We aimed to determine the presence of subclinical atherosclerosis using carotid intima-media thickness (CIMT) and biochemical parameters in children and adolescents with congenital adrenal hyperplasia (CAH).. The patients were divided into two groups according to the degree of control of the clinic, laboratory, and radiological parameters as a) “uncontrolled”  or b) “controlled” . Ultrasonographic examination of carotid artery was performed by the same radiologist using a B-mode ultrasound system. Thirty-four patients with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency on regular glucocorticoid treatment for ≥3 years and 31 healthy subjects were included in the study There was no significant difference between the CAH and control groups in terms of median (IQR) CIMT values . When subgroup comparisons were done in terms of median (IQR) CIMT values, there was no significant difference among the controlled, uncontrolled, and healthy control groups . In addition, CIMT levels were similar according to sex and disease control status. In this study, the CIMT values of CAH cases were similar to those of healthy subjects.  Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by mutations in genes encoding the enzymes that are responsible for the biosynthesis of cortisol. Deficiency of 21-hydroxylase (21-OH) enzyme is the most common type of CAH, accounting for >%95 of all CAH cases [1]. This type of CAH is characterized by a decrease in the production of cortisol and/or aldosterone and the accumulation of precursor steroid hormones in the steroidogenic pathway [2]. This disorder has a quite broad spectrum of clinic form and is classified as classical type [salt-wasting (SW) and simple virilizing (SV)] and mild nonclassic or late-onset CAH according to the degree of enzyme deficiency. The SW type has the most severe phenotype and accounts for 75% of the classic type. The SW type, usually with <2% residual enzyme activity of 21-hydroxylase, is presented by insufficient cortisol and aldosterone synthesis, and excess androgen production. In SV type, unlike SW, mineralocorticoid synthesis is sufficient. The nonclassic type is characterized by an overproduction of androgens only in adulthood . The main goal in the treatment of the disorder is to replace the hormones that are deficient  and to suppress the androgen hormones that are increased excessively [3]. For this purpose, in clinical practice, hydrocortisone and fludrocortisone are frequently used for treatment.Congenital adrenal hyperplasia due to 21-OH deficiency is often associated with risk of obesity, insulin resistance, hypertension, hyperandrogenism, and dyslipidemia among children and adolescents . Intermittent hypercortisolism associated with treatment and adrenomedullary dysfunction lead to these metabolic disorders, resulting in an increased risk of developing metabolic syndrome and atherosclerosis in CAH [6]. Although various organizations have different approaches in defining metabolic syndrome, it is generally defined as the presence of three of the following five criteria; increased waist circumference, glucose intolerance, high triglyceride, low high-density lipoprotein cholesterol, and hypertension [7]. Metabolic syndrome components that occur in this disease may cause endothelial dysfunction and predispose patients to coronary artery disease and stroke at an early age [6]. Carotid intima-media thickness (CIMT) is a noninvasive test that is widely used to define early subclinical atherosclerosis. Increased CIMT is a parameter to predict increased risk of cardiovascular events such as coronary artery disease and stroke [8]. Increased CIMT has previously been reported in children with obesity, type 1 diabetes mellitus and hyperlipidemia . However, there are only a limited number of studies evaluating CIMT in children and adolescents with CAH [11–15].The present study aimed to determine the presence of subclinical atherosclerosis using CIMT and biochemical parameters in children and adolescents with CAH.This prospective study was conducted in 2014–2015, at Dokuz Eylül University in İzmir. Thirty-four patients with classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency and 31 healthy subjects were included in the study. Age, pubertal status, and sex-matched healthy controls were recruited from the general pediatric outpatient clinic. Those with chronic diseases were excluded from both CAH and the control groups. The inclusion criteria for the CAH patients were as follows i: having regular follow-up every 3 months after diagnosis at our institution, ii: using hydrocortisone ± fludrocortisone treatment for at least 3 years. In this study, which included classical type CAH cases, SW and SV classification was determined according to clinical and laboratory findings. Accordingly, patients who presented with severe cases of salt wasting  in the early period (2 weeks–1 month) were evaluated as SW, and who were admitted with signs of androgen excess  at a later age (2–3 years) and without salt loss were evaluated as SV [1–3]. The children’s legal guardian(s) provided written informed consents. The study was conducted according to the Declaration of Helsinki, and the study protocol was approved by the local ethics committee.2/day) and fludrocortisone (mg/m2/day), weight standard deviation score (SDS), height, height SDS, body mass index (BMI) SDS, height velocity (HV), height velocity SDS, blood pressure (BP), and pubertal status  were collected. Measurement of height, weight, blood pressure, and bone age (BA) evaluation are stated in the following source [16]. The auxological evaluation was done according to Turkish National Growth Charts [17]. Blood pressure SDS was calculated according to Child Metrics www.ceddcozum.com, www.childmetrics.org) [18]. Obesity was defined as a BMI at or above the 95th percentile of children of the same age and sex.Demographic and clinical data including chronological age (CA), sex, type of CAH, doses of hydrocortisone , high-density lipoprotein (HDL)-C, low-density lipoprotein (LDL)-C and triglycerides (TG), fasting blood glucose (FBG), 17-OH progesterone and androstenedione were taken between 08:00 and 09:00 from all CAH patients. Androstenedione and 17-OH progesterone measurements were performed with commercially available radioimmunoassay kits. The patients were divided into two groups according to the degree of control of clinic and laboratory parameters as a) “uncontrolled”  or b) “controlled” . Ultrasonographic examination of carotid artery was performed using a B-mode ultrasound system with a high-definition L12-5 linear wide band transducer . Both left and right common carotid arteries (CCA) were scanned. The longitudinal plane images of the CCA at 1–2 cm proximal to the carotid bulb were obtained and digitally archived. All ultrasound examinations were performed according to a standardized protocol by the same radiologist (TO) who was blinded to the clinical details of the participants. Archived images were transferred to a workstation, and intima-media thickness (IMT) values were automatically measured using a computer software . The measurements were performed retrospectively in a single-blind manner. The mean left and right CCA measurements were obtained, and the mean IMT values were recorded. A radiograph of the left hand was used to determine BA using the Greulich-Pyle atlas. All bone ages were estimated by the same endocrinologist (HT). BA/CA rates greater than 1.2 were defined as advanced bone age.Statistical analyses of the data were conducted using SPSS 21.0 . Distribution of data was evaluated with the Kolmogorov–Smirnov test. For numerical comparisons between the study groups, the data was compared using Student’s t-test  and the Mann–Whitney U test . Pearson’s chi-squared test was used for the comparison of categorical variables. The Kruskal–Wallis test was used to compare CIMT differences between more than two groups . Spearman’s or Pearson’s correlation analysis was used to investigate the association between two variables. Data are expressed as mean ± standard deviation (SD) or median (IQR). p < 0.05 was considered statistically significant.The clinical and laboratory characteristics of the children and adolescents are summarized in Table 1. A total of 34 patients with CAH  and 31 healthy subjects  were included in the study. The SW type of CAH represented 64.7% (n= 22) of the studied patients, while the SV type represented 35.3% (n= 12). All patients were receiving glucocorticoid substitution therapy with hydrocortisone; in addition, 22 were receiving fludrocortisone. None of the patients was receiving any additional medication.CAH patients had significantly lower height SDS and significantly higher BMI SDS compared to healthy subjects . No significant difference was detected between CAH patients and healthy subjects in terms of age, sex, puberty, weight SDS, systolic blood pressure (SBP) SDS, diastolic blood pressure (DBP) SDS, FBG, HDL, LDL, TC or TG (p > 0.05). None of the children in the CAH and control groups had hypertension. Eleven (32.3%) patients in the CAH group were obese. Median age at diagnosis of CAH was 0.14 years . Mean duration of the treatment was 8.5 ± 3.7 years. While the median age of diagnosis of SW patients was 0.04 years , the median age of diagnosis of SV patients was 6.0 years . Mean serum 17-OH progesterone and androstenedione concentrations in the CAH group were 13.58 ± 12.85 ng/mL and 2.88 ± 3.44 ng/mL, respectively. Median (IQR) CIMT values of CAH and control groups were 0.47 (0.05) mm and 0.47 (0.07) mm, respectively. There was no significant difference between the CAH and control groups in terms of CIMT values (p = 0.692). When compared according to sex, males with CAH had significantly higher BMI SDS than healthy males, and females with CAH had significantly higher BMI SDS and height SDS than healthy females (p < 0.05); however, there was no difference in terms of CIMT values (p > 0.05) (Table 1). When we compared the clinical and laboratory characteristics of CAH patients according to metabolic control, no difference was found in terms