Clinical symptoms and QoL were monitored using the measurement of leg circumference, visual analogue scale (VAS) for pain, Global Index Score (GIS) and Venous Clinical Severity Score (VCSS). A total of 72 subjects completed the study. From week 4, leg edema was significantly decreased in the active group (p < 0.001). An improvement in the VAS score was observed in the active group compared to placebo at the end of treatment (p < 0.05). GIS and VCSS scores were significantly improved in the active group at week 8 (p < 0.001). No treatment related-side effects were recorded. The results of this study showed that the administration of low-dose μsmin® Plus was safe and effective in relieving symptoms and improving QoL in subjects with CVD.Chronic Venous Disease (CVD) is a common medical condition affecting up to 80% of the general population. Clinical manifestations can range from mild to more severe signs and symptoms that contribute to the impairment of the quality of life (QoL) of affected patients. Among treatment options, venoactive drugs such as diosmin are widely used in the symptomatic treatment in all clinical stages. The aim of this study is to determine the effectiveness of a new formulated diosmin in relieving symptoms and improving QoL in patients suffering from CVD. In this randomized, double-blind, placebo-controlled, multicenter clinical study, CVD patients with a Clinical-Etiology-Anatomy-Pathophysiology (CEAP) classification system between C2 and C4 were randomized to receive a bioavailable diosmin (as μsmin Chronic Venous Disease (CVD) is a very common clinical condition affecting the Western world. The general prevalence ranges from 10% for adults aged below 30 years up to nearly 80% for subjects aged >70 years old .Several genetic and molecular alterations have been found responsible for the onset of CVD, including morphological and functional abnormalities and progression from mild clinical signs, such as reticular and trunk varicose veins, to more advanced and severe signs including leg edema, skin changes and venous ulcers of lower limbs that commonly define the chronic venous insufficiency (CVI) stages ,4,5,6,7.At different stages of this disorder, general practitioners and specialty doctors have to deal with a wide range of symptoms such as leg pain, swelling, itchiness, restless leg syndrome, a burning sensation, and heaviness. These symptoms may be persistent and have a considerable impact on the quality of life (QoL) of many individuals ,9,10. ThIn the clinical practice, treatment options are based on the individual’s severity of CVD and include non-invasive treatment such as lifestyle changes , drug administration, surgical and endovascular procedures as well as compression therapies (bandages and elastic stockings) ,11,12,13The focus of available pharmacological treatments is on preventing or resolving the most serious complications such as leg ulceration ,16,17,18® Plus , was developed to overcome diosmin’s poor oral bioavailability. This product exhibited a 4-fold increase in relative bioavailability compared to micronized diosmin in rats [Recently, a newly formulated diosmin ingredient, μsmin in rats , and res in rats .® Plus in a setting of standard clinical practice where the administration of VADs in patients suffering from CVD is often integrated with lifestyle counselling.In light of these promising results, this study was designed to test the efficacy and safety of µsmin® Plus, a diosmin preparation with enhanced bioavailability) in alleviating symptoms of CVD. The main objectives of the study were the changes in calf circumference and the impact of the active treatment on quality of life, while secondary objectives were the evaluation of CVD-related symptoms including pain, investigator/patient treatment satisfaction and safety. During the study, a total of 3 visits were scheduled. Screening and baseline assessment visit : vital signs and general health condition were checked through physical examination; medical history, concomitant medications and baseline characteristics of each patient  score, Venous Clinical Severity Score (VCSS) and quality of life) were recorded; eligible patients were asked to sign the informed consent. This randomized, double-blind, placebo-controlled, multicenter, 8-week clinical trial was aimed at the evaluation of the efficacy and safety of a commercially available dietary supplement : subject evaluation and symptoms assessment were performed. Adverse effects, if any, were recorded. End of the study visit : final subject evaluation and symptoms assessment were performed. Adverse effects, if any, were recorded. ® Plus  equivalent to 450 mg diosmin. µsmin Plus® is a well-established oral flavonoid with venoprotective properties consisting of 80% micronized diosmin and 90% total flavonoids.The product administration protocol consisted of one coated tablet, once daily, of µsminThis study was conducted according to the guidelines included in the Declaration of Helsinki and all the procedures were approved by the Institutional Review Board SCM Gados, Timisoara, Timis, Romania . Written informed consent was obtained from all subjects before inclusion in the study. The trial was registered in the clinicaltrials.gov database (NCT04101201). Eligible patients were male and female aged ≥18 and ≤60 suffering from CVD with CVI grade C2–C4 on the Clinical-Etiology-Anatomy-Pathophysiology (CEAP) classification system . Exclusi® Plus or matched placebo tablets. According to the summary of product characteristics provided by the manufacturer, one tablet once daily was administered to each subject for 8 consecutive weeks. Patients fulfilling the eligibility criteria were enrolled and randomized by a computer method with equal distribution  to receive either diosmin 450 mg tablets as µsminPhlebotonics and other CVD-related treatments were not allowed during the study. There was no restriction on treatments taken previously by subjects for medical conditions not related to this study protocol.Primary outcomes investigated were change in mid-calf circumference (measured by investigator in cm) of each affected leg and quality of life (QoL) assessed by Global Index Score (GIS) calculated through the CIVQ-20 questionnaire . The ChrVAS scale for pain . Using aThe Venous Clinical Severity Score (VCSS)  includesInvestigator Global Assessment of Efficacy—Investigators made the global assessment regarding the treatment efficacy using a 4-point scale , at visit 2 and 3.Patient Global Assessment of Efficacy—Patients made the global assessment regarding the treatment efficacy using a 5-point scale , at visit 2 and 3.Continuation with investigational product—The evaluation of subject options about continuing with the investigational product after the study termination was assessed at week 8  for both groups. This variable is treated as a categorical variable and described by percentages and frequencies for each allocation group.Evaluation of symptoms relief—Evaluation of symptoms relief was assessed by evaluating the percentage of subjects who experienced symptoms relief within the first week of intake, within 2 weeks of intake, or more than 2 weeks of intake.Investigator Global Assessment of Safety—Investigators made the global assessment regarding the safety of treatments using a 4-point scale , at visit 2 and 3.The following outcomes were recorded:t-tests (if applicable) or Wilcoxon’s signed-rank sum tests were applied for quantitative variables, while McNemar’s tests were used to evaluate changes for binary variables and symmetry tests were performed to evaluate changes for qualitative (not binary) variables. The quality and completeness of the collected data was evaluated preliminarily compared to data analysis. If a subject was missing information for one or more variables, even after the resolution of its query, the missing data were not replaced. If a subject had been involved in the violation of inclusion/exclusion criteria, the respective data were excluded from the analysis. Quantitative variables  if normally distributed were described through mean ± Standard Deviation (SD), otherwise median, minimum, maximum, and interquartile ranges were shown. Qualitative variables were evaluated using frequencies and percentages. To evaluate changes over time before and after the administration, Paired ® Plus group (n = 37) or the placebo group (n = 35). All 72 subjects enrolled completed the study and all of them were included in the intention to treat (ITT) and safety population analysis. Subject demographics are presented in p > 0.05) in baseline demographic and anthropometric measures. It is noteworthy that one subject from the μsmin® Plus group and one subject from the placebo group could not be investigated at visit 2 and 3 due to travel restrictions caused by the COVID-19 pandemic. Therefore, 36 subjects in the μsmin® Plus group and 34 subjects in the placebo group were investigated at visit 2 and 3.Out of 73 subjects screened, 72 were considered eligible for the completion of this study. Subjects were randomly assigned either to the µsminp = 0.033) or 8 weeks (p = 0.016)  ; in partp < 0.001) was found in the active group in comparison to the placebo group. Results are shown in In both groups of patients, GIS improved from baseline to the end of treatment from 58.48 (±14.55) to 84.43 (±12.95) and from 61.96 (±14.13) to 67.46 (±13.77) in the active group and placebo group, respectively. At the end of treatment (week 8), a significant increase in GIS (p = 0.014) in the µsmin® Plus group compared to the placebo group. Results are shown in VAS scores decreased progressively from baseline to end of the study both in the active and placebo group. At the end of treatment, a reduction in VAS score was found statistically significant (® Plus and placebo treatment reduced the symptoms’ severity significantly after 4 weeks and 8 weeks compared to baseline (p < 0.001). At the end of treatment, µsmin® Plus showed a significant improvement in Venous Clinical Severity Score (VCSS) compared to the placebo (p < 0.001). Results are shown in Both µsmin® Plus was already evaluated as excellent or good by the investigators compared to the placebo after 4 weeks.The Investigator Global Assessment of Efficacy (IGAE) between groups is summarized in ® Plus. The evaluation of Patient Global Assessment of Efficacy (PGAE) is reported in The evaluation of symptoms relief was assessed by evaluating the following symptoms: pain, varicose veins, edema, inflammation, skin pigmentation, induration, number of active ulcers, active ulcers duration, active ulcer diameter. Symptoms relief is summarized in No adverse events or serious adverse events have been reported during the clinical trial.The Investigator Global Assessment of Safety (IGAS), which is summarized in Chronic Venous Disease (CVD) determines morphological and functional abnormalities of the leg venous