides, whereas in more advanced CKD, peptides deriving from plasma proteins were abundant and associated with faster progression. When peptide overlap with CKD273 was examined, common peptides varied according to the eGFR stratum. The highest number of common peptides was found for stratum 1 (eGFR ≥ 80 mL/min/1.73 m2), whereas in stratum 6 (eGFR 30–39 mL/min/1.73 m2) there was no peptide overlapping [More recently, CKD subclassifiers specific for CKD stages were generated, allowing early identification of patients at high risk of CKD progression . Urinaryrlapping .2 and at least 3 years of follow-up into non-progressors or stable  as opposed to those with an improved eGFR  [A different approach to evaluating CKD progression was used in a recent study which focused on investigating molecular alterations in CKD patients presenting an improved GFR . The facn = 177) . Out of n = 177) . Only 29n = 177) . These rn = 177) . In addiIn summary, the available evidence underscores the utility of urinary peptidomics in the detection and stratification of patients suffering from CKD. Also, peptides identified by the peptidome analysis may point to abnormal biological pathways and hence lead to new therapeutic targets.Acute kidney injury (AKI) is defined as a rapid decrease in glomerular function and/or urine output. AKI is the most frequent acute renal condition, is associated with significant morbidity and mortality, and increases the risk of CKD ,48.Current diagnostic tools do not allow early detection or prediction of the course of AKI, thereby hampering any improvement in patient outcome . The sean = 20; AUC = 0.84) and in leukemia patients after hematopoietic stem cell transplantation  [Metzger et al.  analyzed = 0.90) . As comp = 0.90) . In a su = 0.90) .CE-MS, while effective in identifying a urinary peptide marker profile for early AKI diagnosis ,52, is aOther clinical studies have identified different biomarkers. The use of serial urinary peptidomics has been employed in subjects undergoing cardiopulmonary bypass surgery, 22 patients with AKI and 22 patients without AKI . This stThere are two clinical trials directly associated with the use of protein biomarkers in acute kidney dysfunction prognosis . In 52 lHopefully, new bioinformatics approaches for the elucidation of the molecular events leading to the development of these molecular species and the mapping of molecular interactions will provide causative insights into the underlying molecular mechanisms and pathways involved in AKI .Kidney transplantation represents the optimal choice of therapy for end-stage renal disease patients. However, long-term graft function is still limited, mainly due to active or latent rejection, viral infections, and drug toxicity. Identification of post-transplantation complications currently requires an allograft biopsy, which however has monitoring drawbacks and is sometimes ambiguous . There iMany proteomic studies have focused on graft rejection, given that, despite improvements in immunosuppressive therapy and patient surveillance after renal transplantation, allograft rejection remains a significant adverse factor for long-term graft survival . Both T Using LC-MS/MS analysis, urine samples from kidney transplant patients with biopsy-proven acute rejection showed significantly different levels in a number of proteins compared to patients with stable graft on a biopsy protocol . SpecifiHowever, detection of T-cell mediated rejection is not the only need in kidney allograft surveillance . The kidWhen evaluating the urinary peptidomic results, the biological significance of identified molecules and identification of the modulated processes which are involved form a key aspect. In a recent review on proteomic markers for T-cell mediated rejection, use of the pathway- and enzyme reaction-related Reactome information resource revealed processes related to platelet degranulation, lipid digestion, keratan sulphate degradation, antigen presentation and interferon gamma signaling to be directly associated with the input proteins . More reGlomerulonephritis represents one of the most common causes of CKD and requires careful assessment of the disease for prognostic and therapeutic purposes. While many diagnostic tests are currently being used to refine the clinical diagnosis, kidney biopsy remains the gold standard for revealing diagnostic and prognostic histological features. However, it is an invasive procedure, cannot be repeated frequently, and does not provide information on a cellular or signaling pathway level. Urinary proteomic analysis has been proposed as a “liquid biopsy”, which can be used to discriminate various types of kidney diseases . In addiProteomic approaches have revealed differences in protein expression among many different glomerular diseases . In addin = 179), membranous nephropathy (n = 77), focal segmental glomerulosclerosis (n = 110), minimal change disease (n = 35), lupus nephritis (n = 92), vasculitis (n = 111), diabetic nephropathy (n = 422) and hypertensive nephrosclerosis (n = 154). Except for the latter two disorders, all the diagnoses were biopsy-proven. Notably, the study was designed to compare one group with all other kidney disease