of age, age at diagnosis, sex, puberty, duration of treatment, hydrocortisone dose, fludrocortisone dose, weight SDS, height SDS, BMI SDS, SBP SDS, DBP SDS, FBG, HDL, LDL, TC, TG, HV SDS, or BA/CA ratio (p > 0.05) (Table 2). When subgroup comparisons were done in terms of CIMT values, there was no significant difference between the controlled, uncontrolled, and healthy control groups . In addition, there was no significant differences between the SW type CAH, SV type CAH, and healthy control groups in terms of CIMT values, SBP SDS, and DBP SDS .There was no correlation between CIMT and age, weight SDS, height SDS, BMI SDS, hydrocortisone dose, fludrocortisone dose, SBP SDS, DBP SDS, FBG, HDL, LDL, TC, TG, 17-OH progesterone, or AS levels (p > 0.05) (data not shown). Blood pressure  was not correlated with hydrocortisone dose, fludrocortisone dose, and duration of treatment (data not shown). Carotid intima-media thickness is an imaging technique used to predict subclinical atherosclerosis, which is an early predictor of future cardiovascular disorders [12]. It has been reported that endothelial dysfunction was associated with CIMT progression [19]. Carotid intima-media thickness has been reported to be associated with family history, ethnicity, advanced age, male sex, high body fat, high blood pressure, presence of diabetes or glucose intolerance, and high serum cholesterol and triglyceride levels [20]. It has been reported that obesity, hypertension, dyslipidemia, insulin resistance and hypercortisolism or hyperandrogenism may cause vascular dysfunction in patients with CAH, resulting in coronary artery disease [21]. Conflicting data have been reported on the effects of CAH on CIMT. Some studies on subclinical atherosclerosis have reported that CIMT was increased in children and adult patients with CAH . Nonetheless, Harrington et al. [19] compared patients with obesity and CAH to healthy subjects in terms of CIMT and found no difference between the groups. In another study, Kim et al. [13] reported that CIMT values were similar between CAH and healthy subjects, but significantly higher in obese subjects compared to nonobese subjects in both the patient and control groups. In the same study, it has been shown that CIMT values in male CAH cases were significantly higher than in females. In subgroup analysis, CIMT values have been reported to be similar in SW and SV type CAH cases . In the study by Metwalley et al. [22], CIMT values have been found to be significantly higher in uncontrolled CAH cases than in well-controlled cases and in both male and female CAH cases compared to healthy subjects. In the same study, it has been shown that CIMT was related to age, duration of treatment, equivalent of hydrocortisone dose, 17-hydroxyprogesterone, and testosterone. In our study, CIMT values were similar between CAH patients and healthy subjects. In addition, CIMT values were found to be similar in subgroups according to sex, CAH forms (SW and SV) and control status (controlled/uncontrolled) compared to healthy subjects. In addition, in our study, no correlation was found between CIMT and age, BMI, hydrocortisone dose, 17-hydroxyprogesterone or androstenedione, conflicting with the findings of some studies . However, these findings do not suggest an increased risk of cardiovascular disease in our CAH patients at the age when they were evaluated. The reasons for the similarity in CIMT values between CAH cases and healthy subjects in the current study may be due to i) the low number of cases, ii) failure to evaluate the familial characteristics of patients or healthy subjects in terms of risk of cardiovascular disease, iii) lack of time for vascular change to occur for CIMT increase to be observed. The prevalence of obesity, hypertension, dyslipidemia, and insulin resistance have previously been reported to be increased in CAH patients. In our study, the height SDS was lower, and BMI SDS was higher in patients with CAH compared to healthy subjects, consistent with the literature . In addition, in our study, 32.3% of the CAH patients were obese. Similarly, increased obesity rates (16.8%–25%) have previously been reported . Obesity in CAH patients has been associated with glucocorticoid therapy, high androgen levels, leptin resistance, and decreased lipolysis due to the reduction in catecholamine secretion [21]. Increased vascular tone due to obesity, hyperinsulinemia, and glucocorticoid treatment may cause hypertension in CAH patients [6]. Hypertension is a major cardiovascular morbidity in children and adolescents with CAH [5]. In our study, none of the children in the CAH group had hypertension. Moreover, in our study, no difference was found between the CAH patients and healthy subjects in terms of SBP or DBP. Some studies have reported normal resting blood pressure values  and 24-h BP profile [29] in children and young adults with CAH compared to healthy subjects, similar to our results. On the other hand, in studies performing blood pressure assessment by 24-h BP profile, Da Silva et al. [30] reported day-time SBP increase (between 90 and 95 percentile range) in 5 of 11 cases with CAH. Völkl et al. [31] reported significantly higher SBP SDSs than healthy subjects in both day-time and night-time, and Roche et al. [4] reported 58% systolic and 24% diastolic hypertension in 38 SW type CAH cases. Interestingly, in CAH cases with normal resting SBP and DBP, peak SBP and DBP values and increases in SBP and DBP values have been found to be significantly higher during exercise than resting or daily routine activity [32]. Although BP profile is not affected in daily routine activities, it seems that BP responses may be impaired during exercise in CAH cases due to their metabolic dysregulation. Finally, contradictory results from different studies could be explained by the facts that a) hypertension may have been missed due to the lack of 24-h blood pressure monitoring in CAH patients b) significant alterations in vascular structure may not yet have developed, and c) impaired exercise performance, which may be an early marker of cardiovascular disease in CAH patients, was not evaluated.Increased frequency of obesity, insulin resistance, hydrocortisone treatment and high androgen exposure is expected to deteriorate the lipid profiles of CAH cases. However, contrary to this expectation, the lipid profiles were similar to healthy controls in this study. Most studies on children and adults have reported similar lipid profiles in CAH cases and healthy control subjects, similar to our study . In their study on prepubertal CAH cases, Botero et al. [36] reported that TC, LDL, and HDL values were similar, but TG values were significantly higher in CAH cases compared to healthy subjects. However, it has been noted that prednisone, which increases the risk of metabolic alteration more than hydrocortisone, was used in treatment in this study. In addition, Zimmermann et al. [37] reported higher small, dense LDL and lower HDL in CAH cases. However, their study groups were quite heterogeneous in terms of age (age range: 4–31), which was one of the main limitations of the study. One study suggested that dyslipidemia may take time to develop, which could be the reason for the lack of changes in lipid profile in children with CAH [21]. In contrast to this hypothesis, Falhammar et al. [35] reported similar serum TC, HDL, LDL, and TG levels between CAH patients aged <30 years and CAH patients aged >30 years. Furthermore, in the same study, the HDL/LDL ratio, which is considered to be protective against cardiovascular disease, has been reported to be significantly higher in CAH patients >30 years of age. In conclusion, contrary to expectations, the current study showed no evidence to support lipid alteration in CAH, as in many other studies. However, the low number of cases in both our study and the previous studies may have prevented us from demonstrating the differences in lipid profiles.The main limitation of our study was that we could not exclude the impact of blood pressure changes. Although there was no difference between the groups in terms of blood pressures, intermittent elevated blood pressure may cause endothelial dysfunction in the congenital adrenal hyperplasia group [32]. Therefore, it would be more appropriate to perform 24-h blood pressure monitoring in congenital adrenal hyperplasia patients. Another limitation was the low number of subgroup cases. In addition, 17-hydroxyprogesterone and androstenedione levels, which are indirect indicators of increase in androgens, were evaluated, but testosterone and DHEAS levels could not be measured. Therefore, we could not investigate whether androgen levels were correlated with CIMT. The effect of genetic factors affecting CIMT and cardiovascular disease risk was not considered in this study, as well. The risk of cardiovascular disease is associated with fat distribution. The risk of this disease is determined by waist circumference regardless of obesity and is more reliable than BMI [38]. We have not performed waist circumference measurements. 17-OH progesterone was not studied in the control group. These are other limitations in this study. The last limitation of the current study is that androstenedione and 17-OH progesterone levels were studied with the radioimmunoassay method. In recent studies, it has obviously been demonstrated that liquid chromatography-tandem mass spectrometry is more reliable in steroid hormones, precursors, and metabolic parameters than the RIA method [39].In conclusion, in the present study, the CIMT values of CAH cases were found to be similar to those of healthy subjects. In addition, CIMT levels were similar according to sex, disease control status, and CAH type and were not associated with any anthropometric or metabolic parameters. Although these findings suggest that the risk of cardiovascular disease is low in CAH patients during childhood and adolescence, it should be kept in mind that clinically significant endothelial dysfunction may not yet have occurred due to early age.This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.