system, causing a broad range of long duration signs and symptoms. Understanding the complex pathophysiology of CVD is pivotal to support the identification of novel therapeutic targets in order to effectively relieve CVD clinical manifestations, thus improving the QoL of affected patients. Due to their potential in inhibiting inflammation pathways and their ability to interact with key molecular targets, VADs have shown consistent efficacy across all CVD stages ,30,31,32CVD related symptoms and signs such as pain, cramps, leg heaviness, paresthesia, itching, and edema were relieved to some extent by treatments with VADs ,26. Diosmin, a flavonoid commonly found in citrus fruits, has been widely used and prescribed for more than 30 years as a VAD in CVD for its phlebotonic effect known to improve venous tone, stabilize capillary permeability and increase lymphatic drainage, and also for its well-established safety profile . It has Flavonoids, including diosmin, are poorly absorbed following oral administration. They are usually hydrolyzed in small phenolic compounds by the enzymes from the intestinal bacteria ; these sFor overcoming diosmin’s poor bioavailability following oral ingestion, several attempts have been made, of which the reduction in particle size  is the most commonly used, to increase flavonoids’ absorption and bioavailability. Although the plasma levels are generally improved after the oral administration of micronized diosmin, concentrations are often low and highly different across published studies ,39,40. A® Plus for alleviating typical symptoms associated with CVD. Subjects were randomized to receive either the treatment formulation (1 tablet/day) or a placebo (1 tablet/day) for 56 consecutive days (8 weeks). Different measurements, scales, and questionnaires were used to quantify the potential beneficial effects of μsmin® Plus on the symptomatology of CVD. Overall, the results clearly indicate that the investigational product is effective in decreasing leg circumference, pain (on moving and standby), and a wide array of symptoms commonly associated with CVD. Nevertheless, following 8 weeks of daily administration, the active product was not associated with any AE/SAEs occurrence, proving its excellent safety. In general, diosmin is considered a safe substance. Mild side effects such as gastralgia, diarrhea, and abdominal pain were only reported in a few cases [® Plus equal to 450 mg diosmin). In fact, typical diosmin-related side effects are usually seen with treatment between 1000 and 3000 mg/day of MPFF.This clinical investigation was designed to investigate the effectiveness of a low-dose diosmin treatment consisting of 450 mg of diosmin delivered once a day as μsminew cases . We specA previous study  performe® Plus has been proven effective at a low-dose regimen (450 mg once per day), thus confirming the data obtained from previous pharmacokinetic studies [Current clinical evidence indicates that MPFF at the dose of 500 mg twice per day or 1000 mg once per day may be sufficient for improving symptoms in CVD patients. Interestingly, μsmin studies ,21. MoreFrom a practical point of view, a single daily dose may improve patient adherence to the medical treatment as already shown in other treatment protocols . This be® Plus is negligible  and significantly lower compared to other studied compounds such as micronized purified flavonoid fraction (MPFF) [It is important to note that the content of hesperidin in μsminn (MPFF) . Therefo® Plus. Chronic Venous Disease has been associated with the significant deterioration of general and health-related QoL. In fact, pain, swelling, leg discomfort and severe skin changes may lead to a progressive impairment of QoL not only associated with physical items but also with emotional/mental health items. As a result, the improvement of QoL can be viewed as the main therapeutic target in CVD . AlthougCVD’s treatment options have evolved in recent years. However, clinicians need to have in their arsenal additional therapeutic products for improving the everyday life of their patients. In this context, several tools can be used to evaluate the safety and effectiveness of new treatments on QoL . In thisOne limitation of the current study was that venous calcifications were not measured, and therefore it is not possible to evaluate this issue within the outcomes of our study. ® Plus, a diosmin preparation with enhanced bioavailability, is safe and effective for improving CVD symptomatology after 8 weeks of treatment in subjects with CEAP clinical classification C2–C4.Based on findings from the current study, we concluded that μsmin


Two different scoring systems were developed to determine the severity of bronchiectasis: FACED scoring and the bronchiectasis severity index (BSI). In this study, we aim to compare these 2 scoring systems according to the 6-min walking distance test and a disease-specific health status questionnaire in patients with noncystic fibrosis bronchiectasis (NCFB).  Smoking history, emergency and hospital admissions, and body mass index were obtained from NCFB patients admitted to our hospitals’ pulmonary rehabilitation unit between 2013 and 2018. Detailed pulmonary function tests were performed for all participants. Dyspnea perceptions were determined according to the mMRC dyspnea scale. The 6-min walking test was used to determine exercise capacity. The Saint George respiratory questionnaire (SGRQ) was applied to determine health status. Both FACED and BSI scores were calculated for all participants.  There were a total of 183 participants, 153 of whom were men. A significant and strong correlation was found between FACED and BSI scores. As the severity of bronchiectasis increased, walking distance was significantly decreased and health status was significantly worse in both FACED and BSI scoring. A statistically significant but weak negative correlation was found between FACED score and walking distance. There was a significant negative correlation between BSI and walking distance, a stronger negative correlation than with FACED. Similarly, there was a significant negative correlation between health status and both FACED and BSI, but this correlation was stronger in the BSI score.  Although both FACED and BSI scores were negatively correlated with walking distance and health status in patients with NCFB, BSI was more strongly associated. Bronchiectasis, which is now termed muco-obstructive lung disease, is actually an orphan disease [1]. Bronchiectasis is a clinical-radiological diagnosis characterized by irreversible airway dilatation with chronic inflammation, which has the clinical features of cough, sputum production, and episodic exacerbations [2]. Pseudomonas aeruginosa, extension of the disease by radiological assessment, and dyspnea [3]. However, BSI score is a 7-point score that determines risk for future mortality and hospitalizations for NCFB patients [4]. In addition to the criteria of the FACED score, the number of emergency visits and number of hospitalizations are also calculated in the BSI score. Bronchiectasis is classified into 2 categories, noncystic fibrosis bronchiectasis (NCFB) and cystic fibrosis bronchiectasis (CFB). NCFB has gained attention with increasing awareness. With the increased use of high-resolution CT scan, more patients have received a diagnosis of bronchiectasis. The severity and the prognosis of NCFB cannot be determined by only one parameter. Therefore, there are several validated scores in determining the severity and prognosis of this disease. Two of these are the FACED score and the bronchiectasis severity index (BSI) . The FACED score is a 5-point score that predicts probability of all-cause mortality. The FACED score consists of forced expiratory volume in 1 sec (FEV1) % predicted, age, chronic colonization byUnfortunately, there are no studies comparing the 2 scores in association with exercise capacity and health status in patients with NCFB. Therefore, in the present study we aimed to compare the results of the assessment of 6-min walking distance and health status (SGRQ) parameters in the same patients with NCFB assessed with both the FACED score and the BSI.We conducted a retrospective database study in our Chest Diseases and Surgery Education and Training Hospital between 2013 and 2018 to compare the results of the assessment of 6-min walking distance and health status parameters in patients with NCFB assessed with both the FACED score and the BSI. The study was approved by the local institutional review board. Patients included in the study completed an informed written consent form. All patients with NCFB were referred to our Pulmonary Rehabilitation Unit for the PR program. Of the 218 NCFB patients, 183 were eligible to participate the study. The inclusion criteria were having bronchiectasis on high resolution computerized tomography (HRCT) of the lungs with or without airway obstruction. The exclusion criteria were having an interstitial lung disease pattern (to exclude traction bronchiectasis) on HRCT, having concurrent lung cancer, or having a history of having had a lobectomy or pneumonectomy for lung cancer. P. aeruginosa, radiologic severity according to HRCT, emergency admission, and hospitalization in the last year) were recorded.Age, sex, and body mass index (BMI) as demographics and clinical history  and carbon monoxide diffusion capacity (ZAN 300) are routinely measured for all patients with NCFB who are admitted to our hospital PR unit [5]. We recorded the % predicted values of FEV1, FVC, FEV1/FVC ratio, and carbon monoxide diffusion capacity (TLCO). We used the modified Medical Research Council (mMRC) dyspnea scale, which consists of 5 items ranging between 0 and 4, to determine the severity of the patients’ shortness of breath. The score 0 represents the best level, while the score 4 indicates the poorest [6].We recorded the walking distance in the 6-min walking test (6mWD) performed according to the American Thoracic Society (ATS) guidelines [7].We used the St. George’s respiratory questionnaire (SGRQ) to determine disease-specific health status [8]. The participants’ psychological status was determined by the hospital’s anxiety depression scale [9]. All of these questionnaires are routinely given to all of our pulmonary rehabilitation candidates.Both FACED and BSI scores were calculated for all participants, and participants were divided into 3 groups as mild  moderate , and severe . All data, especially walking distance and health status data, were compared between these 2 scoring systems. A bronchiectasis exacerbation was defined as a patient with bronchiectasis with deterioration for at least 48 h in ≥3 of the following symptoms: cough, sputum volume and/or consistency, sputum purulence, breathlessness and/or exercise intolerance, fatigue and/or malaise, hemoptysis [10]. The Statistical Package for the Social Sciences  v. 