types, without involvement of healthy individuals. Analysis by CE-MS defined several potential urinary biomarker peptides (ranging from 116 peptides for IgA nephropathy to 619 for diabetes and hypertensive nephrosclerosis), which were combined into support vector machine-based classifiers specific for each kidney disease. The classifiers were validated and proved to discriminate among disease types with good to excellent accuracy. Importantly, several of the biomarkers identified were associated with apparent mechanisms of disease [The value of urine peptide-based signature panels in the differential diagnosis of certain kidney diseases has been suggested by a recent study of 1180 urine samples . The stu disease  and are  disease  and guid disease . IgA nep disease . This prFor better management of kidney diseases, we urgently need to develop highly accurate biomarkers for use in clinics. Proteins and peptides are the main functional and structural units of the cell and hence of great biomarker potential, since qualitative and quantitative differences in the proteome and peptidome composition reflect pathologic conditions. Based on current evidence, mostly in the context of CKD, urinary peptidomics may bring a significant improvement in kidney disease management by supporting earlier and more accurate detection, prognostic assessment, and prediction of response to treatment. Peptidomics/proteomics is expected to develop into the key technology to guide personalized intervention, and CKD273 classifiers may serve as a first example for personalized medicine in nephrology .However, despite the abundance of publications, the clinical translatability of peptidomic studies, already appearing in the literature, has some major limitations which should be considered. One major argument against implementation of urinary peptidomics in daily clinical practice is the lack of guidance on how to place the biomarkers in the clinical context . Clinicawww.peptiCKDdb-com) is expected to facilitate modeling of molecular mechanisms underlying CKD and identification of relevant biomarkers [Sharing of data obtained in peptidomic studies among the scientific community is also of critical importance, since it will ensure data are available, encourage collaboration, and facilitate integration . Specialomarkers .Additional issues blocking clinical implementation regard acceptance by the medical community and the cost-effectiveness of the approach. Urinary proteome analysis is complex, requires expertise, and is not without significant costs. The current technological platforms have been showing limitations in their implementation in laboratory medicine facility, providing ground for the development of centralized facility service over the wide territorial area. If this model proves to be cost effective in terms of laboratory production, it also demonstrates that the current results are not of direct use in taking an immediate medical decision as often required in kidney disease healthcare. The development of more robust analytical platforms is a fundamental prerequisite to explore effectively the clinical application of peptidome analysis, which necessarily requires the molecular discriminatory capacity of mass spectrometer analysers.However, a recent health economy assessment indicates that early detection in type 2 diabetic patients, as achievable by peptidomic analysis, is cost-effective . In a MaIn conclusion, urinary proteomics is one such promising technology that could help to personalize treatment in patients suffering from kidney disease , though


Asthma is a heterogeneous disease with varying severity. Severe asthma is a subject of constant research because it greatly affects patients’ quality of life, and patients with severe asthma experience symptoms, exacerbations, and medication side effects. Eosinophils, although at first considered insignificant, were later specifically associated with features of the ongoing inflammatory process in asthma, particularly in the severe case. In this review, we discuss new insights into the pathogenesis of severe asthma related to eosinophilic inflammation and the pivotal role of cytokines in a spectrum that is usually referred to as “T2-high inflammation” that accounts for almost half of patients with severe asthma. Recent literature is summarized as to the role of eosinophils in asthmatic inflammation, airway remodeling, and airway hypersensitivity. Major advances in the management of severe asthma occurred the past few years due to the new targeted biological therapies. Novel biologics that are already widely used in severe eosinophilic asthma are discussed, focusing on the choice of the right treatment for the right patient. These monoclonal antibodies primarily led to a significant reduction of asthma exacerbations, as well as improvement of lung function and patient quality of life. It is well established that asthma is a disease with a great spectrum of symptoms among patients and wide differences in treatment efficacy. In particular, severe asthma is noted to include various specific phenotypes and endotypes, which differ in their clinical presentation, their unique pathogenetic mechanisms, and their responsiveness to treatment . In orde+ T cells and more eosinophils to the site of damage in this aberrant reaction, leading to the Th2-high endotype of severe asthma with high blood and sputum eosinophils [Eosinophils were described almost 150 years ago by Paul Ehrlich as granulocytic leucocytes with a bilobed nucleus. Their primary location is within tissue and not in the bone marrow, residing mostly in the gastrointestinal tract in normal conditions . This cainophils .