Based on resting BP, the fractions of boys and girls with normal BP and high BP were identified and divided into experimental (EG) and control (CG) groups. EG completed a 10-weeks HIIT program (three cycles of Tabata protocol) implemented in one PE lesson during a week. The duration of the effort was 14 min. The intensity was at 75–80% of maximal heart rate. Changes in systolic and diastolic BP after the experiment were examined. The results indicated the improvement in SBP in EG with high BP compared to the rest of the groups . The EG normotensive had a statistically significant higher reduction of SBP comparing CG normotensive . HIIT effectively decreases BP in adolescents. Implementing HIIT in PE lessons in secondary school is recommended to improve BP parameters.Increased resting blood pressure (BP) is a risk factor for many health complications. The prevalence of elevated BP is growing among adolescents. There is a need to investigate effective ways of decreasing excessive blood pressure in this age group. The study aim was to determine the effect of 10-weeks High-Intensive Interval Training (HIIT)—Tabata protocol—introduced in physical education (PE) lessons on resting blood pressure in adolescents. The sample included 52 boys aged 16.23 ± 0.33 years body height176.74 ± 6.07 (m), body weight 65.42 ± 12.51 (kg), BMI 20.89 ± 3.53 (kg/m These health problems concern not only older people but even at school age. More and more young people are at risk of blood pressure abnormalities4. Song et al.5 indicated a significant increase in the prevalence of high blood pressure among children and adolescents during the last 2 decades. This observation confirms the need to search for effective ways to reduce excessive blood pressure and prevent hypertension. The cardiovascular problem at a young age could become a significant health problem in the future7. Considering the preceding, young people should apply physical activity that leads to normotensive, which could help maintain good health status9. The need for physical activity among youth is obvious, but it is a truism to say that adolescents are generally reluctant to undertake PA in their leisure time.Blood pressure is one of the most critical indicators of a normal cardiovascular state. Hypertension is intricately associated with numerous diseases and could lead to premature mortality10. The duration of physical education lessons  suggests the vital need to perform short, high-intensity efforts to increase physical activity benefits from PE lessons11. Bond et al.12 showed promising effects of High-intensity interval training (HIIT) introduced in physical education lessons. HIIT is time-efficient and effective for decreasing the risk of cardiovascular diseases (CVD)15. HIIT intervention positively affects cardiorespiratory fitness and body composition improvements in the adolescent population16.Physical education (PE) lessons are relevant settings to implement physical activity for adolescents18, whereas less is known about adolescents19. Morales-Palomo et al.15 established the positive effects of HIIT among men with metabolic syndrome. After HIIT intervention, Grace et al.17 showed a significant improvement in blood pressure (both systolic and diastolic) in older men leading a sedentary lifestyle. Eddolls et al.20 concerning HIIT effects on children and adolescents show promising data with blood pressure parameters.However, in the HIIT effect on blood pressure, many studies concern adults21. Martin-Smith et al.22 showed that HIIT during PE could be highly effective for cardiorespiratory status improvement among overweight adolescents. The same study demonstrated that short intensive training interventions could be as effective as prolonged ones during PE, saving time. Moreover, HIIT improves endurance, reduces body fat, increases muscle mass or cardiovascular parameters22. Similar effects are observed among obese adolescents, where HIIT improved anthropometric and cardiovascular parameters23. Delgado-Floody et al.24 found significant improvement in anthropometric and cardiovascular parameters after 28 weeks of HIIT intervention. Still, there were no significant differences in the duration of intervention between the middle and extended periods of HIIT training25.Nevertheless, it is crucial to determine the possibilities and effectiveness of introducing short, high-intensity exercises during physical education lessons and their effect on blood pressure26. Especially interesting to adopt the TABATA protocol27, a short and intensive protocol suitable for physical education lesson conditions. This effort showed a promising, broad effect on body composition and physical performance29. Less is known about the potential impacts of HIIT effects according to Tabata protocol on blood pressure among adolescents. Since hypertension is a growing health problem in the adolescent population5, there is a strong need to involve school youths in physical activity during PE lessons. Many evidence indicated a broadly positive effect of HIIT on health status. Therefore, this study was aimed to examine the effect of 10-weeks High-Intensive Interval Training (HIIT)—Tabata protocol27—introduced in physical education (PE) lessons on blood pressure in normotensive and high blood pressure adolescents concerning intersex differences.The above studies showed overall positive effects on young people's health status, confirming a need to explore this area and implement HIIT in physical education lessons among adolescentswww.randomization.com. Initially, the study sample included 187 individuals. The participants were in six separate classes, out of which three were randomly assigned to experimental and three to the control group. Each class was coded with the number from 1 to 6. Students in each class were on the same education level with the same PE education program. Before allocation to the experimental or control group, 27 subjects were excluded due to medical contradiction (n = 10) or participation in additional sports activities in the last 6 months (n = 17). The next 19 subjects were excluded during the intervention due to absence in physical education lessons. No adverse effect was noted due to HIIT intervention. The final analysis involves 141 adolescent students—52 boys aged 16.24 ± 0.33 and 89 girls 16.12 ± 0.42 from a secondary school in Wroclaw, Poland. The flow of the participants is presented in Fig. Simple, non-returnable group randomization was conducted using the tool provided on the website 2) was calculated based on the received data.Swiss anthropometer  was used for height measurements. InBody 230 machines , was used for bodyweight measurements. The body mass index (BMI)  program based on the Tabata procedure was conductedThe tests took place in the certified Laboratory of the Physical Education Faculty at the Wroclaw University Health and Sport Sciences; ISO number: PN-EN ISO 9001:2009 and Certificate number: PW-48606-10E.All blood pressure measurements were carried out by an Omron BP710 Automatic Blood Pressure Monitor. A cuff of the appropriate size was selected based on upper arm circumference. Participants were sitting quietly for 10 min. Next, the readings were taken three times in 10-min intervals. The results introduced in the analysis were means of the three measurements.29 According to IVR, in normal blood pressure, both: systolic blood pressure (SBP) and diastolic blood pressure (DBP) are lower than the 90th percentile for both blood pressure (concerning sex and age). In a pre-hypertension state, average SBP and DBP are between 90 and 95th percentile. In hypertension state, average SBP and DBP are over 95th percentile. In addition, two stages of hypertension were distinguished: 1°—with SBP and DBP between 95th and 99th percentile and 2°—with SBP and DBP above 99th percentile.The procedure of classifying the adolescents based on the systolic and diastolic blood pressure measurements met the criteria contained in the National High Blood Pressure Education Program (NHBPEP) Working Group published in the 4th Report (IVR)30. In boys, we adopted the following (mm of Hg) values: C90/SBP = 127, C90/DBP = 77, C95/SBP = 133, C95/DBP = 82. Similarly, we adopted the following (mm of Hg) values for girls: C90/SBP = 123, C90/DBP = 77, C95/SBP = 130, C95/DBP = 81. Due to the small size of groups with hypertensive adolescents, prehypertensive and hypertensive individuals were joined together. Therefore, the group was named high blood pressure. Group sizes are as follows: in experimental boys' groups, there were 14 normotensive and 17 high blood pressure subjects, and in the control group, there were 7 normotensive subjects and 14 high blood pressure subjects. In experimental girls' groups, the were 29 normotensive subjects and 13 high blood pressure subjects, and in the control group: there were 34 normotensive and 13 high blood pressure subjects.An essential part of the procedure is using proper and adequate percentiles. Adolescents examined in this study lived in Wroclaw—a big city in the southwest of Poland. Considering that, the blood pressure standards for Polish children and youth from big cities  were used in our study27. In the remaining PE lessons, students improved their physical skills in various sports.In one of the three 45-min weekly PE lessons, students in the experimental groups performed a TAP28. After a 10-min standardized warm-up, students performed a 14-min TAP consisting of three sessions of 4 min each. There was a 1-min break between each session during which no exercise was performed. The Tabata protocol of each session consisted of eight cycles of two exercises. Each cycle started with a maximum intensity exercise lasting 20 s with as many repetitions as possible. This was followed by a 10-s active rest (low-intensity exercise). The following exercises were used in the first session: push-ups, high knees. In the second session—dynamic lunges, spider crawling, and in the third—plank to push-ups side squeeze. The exercises used did not require any specific exercise equipment and were aimed at working with large muscle groups. The authors prepared the exercises (for the experiment), recorded and played them back during PE lessons on the screen to accurately measure the time of exercise and rest. After TAP, flexibility and relaxation exercises were performed as a permanent program for several minutes.The PE lesson, in which exercises based on the Tabata protocol were introduced, was carried out according to the procedures presented by Domaradzki et al.28. It was assumed that TAP exercise should be performed at high intensity, 75–80% of maximum heart rate (145–157 bpm)22. Experimental group EG achieved HR = 155.8 bpm . In subsequent TAP sessions, exercise intensity was reached by using the video of study protocol what let to maintain the same pace of exercise, the frequency of a single movement, as obtained in the first lesson with TAP.The HIIT protocol was presented in video form. The intensity of exercises carried out was verified during the first PE lesson based on the procedure presented by Domaradzki et al.The control group realized a standard physical education program during the intervention.F-ratio), detailed comparisons of post-hoc tests (Tukey's HSD test) were used to determine pairwise differences. The significance level was set at α = 0.05. All the calculations were carried out using Statistica 13.0 .Descriptive statistics were calculated: means, standard deviations (SD), and 95% coefficient intervals (CI). Changes between post-intervention and baseline measurements were calculated (Δ). Both—baseline and changes results are presented in Table The project met the ethical standards for sports medicine. The Ethics Committee of the Wroclaw University Health and Sport Sciences approved the project (ECUPE no: 19/2019). The study was conducted following the Declaration of Helsinki by the World Medical Association for research with humans.Descriptive statistics of the parameters for boys and girls from experimental and control groups regarding BP status are presented in Table Mean square values presented in Table The boys and girls with high blood pressure in experimental groups were taller and heavier than normotensive peers at the baseline. Therefore, body height and body weight measurements were added to the analysis of covariance (ANCOVA) as covariates. There were no statistically significant differences between the sexes. Sex was not a factor that interacted with the effects of HIIT and blood pressure