We evaluated a subset of two subgroups (n = 36 each) for intra and inter-observer (IA/IE) error. From the whole cohort, the best IOP agreement was observed between GATpre and CT1post surgery: 16.09 ± 2.92 vs 16.42 ± 2.87 (p < 0.001); ICC = 0.675 (95% CI: 0.554–0.768). In the analysis of LASIK vs PRK, GATpre and CT1post showed the highest agreement, although LASIK measurements were more accurate than PRK, as the ICC = 0.718 (95% CI: 0.594–0.812) and ICC = 0.578 (95% CI: 0.182–0.795) respectively. Excellent agreement was observed for IA/IE, and there was an ICC > 0.8 (95% CI) in all cases. CT1 proved more accurate in the LASIK subgroup. In conclusion, our new version of GAT could be used with post-surgery LASIK patients as a more accurate measurement device compared to the current reference tonometer.This study assesses the agreement between intraocular pressure (IOP) measurements taken with the Goldmann applanation tonometer (GAT) and a new experimental applanation tonometer with a convexly shaped apex (CT) after laser myopic refractive surgery. Two different CT radii (CT1 and CT2) were designed with a finite element analyser, and a prospective double masked study on 102 eyes from 102 patients was carried out. A Bland-Altman plot and intra-class correlation coefficient (ICC) were calculated to assess the agreement between GAT measurements and the measurements of both CT1 and CT2 before and after myopic laser assisted  However, GAT readings are influenced by corneal central thickness (CCT) and corneal biomechanics (CB), which vary widely among normal individuals6. In myopic laser refractive surgery (LRS) patients, variations induced in both CCT and CB lead to differences in GAT readings. This change in IOP has been inconsistent in previous studies regardless of the type of surgery: Mardelli et al.7 found a significant reduction of 1 ± 2.8 mmHg in IOP measurements after ablating 23 μm in photorefractive keratectomy (PRK) patients; while Duch et al.8 found an underestimation of about 2.9 mmHg per 70 μm ablation in CCT after laser assisted in situ keratomileusis (LASIK). These IOP underestimations affect the reliability of GAT as gold standard after LASIK and PRK procedures11.The Goldmann applanation tonometer  is the current reference tonometer for measuring intraocular pressure (IOP) in daily clinical practice due to its acceptably accurate measurements, reproducibility and reliability12. Moreover, the prevalence of glaucoma is 2 to 4 times higher among the myopic population13. Thus, it is very important to obtain an accurate and real estimation of IOP in order not to miss ocular hypertension after myopic LRS.LASIK is one of the most frequent types of eye surgery and a total of 40 million people had this operation worldwide between 1991 and 2016The aim of our study is to describe a new modified GAT as well as its translational application and clinical outcomes by evaluating IOP measurements before and after myopic LRS. We also evaluate the reliability of the intra and inter-observer agreement.in vivo14. In our study, we used 2D . Three-dimensional (3D) modelling is more accurate than two-dimensional (2D) modelling for predicting what could occur to tissue  2D Fig. –S4.Figur6, with CCT = 520 μm, an anterior corneal curvature of 7.8 mm, and assuming radial symmetry for a cornea-GAT contact of 3.06 mm , as described in the Orssengo-Pye algorithm mm Fig. . The sec mm Fig.  and 2.Fi15 was applied, and every simulation was calculated with Young’s Modulus (Y) = 0.5 MPa. Inverse modelling was performed to account for the IOP: a step-wise computing initial stress state of the cornea was determined by assuming IOP = 15 mmHg16.In all cases, a nonlinearity material model with Mooney-Rivlin parametersDue to pachymetric and elastic corneal variability in our population, we considered different radii for the CT devices (radii varying from 11.5 to 14 mm) to compare contact pressure profiles (CPP) with different corneas, until a similar corneal modelling behaviour was observed between GAT and CT Fig. .Finally, two different tonometers with different radii (r) Fig.  were creA prospective double-masked comparative study was carried out on a sample of 102 myopic subjects who were going in for LSR (LASIK or PRK) at the Barraquer Ophthalmology Center in Barcelona. These subjects first underwent a general medical history review, and a detailed eye examination. After meeting the inclusion criteria and having none of the exclusion criteria, the subjects were included and their informed consent was obtained for study participation. The study protocol is in accordance with the Declaration of