22 was used for data analysis. Before the statistical analysis, the parameters were tested for normal distribution with Kolmogorov–Smirnov and Shapiro–Wilk tests. Results are presented as mean ± standard deviation, median (minimum-maximum), number (n), or percentage (%) according to the statistical method. One-way ANOVA test was used for continuous variables when comparing clinical data of patients between the BSI and FACED groups. As the number of patients differed between the groups and homogeneity of variance was provided, Hochberg’s test was used for posthoc analysis. Hochberg’s test allows for clearly unequal sample sizes. The relationship between the BSI and FACED scores of the patients and the relationship between the 6-min walking distance measurement values and SGRQ health status scores of the patients were demonstrated by simple linear regression analysis and Pearson’s correlation test. P values <0.05 were considered statistically significant.There were 183 participants, 153 of whom were men. The mean age of the study population was 63.1 (±10.4). The mean FEV1 % was 33.5 ± 17.6 and the mean FEV1/FVC ratio was 56.8 ± 14.1. There were 64 (34.9%) patients who had the diagnosis of bronchiectasis with COPD, 7 (3.8%) with a diagnosis of bronchiectasis with asthma; the remaining were classified as isolated bronchiectasis before referral. Nearly half of the patients (46.8%) had at least one comorbidity and 8 (4.3%) patients had sequel tuberculosis. The mean FACED score of the population was 3.4 ± 1.5 while the mean BSI score was 8.9 ± 4.6. The distribution of severity according to FACED and BSI scores is shown in Table 1. The mean 6mWD was 331.8 ± 114.6 m. The other demographic characteristics and the QoL scores are shown in Table 1. According to FACED bronchiectasis classification, patients in the severe group were older and had higher SGRQ scores and a lower 6mWD compared to those of the patients in the mild and moderate groups; all differences were significant  (Table 2). HAD scores were not significantly different between FACED severity groups (P = 0.97 for anxiety and P = 0.91 for depression) (Table 2). According to the BSI index, patients in the mild group were younger, had higher 6mWD, and had lower SGRQ scores; all differences were significant  (Table 3). A statistically significant but weak negative correlation was found between FACED score and distance values; a statistically significant negative correlation was found between BSI and distance values, with a stronger correlation than FACED scores (Table 4). A 1-point increase in FACED score corresponds to a 28.4-m decrease in distance measurement; a 1-point increase in BSI score corresponds to a 13.2-m decrease in distance measurement. There was a statistically significant but weak correlation between FACED score and SGRQ total, SGRQ symptoms, activity, and impact scores, and there was a statistically significant but weak negative correlation between FACED score and walking distance. There was also a statistically significant correlation between BSI and SGRQ total SGRQ symptoms, activity, and impact scores, and there was a statistically significant but weak negative correlation between BSI score and walking distance. The correlation between BSI score and SGRQ total score was stronger than the correlation between FACED score and SGRQ total score. A 1-point increase in FACED score corresponds to a 4.56 points increase in SGRG total score; a 1-point increase in the BSI score corresponds to 1.96 points increase in SGRQ total score (Table 4). There was a statistically significant and strong correlation between FACED and BSI scores . The increase in FACED accounted for about 41% of the increase in BSI (R2 = 0.409). When we compared the 6mWD among the mild, moderate, and severe FACED subgroups, there was no statistical significance between the subgroups (Table 5). However, in moderate and severe BSI subgroups, the 6mWD was significantly lower than that of the mild BSI subgroup  (Table 6).In our cohort, 183 patients with NCFB were reviewed; comparison was performed of the 2 scores in association with exercise capacity and health status. This study showed that as the severity of bronchiectasis increased, walking distance significantly decreased and health status was significantly worsened in both FACED and BSI scoring. Furthermore, there was a significant negative correlation between BSI and walking distance, which was a stronger negative correlation than with FACED. Similarly, there was a significant negative correlation between health status and both FACED and BSI, but this correlation was stronger in the BSI score.In McDonnell et al.’s European bronchiectasis cohorts, nearly 60% of the patients were female  [11]. However, 83% of the patients in our cohort were male. Our hospital is an education and training hospital which serves many districts and generally serves severe patients. Additionally, all of the patients were referred to our pulmonary rehabilitation clinic for consideration and evaluation of a PR program. According to our country’s economic and social status, male patients may have more opportunity to obtain the best health care in the intercity referral system between hospitals. Therefore, this male predominance may be due to the patient referral system in our hospital’s responsive region. In this cohort, we have taken radiologically diagnosed NCFB patients with or without airway obstruction. It is quite hard to distinguish pure COPD, bronchiectasis, and overlap syndrome . Some questions still remain unanswered in this specific group, as bronchiectasis is frequently diagnosed radiologically in patients with COPD, with different clinical phenotypes. However, for distinguishing these 2 entities, an endotype approach is suggested [14]. According to this approach, a combination of imaging parameters, airway inflammation markers, and microbiology would be used to distinguish between true COPD, bronchiectasis, and the overlap syndrome [14]. In our hospital, we use a combination of imaging parameters, inflammation markers, and sputum cultures for identification of pure bronchiectasis. However, our cohort may have patients overlapping with COPD. In this cohort, the mean FEV1/FVC ratio was 56.8. The majority of the patients had airway obstruction, hence some patients may have overlap syndrome. Machado et al. conducted a prospective cohort analysis of 70 patients with NCFB recruited from May 2008 to August 2010 for determining prognostic factors; they found that the mean FEV1% was 48.0 ± 14.8 [15]. In the present study, patients with NCFB had lower FEV1 levels. As we mentioned previously, our hospital is a Step 3 education and research hospital that takes referred severe patients. Therefore, our results may not generalize all NCFB patients in our region. In the European cohort, the mean BSI scores ranged from 6 to 9.7 while the mean FACED score was between 1.5 and 2.3 [13]. In the present cohort, the mean FACED score of the population was 3.4 ± 1.5 while the mean BSI score was 8.9 ± 4.6. Our results are quite similar with those of the existing literature. In McDonnell et al.’s study consisting of 7 cohorts, the cohorts were primarily classified as moderate-to-severe bronchiectasis based on mean BSI scores (6.0–9.7); however, in contrast, the majority were classified as mild bronchiectasis according to the FACED score (mean 1.5–2.3). In our study, the majority of the patients were moderate based on the FACED score; however, based on the BSI scores, the majority of the patients were severe. When we add ER visits and hospitalizations to the scoring system, it is seen that there is a tendency for more patients to settle in the severe group. These 2 scoring systems were developed for obtaining the risk of mortality; however, these scores also correlate well with exercise capacity. In this present cohort, according to FACED bronchiectasis classification, severe patients had significantly lower 6mWD scores compared to mild and moderate groups. The same was true for the BSI score: according to the BSI index, patients in the mild group had significantly higher 6mWD scores. Thus, we believe that classifying the severity of the patients according to these scoring systems may reflect the exercise capacity of NCFB patients. In the same European cohort study, McDonnell et al. suggest that the BSI is superior to FACED in predicting clinically important disease-related outcomes, including hospital admissions, exacerbations, QoL, respiratory symptoms, 6mWD, and lung function decline in bronchiectasis [11]. In this present cohort, the correlation between BSI and SGRQ total score was stronger than for FACED, which is similar to the European results. However, the literature shows different results in different etiologies. In a study conducted by Wang et al. to evaluate the clinical characteristics and validation of bronchiectasis severity score systems for posttuberculosis bronchiectasis, the authors found that both FACED and BSI can predict mortality in posttuberculosis bronchiectasis [16]. These data confirm that both scoring systems are excellent predictors of medium-term mortality in subjects with bronchiectasis; however, in a single center retrospective cohort study by Ellis et al., both scores were able to predict 15-year mortality, with the FACED score showing slightly superior predictive power  [17]. Although BSI is more closely correlated with health status and exercise capacity in the present study, FACED may be better than BSI in some aspects, among them being the simplicity of its use and its clinical applicability, as shown in the literature [17].When dealing with bronchiectasis in our daily clinical practice, it is sometimes difficult to decide which score is the best for the individual patient [18]. With our results, we may say that both scoring systems predicted the exercise capacity and health status correctly in NCFB patients. However, if we would like to make a comprehensive and detailed exercise program for NCFB patients, we would prefer to use the BSI score because of its stronger relation with 6mWD and health status scores.One of the limitations of our study is its retrospective design. However, we have used a detailed data recording system in order to eliminate this limitation. Additionally, NCFB patients referred to our PR unit were symptomatic patients with airflow limitation in need of pulmonary rehabilitation; therefore, our findings may not be generalized to all NCFB patients.This study showed that as the severity of bronchiectasis increased, walking distance significantly decreased and health status significantly worsened, which was reflected in both FACED and BSI scoring. However, the relationship between 6mWD and health status scores was stronger in the BSI scoring system compared to the FACED bronchiectasis severity score. The study was approved by the local institutional review board in January 2017.