+ progenitor stem cells and specialized CD34+ interleukin-5 receptor (IL-5R)+ hematopoietic progenitor cells via a complex activation of transcription factors, the most important being GATA-1, PU.1, and CAAT enhancer-binding proteins α and ε [First of all, it is important to distinguish the two major types of eosinophils in the lungs. Even though the simplistic view we had in the past about eosinophils is still viable, recent studies showed that, except for eosinophils that emerge from the bone marrow and are directly recruited to sites of inflammation, a distinct type of eosinophil with different characteristics resides in tissues in homeostatic conditions. This eosinophil population is called “homeostatic eosinophils” (hEos) . These h α and ε . Notably α and ε .+ progenitor cells with IL-3, IL-5, and GM-CSF resulted in an upregulation of the IL-5R in these stem cells, thus prolonging eosinophil differentiation as long as they were stimulated by IL-5 [Several cytokines also take part in the development of eosinophils apart from the transcription factors previously mentioned, IL-5, IL-3, and GM-CSF(Granulocyte-macrophage colony-stimulating factor). IL-3 and GM-CSF are not selective, and they stimulate the development of other leucocytes such as neutrophils and macrophages more efficiently; however, IL-5 solely affects eosinophils and basophils . The maj by IL-5 . However by IL-5 .Even more interesting is the observation that hEos do not take active part in the allergic inflammation, as well as halting this aberrant response. They express several genes that cannot be found in the normal iEos that take part in the immunoregulation of lung and reduce the Th2 response after contact with allergens. Mice who were stripped of the prevalent eosinophil production gene (ΔdblGATA) showed a more severe allergic reaction after contact with dust mites, proving that hEos do not participate in the inflammatory process and they also downregulate the Th2 response, most probably by inhibiting the functionality of dendritic cells .Eosinophil trafficking from the bone marrow to the lungs is the first major step of the blooming inflammatory process. Even though various chemoattractants were discovered, most of them are not selective and can also draw other leucocytes. Activated Th2 cells and type 2 innate lymphoid cells (ILC2) synthesize IL-4, IL-5, and IL-13, while eotaxin-1 (CCL11) is produced by epithelial and endothelial cells after an allergen challenge. Specifically, IL-5 and eotaxin-1 play a pivotal role in eosinophil trafficking and synergize in promoting lung eosinophilia . Althoug1/2) by almost 50% [IL-5 is the most crucial cytokine not only in recruiting eosinophils but also in prolonging their survival in tissues. This was observed in IL-5 KO mice which showed a greatly reduced number of eosinophils in the lungs when compared to IL-5 transgenic mice ,20. Thismost 50% . IL-5 wamost 50% . It is smost 50% .Eotaxin-1 was first described as a novel component in the bronchoalveolar lavage (BAL) of guinea pigs sensitized and challenged by ovalbumin and was later isolated from human tissue as well. It was the first eosinophil-specific chemoattractant discovered until two more CC chemokines named eotaxin-2 and eotaxin-3 were isolated later on . EotaxinEotaxin-1 is needed in the first steps of the inflammatory response, whereas eotaxins 2 and 3 are needed to prolong eosinophil survival later on . Their sEosinophils, which are found in circulation after being mobilized mostly by IL-5 and eotaxin-1 as previously mentioned, still need to migrate from the vasculature to the lung tissue. In this phase, several eosinophil-specific adhesion molecules with the most important being the β1 integrin very late antigen (VLA-4), the vascular cell adhesion molecule (VCAM-1), and the P-selectin glycoprotein ligand (PSGL-1) play an important role . VLA-4 i+ and CD34+ IL-5Rα+ hematopoietic progenitor cells was much higher in this population’s sputum when compared to mild asthmatics. Even more interesting was the fact that eosinophil progenitor cells did not vanish after anti-IL-5 treatment in these patients, which means that in situ eosinophilopoiesis is an important mechanism of persistent eosinophilia in the airways [Recent studies highlighted the fact that eosinophilopoiesis can also occur in situ in the airways of severe asthmatics, since the number of CD34 airways . This co airways . The T2  airways . This pe airways .Eosinophils are the predominant cells of the inflammatory response in the lungs, contributing greatly to two major events: the remodeling and the hyperresponsiveness of the airways (AHR). Persistent inflammation caused by eosinophils leads to constant damage of the airways. The regeneration process is not flawless and results