Helsinki and the Institutional Ethics Committee of the Barraquer Ophthalmology Center approved this study.The right eye of each subject was randomly selected adding up to a total of 102 eyes. The inclusion criteria were: myopic patients aged above 18 years with a stable myopic refractive error less than spherical −9 diopters (dpts), and less than myopic astigmatism −4 dpts. Subjects who had previously been diagnosed with an ocular pathology before ocular surgery or treated with medications that could affect IOP levels were excluded from the study.All patients underwent a standardized examination that included measuring visual acuity , slit-lamp anterior biomicroscopy, posterior segment ophthalmoscopy and IOP measurements. Age, gender and refractive error were also recorded.Before IOP measurements were recorded, the corneal topography was determined with a Pentacam  to obtain anterior simulated keratometry and posterior corneal curvature , corneal volume (VOL) and CCT. In addition, the corneal hysteresis (CH) and corneal resistance factor (CRF) were obtained by Ocular Response Analyser . The maximum ablation depth (Max.Abl) and percentage of ablated tissue (PTA) were also analysed. Postoperative treatment included ofloxacin 0.3% and fluorometholone (FML) 0.1% 4 times a day for 1 week in both groups, and an additional 3 weeks of FML in the PRK group.17. The IOP measurements were taken between 10:00 and 13:00 hours (10 am and 1 pm), similarly to the 3rd month after surgery readings, which were performed at approximately the same time as the pre-operation readings19.IOP was measured before and three months after surgery. Each measurement was carried out by the main observer (MI) using 3 different devices  and leaving 2 minutes between taking measurements with each tonometerIn order to assess intra-observer (IA) error, a subset of 36 patients were randomly double-masked measured by the main observer MI in the presence of a second observer (AL). To assess the inter-observer (IE) error, a different subset of 36 patients were also evaluated randomly by the main observer MI and by a second observer AL. In these cases, IOP measurements were carried out consecutively, were double-masked and after an interval of 5 minutes. In all cases, a third observer (BK) was present to ensure the double-masking.® . A significance level of 5% was considered in all the analyses. All quantitative variables were tested for normality with the Kolmogorov-Smirnov or the Shapiro-Wilk tests. We conducted descriptive analyses for all the variables preoperatively and 3 months after surgery. The descriptive values are presented as mean ± standard deviation unless stated otherwise.The statistical analysis was performed using SPSSt-test or the Wilcoxon test depending on data normality. In addition, we compared the eye characteristics of patients undergoing surgery with LASIK. PRK was performed using the independent t-test or the Mann-Whitney test depending on data normality.Pre- and post-surgery values were compared considering all the cases using the paired To evaluate the relationship between IOP and the corneal characteristics, IOP values obtained with GAT, CT1 and CT2 in pre- and post-surgery were correlated respectively with clinical characteristics of pre- and post-surgery eyes using the Pearson or Spearman’s correlation coefficients depending on data normality.20. To determine which factors explain the bias trend in the Bland-Altman analysis, multiple stepwise regression was used to relate the differences in IOP and the change in corneal characteristics from pre- to post-surgery.We considered GATpre as the current reference for evaluating the concordance between IOP measures taken with the different tonometers. Different approaches were used to evaluate the differences between GATpre IOP and IOP determined with GAT, CT1 and CT2post respectively. In short, 1) the mean differences between GATpre measurements and the measurements taken with all the devices in post-surgery were calculated and the absence of differences was tested with the Wilcoxon test; 2) the Bland-Altman analysis was used to compare the agreement of measurements taken with the different tonometers; and 3) the ICC was calculated based on absolute-agreement. Values lower than 0.5, between 0.5 and 0.8, and greater than 0.8 were indicative of poor or weak, good, and excellent reliability, respectivelyt-test. The Bland-Altman analysis was used, and the ICC was calculated based on absolute-agreement.To analyse IA/IE, we calculated the mean difference between measurements and tested the absence of differences with the Wilcoxon or paired