status, which confirmed the lack of statistically significant interactions Table . A statiThe results presented in Table 31. Because of that, it is crucial to engage adolescents in physical activity, which is the best tool to defeat it. PE lessons seem to be the best settings.A sedentary lifestyle, bad nutrition habits, and lack of physical activity are the main determinants of many health disturbances. This problem affects young people who risk many metabolic diseases by giving up physical activity. Even they could suffer abnormalities connected with high blood pressure14. Therefore, this study aimed to determine the effect of 10-week HIIT (Tabata protocol), introduced in physical education (PE) lessons, on blood pressure in 16-years girls and boys. The study sample was at the age of dynamic adolescence, which indicated changes in body height during 10 weeks of intervention. High-intensity intermittent efforts have been demonstrated as a promising tool in improving adolescents' health18. This effort significantly affects metabolism even a few hours after finishing the session33. This phenomenon is associated with excess post-exercise oxygen consumption (EPOC), which demands an increased commitment of the cardiorespiratory system and has more impact on it and develops heart, blood vessels, and lunges function35.However, limitations in duration lessons and the need to complete other curricular items force the introduction of effective and time-saving methods of physical activity, like HIIT, with its well-known positive effect on the cardiovascular system and endurance18.The present study's main findings are that HIIT significantly reduced systolic blood pressure among high blood pressure adolescents and with a slightly smaller decrease in systolic blood pressure among normotensive. The obtained results show an excellent product that could be widely implemented in schools, aimed at blood pressure adjustment and CVD risk prevention35 indicated a significant reduction of cardiovascular risk among overweight adolescents after HIIT program intervention through reducing blood pressure parameters. In another study, improvements in physical performance among adolescents after the HIIT program were also observed, additionally constituting a factor that keeps youth interested in physical activity29. Similar Farach et al.36 confirm positive effects of HIIT on blood pressure parameters. Moreover, it was noted improvement in fat reduction. The study by Corte de Araujo et al.37 showed significant improvement in systolic blood pressure comparison to continuous endurance training among obese youth.Similar to our observation, Tjonna et al.38. Nevertheless, intensive interval training may produce more positive effects on health than moderate-intensity training39. It was clearly indicated by Kouuba et al.40, which demonstrated more benefits from intermittent physical efforts than from continuous ones. Other studies showed similar effects between HIIT and continuous one41. But there must be remembered single physical education lesson is slightly short (45 min), so implementation of HIIT could be more suitable28.Studies mentioned above showed excellent positive effects on adolescents. Therefore, engaging children and adolescents in any physical activity positively influence cardiometabolic risk is crucial. It is based on PE lessons, which can positively affect physical activity on health38. However, some positive metabolic effects of HIIT are observed after 2 weeks42. Our study revealed a significant impact on SBP, with no impact on DBP among subjects with high blood pressure. A similar effect on BP through HIIT was observed by Olea et al.43 in an experiment conducted among adult women. Despite a different study sample, it confirms the positive effect of HIIT on blood pressure.Still, the duration of this type of program should typically be approximately 8 weeks or more to reach more positive effects44. The HIIT program duration of 8 weeks seems to be enough to achieve this effect in a group of obese young women. Furthermore, implementing HIIT in PE lessons influences the general level of physical activity. Costigan et al.45 indicated that adolescents were more active when they had HIIT than on other days. It is also worth noting the positive effect of HIIT regardless of body composition. It conditions the possibility of introducing this type of intervention regardless of the BMI status. It, therefore, enables a broader implementation of such efforts in various groups22. HIIT training seems to be an effective solution to counteract the risk of developing cardiovascular and metabolic health problems.The effect of HIIT is more comprehensive than only BP effects. There was proof of a vast influence on plasma lipids strongly associated with cardiovascular disease risk46. It supports overall health effects, which could be introduced among adolescents in PE lessons at school, with success. HIIT requires a short time and enables conducting it during the 45-min lessons while being an attractive and effective way of improving health conditions.Even though HIIT constitutes a heavy and demanding activity, it is not only sufficient for athletes focused on developing physical performanceWe are aware of some limitations of our study. Due to small groups of hypertensive prehypertensive and hypertensive individuals were joined together. Only one secondary school took part in the experiment. We did not have nutrition control and average daily physical activity monitoring. There was no continuous heart rate monitoring in the following lessons.On the other hand, this study has some strengths. It was conducted in natural school settings, indicating the possibility of implementing HIIT in the physical education program. Moreover, our study sample was heterogeneous. They were on the same level of education at a similar age.The 10-week HIIT intervention introduced in PE lessons resulted in a significant reduction of systolic blood pressure in the group with high blood pressure and slightly among normotensive groups. Changes in blood pressure values were not observed in the groups following the only traditional PE program. The Tabata training positively affects blood pressure and can effectively prevent hypertension in adolescents. It suggests that implementing HIIT in physical education lessons is an effective tool for improving blood pressures parameters.Further studies on a bigger study sample are required, and adolescents of various ages should be studied. There is a need to determine the sustainability of the induced changes. Dietary control should be considered. Participants should use accelerometers to monitor their daily physical activity in the future.


Cardiovascular disease and especially hypertension are a growing problem among people living with HIV (PLHIV) on antiretroviral therapy (ART) in sub-Saharan Africa.As robust data on hypertension phenotypes associated with distinct cardiovascular risks among PLHIV are limited, we aimed to assess the frequency of white-coat (WCH), masked (MH) hypertension, and blood pressure dipping-patterns in a group of Malawian PLHIV.As part of the prospective Lighthouse-Tenofovir-Cohort-Study, we analyzed clinical, laboratory and 24-h-ambulatory blood pressure monitoring (ABPM) data of PLHIV from urban Lilongwe with treated or untreated hypertension or raised office blood pressure (OBP) during routine study-visits.118 PLHIV were included and data of 117 participants could be analyzed. Twenty–four-hour ABPM normotension was found in a total of 73 PLHIV including 14/37 on antihypertensive treatment (37.8%). Using strict definitions, i.e. normal OBP plus normal mean BP for all periods of ABPM, controlled hypertension was found in only 4/37 (10.8%) PLHIV on antihypertensive treatment while true normotension was observed in 10/24 untreated patients (41.7%) with previously diagnosed hypertension and 22/56 patients (39.3%) without a medical history of hypertension. WCH with normal BP during all periods of 24-h-ABPM was identified in 12/64 OBP-hypertensive PLHIV (18.8%), primarily in patients with grade 1 hypertension . MH was found in 17/53 PLHIV with OBP-normotension (32.1%), predominantly in patients with high normal BP . The estimated glomerular filtration rate tended to be lower in MH compared to strictly defined normotensive PLHIV (92.0±20.4 vs. 104.8±15.7 ml/min/m²). 64.1 percent of PLHIV (59.5% with 24-h hypertension and 66.7% with 24-h normotension) had abnormal systolic dipping.The high prevalence of WCH and MH with signs of early renal end-organ damage and an abnormal dipping in approximately 2/3 of PLHIV warrants further investigation as these factors may contribute to the increased cardiovascular risk in PLHIV in resource-limited settings like Malawi.https://clinicaltrials.gov (NCT02381275), registered March 6th, 2015. The increase in survival of people living with HIV (PLHIV) is accompanied by a rising number of patients suffering from non-communicable diseases (NCD) 23. In pa248Twenty-four-hour ambulatory blood pressure monitoring (ABPM) has been established as the most accurate non-invasive method to diagnose arterial hypertension and therefore is recommended in the European and latest US guidelines 11. ABPM 121317A recently published systematic review revealed a high burden of hypertension in Malawi with prevalence estimates ranging from 15.8 to 32.9% and in particular hypertension control has been declared as one major priority in a current NCD-focused implementation research plan in Malawi 20. As paClinicalTrials.gov NCT02381275), which has been conducted since 2014 at the Lighthouse Clinic in Lilongwe, Malawi, analyses changes in kidney function of PLHIV after initiating a tenofovir-based ART. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethical Committees of the involved German universities of Cologne and Heidelberg and the National Health Sciences Research Committee of the Ministry of Health, Malawi. Adult, ART-naïve patients (≥18 years) with confirmed HIV infection were included after they had given written informed consent. Patient demographics, medical history, laboratory values, current medication, and anthropometric data were recorded at study entry as well as during regular study visits. The present study is a subgroup analysis of participants with self-reported hypertension (treated or untreated), newly detected hypertension (BP ≥ 140/90 mmHg on ≥2 measurements at ≥3 occasions) or raised BP during ≥2 routine study visits. At the time when 24-h-ABPM equipment became available at the Lighthouse Clinic in 2017, participants still active and fulfilling these criteria were contacted and asked to participate. 