in hypertrophy of the smooth muscles, hyperplasia of goblet cells, and deposition of extracellular matrix proteins, causing membrane thickening and fibrosis .The damage caused at the bronchial level is attributed to the degranulation of eosinophils and the release of their toxic proteins. Degranulation can occur in three different ways: (i) exocytosis, (ii) piecemeal degranulation, and (iii) cytolysis. In exocytosis, most specifically the subtype compound exocytosis, multiple granules inside the cell fuse and are then secreted to the extracellular space. This is the classic way that eosinophils act against helminths . PiecemeGATA-1 gene showed no protection from AHR when compared to normal mice in asthmatic models [The release of eosinophilic granules and other mediators was proven to damage the airways in multiple ways. The smooth muscles of the airways contract via the M3 receptor after being triggered by acetylcholine. The M2 receptor limits its release and acts as a regulatory mechanism . Eosinopc models . TherefoNevertheless, eosinophils were proven to be one of the main factors behind airway remodeling. In a study designed with the same concept as the previous one mentioned, Δdbl-GATA mice were challenged by allergens and compared with wild-type mice. The latter group was found to exhibit all the features of airway remodeling, whereas the eosinophil-naïve mice were protected from it . SimilarThere was always a notion that the heterogeneity of asthma is due to the different phenotypes and endotypes of the disease. Nevertheless, endotyping became a necessity throughout the years; therefore, the need for specific biomarkers of every distinct type increased. These biomarkers include serum IgE, blood eosinophil levels, sputum eosinophils, and levels of exhaled nitric oxide in breath (widely known as FeNO) .Sputum eosinophils are the most interesting biomarker in severe eosinophilic asthma due to the insight they provide into airway eosinophilia, despite the difficulty of collecting and analyzing them in every patient routinely. Treatment of patients based on sputum eosinophils showed a reduction of the rate of exacerbations, especially in those with severe asthma.  Both EurBlood eosinophils were used in the past few years as a marker for severe eosinophilic asthma requiring biological treatment with an anti-IL-5 agent, since they are correlated with sputum eosinophils. The threshold was put in several counts during trials, with the most often picked numbers being 150 cells/μL or 300 cells/μL; however, the most important from a clinical point of view is that blood eosinophil count—an easy and inexpensive biomarker—was chosen over sputum eosinophil number for eligibility for anti-IL-5 therapy . During FeNO is another marker that is used commonly and can inform us about the ICS response we should expect from a patient . NeverthVolatile organic compounds (known as VOCs) are a modern biomarker also found in exhaled breath, like FeNO, and they are bound to predict with great accuracy both eosinophil and neutrophil counts in blood, while they are also correlated with eosinophil number in BAL . They arIL-13 genes in epithelial cells of the bronchial tree.A combination of biomarkers may be better than using one alone, and this trend was followed in many studies. In the U-BIOPRED cohort study, a specific endotype of severe asthma involving eosinophils was described as “late-onset asthma with past or current smoking and chronic airflow obstruction with a high blood eosinophil count” . A similConcluding, it is clear that biomarkers have a role to play in guiding therapy of severe asthma. However, a combination of biomarkers may be used in order to achieve a greater predictive value. Also, new biomarkers with better correlation to specific endotypes and their respective molecular pathways need to be discovered in order to achieve optimal therapy.The story of anti-IL-5 treatment in asthma is definitely a fascinating one. Given the central role of eosinophils both in the allergic and non-allergic cascade of asthmatic inflammation, along with the fact that IL-5 is the cytokine mainly responsible for the differentiation, maturation, airway trafficking, and survival of eosinophils, the development of monoclonal antibodies against IL-5 raised high expectations for new treatment approaches, primarily in severe asthma.However, the first studies were somewhat disappointing. In one study, mepolizumab prevented the rise in eosinophil numbers both in blood and sputum after inhaled allergen challenge, but it did not ameliorate allergen-induced asthmatic responses . In anotHowever, in 2009, two small but well-designed randomized controlled trials were contracted that meant to change the road of anti-IL-5 treatment in asthma. In the first study, 20 asthmatics received either mepolizumab or placebo at five monthly intravenous infusions. These patients had corticosteroid-resistant eosinophilic asthma, and it is important to note that, although they were receiving a median dose of 10 mg of prednisone for a mean time of nine years and a high ICS dose, they still had >10% sputum eosinophils . In the Apart from a clear link with exacerbations, eosinophils are also important in