sample CPP comparisons can be helpful for understanding how corneal tissue will react to contact with different tonometers, such as GAT or CT. To compare these behaviours easily, we developed a 2D graphic diagram in which a cross sagittal section of corneas and tonometers are shown with their corresponding graph below Fig. .2. The maximum corneal deformation is (MCD) = 8 × 104 N/m2. This contrasts with a lower initial arc length converging in the centre that is observed if the GAT contacts an OC, and its ICP = 1600 N/m2  when an external force with a convex surface (CT) contacts the anterior corneal surface after surgery  with an initial IOP of 15 mmHg Fig. , as the A total of 102 eyes from 102 patients  were included in the study. Seventy-three (71.6%) patients underwent LASIK and 29 (28.4%) PRK. The mean age was 31.6 ± 6.1 years. The descriptive statistics of variables in the pre- and post-surgical evaluation are normally distributed and are shown in Table IOP values obtained with GAT, CT1 and CT2 in pre- and post-surgery show a significant correlation with CRF for all patients .We evaluated IOP for different tonometers by analysing the concordance of the CT1 and CT2post measurements. Considering GATpre the main reference, CT1 and CT2 in pre-surgery appear to overestimate IOP Fig. . Howeverp < 0.001; limits of agreement: −8.10–0.96). However, a much better agreement is observed between GATpre and CT1post  and CT2post .Considering LASIK and PRK patients separately, CT1post (−0.19 mmHg) showed the smallest differences with GATpre (1.62 mmHg) for both LASIK and PRK Table . Consideost Fig.  . A better agreement was found for CT1  and CT2post  respectively. The PRK group also showed poor agreement for GATpost . A better agreement was found for CT1post  and CT2post   , differences between GATpre and the measurements in post GAT, CT1 and CT2 appeared to be correlated with the differences in CRF before and after surgery (p < 0.001)  in the LASIK soubgroup , and the values are lower in the postoperative period: GATpost ICC = 0.833 (95% CI: 0.697–0.911), CT1post ICC = 0.857 (95% CI: 0.738–0.924), and CT2 post ICC = 0.833 (95% CI: 0.698–0.911). Concerning IA error measurements, in all cases the ICC is higher than 0.9 (p < 0.001) and the values are lower in the postoperative period: GATpost ICC = 0.948 (95% CI: 0.895–0.975), CT1post ICC = 0.933 (95% CI: 0.866–0.967), and CT2post ICC = 0.966 (95% CI: 0.927–0.984)  is consistent with the idea of Imbert-Fick behaviour24, but not the edges when the GAT is applied. Shih23 also described the area of highest stress to be located around the ablated edge under applanation when CB and IOP were compared between LASIK and PRK. However, we have demonstrated that a different phenomenon can be observed when a convex force (CT) is applied towards the centre of an ablated zone: the initial contact pressure increases in the centre, resulting in a balance of forces similar to that which existed before surgery.The applicability of the Imbert-Fick law for applanation tonometry is compromised after myopic LRS25. Hamilton6 found a 5.35 mmHg error caused by an increase from 0.16 MPa to 0.40 MPa, and a 4.67 mmHg IOP difference across the CCT sample (487.7 to 599.9 μm). This means that both Y and CCT influence IOP measurements in vivo estimations. In our Y = 0.5 MPa ex vivo calculations we found some mathematical results that, without being absolute, correspond to what was obtained in the clinical study: FEA analysis showed that GATpost recorded a −4.4 mmHg as CCT was ablated 80 μm. Considering that LASIK reduces CCT more than PRK (with Max.Abl = 71.23 μm in our sample) this value nearly corresponds to the GATpost underestimation of −3.94 mmHg shown in our LASIK results.Multiple theoretical models have been described with very different Y estimations (0.1–1.24 MPa)The FEA analysis of CT1post and CT2post compared to GATpre showed IOP differences of 0.4 and 0.0 mmHg respectively, which correlates similarly to our clinical results of a CT1post of −0.19 mmHg and a CT2post of 0.42 mmHg. According to these comparisons, this should have a large impact on the model for explaining what may be truly measured in real life. Hence, these simulations seem valid to compare differential behaviours among different tonometers or between corneas with different thicknesses.26. The main limitation of our numerical models is that they do not take into account the viscoelastic behaviour of the cornea. In addition, they were designed for PRK and not LASIK. Nevertheless, previous results23 have found similar corneal deformation patterns in 3D and 2D FEA between PRK and