24-hour-ABPM measurements were performed between August 2017 and December 2018. Reimbursement for the cost of the additional trip to and from the clinic and a meal was granted to the study participants. The recruitment had to be stopped because of financial constraints. However, the enrolled study participants were similar to not-enrolled patients with respect to demographic and clinical characteristics .Study participants underwent ABPM using a non-invasive oscillometric device . This device had been validated and received the highest A/A grade according to the British Hypertension Society protocol . The cufDaytime (BP-measurements every 15 minutes) and nighttime (BP-measurements every 30 minutes) were originally programmed from 6:00 a.m. to 10:00 p.m. and from 10:00 p.m. to 6:00 a.m., respectively. Already during the feasibility phase of the study only a few individuals marked the time of going to bed and getting up in the morning . Hence, OBP was determined as the mean of the following measurements: 1) oscillometric BP measurement (Rossmax CF115f and Omron M300) as part of anthropometric measurements after entering the clinic, 2) additional measurement by a qualified nurse if BP >140/90 mmHg and 3) manually induced measurement after positioning of the cuff for the ABPM to the non-dominant upper arm. All measurements were done in sitting position after 5 minutes rest 15. A sep10True normotension as well as controlled hypertension were defined as mean OBP <140/90 mmHg plus mean values below all ABPM thresholds, while sustained hypertension was defined as mean OBP ≥140/90 mmHg plus mean 24-h BP ≥130/80 mmHg.MH was defined as normal OBP with hypertension according to one of the aforementioned ABPM thresholds and WCH was defined as OBP ≥140/90 mmHg with mean values below all ABPM thresholds . PatientSince the nocturnal fall in BP is known to be impaired in diabetes , the dip24Laboratory data were taken from the electronic LighTen-study database including CD-4 cell counts , HIV-RNA plasma levels , and creatinine plasma levels . The estimated glomerular filtration rate (eGFR) was calculated according to the CKD-EPI equation .p-value <0.05 was considered as statistically significant.Descriptive statistics were undertaken. They included the median with interquartile range (IQR) for continuous variables and frequencies for categorical variables. Differences between two groups were analyzed using Student’s t-test for continuous variables and Chi²-test for categorical variables. Differences in mean eGFR between the four hypertension phenotypes were analyzed using Welch’s ANOVA. Statistical analyses were performed using SPSS . A two-sided A total of 118 PLHIV were enrolled in the LighTen-ABPM sub-study. One patient had stopped the 24-h recording prematurely due to discomfort. Thus, 117 PLHIV were included for further analysis .3 and all but four patients had viral loads below the lower limit of detection. Study cohort characteristics are shown in Table The median age of the PLHIV cohort was 45.6 years (IQR: 38.0–51.4) and 51.3% were female. About two-thirds of the PLHIV were classified as overweight  or obese . At the time of ABPM, all PLHIV received ART, in all but four patients consisting of tenofovir/lamivudine/efavirenz in a fixed drug combination and the median duration on ART was 26.0 (IQR: 19.0-33.5) months. The median CD4-count was 392 (IQR: 241–528) cells/mmst, 2019, none of the included patients had died.At the time of ABPM 52.1 % of PLHIV (n = 61) had a prior diagnosis of hypertension, of whom 37 (60.7%) took antihypertensive medications as prescribed. When follow-up of the LighTen Cohort study ended on October 31Elevated OBP was found in 64 of 117 PLHIV (54.7%) Table . Grade 124-h ABPM normotension was confirmed in only 14 of 37 PLHIV (37.8%) with treated hypertension  vs. true normotension/controlled hypertension (104.8 ± 15.7 mL/min/1.73m2), WCH (93.3 ± 11.4 mL/min/1.73m2), or sustained hypertension (95.3 ± 22.8 mL/min/1.73m2). Data sets from 103 PLHIV were eligible to analyze the dipping status , the prevalence of WCH among PLHIV was 39% and 26%, respectively 42. WhileThe clinical management of WCH is debatable. While previous data suggested that patients with untreated WCH (defined by elevated OBP and normotensive daytime ABPM) and normotensive patients were at a similar risk of cardiovascular disease , a comprBesides MH and WCH, an abnormal systolic dipping pattern of BP is also associated with more severe target-organ damage and increased cardiovascular risk 25. In liUncontrolled hypertension and hypertension unawareness turned out to be further significant problems in the investigated study cohort, both of which are particularly prevalent in rural areas of sub-Saharan Africa 49. A lar5052Self-reported hypertension represents another important source of bias and seems to be considerably flawed as can be seen in the present study where 10/24 PLHIV with known but untreated hypertension were truly normotensive according to all ABPM thresholds .This study has several strengths, including the availability of data from a comparably large PLHIV cohort from SSA. In addition, ABPM and OBP were measured following standardized protocols. However, there are also several limitations. Firstly, the absolute number of patients with MH or WCH was overall low, and follow-up was limited with respect to clinical endpoints. More and reliable data on MH and WCH among PLHIV are needed to estimate the true prevalence of hypertension subtypes in this population at increased cardiovascular risk. Secondly, the LighTen study protocol did not include imaging procedures, thus, hypertensive vascular or cardiac damage could not be determined. Thirdly, as ABPM was obtained only on a single occasion and at least for treated patients, the recently shown poor long-term reproducibility of masked or white-coat uncontrolled hypertension has to be taken into account . FourthlABPM helped to identify MH with its high cardiovascular risk in a considerable number of PLHIV with normal or high normal BP, while WCH was confirmed in a quarter of patients with grade 1 office hypertension, whose therapy can be postponed in view of a probably favorable cardiovascular prognosis. In addition, a lack of normal nocturnal BP dipping with its inherent unfavorable effects on cardiovascular disease was identified in the majority of PLHIV from urban Malawi.Implementation of ABPM into HIV care programs in resource-limited settings represents a promising approach to affirm a diagnosis of hypertension and allocate antihypertensive treatment to those who need it the most among in the high-risk population of PLHIV.Data available on request.The additional files for this article can be found as follows:10.5334/gh.945.s1Supplementary Table 1.Enrolled vs. not enrolled PLHIV for the LighTen ABPM sub-study.10.5334/gh.945.s2Supplementary Material.LighTen-24-h ABPM sub-study event diary and patient instructions.


Cardiorenal syndrome refers to combined cardiac and renal dysfunction that adversely impacts both organs and is also associated with severe clinical outcomes. The pathophysiology is believed to be multifactorial and complex. Increased central venous pressure and intra-abdominal pressure, overactivation of the Renin-Angiotensin-Aldosterone System (RAAS), systemic illnesses like sepsis, amyloidosis, diabetes are important factors in developing the cardiorenal syndrome. Our review article attempts to review the pathophysiology and treatment aspect of cardiorenal syndrome and explores potential therapeutic strategies that can be adopted for the management.We searched PubMed, EMBASE, Google Scholar for relevant articles using different keywords and Medical Subject Headings, and finalized 38 articles to be included in our study. Cardiorenal syndrome management aims to eliminate venous congestion and fluid retention, which leads to improved cardiorenal status. This is usually achieved using pharmacologic agents like diuretics, vasodilators, inotropes, angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin II receptor blockers (ARBs), neprilysin inhibitors, and extracorporeal methods like ultrafiltration. The use of therapeutic agents such as sodium-glucose co-transporter 2 inhibitors and tolvaptan (a vasopressin V2 receptor antagonist), and cardiac resynchronization therapy has also been shown to have potential benefits in managing the disease. These agents can be instrumental in the management and require large-scale clinical trials specifically aimed at improving cardiorenal outcomes based on severity and type of cardiorenal syndrome. The symbiotic relationship of the heart and kidney function was recognized in the 19th century by Robert Bright, but the term Cardiorenal Syndrome(CRS) was coined in 2004  in 10.5% of patients receiving empagliflozin compared to 12.5% of patients on placebo. In contrast, the composite renal outcome  was reported to be 1.7% compared to 3.1% in placebo. The study also showed that the drug resulted in a significant reduction of cardiovascular risk factors like weight, waist circumference, uric acid level, and systolic and diastolic blood pressure without increasing heart rate Another trial conducted by Neal et al., called the CANVAS trial (Canagliflozin Cardiovascular Assessment Study), a set of two sister trials to study the efficacy of canagliflozin on cardiorenal outcomes, showed a composite CV outcome of 26.9 per 1000 patients compared to 31.5 per 1000 of placebo patients and renal outcome of 5.5 versus 9 per 1000 patients in placebo group conducted over 10,142 patients overall. The study attributed the benefits to improved glycemic control, reduction in blood pressure (BP), decreased intra-glomerular pressure, reduced albuminuria, and amelioration of volume overload .Wiviott et al., in DECLARE-TIMI 58 trial ( Dapagliflozin Effect on Cardiovascular Events), showed composite CV outcome in 8.8% of patients receiving dapagliflozin compared to 9.4% of placebo patients and renal outcomes in 1.5% on the drug compared to 2.8% placebo, which included a total of 17,160 patients . CREDENCA review by Kluger et al. mentioned that the four trials on SGLT2i showed reliable cardiorenal benefits and similar safety profiles. The potential benefits of SGLT2i were found to be due to a reduction in systemic and renal artery stiffness, hyperglycemia, hyperlipidemia, and decreased expression of inflammatory molecules. Increased expression of sodium-glucose co-transporter-1 inhibitors (SGLT1) receptors in cardiomyocytes may have represented a potential pharmacological target for cardio-protection . EffectsCardiovascular protection is also partly due to caloric restriction and adaptive ketogenesis. Increased glucose excretion causes a shift to fat utilization, which improves peripheral insulin sensitivity and glucagon secretion, which subsequently releases free fatty acid and increased ketogenesis, enhancing cardiac metabolism. This shift in metabolism lowers renal oxygen consumption alleviating hypoxic stress and thereby slowing the progression of the disease .Besides, Zelniker et al., in their study, mentioned the benefits of SGLT2i in reducing epicardial fat, which might result in decreasing noxious stimuli like leptins and components of RAAS involved in cardiovascular inflammation, and fibrosis. SGLT2i mediated diuresis and natriuresis are also observed mechanisms of renoprotection . The natTable TolvaptanElevated arginine vasopressin levels in heart failure due to activation of RAAS pathway have adverse effects, resulting in deterioration in cardiac function and leading to peripheral vasoconstriction and increased afterload through V1a receptors (Vasopressin 1). Vasopressin V2 receptor stimulation leads to water retention and elevation in preload ,29. TolvIn EVEREST TRIAL (Efficacy of Vasopressin Antagonism in Heart Failure Outcome Study with Tolvaptan) conducted on 4133 patients, it was found that tolvaptan effectively reduced volume overload and provided symptomatic relief but failed to improve mortality and morbidity . It has Cardiac resynchronization therapyCardiac resynchronization therapy (CRT) has been used for the treatment of heart failure. It has also improved renal function by improving cardiac output, increased mean arterial pressure, and decreased central venous pressure . In a reHowever, a retrospective study including 482 patients reported increased incidence of renal dysfunction at the time of CRT implantation and was usually associated with poor survival outcomes compared to patients with preserved renal function and reported increased mortality in patients with CKD. The authors attributed the findings to a higher prevalence of anemia in CKD . Truong Limitations Most of the available data was inaccessible due to institutional fees or citing confidentiality. There was a lack of large-scale randomized controlled trials aimed at first-hand understanding of cardiorenal syndrome pathogenesis and the effects of different treatments on morbidity and mortality outcomes. Some of the included studies were primarily designed to understand the effects of the therapies mentioned above on heart failure prognosis. However, they did highlight benefits in cardiorenal syndrome and thus call for further research of the topic.In this review, we strived to elaborate on the pathophysiology of cardiorenal syndrome with special emphasis on management and newer treatments of cardiorenal syndrome. The impact of RAAS activation on systemic blood pressure combined with oxidative and inflammatory damage to renal vasculature results in cardiorenal syndrome. ACE inhibitors, ARBs, and diuretics countering the effects of RAAS activation were concluded as initial management pillars of these patients. Furthermore, various trials were cited to highlight the efficacy of SGLT-2 inhibitors on morbidity and mortality of these patients when compared to placebo. Tolvaptan, offsetting the effects of elevated vasopressin, has also been demonstrated to have symptomatic benefit in cardiorenal syndrome. Moreover, CRT, which has proven beneficial in heart failure, has also shown improvements in renal function by improving cardiac output.In our opinion, these newer treatments can prove to be instrumental in the management of cardiorenal syndrome and calls for large-scale trials specifically aimed at understanding these drugs in improving outcomes of patients with cardiorenal syndrome.