Titanium is one of the most commonly used biomaterials for implantation as a part of the orthopedic procedures. However, this biomaterial can cause an excessive inflammatory response, even leading to rejection of the implant. Therefore, the aim of our study was to assess the overall organism response after insertion of Ti implant and the activity of neutrophils and monocyte-derived macrophages (MDM), to evaluate the possible negative effect of this biomaterial on the host cells. Our study revealed that insertion of the Ti implant did not evoke systemic inflammatory response or activation of leukocytes. Additionally, we evaluated the activity of neutrophils and MDM after stimulation with autologous neutrophil products, namely, antimicrobial neutrophil extract and neutrophil degranulation product as two potential regulators of inflammatory response. Antimicrobial neutrophil extract appeared to be a factor causing the decrease of secretory neutrophil response and polarization of MDM towards pro-resolving phenotype, whereas the neutrophil degranulation product acted as pro-inflammatory.Titanium (Ti) is currently the most common biomaterial used for orthopedic implants; however, these implants may cause deleterious immune response. To investigate the possible mechanisms involved in excessive inflammation, we assessed the activity of neutrophils and monocyte-derived macrophages (MDMs) during the insertion of the Ti implant in a sheep model. The study was conducted on 12 sheep, 4 of which were control animals and 8 were in the experimental group with inserted Ti implant. Neutrophil secretory response was estimated at two time points T0 before surgery and T1 1 h after implantation and was based on the release of enzymes from neutrophil granules and reactive oxygen and nitrogen species (RONS) generation. MDM function was evaluated 5 months after implantation, on the basis of RONS generation arginase activity and morphological changes. Moreover, the influence of some autologous neutrophil derived products, namely, antimicrobial neutrophil extract (ANE) and neutrophil degranulation products (DGP) on leukocytes was estimated. Our study revealed that Ti implant insertion did not cause any adverse effects up to 5 months after surgical procedure. Stimulation of neutrophil cultures with ANE decreased the enzyme release as well as superoxide generation. Treatment of MDM with ANE diminished superoxide and NO generation and increased arginase activity. On the other hand, MDM stimulated with DGP showed elevated superoxide and NO generation as well as decreased arginase activity. To summarize, ANE exerted an anti-inflammatory and pro-resolving effect on studied leukocytes, whereas DGP acted as pro-inflammatory. Using various metal implants is necessary to improve healing and restore tissue integrity in many orthopedic disorders. Implants may, however, spark an excessive immune response, leading to high failure rates accompanied with immune implant rejection . DespiteThe role of neutrophils in the bone healing process remains controversial; some authors postulated a negative influence of neutrophils on bone regeneration because their depletion improved fracture healing. They proposed that neutrophils could cause tissue damage by release of collagenase, elastase, free radicals and arachidonic acid. Indeed, this activity might be beneficial for intramembranous bone formation, but diaphyseal fracture healing might be delayed, because cartilaginous callus formation is essential for this process. More recent studies indicated that a balanced neutrophil activation is important for fracture healing . TherefoNeutrophils and blood monocytes invade the injury site during the course of tissue repair, where they participate in host defense, phagocytosis of necrotic tissue, and secretion of paracrine factors. Macrophages, in turn, are essential for the regeneration, repair, and remodeling in many tissues. However, chronic inflammation and persistent macrophages accumulation are often associated with tissue destruction and fibrosis .Metal materials are commonly used in orthopedic and dental implants; however, recent evidences suggest that these biomaterials induce an immunomodulatory interaction with the host. Therefore, it was assumed that materials such as titanium (Ti) are not inert upon contact with host bone ,6. Ti isMeasuring the response of some immune cells to foreign biomaterials, including cell viability and activation studies at different time points, is the most common method for characterizing in vitro immunogenic response, with a pivotal role for clinical outcomes and potential therapeutic application .Titanium is a generally accepted biomaterial as a relatively inert substance with minimal side effects, and it is used in many medical fields among other in orthopedics implants. However, with the increased application of Ti, the concerns over the safety of this biomaterial also increases. Some recent studies suggest that, under some circumstances, the presence of Ti particles may be harmful, especially following frictional wear of medical prostheses or of screws used in plate fixation during orthopedic procedures. The particles and ions released from Ti implant can be deposited in surrounding tissues causing inflammatory reactions, hypersensitivity, and/or can lead to toxic reactions . Thus, tn = 4) and an experimental group (n = 8). All sheep from the experimental group had a Ti implant inserted into the tibia; sheep from the control group were used for comparison on long-term effect of Ti implant on MDM population.The study was conducted in a group of 12 sheep, females, BCP local breed, 4 months old, approximately 20 kg body weight, from the Bezek Experimental Farm, University of Life Sciences in Lublin. Animal management and surgical protocol were approved by the local Ethics Committee number II in Lublin (No 84/2019). The animals were randomly divided into two groups, a control group (After premedication with intramuscular injection of xylazine (0.1 mg/kg) and Butorphanol (0.1 mg/kg), the surgical area was aseptically cleaned, and the standard surgical approach for proximal tibia was performed. Then, the commercial sterile eight plate system for pediatric usage with elastic plate and two screws was implanted  Figure .Postoperatively, all sheep received antibiotic  intramuscularly as well as Melovem  as analgesic. For two weeks after surgery, a full clinical examination with routine post-operative inspection of surgical wounds and vital parameters recording was conducted every day. Thereafter, these procedures were conducted once a week up to 5 months after surgery.Blood for hematological assays was collected from the jugular vein seven days before the experimental procedure. Blood was collected into tubes containing EDTA as an anticoagulant. A complete blood count was performed using an Abacus Junior Vet analyzer  and hematological parameters in all animals were within the reference ranges. At the same time, blood aliquots of approximately 25 mL with 3.8% sodium citrate as anticoagulant were obtained for preparing both blood-derived products, namely, ANE and DGP.ANE was prepared as previously described . Blood wThe obtained ANE was used for stimulation of neutrophil or MDM cultures to assess their activity. To achieve this, immediately before the experiments appropriate amounts of lyophilized samples were resuspended in a sterile medium to final desired concentrations and added to cultures. ANE concentrations of 0, 4, 40, and 100 μg/mL were used to assess potential toxicity against ovine cells; for assessment of leukocytes in vitro activity, the concentrations of 0 and 40 μg/mL were used.N formylmethionyl-leucyl-phenylalanine . After 10 min of incubation at 37 °C, neutrophils were centrifuged  and cell free supernatant containing DGP was collected and stored at −20 °C until later use [DGP preparation relied on the degranulation of isolated neutrophils, induced by ater use .A 20 mL blood sample from each sheep was used for preparing approximately 4 mg of neutrophil extract (ANE), which was lyophilized. A part of this lyophilizate was used to test for potential cytotoxic and hemolytic activity against host cells.2 for 24 h, then the cells were incubated with 5 mg/mL MTT in PBS for 3 h, lysed in 10% SDS overnight, and optical density was measured. The obtained values were calculated as a percentage of 100% activity of cells without stimulation. Cell viability was assessed in triplicate.The effect of ANE on neutrophil viability was determined using an MTT test , which is based on the conversion of MTT tetrazolium salt by mitochondrial dehydrogenase to a formazan color product as measured at an absorbance 169 of 565 nm . Followig, 10 min). Erythrocytes were rinsed three times with PBS and resuspended at a concentration of 2%. Then, 200 μL of erythrocyte suspensions with various ANE concentrations  were incubated at 37 °C for 30 min in 96-well microtiter plates. Triton X-100  was used as the positive control for 100% hemolysis and PBS was used as the negative control. Absorption of supernatants was measured at 405 nm, using a BioTek EL800 microplate reader . All assays were performed in triplicate [To assay hemolytic activity, freshly drawn ovine blood was collected into an EDTA tube and plasma was removed by centrifugation  and both groups were incubated for 30 min and for 22 h at 37 °C in the presence of 5% CO2.The cell suspensions prepared at a density of 2.0 × 10Enzyme release from azurophilic granules was estimated on the basis of elastase activity and was measured spectrometrically on the basis of cleavage of azocasein (Sigma-Aldrich) using a microplate reader BioTek EL800 . Alkaline phosphatase (ALP) release, as a marker of specific granule response, was determined after 10 min incubation at 25 °C with substrate 4-nitrophenyl phosphate disodium salt hexahydrate . Nitric oxide (NO) level was determined by means of the Griess reaction, and superoxide anion generation was measured as described previously .n = 4) was sampled to obtain a control group for comparison. Mononuclear cells fraction (MNC) was isolated by gradient centrifugation over Histopaque-1077 . To obtain macrophages, MNC were immediately seeded at a concentration of 1.0 × 106 cells/mL into 96-well flat-bottomed tissue culture plates  and cultured at 37 °C and 5% CO2 for 72 h in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% bovine calf serum  [Escherichia coli serotype 055:B5 , as an LPS group, or with dexamethasone (DEX group) at a concentration of 100 nM. All these cultures were incubated for 3 days at 37 °C and 5% CO2. During this period, cultures underwent daily functional analysis on the basis of NO generation and superoxide generation. Arginase activity was assessed at the end of the experiment (after 72 h of incubation).Approximately 10 mL of blood was taken from each sheep 5 months after implantation of biomaterial to obtain MDM; additionally, the blood from healthy sheep ( Poland) . The culSuperoxide production was measured using the method described previously . The NO Fibrinogen and haptoglobin plasma concentrations were evaluated as described previously  at threep < 0.05.Data from neutrophil and MDM activity, as well as assessment of APP response were analyzed using Statistica 13.1 . All results represented the mean and standard error (SE) of their respective groups. Significance was calculated using a one-way ANOVA followed by a Tukey’s test. Differences were considered significant at No adverse effects were observed in the course of the experiment up to 5 months after implantation of Ti implant into ovine tibia.Our study revealed low cellular toxicity of ANE measured as a decrease in the viability of ovine neutrophils. At the highest concentration, namely, 100 μg/mL, cell viability was reduced to 72%. During hemolytic activity studies, we estimated that ovine erythrocytes were resistant to ANE at concentrations of 4 and 40 μg/mL, the concentration of 100 μg/mL only caused a slight lysis, reaching up to 3% .p < 0.05) decrease of elastase release from 50.7 ± 0.8 to 48.2 ± 0.48% of maximal enzyme release. In cultures after 22 h incubation, changes