LASIK. Therefore, the two surgical approaches are comparable in terms of central corneal displacement. Since our goal was not to compare different corneal biomechanics between LASIK and PRK, but rather to evaluate areas of applanation  contacted by different external tonometers after LRS, we decided to simplify our comparisons and use a PRK shaped cornea.However, we must make a conservative interpretation. FEA has several limitations, mainly due to the complexity of representing ocular tissues mathematically, which affects the range of applicability of the Imbert-Fick law. Furthermore, from a mechanical point of view, the structural geometry of the eye cannot be perfectly represented as it is limited by the numerical viability of the simulation. FEA simulations are complex and require multiple models to achieve hypothetical geometric pre- and postoperative corneas27, it is almost impossible to find a single universal number for corneal clinical properties. Assessing how the strength or weakness of a cornea can influence IOP readings seems unrealistic beyond a single pachymetric or biomechanical parameter. This implies that tonometry is not reliable, not only due to CCT variability28, but also due to the alteration of the corneal structure. Therefore, correction algorithms for GAT are not reliable since they are based only on changes in CCT29.Besides, another significant limitation still affects the tonometry: the internal variability in corneal idiosyncrasies. As discussed previously32 that the bias in internal patient variability in CCT can be minimized with modified versions of GAT, which opens a new horizon for GAT modifications in the near future. Mccafferty et al. demonstrated that a bi-curved concave-convex surface modified GAT can significantly reduce GAT prism sensitivity to CCT in standard corneas. However, our FEA analysis and clinical results show that a central convex modification of GAT seems to be accurate for measuring IOP in OCs. Therefore, this study is the first to show that a unique convex surface modified version of GAT could be reproducible for post operated LASIK or PRK IOP measurements.It has been described33. After reducing the mean corneal pachymetry of 90.2 μm, Pepose et al. found no statistically significant differences in pre- and post-surgery IOP measurements taken with PDCT , compared to pre-and post-measurements taken with GAT (−1.8 ± 2.8 mmHg p < 0.01). Sales-Sanz34 found that the Schiøtz tonometer has less disparity in terms of coefficient ocular rigidity (Ko) than PDCT and GAT after the LASIK procedure, although it is not commonly used in clinical practice. There are other devices that are more extensively used, such as the rebound tonometer iCare ; however, it underestimates IOP in post LASIK like GAT. Previous reports35 described no statistically significantly difference between iCare and GAT in 96 myopic patients after LASIK with a mean underestimation of −4.9 ± 2.5 mmHg and −3.4 ± 2.5 mmHg, respectively.Concerning other devices, the Pascal tonometry (PDCT) does not appear to be as influenced by CB after LASIK as other tonometers36, different IOP measurements between the Corvis ST tonometer, GAT and ORA  have been described in 50 myopic patients after LASIK: 3.4, 1.0 and 3.8 mmHg Bland-Atlmann bias respectively . In our LASIK subgroup of 73 patients, the mean difference for CT1post was 0.19 mmHg (limits of agreement: −4.30–3.92), and 0.42 mmHg for CT2post (limits of agreement: −3.77–4.62), indicating that IOP estimations with CT1post are similar to those provided with GATpre. Therefore, it seems that CT1 offers a more accurate estimation in patients with LASIK than GAT. Nevertheless, tonometry after LRS may not be interchangeable due to the diversity in the results. We believe it is relevant which tonometer and which IOP are taken as baseline after LRS, and that GATpost should not be taken as IOP reference. Our new device correlated with GATpre in our LASIK group, but an important remark of our study is that GATpre is an estimation of IOP and could not be accurate. Besides, CT must not be used in subjects whose CB properties have not been strongly modified by laser -as in PRK corneas- because it seems to overestimate IOP. On the other side, we have not compared it with other non-applanation tonometers in pre or post-surgery. Future studies are necessary to evaluate agreement between CT and PDCT, Corvis ST, or corneal compensated IOP in this subgroup of patients.With respect to tonometers that are less influenced by CB7, which found minimal changes with GAT  in 111 PRK patients with a mean ablation of 23 ± 23 μm in corneal