airway remodeling in asthma. TGF-beta derived from eosinophils is involved in this process. In a study including 24 atopic asthmatics, anti-IL-5 treatment with mepolizumab reduced airway eosinophil numbers and significantly decreased the expression of three extracellular matrix proteins  in the reticular basement membrane. It also reduced the percentage and the number of eosinophils expressing TGF-beta 1. These findings are extremely important, especially taking into consideration that the asthmatics included in this study were mild and received only short acting beta agonists (SABA) and not ICS. Firstly, these findings indicate that remodeling is present even in mild asthma, and it is driven to some degree by eosinophil-derived TGF-beta 1; secondly, anti-IL-5 can prevent this process by regulating the TGF-beta-enhanced deposition of matrix proteins through the reduction of eosinophils .6/L, FeNO > 50 ppb, or loss of asthma control after a ≤ 25% reduction in regular corticosteroid dose . The study had a duration of 52 weeks, and patients received 13 infusions of one of three doses of IV mepolizumab . All three doses equally and significantly reduced the rate of asthma exacerbations. Moreover, they reduced the number of blood and sputum eosinophils and they were well tolerated. No improvements in FEV1 and AQLQ  were observed, and this, in accordance with previous studies, indicated the dissociation of measures of control and exacerbations. This study also provided clinically valuable information regarding predictors of efficacy of mepolizumab treatment. The two main determinants were the baseline peripheral blood eosinophil number and the number of exacerbations in the previous year. Higher numbers indicated a more likely response to treatment. Other factors such as baseline FEV1, acute response to bronchodilators, IgE level, and atopic status were not associated with probability of response to mepolizumab [One of the largest studies in severe asthma, the DREAM study, including 621 patients was undertaken in order to examine the effect of mepolizumab in reducing the rate of clinically significant exacerbations. As such were defined the exacerbations that required oral corticosteroids or visit to an emergency department or hospitalization. All asthmatics had a history of at least two exacerbations requiring systemic corticosteroids in the previous year and signs of eosinophilic inflammation despite treatment. These signs were either sputum eosinophils >3%, peripheral blood eosinophils > 300 × 10olizumab .In a following study (MENSA), 576 asthmatics treated with high-dose ICS with or without oral corticosteroids were randomized to receive either 75 mg of mepolizumab IV, 100 mg of mepolizumab subcutaneously (SC), or placebo every four weeks for 52 weeks. These asthmatics had at least two exacerbations requiring systemic corticosteroids the previous year and evidence of eosinophilic inflammation reflected by an eosinophil count of 150 cells/μL at screening or above 300 cells/μL at some time point in the previous year. The primary outcome was the annualized rate of exacerbations, and they were significantly reduced by both IV and SC mepolizumab by 47% and 53%, respectively. This was the first study to show that mepolizumab was associated with a significant improvement in lung function (FEV1), quality of life (AQLQ), and asthma control (ACQ-5) .Another study (SIRIUS) explored the systemic corticosteroid-sparing effect of mepolizumab. In total, 135 asthmatics with severe eosinophilic asthma were randomized to receive either mepolizumab (100 mg SC) or placebo every four weeks for 20 weeks, and the primary outcome was the percentage reduction of the oral corticosteroid dose. The evidence of eosinophilic asthma was determined—similar to MENSA—by an eosinophil count of 150 cells/μL at screening or above 300 cells/μL at some time point in the previous year. In contrast to the MENSA study where 25% of asthmatics received oral steroids, in SIRIUS, all of the included patients received a mean dose of 10 mg of prednisone. This study involved a so-called optimization phase, in which a reduction of the dose of oral steroids was attempted before the start of mepolizumab, so as to establish that the patients genuinely needed this dose for their asthma control. The study showed that mepolizumab permitted the reduction of oral corticosteroid dose; moreover, despite this reduction, it also significantly reduced the rate of exacerbations and improved asthma control and quality of life (secondary outcomes in this study) .In a 12-month open-label extension study of MENSA after the cessation of mepolizumab treatment, it was found that eosinophils increased both in blood and sputum, returning to pre-treatment levels within three months of cessation. As for asthma control, 12 months after the stop of medication, the exacerbation rates were similar to the pretreatment levels . This stIn a post hoc analysis of the DREAM and MENSA studies, patients were stratified according to baseline blood eosinophil count in order to evaluate whether this biomarker could be used to predict response to mepolizumab. It was shown that using a threshold of 150 cells/μL could predict a favorable