were insignificant , the stimulation with ANE for 30 min caused significant  with MDM from a group of sheep 5 months after implantation . We did not observe any significant differences between MDM isolated from the control and experimental sheep. Then, we evaluated differences between both groups after treatment with different stimulations. Part of the mature macrophages was described as BCS and were left without any additional stimulation for comparison with other cultures treated with different stimulators. One part of MDM was stimulated with 40 μg/mL of ANE to obtain the ANE group, another part was stimulated with DGP (DGP group), or with 1 μg/mL LPS (LPS group), or with dexamethasone (DEX group) at a concentration of 100 nM. We found no significant differences between MDM from the control and the experimental sheep. However, there were noticeable differences in MDM activity and morphological features that only resulted from the type of stimulus used.Initially, we compared MDM obtained from healthy sheep (p < 0.05) lower after 48 and 72 h incubation in sheep from both the control and experimental group, in comparison with MDMs without additional stimulation (BCS group) (Superoxide generation by macrophages treated with ANE was significantly (S group) .We also observed a decrease in the generation of NO in MDM stimulated with ANE in both groups obtained from the control and the experimental sheep .p < 0.05) in the sheep from the control group, whereas in the experimental group it increased from 38.81 ± 2.63 μg urea to 42.98 ± 3.85 μg urea (p < 0.05) (Together with a decrease of reactive oxygen and nitrogen species (RONS), an elevated arginase activity was noted after MDM stimulation with ANE. We found that arginase activity increased from 38.00 ± 2.2 μg urea to 42.00 ± 3.00 μg urea ( < 0.05) .Morphological changes were suggestive of an anti-inflammatory pro-resolving phenotype .p < 0.01)  . The mor < 0.01) .Immediately after surgery (B), plasma concentration of both evaluated APP—fibrinogen and haptoglobin—increased compared to the values before surgery (A), and 7 days after insertion of the Ti implant (C) the level of both APP decreased to values similar to range before procedure .In this study, we used an ovine biomaterial implantation model, and the primary aim of the experiment was to evaluate response of two subpopulations of the white blood cells system: neutrophils and MDM, to the implantation of a Ti plate into the tibia. We evaluated elastase and ALP release as markers of degranulation of azurophilic and specific granules, respectively. Additionally, RONS generation was a marker of neutrophil activity. The enzymatic response remained unchanged after implantation; thus, circulatory activation of neutrophils did not appear.Previously, neutrophil response to implantation was assessed in rabbit neutrophils during osteochondral grafting, when increased activity of these cells was noted two hours and three days after surgery and was related to increased acute phase response . Some auEarly response of MDM on Ti implant insertion was evaluated previously . In the The second part of the experiment included preparation of the autologous neutrophil derived product ANE , which was then used for in vitro stimulation of neutrophils and MDM. Numerous antimicrobial peptides (AMP) from a large variety of animal sources have been isolated and evaluated to date. It turned out that these peptides play an important role in host defense. Ovine extract has previously been considered a therapeutic agent against some ovine pathogens and its possible use in humans against cystic fibrosis or oral infections has been considered . HoweverResults clearly indicate that ANE demonstrated low hemolytic activity at the doses used, with the highest hemolysis reaching 3% at 100 μg/mL. Hemolytic effects of ANE on ovine erythrocytes have not been previously evaluated. According to Skerlevaj et al. , ovine eThe influence of ovine ANE on neutrophil secretory activity has not been previously evaluated, our study constitutes the first research on this topic. Other studies showed that an autologous rabbit extract of antimicrobial peptides decreased neutrophil activity , whereasAccording to Mansour et al. , AMP disThe last part of this experiment included stimulation of cultured MDM with an autologous neutrophil degranulation product (DGP). As estimated previously, extracellular neutrophil products are efficient regulators of many cellular functions and interactions between inflammatory cells. For example, neutrophil antimicrobial functions are not required during the implantation of a sterile implant and these cells play an immunomodulatory role rather than antimicrobial one. Therefore, the major role of activated neutrophils at inflammatory sites is to regulate and shape the inflammatory response. Neutrophil proteases may positively or negatively influence inflammatory reactions, among others, by modulating the activity of mediators released from necrotic cells. Thus, these neutrophil compounds may represent promising therapeutic targets for the treatment of conditions characterized by extensive neutrophil activity . For thiIn the recent study Ng et al.  summarizIn another study on rabbit model, the Ti plate was removed after 1, 3, 6, 12, or 24 months after implantation and the obtained results revealed that Ti accumulation in the organs was highest at 3 months after implantation, and then gradually decreased. Ng et al.  conducteThis study revealed the lack of any undesirable effects either local, systemic, or involved in activity of some components of WBC up to 5 months after Ti implant insertion. This is a continuation of previous investigations, and these findings completed the knowledge about host–biomaterial interactions and potential mechanisms involved in these interaction.Our findings provide knowledge about possible medical applications of neutrophil derived products.Ovine antimicrobial neutrophil extract (ANE) did not exert any deleterious effect in contact with studied cells and may be a promising tool for the enhancement of the healing process through restriction of an undesirable inflammatory response.The obtained results have shown that ANE can act as modulator of the secretory activity of neutrophils, and as anti-inflammatory stimulator of MDMs. These differences in cellular responses could be used in new autologous blood-derived products for regenerative medicine, since decreased generation of RONS with increased arginase activity are known to be essential for healing.DGP caused macrophage polarization to pro-inflammatory phenotype for enhancement of host defense mechanisms.


The lacking of laboratory black carbon (BC) samples have long challenged the corresponding toxicological research; furthermore, the toxicity tests of engineered carbon nanoparticles were unable to reflect atmospheric BC. As a simplified approach, we have synthesized artificial BC (aBC) for the purpose of representing atmospheric BC. Surface chemical properties of aBC were controlled by thermal treatment, without transforming its physical characteristics; thus, we were able to examine the toxicological effects on A549 human lung cells arising from aBC with varying oxidation surface properties. X-ray photoelectron spectroscopy, as well as Raman and Fourier transform infrared spectroscopy, verified the presence of increased amounts of oxygenated functional groups on the surface of thermally-treated aBC, indicating aBC oxidization at elevated temperatures; aBC with increased oxygen functional group content displayed increased toxicity to A549 cells, specifically by decreasing cell viability to 45% and elevating reactive oxygen species levels up to 294% for samples treated at 800 °C. As aerosols . Besidesaerosols ,7.BC is characterized as having fractal agglomerates, being insoluble in water or common organic solvents, and being refractory and potential toxic ,8. The fOn the other hand, engineered carbon nanoparticles with corresponding toxicity were widely carried out ,12. CarbThe diesel soot particulate matter is produced by the National Institute of Standards and Technology (NIST), which can mimic real-world diesel soot, however, the existence of polycyclic aromatic hydrocarbon (PAH) with other chemical compositions renders the identification of specific parameters responsible for inducing toxicity and the mechanisms by which they cause harm to the human body highly challenging . MoreoveHerein, we have synthesized aBC using an aerosol generator. We defined aBC in this study as the ultrafine particles generated from one origin source, graphite, to simplify the test of relation between toxicity and the chemical surface of aBC; aBC must possess a controllable chemical surface and common physical properties. Thermal treatment is a straightforward method for manipulating the chemical properties of aBC surfaces, especially to control oxygen functional group content while maintaining its physical characteristics. Because a dominant cytotoxicity mechanism of ultrafine particles is a cell–particle interaction accompanied by the overproduction of ROS ,24,25, RGraphite was selected to produce aBC generated from a spark discharge soot generator . It operates on the principle of spark discharge to produce nanoscale particles of consistent concentration in high yields and negligible volatile content. The generator maintains a jump spark between two graphite electrodes at a high voltage of 2500 V. Graphite is vaporized by this spark, and then condenses to form particles with a size distribution and structure similar to those of diesel soot. The generator was operated under a constant flow of carrier inert Ar gas and purified dilution air at flow rates of 5 and 10 LPM, respectively (gas streams were controlled with a series of mass flow controllers).The generated particles were produced in a tubular furnace at different ranges of temperatures. The first experiment was performed with the furnace temperature increasing continuously from room temperature (RT) to 800 °C to investigate particle properties as a function of the treatment temperature. Five treatment temperatures  were applied separately in the second experiment to evaluate the effects of temperature on the physicochemical properties of aBC. A schematic of the experimental setup, including the sampling and measuring devices, is presented in TM, DEKATI, Finland) was employed to measure the concentration of generated particles in the range of 6 nm to 10 μm with a time resolution of 1 s. Furthermore, we estimated the surface area concentration of the deposited particles in the human respiratory tract based on ELPI+ stage-specific conversion factors. Teemu et al. estimated the ELPI+ response coefficient (3) for spherical particles, with a possible error in the mean diameter arising at each stage of each instrument 2% [dep) was estimated for three regions, head airways, tracheobronchial, and alveolar, according to the following equation:An electrical low-pressure impactor  with a diameter of 25 mm  and Teflon filters  with a diameter of 47 mm using filter holders at five different temperatures ; these samples were utilized for chemical analysis and the in vitro test.High-resolution transmission electron microscopy  was employed to observe the size and morphology of the aBC at an accelerating voltage of 200 kV. The aBC was collected on holey carbon TEM grids using a mini particle sampler . For SEM analysis, aBC was dispersed on a heated (100 °C) silicon wafer by depositing a drop of particle suspension, created by sonicating aBC collected on a quartz filter in DI water. These samples were coated with a thin film of Pt and then observed by field emission-scanning electron microscopy . The surface chemical compositions of the aBC samples were analyzed by X-ray photoelectron spectroscopy . Additionally, Raman spectra were acquired using an Xplora spectrometer  equipped with a 532 nm solid state laser source. Fourier transform infrared vacuum spectroscopy  was employed to investigate structural changes occurring during the heating process. 