thickness. Furthermore, the PRK group received FML during 4 weeks after surgery. Despite topical steroids (TS) could have elevated IOP37, we believe that differences in IOP in both groups are due to CB changes, not to TS effect. FML has proven to be less ocular hypertensive than other topical steroids37, and TS effect on IOP recovers to baseline within 1 to 3 weeks of discontinuing treatment38. Our PRK IOP was measured after 8 weeks of stopping TS, so it seems steroids could not influence the IOP measurements at this point.In contrast to the LASIK group, our PRK patients showed greater deviation, indicating that the CT device is not as accurate as for LASIK patients. In our Bland-Altman analysis, the IOP readings obtained with CT1post were similar to those obtained with GATpre for all patients. However, for PRK patients the deviation was higher than in LASIK patients, indicating that CT performed less accurately for the PRK approach. Moreover, GATpost showed a relatively good agreement in the PRK group, which means GAT could be reproducible in PRK patients. This is consistent with previous research39 due to its invasive nature. Second, the sequence in which we measured IOP could imply certain bias in the second and third IOP measurements: repeated tonometry may induce changes in the anterior chamber volume and thus, in the registered pressure. AlMubrad et al.40 found a statistically significant IOP reduction  on subsequent measurements performed with a non-contact tonometer (Topcon CT80) after GAT in 65 patients. Gaton et al.41 recorded a significant IOP decrease between first and second successive measurements with GAT  in 70 glaucomatous eyes. We believe that repeated contact of any external force with the eye may produce occasional IOP fluctuations. This would lead to significant underestimations that could be transcendent regarding glaucoma diagnosis. However, other considerations should be taken into account, such as IOP levels beyond the normal range or CB. A third source of bias in our study could be related to IOP diurnal fluctuation in time. Baseline IOP could be unbalanced across measurements after 3 months even if measurements are taken at the exact same time42. Further research could determine whether CT performs accurately.Other limitations affect our research: First, post-surgery IOP was not validated by comparing it with intracameral IOP readings43, CRF decreased after both procedures but mainly in LASIK as opposed to PRK. It is evident that PRK is less invasive than LASIK, which implies that CB properties are better conserved. We believe this could be the reason why CT overestimates IOP before surgery and performs less accurately in PRK corneas. This would coincide with our clinical findings for CT1 and CT2pre, which were not useful in non-operated corneas since they overestimated IOP measurements.Regarding variables that could influence our measurements, CRF showed a significant correlation for all the tonometers pre- and post-surgery. As in other reported studies46. However, no previous studies have specifically addressed how PCC changes could influence GAT IOP readings after LRS procedures. In 50 normal subjects, Firat et al.47 found that anterior and posterior curvature values and corneal volume do not influence IOP readings made with GAT. These results coincide with our pre-surgery results in which no IOP reading of any tonometer was correlated with PCC, simK or VOL in 102 normal patients. In the post operated subgroups, PCC and VOL also did not seem to influence IOP readings. We believe this could be related to posterior corneal curvature changes recovering three months after surgery46, which was the time our measurements were taken. On the other hand, CCT, Max. abl, and PTA could explain the differences among GATpre, CT1 and CT2post in the LASIK group. As more corneal tissue is removed in high myopia in the anterior stroma, it is expected that CB is more altered. Therefore, a bias could be expected for all the tonometers, which would have a significant impact on GAT readings, compared to the PRK group.The posterior corneal shift after LRS procedures has been widely evaluated to detect possible ectasiaBoth pre- and post-surgery tissue characteristics should be taken into account when IOP measurements are considered in post LRS corneas. Although we could expect post-surgery measurements to be close to those prior to surgery, a range of known variability  can be expected because tonometry is not personalized. In addition, the most accurate options for measuring IOP in LRS patients are not usually available or accessible to all ophthalmologists. This