outcome in reducing exacerbations. Most importantly, this reduction was higher with increasing baseline blood eosinophil count (52% versus placebo for those with baseline blood eosinophils >150 and 70% for those with baseline blood eosinophils >500 cells/μL) . In a suIn patients with severe eosinophilic asthma previously treated with omalizumab, a post hoc analysis from MENSA and SIRIUS demonstrated that the response to mepolizumab was the same regardless of previous use of omalizumab . This isAnother 32-week study (OSMO) included 145 patients who were eligible for both omalizumab and mepolizumab and were not controlled with omalizumab . These asthmatics were switched immediately after the last dose of omalizumab to mepolizumab and achieved significant improvement, reflected by a 64% reduction in exacerbations compared to the previous year, better asthma control (measured by ACQ-5), and better quality of life (measured by Saint George’s respiratory questionnaire—SGRQ). These outcomes were achieved early within 8–12 weeks and were kept or even improved during the study, indicating no evidence of possible additional action of the two antibodies until the wash-out of omalizumab. This study provided support to clinical practice in terms of switching from one biologic agent to another .A study (MUSCA) assessed the effect of mepolizumab in the quality of life of patients with severe eosinophilic asthma and found a significant improvement in SGRQ of 7.7 , with a safety profile comparable to placebo .Regarding safety, in a 52-week, open-label extension study of MENSA and SIRIUS (COSMOS study), mepolizumab had a favorable long-term safety profile, without any increase in the rate of adverse events . SimilarUsing data from five phase III studies with mepolizumab, it was shown that few patients developed anti-drug antibodies that had no impact on safety or efficacy of mepolizumab. Only one patient (from the SIRIUS study) was positive for neutralizing antibodies, but pharmacokinetic samples were not quantifiable during follow-up. These data show the low immunogenic response of mepolizumab .Reslizumab is a humanized anti-IL-5 IgG4 monoclonal antibody that binds with high affinity to the alpha subunit of the cytokine IL-5, thus preventing the interaction with its receptor .Initially, a pilot safety study including 32 asthmatics showed that reslizumab at a dose of 1 mg/kg given intravenously reduced blood and sputum eosinophils but had no effect in lung function and airway hyperresponsiveness . In the The two main phase III studies included 953 asthmatics that were randomized to receive either reslizumab (3 mg/kg IV) or placebo. All included patients had a baseline peripheral blood eosinophil count of > 400 cells/μL, ACQ-7 >1.5, at least 12% FEV1 reversibility, and at least one exacerbation requiring OCS in the last year; they were also on regular treatment with high-dose ICS plus additional controller with or without OCS (up to 10 mg of prednisone). The duration of the studies was 52 weeks, and the primary outcome was the rate of exacerbations defined either as need for OCS or doubling the ICS dose. Reslizumab was effective in reducing exacerbations significantly, improving FEV1, ACQ-7, and AQLQ, as well as reducing rescue medication and blood eosinophils . In a poIn conclusion, these studies showed that reslizumab at a dose of 3 mg/kg IV is safe and more effective in patients with severe eosinophilic asthma and a peripheral blood eosinophil count > 400 cells/μL.In another study including 10 patients with oral corticosteroid-dependent asthma, weight-adjusted intravenous reslizumab was more effective in reducing sputum eosinophilia compared to fixed-dose SC mepolizumab that was administered for at least one year with inadequate response. This was associated with a greater improvement in asthma control measured by ACQ-5 .Benralizumab is a humanized, afucosylated, monoclonal antibody targeting the IL-5α receptor. In comparison to anti-IL-5 monoclonal antibodies, benralizumab induces a direct, fast, and nearly complete depletion of blood eosinophils through enhanced antibody-dependent cell-mediated cytotoxicity, via natural killer cells . As IL-5In both studies, benralizumab produced a direct, rapid, and nearly complete depletion of eosinophils as early as four weeks, providing support for its mechanism of action directly on the IL-5α receptor, causing eosinophil apoptosis. Benralizumab depletes eosinophils directly, whereas mepolizumab and reslizumab reduce eosinophil number rather than deplete them entirely. This way, benralizumab is likely to overtake potential issues such as the induction of increased cytokine production due to cytokine-directed antibodies. A pooled analysis from the SIROCCO and CALIMA studies demonstrated that benralizumab was safe and effective in patients with severe eosinophilic asthma and blood eosinophils > 150 cells/μL .A subsequent pooled analysis of the SIROCCO and CALIMA studies stratified patients according to baseline blood eosinophil count and by number of exacerbations (two and three or more). In this analysis, the rates of asthma exacerbations were increasingly reduced with increasing blood eosinophil thresholds and with greater