PTGDR2 that encodes for CRTH2 receptor, expressed in cells involved in T2 inflammation, was developed in a cohort of 361 independent subjects: 94 non-asthmatic non-atopic controls, 187 asthmatic patients [including 82 with chronic rhinosinusitis with nasal polyposis (CRSwNP) and 24 with aspirin-exacerbated respiratory disease (AERD)], 52 with allergic rhinitis, and 28 with CRSwNP without asthma. Results: PTGDR2 was one of the most differentially overexpressed genes in asthmatic patients’ peripheral blood . These results were confirmed by qPCR in the validation study, where PTGDR2 transcripts were significantly upregulated in asthmatic patients (p < 0.001). This upregulation was mainly detected in some subgroups such as allergic asthma, asthma with CRSwNP, AERD, eosinophilic asthma, and severe persistent asthma. PTGDR2 expression was detected in different blood cell types, and its correlation with eosinophil counts showed differences in some groups of asthmatic patients. Conclusions: We found that PTGDR2 expression levels could identify asthma patients, introduce a minimally invasive biomarker for adult asthma molecular phenotyping, and add additional information to blood eosinophils. Although further studies are required, analyzing PTGDR2 expression levels in peripheral blood of asthmatics might assist in selecting patients for treatment with specific antagonists. Background: Precision medicine is a promising strategy to identify biomarkers, stratify asthmatic patients according to different endotypes, and match them with the appropriate therapy. This proof-of-concept study aimed to investigate whether gene expression in peripheral blood could provide a valuable noninvasive approach for the molecular phenotyping of asthma. Methods: We performed whole-transcriptome RNA sequencing on peripheral blood of 30 non-atopic non-asthmatic controls and 30 asthmatic patients. A quantitative PCR (qPCR) validation study of  Asthma is a heterogeneous disease, characterized by chronic airway inflammation with variable respiratory symptoms, affecting around 358 million people worldwide ,2. Type In the precision medicine era, it is necessary to identify inter-individual differences in response to drugs to better stratifying patients finding the most effective treatments for each patient . In thisIn summary, different transcriptomic studies in asthma are adding information on novel genes and related pathways involved in the numerous pathophysiological mechanisms of this heterogeneous disease. In this work, we performed a transcriptomic analysis in whole blood samples of patients with allergic asthma and non-allergic non-asthmatic controls. The study aimed to find differentially expressed genes and analyze whether the determination of any of these gene expression levels could be employed as a biomarker to provide additional information for the phenotypic characterization of patients with asthma.A total of 421 subjects participated in the study, including 60 subjects who participated in the transcriptomic analysis, and 94 controls and 267 patients involved in the validation study. In the transcriptomic analysis, 30 patients with allergic asthma diagnosed by an allergist, and 30 non-asthmatic non-atopic controls were included. For the validation analysis, 361 Caucasian individuals were recruited from the Allergy Department of the Salamanca University Hospital. The Clinical Research Ethics Committee of the Institute for Biomedical Research of Salamanca (IBSAL) approved the study (PI 2020-02-433) and informed written consent from study subjects was obtained. Ninety-five individuals were enrolled as controls. Controls fulfilled the following criteria: (i) no symptoms or history of fulfilled the following criteria: (i) no symptoms or history of asthma or other pulmonary diseases, (ii) no symptoms or history of rhinitis, (iii) no symptoms or history of allergic diseases, (iv) negative skin prick tests to a battery of common aeroallergens; (v) absence of a family history of asthma, rhinitis, or atopy, and (vi) age > 16 years old. Age was significantly higher in controls than in patients in order to permit a more extended period for asthma to have appeared. In addition, the patients were recruited meeting the following criteria: (i) physician-diagnosis of respiratory allergy , (ii) age > 16 years. Skin prick tests were performed from a battery of common aeroallergens previously described , allergiTotal RNA was isolated from peripheral blood samples stored in RNA later at −20 °C using the Ambion RiboPureTM Blood kit . Peripheral blood was selected due to its accessibility. RNA was treated with Ambion DNAse I and concentrated with the RNeasy MinElute Cleanup Kit . All purification protocols were carried out following the manufacturer’s instructions. RNA was quantified with a Nanodrop 1000 instrument, and the RNA quality was assessed by measuring the RIN (RNA Integrity Number) in a Bioanalyzer Agilent 2100 expert equipment using the Eukaryote Total RNA Nano kit . 1 µg of RNA samples with an RIN higher than 8.0 was used for RNAseq. Globin transcripts and ribosomal RNA were removed, and RNA was cleaved to prepare RNA strand-specific libraries, generate clusters from each library, and perform a high-throughput sequencing using the Illumina HiSeq 2500 ultra-sequencing platform .http://www.bioinformatics.babraham.ac.uk/projects/fastqc (accessed on December 2019)). The reads were then mapped against the human genome version hg39 using the tophat2 algorithm  (p < 0.05). However, when establishing a positivity cutoff of ≥250 eosinophils peripheral blood eosinophil (specificity ≥ 95%), 46.5% (n = 114) of patients of the global sample were erroneously classified as false negatives. In this case, using the PTGDR2 expression test with a cutoff of 9.3, which also has a specificity of ≥95%, we were able to identify 11 patients (10%) from the total of false negatives. In PTGDR2 expression) determine four quadrants ; these patients were located in the Q2 quadrant and were distributed into the following groups: Rhinitis (4 patients), asthma with polyposis , asthma without polyposis (3 patients) and polyposis without asthma (one patient). This result suggests that the PTGDR2 expression test is a more specific biomarker in patients with low eosinophil values (<250 eosinophils), giving additional information to eosinophil levels.As PTGDR2 [0.612 (0.533–0.691)] is higher (not significant p > 0.05) than for eosinophils [0.587 (0.511–0.663)]. Exploring the CRSwNP patients with or without asthma, we also found a higher (not significant p > 0.05) AUG for PTGDR2 [0.672 (0.566–0.778)] than for eosinophils [0.597 (0.486–0.707)]. Finally, in the case of asthma with CRSwNP, with or without NSAID intolerance respiratory, a higher (not significant p > 0.05) AUC was also found for PTGDR2 than for eosinophils  for inophils .p < 0.01) between PTGDR2 expression and eosinophil counts was found in the group of asthmatics. However, streaking differences in PTGDR2 expression were detected in asthmatic patients with similar baseline blood eosinophil levels (PTGDR2 expression (quadrant 2) and those with high eosinophil counts and low PTGDR2 expression (quadrant 4) (PTGDR2 expression among different quadrants using post hoc tests (p < 0.001). The distribution among the four quadrants is shown in p < 0.05; using χ2 test). It suggests that the combination of the selected cutoffs could efficiently discriminate the patient groups studied. A moderate correlation  . This fadrant 4) . The phePTGDR2 PBMCs were labeled using specific antibodies for lymphocytes T (CD45+CD3+) and basophils (CD123+). Eosinophils were identified by their autofluorescence. Cells were also labeled with anti-PTGDR2 (CD294) to determine the protein expression on the cell surface. Results from four representative patients are shown in PTGDR2 gene expression was found (R2 = 0.6077). Eosinophils were, as expected, the most abundant CD294+ population. Eosinophil count (as per hemogram) was found to correlate with the percentage of CD294+ eosinophil . Thus, patients with the highest eosinophil counts in peripheral blood showed the highest CD294+ labeling on eosinophils . However, some differences could be appreciated between these two patients. While the eosinophil CD294+ labeling was quite similar (11.8% and 11.7%), total CD294+ labeling differed due to differences in CD3+CD294+ cell percentage, as the level in patient 2 was three-fold that of patient 1  when adjusted for white blood cell subtypes  remains unknown. Interestingly, the highest PTGDR2 levels (54.1-fold change) corresponded to a young male patient with intermittent asthma and CRSwNP who had asthma exacerbations only when aspirin was taken, without symptoms or needed medication out of these episodes. Although it is highly speculative, maybe the high levels of PTGDR2 expression could suggest a predisposition for evolution to more severe forms of asthma. In any case, these patients should be monitored to evaluate whether the severity of asthma will increase in the future. If this were to occur, the use of PTGDR2 as a biomarker of asthma severity could identify patients long before they have severe asthma episodes.Access to high-throughput technologies has made transcriptomic approaches being increasingly applied in the field of allergic diseases. RNAseq technology can thus find transcripts that might become future biomarkers . In the xpressed . Interesxpressed . The genreatment . PTGDR2 controls . PTGDR2  in vivo ,51,52. F in vivo ,54, sugging qPCR , showingx asthma . These dpatients . We alsoPTGDR2 expression and peripheral blood eosinophils count in most asthma groups , and two asthmatic patients with CRSwNP and sensitivity to NSAIDs (AERD)  (There was a moderate positive correlation between a groups . Interesrhinitis . The higctively) . On the ctively) . WhetherPTGDR2 expression levels correlated with PTGDR2 mean fluorescent intensity on CD4+CRTH2+ T cells but not with CRTH2+ eosinophils assessed by flow cytometry [PTGDR2 mRNA had significantly more surface PTGDR2 in CD4+ T cells compared with those with low levels of PTGDR2 mRNA, suggesting that the high level of PTGDR2 mRNA in severe asthmatics could be driven by the CD4+ T cell population [PTGDR2 expression. Moreover, CD3+CD294+ cell percentages in some AERD patients  were higher than in others , showing that this cell population could be relevant at least in a group of AERD patients. Also in these patients, levels of PGD2 are high, both basally and during acute reactions to NSAID [PTGDR2 mRNA expression levels could be particularly helpful in AERD patients.We found that PTGDR2 (CD294) positive lymphocytes should also be considered when studying PTGDR2 expressing cell populations. Although eosinophils were the most abundant CD294+ cells, some patients exhibited higher levels of CD3+CD294+ than others with similar CD294+ eosinophil levels, showing that T cells could also be relevant. Accordingly, Palihke et al. reported that ytometry . The autpulation . In thisto NSAID . PGD2 ovto NSAID . TherefoPTGDR2 levels could have provided better patient selection and given better results. A clear example is what happened with mepolizumab, which failed in the initial studies due to an inappropriate patient selection [PTGDR2 mRNA and blood eosinophils count could provide an optimal predictive value for asthma, CRSwNP, and AERD. That might be particularly relevant in those patients with low eosinophils and high PTGDR2 expression levels.In recent years there has been a significant research effort in the field of PTGDR2 antagonists ,5,40,58.election . Kerstjeelection , commentPTGDR2, and other genes may also be relevant in response to treatments. Nevertheless, we selected PTGDR2 by its crucial implication in the immunology of type 2-asthma and because different antagonists are directed against this molecule. One of the limitations of the study is that it is a unicentric study. Nevertheless, this fact gives uniformity to the study. Additionally, we have mainly focused on PTGDR2 levels in peripheral blood as a possible biomarker demonstrating differential expression levels between asthmatic and controls and progressively increasing PTGDR2 levels as the severity of asthma raised. Finally, we found patients with low eosinophil counts and high PTGDR2 levels and vice versa, thus providing additional information to peripheral eosinophil counts. Concerning these findings, we propose that peripheral blood mRNA levels of different genes (such PTGDR2) should be explored as new minimally invasive biomarkers that might assist in the molecular phenotyping of asthma and selecting patients for treatment with specific antagonists.In the present work, we explored


Fractional anisotropy (FA), and mean, radial and axial diffusivity were evaluated from renal cortex and medulla. Moreover, the corticomedullary differentiation of each DTI parameter was compared between groups. Only cortical FA statistically differed between BBS patients and controls (p = 0.033), but all the medullary DTI parameters discriminated between the two groups with lower FA (p < 0.001) and axial diffusivity (p = 0.021) and higher mean diffusivity (p = 0.043) and radial diffusivity (p < 0.001) in BBS patients compared with controls. Corticomedullary differentiation values were significantly reduced in BBS patients. Thus, DTI is a valuable tool for investigating microstructural alterations in renal disorders when kidney functionality is preserved.Kidney structural abnormalities are common features of Bardet-Biedl syndrome (BBS) patients that lead to a progressive decline in renal function. Magnetic resonance diffusion tensor imaging (DTI) provides useful information on renal microstructures but it has not been applied to these patients. This study investigated using DTI to detect renal abnormalities in BBS patients with no overt renal dysfunction. Ten BBS subjects with estimated glomerular filtration rates over 60 ml/min/1.73m BBS 1–24), most encoding proteins localised within the basal body of the primary cilium1, have been associated with human BBS. BBS patients are prone to develop renal failure2. Abnormal renal and urinary tract structures have been reported4; accordingly, chronic kidney disease (CKD) is considered the most common cause of morbidity and mortality. The renal phenotypes of both patients and mouse models are variable, although a tubulointerstitial defect is postulated to be a common substrate5 because of the low incidence of proteinuria and the high rate of urine concentration defects7.Bardet-Biedl syndrome (BBS) is a rare inherited disorder characterised by the dysfunction of several organs. To date, at least 24 genes , recent studies have focused on the ability of kidney MRI to provide quantitative functional biomarkers for both diagnostic and prognostic purposes16. In this field, diffusion tensor imaging (DTI) provides useful insights into in vivo kidney analyses by measuring, noninvasively, physical properties related to kidney tissue microstructural integrity17. Indeed, the water movement analysis provided with the DTI allows the measurement of quantitative parameters, such as mean diffusivity (MD) and fractional anisotropy (FA), which reflect relevant features strictly related to the functional integrity of renal structures, such as tubules, collecting ducts and vessels19. In addition to the characterisation of healthy kidneys22, DTI has been successfully applied to detect renal damage in different kidney disorders, such as CKD25, kidney transplant28, diabetic nephropathy31, renal clear cell carcinoma32 and glomerulonephritis33. In these contexts, the absence of a contrast media makes the DTI analysis particularly suitable for subjects with kidney failure in which contrast media are highly contraindicated.In the last decade, magnetic resonance imaging (MRI) has been increasingly used for in vivo kidney studies. In addition to the clinical utility of MRI examinations for the assessment of several structural kidney abnormalities  obtained from DTI parameters was measured in patients and controls, and the right and left kidney were analysed separately using DTI parameters to assess possible asymmetrical alterations.The principal aim of the present study was to investigate how microstructural parameters derived from DTI can be used as markers of kidney impairment in patients with BBS. Specifically, we assessed the utility of DTI in detecting early kidney alterations in a subset of BBS individuals with preserved renal function. To date, no dedicated quantitative MRI kidney studies in BBS are present in the literature. Indeed, only structural macro-abnormalities  have been detected by the application of MRI in BBS36. Retinal degeneration was assessed by ophthalmologic evaluation and learning disabilities were defined as defects in writing, reading and spelling, as well as deficiencies in memory, coordination and emotional maturity. Obesity was defined as a body mass index (BMI) higher than 30 kg/m2. Non-hypertensive subjects were defined as people with blood pressure < 140/90 mmHg in the absence of dedicated treatment. Non-diabetic individuals were defined as those with plasma HbA1c level < 7%, without a personal history of diabetes mellitus. Genetic validation was available for all the enrolled BBS patients. Renal function was assessed by estimating the glomerular filtration rate using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation with standardized plasma creatinine measurements. Genetic validation was available for all the enrolled patients.This study was approved by the local ethics committee  with protocol number 02/17. All the procedures adopted in this study were performed in accordance with the approved guidelines and regulations. Written informed consent was obtained from all the subjects enrolled in this study. From May 2018 to August 2019, 10 BBS patients referred to the Nephrology Unit of the University of Campania L. Vanvitelli were enrolled in the study. The diagnosis was based on Beales clinical criteria2.The inclusion criteria were as follows: more than 18 years old, no contraindications to MRI examinations , estimated glomerular filtration rate (eGFR) greater than 60 ml/min/1.73 m2, absence of renal pathologies, diabetes, hypertension or metabolic abnormalities. Moreover, control subjects were selected by matching the gender, age range and BMI of the BBS group members.In total, 14 subjects with no history of renal diseases, diabetes or hypertension were enrolled as the control group. The inclusion criteria of the controls were as follows: no contraindications to MRI examinations, eGRF greater than 60 ml/min/1.73mBoth BBS and control subjects underwent MRI examinations with the same acquisition protocol using a 3 T scanner , equipped with a 4-channel phased-array body coil.Anatomical T1- and T2-weighted images, covering both the right and left kidney, were acquired to examine kidney morphology. The T1-weighted images were acquired with the 2D Fast Low Angle Shot (FLASH) sequence, whereas T2-weighted images were obtained with the half-Fourier acquisition single-shot turbo spin echo (HASTE) sequence. Both FLASH and HASTE images were acquired with a coronal orientation in breath-hold.2 as diffusion weighting and three averages each of which with six different diffusion directions at b = 500 s/mm2 and two volumes at b = 0 s/mm2. A diffusion weighting of b = 500 s/mm2 was chosen as a good compromise between image quality and pure diffusion measurement  and considering the mean diffusivity of the kidneys as reported in the literature39. Additional parameters of the EPI sequence were as follows: oblique-coronal orientation, repetition time 1800 ms/echo time 74 ms; 152 × 152 matrix; 319 × 319 mm2 field of view; slice thickness of 3 mm; 2.1 × 2.1 mm2 pixel size; 36 slices; 2 as the parallel imaging accelerator factor and left to right as the phase-encoding direction. Another set of DTI images was acquired with the same parameters by only blipping the phase-encoding direction (right to left) for the subsequent geometric distortion correction39. The total scan time varied from 15 to 20 min, depending on the individual’s respiratory cycle.For DTI images, a fat-saturated twice-refocused EPI sequence was performed with a respiratory triggering, 500 s/mmPrior to image analyses, the images were de-identified and visually checked for image artefacts. In particular, the raw image data in the DICOM format were elaborated with the software provided with the scanner to remove the DICOM tags correlated with patient’ data, and then, they were transferred to a workstation for the subsequent image analyses.The acquired images were analysed