new simple and affordable option could solve a problem that has not yet been solved and make it available for universal use. Notwithstanding, new studies will be necessary to confirm the data analysis, make comparisons with other tonometers, and verify whether CT could also be used in patients with hypermetropic LRS, keratoconus, or after corneal transplantation.11. In conclusion, we have designed a new version of the applanation tonometer that could be used after LASIK instead of the current tonometer reference. This provides a new applanation tonometry option that is appropriate for supporting the diagnosis of ocular hypertension in this subgroup of patients.Nevertheless, our device has demonstrated good agreement between GAT and CT1post in the LASIK subgroup, and thus minimizes the effect of the loss of central tissue in this type of surgery. The IA/IE results also indicate that there were no significant differences between observers, and therefore it could be a reproducible and convenient alternative for any ophthalmologist, and suitable for a currently very frequent and specific patient profileSupplementary material.


Chlamydia pneumoniae and Mycoplasma pneumoniae have been implicated in the pathogenesis of asthma and are responsible for chronic inflammation when host immune system fails to eradicate the bacteria.C. pneumoniae and M. pneumoniae.We performed a prospective study on 410 patients who underwent a visit at the asthma clinic of CHU of Liege between June 2016 and June 2018 with serology testing for C. pneumoniae, while only 12.6% had IgM and/or IgG against M. pneumoniae. Compared to seronegative asthmatics, asthmatics with IgA+ and IgG+ against C. pneumoniae were more often male and older with a higher proportion of patients with smoking history. They received higher doses of inhaled corticosteroids (ICS) and displayed lower FEV1/FVC ratio, higher RV/TLC ratio and lower conductance. They had higher levels of fibrinogen, though in the normal range and had lower sputum eosinophil counts. Patients with IgA− and IgG+ against C. pneumoniae were older and had higher blood monocyte counts and alpha-1-antitrypsin levels as compared to seronegative patients. Patients with IgM and/or IgG towards M. pneumoniae were more often males than seronegative asthmatics. In a subpopulation of 14 neutrophilic asthmatics with Chlamydia pneumoniae IgA + /IgG + treated with macrolides, we found a significant decrease in blood neutrophils and normalization of sputum neutrophil count but no effect on asthma quality of life and exacerbations.65% of our asthmatic population had serum IgA and/or IgG towards C. pneumoniae have more severe disease with increased airway obstruction, higher doses of ICS, more signs of air trapping and less type-2 inflammation.Positive Chlamydia serologic test is more common than positive Mycoplasma serology. Asthmatics with IgA and IgG against  Asthma is an inflammatory disease of the airways that is characterized by airway hyperresponsiveness towards various environmental factors. Previous studies suggest that the respiratory tract microbiome may underlie the development of asthma  and clinChlamydia pneumoniae and Mycoplasma pneumoniae are intracellular and extracellular bacteria respectively, both causing respiratory tract infection. Chlamydia and Mycoplasma pneumoniae have been implicated in the pathogenesis [Chlamydia pneumoniae is thought to promote asthma by inducing both airway eosinophilia and neutrophilia with concomitant enhancement of the severity of airways disease [C. pneumoniae can lead to chronic infection, where C. pneumoniae enters a state of “latency” in which it is viable but dormant and does not multiply. It however continues to synthetize heat shock protein 60 [Chlamydia pneumoniae has also been shown to induce secretion of TNF-alpha and IL-8 [Chlamydia pneumoniae seems to be able to impair apoptosis of infected cells leading to chronic infection and to induce ciliostasis in the bronchi. On the other hand, Mycoplasma pneumoniae attaches to and destroys ciliated epithelial cells of the respiratory tract mucosa. It induces the secretion of IL-8 and TNF-alpha in vitro [ogenesis  of asthmogenesis –7, especogenesis . Chlamyd disease  and asth disease . Failureotein 60 , able tootein 60 . Chlamydand IL-8 . It has and IL-8  by inducand IL-8 . Moreovein vitro , Type-2 in vitro  and incrin vitro  and IgE in vitro .As atypical bacterial infection of epithelial cells induces the production of a cascade of cytokines that recruit and activate immune cells, we wanted to evaluate if patients with signs of chronic infection had different