exacerbation history. These reductions were even greater with a combination of high blood eosinophils and a history of more frequent exacerbations .Another phase III study, ZONDA, showed that benralizumab significantly reduced the dose of oral prednisone in OCS-dependent patients, while also reducing the rate of exacerbations. All patients received oral corticosteroids for at least six months prior entering the study. The study included a run-in phase where the dose of prednisone was reduced to the minimum while maintaining asthma control, and this preceded the first administration of benralizumab. After 28 weeks, 50% of the patients managed to stop oral corticosteroids, while the likelihood of reducing the dose was four times higher in benralizumab-treated than in placebo-treated asthmatics .A phase III extension study, BORA, included patients who completed the SIROCCO and CALIMA studies and evaluated the safety and tolerability of benralizumab. Interestingly, patients that received placebo in SIROCCO and CALIMA were randomized to receive benralizumab either every four weeks or every eight weeks (after administration of the first three doses every four weeks). The study confirmed the two-year safety of benralizumab as the percentage of patients experiencing adverse events was not different between BORA and the SIROCCO and CALIMA studies. No increased risk of infection was observed in patients receiving benralizumab for two years despite the long-term depletion of eosinophils. Moreover, asthmatics who were treated with benralizumab in BORA but received placebo in SIROCCO and CALIMA showed a comparable reduction in exacerbation rate with those receiving the active drug from the first year .It is of high clinical importance to assess baseline characteristics in patients with severe eosinophilic asthma that may predict the response to treatment with benralizumab. In a study including patients from the SIROCCO and CALIMA phase III studies, it was shown that OCS use, nasal polyps, forced vital capacity (FVC) < 65% pred adult onset of asthma (>18 years), and three or more exacerbations in the previous year were associated with a greater response to benrlizumab, measured either as annual exacerbation rate or change in pre-bronchodilator FEV1 for those with > 300 eosinophils/μL. Interestingly, OCS use, nasal polyps, and FVC < 65% pred could predict a better response to benralizumab in decreasing the rate of exacerbations, even in patients with < 300 eosinophils/μL . This stAnother study assessed the effect of benralizumab treatment by stratifying patients according to atopic status (atopic or non-atopic) and IgE level (high > 150 kU/L or low < 150 kU/L). The study again included patients from the phase III SIROCCO and CALIMA studies and demonstrated that the efficacy of benralizumab in reducing the exacerbation rate and improving lung function was not affected by atopic status and serum IgE level . This isThere are no head-to-head trials for direct comparison between the different anti-IL-5 biologics. In a matching-adjusted indirect comparison, benralizumab and mepolizumab similarly reduced exacerbation rate and improved lung function. No comparison could be made between benralizumab and reslizumab due to differences in study populations . AnotherThere are no studies evaluating possible co-administration of biologics with different mechanisms such as anti-IgE and anti-IL-5 for those who present a mixed phenotype . In these patients, it is logical to assess the predominant characteristics and decide which biologic to start .It is suggested that anti-IL-5 antibodies be administered for at least 16 weeks in order to assess efficacy. However, this time may be extended up to 12 months as there are some late-responders, and 16 weeks is possibly too short a length of time to evaluate reduction in exacerbations .There are still some unanswered questions with major clinical importance. How long should an anti-IL-5 be prescribed in a patient with severe eosinophilic asthma? It seems that, after stopping it, there is a relapse of exacerbations following an increase in blood and sputum eosinophils, and this was shown with mepolizumab and benralizumab. This relapse of eosinophilic inflammation is compatible with a lack of long-term bone marrow suppression after discontinuation of medication ,119. Is Dupilumab is a fully human anti-interleukin-4α receptor monoclonal antibody, recently approved for moderate-to-severe eosinophilic asthma or oral steroid-dependent asthma. It blocks interleukin-4 and interleukin-13, which are key mediators in type-2-mediated inflammation.The first study on dupilumab included 52 asthmatics with severe eosinophilic asthma and a baseline blood eosinophil count of > 300 cells/μL or sputum eosinophils > 3% who were treated with medium-to-high-dose ICS plus LABA. The patients received dupilumab (300 mg SC) or placebo weekly for 12 weeks or until an exacerbation occurred. The design of the study was provocative since asthmatics discontinued LABA by week four and gradually tapered and discontinued ICS at weeks 6–9. Dupilumab was associated with an 87% reduction in exacerbations compared to placebo and also improved lung function and reduced markers of Th2 