by an expert radiologist (C.C.) with more than 8 years of experience in abdominal imaging. In particular, the radiologist assessed the image quality by visually checking for the absence, or low presence, of image artefacts such as gross movement, ghosting and signal loss. Moreover, both the FLASH and HASTE images were inspected to detect renal structural abnormalities .39. In particular, the movement between and within the DTI series and the distortions induced by eddy-currents were corrected using the eddy command, whereas the susceptibility-induced geometric distortions were corrected using the topup function. Both dedicated routines were implemented in the FMRIB-FSL library (v. 6.0.0)40. For the geometric distortion correction, an approach based on the estimation of the susceptibility off-resonance field41 was adopted using additional DTI data acquired with the opposite phase-encoding direction.The DTI images were pre-processed for mitigating image artefacts such as movement, eddy-currents and geometric distortion. The pre-processing procedure was chosen to provide the highest test–retest reproducibility results, as reported in Borrelli et al.42. In particular, MD, FA, radial diffusivity (RD) and axial diffusivity (AD) were evaluated from the eigenvalues of the calculated diffusion tensor. Moreover, the mean image among b = 0 s/mm2 DTI images (denoted as b0) was calculated for further processing.Parametric maps derived from the pre-processed DTI images were evaluated using MRtrix3 toolbox43 was used to annotate ROIs. The ROI generation procedure consisted of manually drawing circular 58 mm2 ROIs at the upper, middle and inferior poles of both cortical and medullary kidney tissues. ROI selection was performed by the radiology technician (L.B.) and confirmed by the radiologist (C.C.). For each subject included in the present study, the ROIs were drawn on coronal slices in the central section of the kidney  paying attention to avoid the renal pelvis, vessels, calyces and, eventually, renal cysts and fetal lobulations. ROI placement was performed on the b0 image and the corresponding FA map was used to confirm the accuracy of the annotation procedure. The purpose of constraining the ROI placement on a single well-identifiable slice was to create the most reproducible procedure for revealing abnormalities in a diffuse  renal disease, such as BBS. Both right and left kidneys were annotated, resulting in 12 different ROIs for each subject For the following statistical analysis, both cortical and medullary means and standard deviations (SD) from each DTI map were calculated by averaging the ROI values from the upper, middle and inferior poles of each kidney. Moreover, the obtained values were averaged for the left and right kidneys, thereby obtaining a single measurement for both kidneys.44, FA and AD maps should result in CMD values less than 1 owing to higher FA and AD values in the medulla compared with the renal cortex. Conversely, because MD and RD values reflect the mean diffusivity and the diffusivity parallel to the principal diffusion direction respectively, the CMD of a healthy kidney should result in a value greater than 1.In addition, the CMD derived from each DTI parameter was calculated as the ratio between cortical and medullary mean values. A value close to 1 reflected poor CMD, whereas values farther from 1 represented good differentiation between renal cortical and medullary tissues. By incorporating DTI values evaluated from healthy subjectsThe statistical analysis was performed using MATLAB r2018a .DTI parameters: for each parametric map, the Wilcoxon rank sum test was performed between mean values of both kidneys, separately for cortical and medullary values, from BBS and control subjects to compare between the two groups.CMD analysis: the Wilcoxon rank sum test was applied to CMD values to investigate CMD differences between BBS subjects and controls.Kidney asymmetry: the statistical difference between measurements from right and left kidneys within both BBS and control subjects was assessed using the Wilcoxon rank sum test to determine whether DTI parameters of the left kidney significantly differed from those of the right kidney.To investigate renal microstructural injuries in BBS subjects, three specific statistical analyses were performed:For all the statistical tests, p < 0.05 was considered statistically significant.2, respectively. Additional clinical features of BBS patients included retinal dystrophy in 9/10 patients and learning disabilities were detected in 6 out of 10 BBS individuals. The mean BMI was of 26.96 kg/m2 in BBS individuals, which was not significantly different from that of the controls. Genetic analysis was available for all the BBS patients, and after a next generation sequencing analysis, 3/10 patients showed no mutations in known BBS-related genes. The remaining patients were homozygous for the BBS4 (n = 2), BBS12 (n = 2), BBS1,10 and 9  variants. None of the controls had signs of functional and/or structural renal anomalies.The details of patients and controls are reported in Table After the preliminary visual assessment of image quality, all the acquired images, both from BBS and controls, were suitable for the image analyses procedures. An example of DTI maps, with related CMD values, for a BBS and a control subject included in the present study is shown in Fig. Renal structural abnormalities were found in six BBS subjects. In particular, three BBS subjects presented renal fetal lobulations Fig.  and fourThe distributions of the medullary and cortical values for both BBS and control subjects are shown in Fig. The details of the CMD values derived from each DTI parameter for both BBS and control subjects are shown in Table Neither BBS subjects nor controls showed statistical differences in DTI parameters from the left and right kidney Table . MoreoveThis is the first study using DTI to characterise renal abnormalities in BBS patients before the onset of clinically evident renal dysfunction. The latter is an important cause of morbidity and mortality in BBS patients and, to date, the exact pathomechanisms leading to kidney disease in these patients is unknown. Moreover, because CKD in BBS, as well as in the general population, is a progressive disorder characterised by a time-dependent progression, the early diagnosis of kidney abnormalities will have great clinical impact, because it allows the determination of patients at risk of progressing renal disease.31. For this purpose, we selected BBS patients with eGFR values greater than 60 ml/min/1.73 m2. This subgroup of individuals included young adult subjects with plasma creatinine level in the normal range. Moreover, consistent with the well-documented theory that kidney disease in this setting is not primarily a glomerular disorder, these patients did not show significant increases in urine protein excretion. Thus, the BBS patients we selected did not show alterations in the major parameters used in the clinical setting to address renal dysfunction.To date, limited information is available in the literature on markers of early CKD in BBS patients. As in non-syndromic individuals, an increased plasma creatinine levels is not an early index of kidney dysfunction. This study demonstrates that the major parameters used to address renal dysfunction are sometimes not sufficient to detect small kidney defects that may predispose a patient to CKD progression45. Among the characteristics, the FA was able to best discriminate BBS from controls because it measures the preferential directionality of water molecules within tissues. In this setting, alterations in FA values may reflect microstructural changes in the kidneys of BBS subjects. Our findings are in-line with a recent study in which FA values identified early renal damage in diabetic patients with normal BMI and eGFR values31.As a primary finding, DTI was able to discriminate BBS patients from age-gender-eGFR-BMI matched controls. Indeed, DTI values differed between BBS and controls, especially in the renal medulla, in which more tubular structures are represented. The altered values in the medullary renal tissues of BBS subjects may reflect the compromised integrity of collecting ducts as a sign of early tubulointerstitial injuries, which is a common feature that causes urine concentration dysfunction in BBS3.Moreover, poorer CMD was revealed in BBS patients, indicating that the CMD may represent an additional index of early renal abnormalities in BBS. The reduction of CMD in BBS patients was likely owing to the significant alterations in the DTI values of medullary tissues compared with the controls. Therefore, the kidney injuries measured using DTI were not specifically related to the left or right kidney, which confirmed the symmetry of CKD-related renal damage in BBS patients46. Finally, the genetic variability in the BBS subjects, together with the small sample size, made it difficult to assess genotype to phenotype correlations. Further studies with larger numbers of BBS subjects may untangle possible associations between specific BBS gene mutations and kidney phenotypes using the DTI technique.The primary limitation of the present study is the small sample size. However, the limited number of subjects is due to the rarity of BBS and the presence of constraints on MRI examinations . As a second limitation, no direct comparison of the presented findings were performed with DTI parameters obtained from subjects with other kidney structural abnormalities . In addition, because the morbidly obese cannot undergo MRI studies, the analysis was applied to mainly overweight BBS patients with no severe obesity. In this scenario, obesity may confound the assessment of kidney diffusion parameters based on the DTIIt is worth underlining the importance of the non-invasiveness of renal DTI. Indeed, as specified in the methodological section, the DTI technique does not require the use of any contrast media and, therefore, is suitable for renal pathologies, thus deserving attention for the translation in clinical practice.In conclusion, this study confirmed the ability of the DTI technique to reveal renal abnormalities before the clinical onset of renal dysfunction, demonstrating its suitability as a tool for detecting early renal injuries.Supplementary Information.