inflammatory phenotypes and severity profile.We conducted a prospective study on a series of 406 patients with asthma recruited from the University Asthma Clinic of Liege between 7th of June 2016 and 26th of June 2018. The patients came from routine practice to University Hospital and were recruited by two clinicians involved in asthma. Entry criteria were any patients with asthma who accepted to undergo detailed investigation at the Asthma Clinic. Their demographic, functional and inflammatory characteristics are summarised in Tables 1) of > 12% and 200 ml following inhalation of 400 µg salbutamol or inhaled concentration of methacholine provoking a 20% fall in FEV1 of < 16 mg/ml. Methacholine challenge was performed according to a standardised methodology as previously described. Subjects were characterised as atopic if they had at least one positive specific IgE  for at least one common aeroallergen . Exacerbation in the previous year was defined by a course of oral corticoids for at least 3 days in case of asthma worsening. Patients experiencing an exacerbation treated with OCS or antibiotics during the last 6 weeks were excluded.Asthma was diagnosed based on the presence of chronic respiratory symptoms such as cough, breathlessness or dyspnoea together with the demonstration of airflow variability. The latter was defined by airway hyper-responsiveness shown by one or more of the following: increase in Forced Expiratory Volume in 1 s . FeNO was first measured and followed by spirometry with bronchodilation, sputum induction and blood sampling. All tests were performed on the same day.Quality of life was assessed using the self-administered Asthma Quality of Life Questionnaire (AQLQ)  and asthSputum was induced and processed as previously reported and was successful in 78% of the patients encountered in our asthma clinic which is similar to previous report. Cell count were estimated on samples centrifuged (Cytospin) and stained with Diff Quick after counting 500 cells .This study was conducted with the approval of the ethics committee of CHU Liege.C. pneumonia-specific antibodies were detected with the aid of a quantitative ELISA-based assay (Savyon® kit) and processed with the Eti-max 3000® analyzer (DiaSorin). Values of IgA greater than or equal to 2 UA/ml were considered positive in the present study. For IgG, a cut-off of ≥ 28 UA/ml was used. The kinetics of IgG and IgA secretion are taken into account when determining acute or chronic infection. Chronic infection diagnosis requires the detection of persistent serum IgG levels and persistence of elevated IgA levels on two samples taken at 2 weeks interval [Due to the difficulties of detecting the bacteria in airway secretions, we used indirect methods to differentiate acute from possible chronic infection. Serum interval . PatientMycoplasma pneumoniae antibodies were analyzed with a quantitative assay by immunoluminometric method, LIAISON®XL analyzer (DiaSorin). IgM were either positive or negative and IgG were considered positive when ≥ 11 UA/ml.Serum Chlamydia pneumoniae received macrolides after the first visit at the Asthma Clinic due to the presence of neutrophilic asthma (sputum neutrophil count ≥ 76%) insufficiently controlled with ICS/LABA treatment. Demographic, functional and inflammatory characteristics of these patients were compared before and after treatment with antibiotics.14 asthmatics with IgA + /IgG + for 1, post-BD FEV1/FVC, RV/TLC, sGaw, ACQ score, AQLQ score, exacerbations during the last year, FeNO, Sputum Eosinophils (%), Sputum neutrophils (%), Blood eosinophils (/mm3), Blood neutrophils (/mm3), total IgE, and ICS dose were included in the univariate model. Positivity to Chlamydia (IgA−/IgG + and IgA + /IgG +) was used as the dependent variable. A multivariable stepwise forward analysis was done including all independent variables. A p value < 0.05 was considered statistically significant. Statistical analysis was done using STATA version 14.0 .The results were expressed as mean ± SD or mean ± SEM for continuous variables; median and interquartile ranges (IQR) were preferred for skewed distributions. For categorical variables, the number of observations and percentages were given in each category. Comparisons between different subgroups were performed with a Kruskal–Wallis test. The Spearman correlation coefficient was used to measure the association between clinical parameters. Paired T-test were used to compare patients before and after treatment with macrolides. Variables independently associated to Positivity to Chlamydia (IgA−/IgG + and IgA + /IgG +) were identified by logistic