inflammation .The following phase IIb study included 769 patients with severe asthma on medium-to-high ICS plus LABA, irrespective of baseline blood eosinophil count. They received 200 mg or 300 mg of dupilumab or placebo every two or every four weeks for a total duration of 24 weeks. Dupilumab improved FEV1 and reduced the exacerbation rate significantly in the total population and also in the subgroups of patients with less than or more than 300 eosinophils/μL . In a poAs with anti-IL-5 antibodies, dupilumab was assessed regarding its efficacy in reducing OCS in asthmatics with oral steroid-dependent asthma. Accordingly, 210 patients received dupilumab (300 mg) or placebo every two weeks for 24 weeks. Oral steroid doses were reduced from week four to week 20 and then remained at a stable dose for another four weeks. Dupilumab reduced oral corticosteroid dose by 70% compared to 42% reduction of placebo, and simultaneously decreased the rate of exacerbation by 59% compared to placebo; this effect was observed despite the reduction in the OCS dose. It also significantly improved lung function . In the A meta-analysis involving 3369 asthmatics from five studies concluded that treatment with dupilumab was effective in reducing exacerbations and improving lung function, asthma symptoms, asthma control and quality of life. Dupilumab was safe and well tolerated, and the most frequent adverse event was injection-site reaction .Chronic rhinosinusitis with nasal polyps (CRSwNP) is often a comorbidity of severe eosinophilic asthma. In a subgroup analysis of a study involving patients with CRSwNP who received dupilumab as add-on therapy to mometasone fuorate nasal spray, those patients with comorbid asthma showed improvements not only in nasal polyp burden but also in asthma control, quality of life, and lung function .Omalizumab is a humanized monoclonal antibody that binds free IgE and prevents it from binding to the high-affinity IgE receptor on basophils and mast cells . OmalizuThymic stromal lymphopoietin (TSLP) is produced by airway epithelial cells in response to inhaled allergens and proinflammatory stressors ,139.Tezepelumab is a human monoclonal antibody that binds to TSLP, inhibiting its stimulatory action on dendritic cells and innate lymphoid cells, thus preventing the induction of type 2 cytokines . One phase II, randomized, double-blind, placebo-controlled trial evaluated the efficacy and safety of tezepelumab in patients with uncontrolled asthma, despite treatment with long-acting beta-agonists and medium-to-high doses of inhaled corticosteroids. Three dose levels of subcutaneous tezepelumab were compared to placebo over 52 weeks. The primary end point was the annualized rate of asthma exacerbations. Exacerbation rates were significantly reduced in tezepelumab groups—regardless of the baseline blood eosinophil count—compared to placebo by 61% in the low-dose group, 71% in the medium-dose group, and 66% in the high-dose group. Lung function was improved irrespective of the dose, while health-related quality of life improved only in the high-dose group .Prostaglandin D2 (PGD2) is mainly released from mast cells, but platelets, alveolar macrophages, Th2 cells, and dendritic cells can also produce smaller amounts of PGD2. Prostaglandin D2 contributes to T2 inflammation through binding of the G-protein-coupled receptor chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) . Fevipip0–24 only in patients with high serum IgE and blood eosinophils > 300/µL [In a phase II study, including 170 patients with mild-to-moderate persistent, allergic asthma, fevipiprant produced a significant improvement in FEV1 AUC> 300/µL .In another phase II study, including 61 patients with moderate-to-severe, persistent asthma and sputum eosinophilia (≥ 2%), fevipiprant produced a significant, 3.5-fold greater decrease in sputum eosinophilia than placebo during the 12-week treatment period. In addition, fevipiprant reduced bronchial submucosal eosinophil numbers in bronchial biopsies compared to placebo. However, no change in blood eosinophil count was observed .Finally, in another phase IIb study, including 1058 patients with allergic asthma uncontrolled with inhaled corticosteroids, fevipiprant—as well as montelukast—improved pre-dose FEV1 compared to placebo. However, no evidence of a higher efficacy in any predefined subgroup, including blood eosinophil count was observed .In order to consider a biologic therapy for severe asthma, it is fundamental to firstly confirm asthma diagnosis and then solve possible problems related to non-adherence to medication, improper inhaler technique, and treatment of comorbid conditions.For severe eosinophilic asthma, targeted therapies directed against IL-5 and IL-4 are available up to date . These aAnother major benefit from the use of biologics in severe asthma is the opportunity for a better insight into asthma pathophysiology mechanisms. An important but still unanswered question is whether biologics have an effect on moderate asthma or produce a disease-modifying effect. Until then, and while expecting more biologics to come , we hope to gain experience and understand more from the longer use of the current anti-T2 biologics.