2.A549 cells were purchased from the American Type Culture Collection . The cells were cultured in Roswell Park Memorial Institute (RABCI) 1640  containing 5% fetal bovine serum  and 1% streptomycin and penicillin at 37 °C in an atmosphere containing 5% CO4 cells per well and left to attach overnight. The cells were then treated with various concentrations (50–2000 µg mL−1) of aBC in the complete medium for 48 h. This was followed by the addition of 3--2,5-diphenyl-tetrazolium bromide  solution to the final concentration of 1.0 mg mL−1. After 3 h of incubation, the medium was removed, the formed blue formazan crystals were dissolved in 100 μL of dimethyl sulfoxide  per well DMSO, and the absorbance at 570 nm (690 nm background control) was measured using a microplate reader . Low toxicity of aBC was observed in most treatments, with 75% of cells remaining viable after treatment with the highest dose (2.0 mg mL−1) of aBC for 48 h. Therefore, these treatment conditions were used for all subsequent experiments. For cytotoxicity experiments, A549 cells were seeded in 96-well plates at a density of 1 × 10The aBC-exposed cells were harvested using 0.25% trypsin-EDTA  from culture plates and cyto-centrifuged  onto glass microscope slides using Cytospin 4 . Cell smears were fixed in methanol for 1 min and stained using Diff-Quik solution . Stained cells were analyzed under a light microscope .−1 of 2,7-dichloro-fluorescein diacetate (DCF-DA)  for ROS detection. The cells were treated with aBC (2.0 mg mL−1 for 48 h), then washed with Dulbecco’s phosphate-buffered saline . The DCF-DA intensity in the cells was immediately measured at 495 nm (excitation)/529 nm (emission) using a microplate reader. ROS production in the cells is represented as a percentage of DCF-DA intensity relative to cell viability in each well, which was defined as 100%.A549 cells were incubated for 30 min at 37 °C in RABCI 1640 medium containing 3.3 μmol L8 cm−3, decreasing with higher treatment temperatures and eventually reaching 2.2 × 107 cm−3 when the furnace temperature was 800 °C. The size distribution is also provided here, with the majority of the produced particles exhibiting aerodynamic diameters smaller than 100 nm. The aerodynamic diameter has been reported as the dictation of particle penetration into the lung, therefore, with those nano-scaled sizes, aBC has a higher chance to travel deep into the human lung, even meeting the bloodstream, cells, and tissues [ tissues ,28. All  tissues . At ever tissues .TEM and SEM images of the particle samples were collected for the five treatment temperatures, as shown in The XPS element and atomic concentration survey a showed −1 and a G peak at 1600 cm−1 [1g symmetry), and the G band is the in-plane stretching vibration of sp2 carbons (E2g symmetry) [To verify the oxidation of aBC, vibrational characterization was performed by Raman and FTIR analysis. Two typical overlapping peaks are visible in the Raman spectra displayed in 600 cm−1 ,35,36,37ymmetry) ,39. The −1, assigned to aromatic C=C. Three vibrations located at 2852, 2922, and 2962 cm−1 were assigned to asymmetric and symmetric C-H stretching of CH3 and CH2 aliphatic groups [−1) shoulder, becoming a notable peak at 800 °C and indicating the presence of oxygen functionalities [c groups ,41. At 6nalities . Exposure to aBC results in harmful effects on human health, causing pulmonary and cardiovascular diseases ,44. The Herein, we selected the A549 human lung alveolar basal epithelial cell line for in vitro testing. To verify whether aBC directly affects epithelial cells, we confirmed endocytosis of aBC in A549 cells. Diff-Quik staining indicated the appearance of aBC in the cytoplasm and in the region near the nucleus , indicatExposure to aBC induces cell damage via oxidative stress mediated by ROS ,51. ExceThe physical characterization illustrated a uniform size and shape of aBC synthesized under varying treatment temperatures. On the other hand, Raman and FTIR analysis supported by XPS data led to the conclusion that a greater number of oxygenated functional groups were present on the surface of aBC treated at elevated temperatures. Cell viability and ROS level results indicated that cytotoxicity increased with increasing oxygenated functional group content on the surface of aBC. This agrees with the findings of Das et al., who investigated the correlation between cellular toxicity and oxygenated functional group density on graphene oxide (GO) . They prThe aBC was successfully generated at various treatment temperatures without transforming the original physical features. The treatment temperature is known to significantly impact the surface chemical structure of aBC; hence, we were able to simplify the investigation into the effect of surface chemical properties of aBC on human epithelial cells; aBC with a nano size effectively penetrated the plasma membrane, leading to cell damage. It was found that an increase in the presence of oxygen functional groups on the surface of aBC directly affected cell viability and oxidative stress in A549 cells. The remaining limitation of this research is that aBC is still far from real-world BC, which has a complex mixture of chemical components, however, the results of the relationship between cytotoxicity and surface oxidation of aBC can be the foundation to conduct further research. Furthermore, aggregation and surface morphological changes of aBC in cell medium culture need further investigation, and aBC-induced bio-toxicity with proof of cell uptake is also needed to improve the quality of the cytotoxicity test. The semi-quantification of cell uptake should be investigated to support the mechanism of cell death. An in vivo test and in vitro test with an air–liquid interface approach would be needed for further toxicological models for our synthesized aBC, rather than a submerged cell culture to minimize the current limitations. Future studies will focus on aBC synthesized with sulfates, nitrates, metals, and organic compounds that are commonly emitted with BC to the atmosphere to better simulate real-world experimentation.


CASR) A990G genetic polymorphism on parathyroid hormone (PTH) lowering response to cinacalcet treatment when controlling for significant influencing clinical factors. This retrospective study was conducted on 135 Thai hemodialysis (HD) patients with secondary hyperparathyroidism (SHPT). CASR A990G genotypes were determined. The patients were identified as either G carriers  or noncarriers (homozygous CASR 990A carriers). Tested covariates were baseline PTH level (bPTH), baseline serum phosphate (bPhos), baseline serum calcium (bCa), baseline calcitriol equivalent dose (bCtriol), baseline ergocalciferol dose (bErgo), and age. The ANCOVA showed that intact PTH levels after 12 weeks of cinacalcet treatment (PTHw12) was significantly lower among G carriers compared with noncarriers after controlling for bPTH, bPhos, bCtriol, and bErgo  = 15.472, p < 0.001), with the adjusted mean difference of 253.7 pg/mL. The logistic regression analysis revealed that the odds of a G carrier achieving 30% PTH reduction after 12-week cinacalcet treatment were 3.968 times greater than the odds for a noncarrier after adjusting for bPhos, bCtriol, and age. In conclusion, the CASR A990G polymorphism significantly influences cinacalcet response in HD patients with SHPT.The objective of this study was to determine the impact of calcium sensing receptor ( In dialysis patients with SHPT, cinacalcet is efficacious in lowering levels of PTH, serum phosphate, and serum calcium as well as reducing the risks of parathyroidectomy, fractures, and cardiovascular hospitalization11.Cinacalcet is an oral calcimimetic agent available for treatment of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). Cinacalcet increases the sensitivity of the calcium-sensing receptor (CASR), the main regulator of PTH secretion, to extracellular ionized calcium ions, resulting in decreased parathyroid hormone (PTH) level2 vitamin D, and hypocalcemia. SHPT is present in the majority of hemodialysis (HD) patients and contributes to bone and cardiovascular disorders, which are referred to as chronic kidney disease-mineral bone disorder (CKD-MBD), a serious complication of CKD13. In dialysis patients, SHPT has been independently associated with all-cause and cardiovascular mortalities17. Optimal levels of intact parathyroid hormone (iPTH) have been correlated with improved survival, decreased cardiovascular hospitalization, and reduced clinical bone fracture20. Maintaining iPTH levels in the range of approximately two to nine times the upper limit for the assay used is suggested for dialysis patients13.SHPT in CKD is mediated by impaired phosphate excretion, hyperphosphatemia, decreased 1, 25 (OH)22. A significantly higher percentage of patients achieved plasma PTH target levels. Also, a significantly greater proportion of patients with at least a 30% reduction of plasma PTH levels from baseline levels have been reported among patients who were treated with cinacalcet, compared with those who received control treatment24. However, interpatient variability in response has been observed and not all patients have a good response to cinacalcet therapy. A reduction of PTH levels after three months of treatment with relatively lower doses has been reported as a prognostic marker of good response to cinacalcet treatment25.Cinacalcet significantly reduces plasma levels of PTH, irrespective of the severity of SHPT in dialysis patients26. Additionally, the association between the A990G SNP of the gene encoding calcium-sensing receptor (CASR) and the biochemical response to cinacalcet treatment has been observed in two previous studies28. Rothe et al.27 reported the findings in seven HD patients with SHPT and first proposed the possibility that the CASR A990G genetic polymorphism may be one of the possible causes of significant interpatient differences in the response to cinacalcet treatment. Later, Jeong et al.28 observed the association between CASR A990G polymorphism and reduction of iPTH during a 3-month cinacalcet treatment among 70 HD patients with SHPT. On the contrary, Moe et al.29 found no association between the CASR A990G SNP and change of iPTH from baseline to 20 weeks after cinacalcet treatment in HD patients of European and African ancestry.Several factors, including high baseline PTH, persistent hyperphosphatemia, and nodular hyperplasia of parathyroid glands have been reported to significantly influence the response to cinacalcetCASR A990G genetic polymorphism on cinacalcet response is inconclusive and needs to be explored. To determine the impact of the CASR A990G polymorphism on PTH lowering effect of cinacalcet in HD patients with SHPT, the primary objective of this study was to compare iPTH levels after 12 weeks of the treatment (PTHw12) between CASR 990G carrier and noncarrier genotypes while considering the variability of other clinical factors that significantly influence PTH level. The secondary objective was to assess the effect of the polymorphism on achievement of at least 30% reduction of iPTH levels from their baselines, after 12-week cinacalcet treatment, comparing between the two genotype groups while controlling for significant factors.Available information regarding the influence of A total of 135 Thai HD patients with SHPT participated in this study  decrease in iPTH level 1262.0  pg/mL from baseline to 602.0  pg/mL at week 12 of cinacalcet treatment ; a median (IQR) reduction of 521.6  pg/mL, with the corresponding median (IQR) percentage of iPTH reduction from baseline of 49.8% . A reduction in plasma iPTH of at least 30% from baseline was observed in 79 patients (58.5%). Sixty-four patients (47.4%) achieved the KDIGO recommended target of two to nine times the upper limit for the PTH assay used for CKD patients on dialysis (130–585 pg/mL). The iPTH of 15 patients (11.1%) increased from baseline even after 12-week cinacalcet treatment.CASR 990A and 990G were 47.8% and 52.2%, respectively. The genotype frequencies were in accordance with the Hardy–Weinberg equilibrium . The AA, AG, and GG genotypes were observed in 32 (23.7%), 65 (48.1%), and 38 (28.1%) patients, respectively. Therefore, 103 patients (76.3%) were identified as G carriers and 32 (23.7%) were noncarriers. Baseline characteristics were comparable between the two groups.The allele frequencies of Absolute change and percentage change of iPTH levels from baseline to after 12 weeks of cinacalcet treatment among the three genotype groups are presented in Fig. r ranged from − 0.262 to 0.175). There were significant linear relationships between log PTHw12 and baseline PTH level (bPTH), baseline serum calcium concentration (bCa), baseline calcitriol dose or relative calcitriol equivalent dose of alfacalcidol (bCtriol), and baseline ergocalciferol dose (bErgo) . The trends of linear relationships between log PTHw12 and the other two factors, baseline serum phosphate concentration (bPhos) and age of patients (AGE), were also observed ; therefore, all six factors were tested as a covariate.Analysis of covariance (ANCOVA) was used to compare PTHw12 between G carriers and noncarriers. Initial data screening led to the logarithmic transformation of PTHw12 for conforming data to the normal distribution . No strong correlation was found among potential covariates (p2 = 0.109). The significant covariates were bPTH, bCtriol, bPhos, and bErgo while AGE and bCa were not significant. The adjusted mean difference of the untransformed PTHw12 between G carriers and noncarriers was 253.7 pg/mL after controlling for significant covariates. The observed and adjusted mean values of log PTHw12, together with untransformed PTHw12 among CASR genotypes, are presented in Table With all test assumptions fulfilled, the ANCOVA revealed that there was a significant difference in log PTHw12 between G carriers and noncarriers after controlling for significant covariates  = 15.472, p < 0.001, ŋp2 = 0.123]. Multiple comparisons revealed a significantly higher adjusted mean of log PTHw12 in AA group compared with those in AG and GG groups ; however, adjusted log PTHw12 means were comparable between AG and GG groups (p = 0.468). The AA group had the highest untransformed PTHw12 (859.0 pg/mL) compared with AG and GG groups .Significant differences in log PTHw12 were also found among the three genotype groups after controlling for bPTH, bCtriol, bPhos, and bErgo [F = 8.817 : 1.262 to 21.179] times greater than the odds for a noncarrier. In addition, the log of the odds of achieving 30% iPTH reduction was positively related to bCtriol and AGE, but negatively associated with bPhos.CASR polymorphism on the efficacy of 12-week cinacalcet treatment, in terms of PTH reduction, was determined in adult HD patients with SHPT in real clinical practice. There were 135 participants, of which 103 were G carriers and 32 were noncarriers, representing 76% and 24% of the study population, respectively. Despite similar baseline characteristics, G carriers had significantly lower PTH levels compared with noncarriers after 12 weeks of cinacalcet treatment, with the adjusted mean difference of the untransformed PTH levels of 253.7 pg/mL, when controlling for significant influencing factors, namely, bPTH, bPhos, bCtriol, and bErgo. Furthermore, the odds of a G carrier achieving at least a 30% reduction in PTH concentrations from baseline to week 12 were approximately four times higher than those for a noncarrier, after adjusting for significant covariates, namely, bPhos, bCtriol, and AGE.In this study, the impact of 30. Activation of the CASR by increased extracellular Ca2+ stimulates multiple G proteins and confers an intracellular signaling cascade, which leads to inhibition of PTH secretion. Cinacalcet, a type II calcimimetic agent, acts as a positive allosteric modulator of the CASR. By interacting with the seven-transmembrane domain of the CASR, cinacalcet lowers the threshold for receptor activation by extracellular Ca2+, presumably by inducing conformational change in the receptor, thereby promoting activation of signal transduction pathways leading to reduction of the circulating PTH level at normal, or even at low, serum calcium levels31. The CASR A990G SNP alters the amino acid sequence and changes the conformation of the CASR intracellular carboxyl-terminal domain, altering its co-operative response to Ca2+ and its binding to filamin-A, a scaffold protein for a signaling cascade33. It has also been demonstrated that the glycine substitution at arginine-990 encoded by the CASR A990G SNP results in a gain-of-function of the CASR34. Although the precise underlying mechanism needs to be further explored, the greater PTH lowering effect of cinacalcet observed in CASR 990G carriers compared with noncarriers could be explained by the combined effect of the CASR A990G polymorphism and cinacalcet treatment.The CASR, a G protein-coupled receptor highly expressed in the parathyroid chief cells, plays an important role as the key modulator of parathyroid gland function by sensing perturbations of extracellular ionized calcium concentration from its narrow physiological range, and regulating PTH secretion, synthesis, and degradation, as well as parathyroid cellular proliferationCASR A986S (rs1801725), CASR Q1011E (rs1801726), and CASR A990G (rs1042636) have been identified. The frequency of these polymorphisms varies between different populations. CASR A986S is the most common SNP of the CASR gene among Caucasians, and CASR Q1011E is a common polymorphism in populations with African ancestry, whereas CASR A990G occurs at high frequency in Asians35. It has been reported that linkage disequilibrium exists among them, indicating that these SNPs are not independent and are highly correlated36. Of note, the frequency of occurrence of both CASR A986S and CASR Q1011E is quite low among Asians, likely limiting the statistical power to detect associations with cinacalcet response (if one exists). In addition, the association between 25 selected SNPs in genes related to calcium regulation, serum phosphate concentration, and bone metabolism were evaluated in Korean SHPT patients on dialysis by Jeong et al28. CASR A990G and CASR rs1802757 were found to be associated with PTH reduction after controlling for age, sex, and baseline calcium level, while no correlations of the tested SNPs, other than CASR with PTH reduction during 3-month cinacalcet treatment, were observed. Linkage disequilibrium test showed high correlation between CASR A990G and CASR rs1802757 (r2 = 0.68) and it was mentioned that the functionality of the CASR rs1802757, which is located in 3’ untranslated regions of the gene, has not been well documented28. Furthermore, the associations of 18 SNPs in CASR and one SNP in the vitamin D receptor gene (VDR) with response to cinacalcet were examined in another study by Moe et al.29. A modest association of the CASR rs937627 genotype with percentage change of PTH in the European ancestry population was observed, but no significant interaction with cinacalcet treatment was found. With consideration to the frequency and functionality of polymorphisms together with the correlation of SNPs due to linkage disequilibrium, the polymorphisms other than the CASR A990G were, therefore, not assessed in the present study.Genetic polymorphisms of several genes related to PTH regulation have been considered as plausible factors influencing cinacalcet response. Three nonsynonymous variants at the intracellular domain of the CASR encoded by CASR 990G allele frequency of 52% in the study patients was similar to that reported among other Asian populations, but higher than those observed among Caucasians and African Americans39. The differences in the 990G allele frequencies observed among ethnicities is a possible explanation for the conflicting results of previous studies assessing the associations between the CASR A990G polymorphism and biochemical response of cinacalcet29.The 11 demonstrated that, among 260 HD treated patients (38% white race and 39% black race), the percentage reduction in mean PTH from baseline was 47% in patients with mild SHPT , 44% in patients with moderate SHPT , and 33% in patients with severe SHPT . Comparing those with the same SHPT severity, our studied patients had a higher median percentage of PTH reduction than that reported by Lindberg et al.11; this might also be at least partly due to differences in ethnic proportions of CASR 990G carriers.Although almost all of the patients in this study had very high bPTH of more than 800 pg/mL, a significant reduction of PTH was observed, with the overall median percentage of PTH reduction from baseline to week 12 of approximately 50%. Cinacalcet efficacy in decreasing mean PTH, regardless of severity of SHPT at baseline, is supported by a previous randomized controlled trial. Lindberg et al.CASR genetic polymorphism significantly influences the response to cinacalcet treatment.Not only were reductions of PTH from baseline observed, but also the proportions of patients with at least 30% reduction of PTH and the proportions of patients who achieve the KDIGO target range of two to nine times the upper limit for the PTH assay were found to be statistically significant between the two groups of genotypes in this study. Of G carriers, 64% had at least 30% PTH reduction, and 55% achieved the KDIGO recommended PTH target of two to nine times the upper limit for the assay used for dialysis patients, while 40% of noncarriers had at least a 30% PTH reduction, and only 22% achieved the KDIGO recommended PTH target. Moreover, increased iPTH from baseline after 12-week cinacalcet treatment was observed in 7.8% of G carriers and 21.9% of noncarriers. These findings support that 24 but lower than the reported 90.3% among patients randomized to cinacalcet in a phase 3 trial among Japanese HD patients, with median baseline PTH levels of 606.5 pg/mL10. This could possibly be explained by differences in baseline PTH levels and ethnicities of the study populations.Notably, although the median baseline PTH level of 1262 pg/mL was observed in this study, the overall proportion of patients who achieved a reduction in PTH of at least 30% from baseline was comparable to results reported in two previous randomized clinical studies, with median baseline PTH levels of approximately 600 to 850 pg/mLCASR polymorphism and cinacalcet response, lower PTH, lower phosphate, higher calcitriol or vitamin D analog dosage, and higher ergocalciferol dosage at the time of cinacalcet initiation were found to be significantly associated with lower PTH after a 12-week cinacalcet treatment, while lower phosphate, higher calcitriol or vitamin D analog dosage, and increasing age at the time of cinacalcet initiation were significantly associated with improving the odds of achieving at least 30% PTH reduction. In concordance with our results, the association of multiple clinical factors, including the severity of SHPT, age, race, serum phosphate, and serum calcium, with PTH control among HD patients treated with cinacalcet has been reported41.Along with the association between 10, and in a post hoc analysis of pooled data from three randomized controlled trials in dialysis patients21, as baseline PTH levels increased, the target PTH of 250 pg/mL was achieved in a lower percentage of patients. Similarly, a recent study in HD patients enrolled in the Dialysis Outcomes and Practice Pattern Study (DOPPS) (Phases 4–6) reported that increased PTH immediately prior to HD initiation predicted a higher PTH level 9–12 months later40 and the severity of SHPT was found to be an important clinical factor in determining the response to cinacalcet therapy among HD patients in a real-life observational study41.The influence of bPTH on the 30% reduction of PTH was not statistically significant, whereas the significant impact of bPTH on PTHw12 was observed in this study. These findings indicate that, although cinacalcet is efficacious at lowering PTH levels irrespective of the baseline severity of SHPT, the higher the baseline PTH concentrations, the higher are the PTH levels after 12 weeks of cinacalcet treatment. In agreement with our findings, it has also been reported that among Japanese HD patients randomized to cinacalcet42. The significance of bPhos influence on cinacalcet response in terms of mean absolute PTH levels and the proportion of patients achieving at least 30% PTH reduction from baseline was determined in this study. According to the regression model, for each 1 mg/dL increase in bPhos, there was a 32% decrease in the odds of achieving a reduction in PTH of at least 30% after 12 weeks of cinacalcet treatment, when all other factors were held constant.SHPT is known to be a compensatory response to phosphate retention. The direct effects of phosphate on PTH secretion and parathyroid cell proliferation have long been demonstrated in experiment studies11. Cinacalcet and active vitamin D sterols have different receptor sites where they exert their pharmacological effects. The possibility of a crossed positive, bidirectional and additive interaction between calcimimetics and vitamin D sterols, probably mediated by the heterologous upregulation of the parathyroid CASR and vitamin D receptors, has been described in experimental conditions44. In addition, cinacalcet and active vitamin D have disparate effects on calcium and phosphate levels. The use of calcitriol or vitamin D analogs, particularly in a high dose or in combination with calcium-containing phosphate binders, may lead to hypercalcemia and hyperphosphatemia as a result of increased intestinal absorption of calcium and phosphate. On the other hand, cinacalcet suppresses PTH while simultaneously reducing serum calcium and phosphate in dialysis patients. Combination usage of cinacalcet and vitamin D has enabled simultaneous attainment of all four KDOQI bone and mineral targets, which has been reported to improve survival in incident HD patients45. Of note, in the present study, the observed serum calcium levels were comparable between G carriers and noncarriers not only at baseline  but also after 12 weeks of cinacalcet treatment .Augmented effects of combination use of cinacalcet with calcitriol or vitamin D analogs for lowering PTH that were observed in this study have also been reported in previous randomized controlled trials13.The dose of ergocalciferol that the patients had been receiving at the time of initiating cinacalcet treatment also significantly influenced PTHw12 in our studied patients. Ergocalciferol is effective in repleting vitamin D levels in HD patients, though there is no conclusive evidence to support a benefit of vitamin D replacement in dialysis patients. The KDIGO guidelines, however, suggest that vitamin D deficiency or insufficiency should be corrected46.Age is considered to be an important determinant of many clinical outcomes. According to the regression model, for each 1-year increase in age, the odds of achieving at least a 30% PTH reduction improved by 5.3%, with a corresponding 68% increase in the odds for each 1-decade increase in age. Of interest, it has been shown that cinacalcet reduced the risk of major cardiovascular events and death among HD patients older than 65 yearsThe high cost of cinacalcet may limit its use in clinical practice. Additional well-designed studies are needed to identify subgroups of patients for whom cinacalcet may potentially provide benefits by preventing SHPT or improving long-term clinical outcomes, and helping with the decision-making process in the management of patients with SHPT.47, cinacalcet does not appear to provide a reduction in risk of mortality or major cardiovascular events in dialysis patients with moderate to severe SHPT. In clinical practice, cinacalcet is generally prescribed when a patient has a very high serum PTH concentration, and where considerable vascular damage may already have occurred. Given data from DOPPS suggesting that PTH levels > 600 pg/mL are associated with higher risk for all-causes and cardiovascular mortality and cardiovascular hospitalizations in dialysis patients48, the question of whether initiating cinacalcet treatment earlier in course of SHPT disease progression would prevent long-term unwanted clinical consequences remains to be answered. Finally, whether or not CASR genetic polymorphism would significantly impact the efficacy of a newer available calcimimetic agent remains an unanswered question.Our study has certain limitations. First, this is a retrospective observational study in a real clinical situation. Our ANCOVA model can explain approximately 35% of the variability in cinacalcet response, implying that there might be other factors, not examined in this study, that influence the variability of cinacalcet response in HD patients. Second, this study is a single-center study. However, the participants were uniformly treated with the same strategies for the management of SHPT. Third, this study focused on a 12-week treatment of cinacalcet; the possible association between the PTH lowering effect and clinical outcomes was not evaluated, given the fact that efficacy of cinacalcet in lowering PTH did not translate into a straightforward improved clinical outcome. According to the unadjusted intention-to-treat analysis of the Evaluation of Cinacalcet Hydrochloride Therapy to Lower Cardiovascular Events (EVOLVE) randomized controlled trialCASR A990G polymorphism significantly influences the PTH lowering effect of cinacalcet treatment in HD patients with SHPT. The magnitude of PTH suppression and the proportion of patients achieving at least 30% PTH reduction from baseline to week 12 of cinacalcet treatment are greater in G carriers as compared to noncarriers.In conclusion, In this observational study, all adult patients (age > 18 years) who visited the King Chulalongkorn Memorial Hospital (KCMH) 1500-bed university hospital in Thailand, having been on HD for at least 3 months and having been treated with cinacalcet for SHPT for more than 12 weeks starting in the period of January 2015 to August 2019, were invited to participate. Patients with evidence of serious hepatic disease with a Child–Pugh score > 7, a history of previous surgical parathyroidectomy or percutaneous ethanol injection therapy into the parathyroid gland, incomplete record data, or those receiving strong inhibitors of cytochrome P450 enzyme that could interfere with cinacalcet pharmacokinetics were excluded. Cinacalcet was initiated at the dose of 25 mg per day at bedtime. Dietary restriction, the dose or type of phosphate binders or vitamin D sterols, and dialysate calcium concentration were adjusted at the discretion of the physicians according to standard of care.The study protocol has been reviewed and approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. The IRB approval number is 672/61. The study was registered at Thai Clinical Trials Registry (TCTR20190820002) and was conducted in accordance with the declaration of Helsinki and its subsequent revisions. Each patient signed an informed consent document before enrollment.13.Patient characteristics and relevant clinical information on the starting date of cinacalcet treatment, both as baseline data and at week 12 of cinacalcet treatment, were retrieved from KCMH electronic medical records. All serum biomarkers were measured at KCMH central laboratory. Serum iPTH levels were determined by using a chemiluminescence immunoassay on a Roche Elecsys 2010 Analyzer. Levels of 25-hydroxy vitamin D were assessed by using chemiluminescent immunoassay . Throughout this study, serum calcium was reported as albumin corrected values when serum albumin was less than 4.0 g/dL [corrected calcium in mg/dL = measured calcium in mg/dL + 0.8*]CASR  was performed using TaqMan® Assay Reagents for allelic discrimination  with Applied Biosystem® QuantStudio 5 Real-Time PCR System . Each 20 μL PCR mixture contained 4 μL of genomic DNA (5 ng/μL), 10 μL of TaqMan® Genotyping Mastermix, 1 μL of allele specific TaqMan® MGB probe and sequence-specific primer kit, and 5 μL of DNase-free H2O. The conditions of PCR analysis were set as 50 °C for two minutes and 95 °C for 10 min, and then 40 cycles of 95 °C for 15 s and 60 °C for one minute.Venous blood samples were collected into tubes containing EDTA during outpatient visits after recruitment. For genotyping, genomic DNA was extracted from the buffy coat that was collected from the peripheral blood using a QIAamp DNA Blood Mini kit for DNA purification . The genotype analysis of CASR 990G allele (the AG or GG genotypes) were classified as G carriers, and those identified as homozygous carriers of CASR 990A allele (the AA genotype) were classified as noncarriers. The physicians were not aware of the genotype of the patients at the time of any drug dosage modifications.Individual patients identified as either heterozygous or homozygous carriers of Distribution of continuous data was assessed by Kolmogorov–Smirnov or Shapiro–Wilk test, and, subsequently, parametric tests or nonparametric tests were applied as appropriate. Continuous variables are presented as either means ± standard deviations when data are normally distributed, or medians with IQR when data are not normally distributed. Categorical data, on the other hand, are presented as counts and percentages. Continuous data were compared using the independent-samples t-test, analysis of variance (ANOVA), paired t-test, Wilcoxon Signed Rank test, Mann–Whitney U test, or Kruskal–Wallis test. Categorical variables were compared by chi-square test. Genotype frequencies of the polymorphism were tested for deviations from Hardy–Weinberg equilibrium using appropriate chi-square testing.Assumptions for the ANCOVA and logistic regression were performed. Where appropriate, data were logarithmically transformed to obtain normal distribution. Bivariate correlations between continuous variables were evaluated by either Pearson correlation test or Spearman rank correlation test as appropriate. The linear relationships between continuous variables were assessed by simple linear regression analysis. The homogeneity of variances was tested by Levene’s test. The homogeneity of regression was analyzed by conducting a univariate ANOVA. The linear relationships between continuous independent variables and the logit transformation of the dependent variable were assessed by Box-Tidwell procedure.CASR genotypes and PTHw12. The potential covariates were selected based on the previous reports of clinical factors that significantly influenced PTH level50. The covariates considered were bPTH in *100 pg/mL, bPhos in mg/dL, bCa in mg/dL, bCtriol in μg/week, bErgo in *20,000 IU/month, and AGE in years. A relative calcitriol equivalent dose of alfacalcidol in μg/week was calculated by dividing the alfacalcidol dose per week by 1.552. Any covariates that were strongly correlated with another covariate at r > 0.8 were eliminated, as they did not add a significant amount of information independent of the related variable. To indicate the degree to which each factor affected cinacalcet response, partial eta squared (ŋp2), a measure of the effect size, was computed. Bootstrapping was performed to obtain 95% CI of the adjusted mean scores. The untransformed iPTH were also determined by backtransforming the adjusted means.The ANCOVA was used to evaluate the relationships between CASR genotypes in terms of achievement of at least 30% reduction of iPTH levels from their baselines between the two studied groups whilst controlling for significant covariates. Overall model evaluation was assessed by omnibus tests of model coefficients. Significance of individual regression coefficients was tested by Wald chi-square statistic. The Hosmer–Lemeshow test and Nagelkerke R square were used to evaluate goodness-of-fit of a logistic model. Bootstrapping was also conducted to derive 95% CI of the odds ratio. The degree to which predictive probabilities, with a cutoff of 0.5, agree with actual outcomes was determined to document the validity of the predicted probabilities.Binomial logistic regression was applied to compare the effect of 53. All statistical tests were performed using IBM SPSS version 22 . Unless stated otherwise, a two-sided p-value of less than 0.05 was considered statistically significant.Power calculation was performed. With an effect size of 0.25, a power of 0.8, and a significant level of 0.05, a required total sample size of at least 128 persons was estimated by G*Power 3.1.9.2 software