regression. Independent variables such as Age, BMI, Gender, Tobacco status, disease duration, post-BD FEVChlamydia pneumoniae and IgG and IgM for Mycoplasma pneumoniae were included in this study.406 asthmatics who underwent a visit to the asthma clinic in stable state with a measurement of serum IgA and IgG for Mycoplasma pneumoniae while 65% had IgA and/or IgG against Chlamydia pneumoniae  and was used as the control group. The second group comprised of patients with a previous infection with Chlamydia pneumoniae (IgG positive but IgA negative). The third group involved asthmatics with chronic infection with Chlamydia pneumoniae (IgG and IgA positive).Only 12.6% of our asthmatic population had IgM and/or IgG against Chlamydia pneumoniae were more often males, were older with a lower proportion of non-smokers. Most of them had moderate or severe asthma and were treated with higher doses of inhaled corticosteroids. They had a longer disease duration as compared to asthmatics without a history of Chlamydia pneumoniae infection. They also had lower FEV1/FVC ratio and more signs of air trapping with a higher residual volume on total lung capacity ratio. Their conductance was lower and they exhibited higher fibrinogen but similar C reactive protein (CRP) levels. We also found lower rates of sputum eosinophilic inflammation in these patients as compared to Chlamydia pneumoniae IgA−/IgG− asthmatics , gender  and FeNO .We performed a stepwise multivariate regression analysis to highlights predictors of positive serology against ae Table . PredictChlamydia pneumoniae had similar demographic, clinical, functional and inflammatory characteristics as compared to those without a history of Chlamydia pneumoniae infection with the only differences being that they were older and had higher blood monocyte counts and alpha-1 antitrypsin levels. It should be noted that both monocytes and alpha-1-antitrypsin levels were within the normal range.Patients with positive IgG but negative IgA for Mycoplasma pneumoniae infection were more often males. This was the only significant difference as compared to asthmatics without history (IgM−/IgG−) or with previous infection (IgM−/IgG +) to Mycoplasma pneumoniae.Compared to the other groups, patients with chronic Chlamydia pneumoniae infection was 84 (median 30 to 500) while IgG index was 20 (0–22) for the negative counterpart. We found a positive correlation between the level of IgG against Chlamydia pneumoniae and age , history of smoking measured in pack-years , air trapping ) and alpha-1 antitrypsin level  while there was a negative correlation with FEV1/FVC ratio , post-BD FEV1  and sGaw .The median IgG titer for patients with a history of 1/FVC  and diffusion capacity .Looking at IgA levels, we found a positive correlation with age , age of onset , history of smoking (in pack years) , the dose of ICS , asthma control , air trapping ) while there was a negative correlation with FEV3 (2999–6627) to 2562 (2240–4692), p = 0.016) and a significant reduction in sputum neutrophils (from 85% (80–95) to 67% (29–87), p = 0.047).14 patients received macrolides after the first visit at the Asthma Clinic and came back 9 to 12 months later for a follow-up visit. After macrolides were initiated, we found a significant decrease in blood neutrophils  and monocytes during acute asthma with Chlamydia pneumoniae infection, but no significant differences in sputum cell count was found one month after acute infection between those who had antibodies and those who had not. In our study, no association was found between chronic infection and sputum neutrophil counts. We however observed significantly lower sputum eosinophil counts in patients with a serologic signature of Chlamydia pneumoniae infection. It could be suggested that the decrease in sputum eosinophils observed in asthmatics with persistent IgA and IgG against Chlamydia pneumoniae might be due to treatment with higher doses of ICS in this subgroup. When focusing on the steroid naïve patients (n = 145) included in our study, we however confirm lower numbers of sputum eosinophils in asthmatics with IgA + /IgG + against Chlamydia pneumoniae (0.6 (0–1.6)) as compared to IgA−/IgG− (1.4 (0.4–4.2), p < 0.05). Additionally, no association was found with IgE, as it has been previously reported [In our study, asthmatics in stable state with chronic ogenesis , chronicogenesis . Wark etogenesis  found threported .1/FVC ratio and sGAW indicating more severe airway obstruction and more signs of air trapping with a higher residual volume on total lung capacity ratio. It might be that the lower FEV1/FVC and higher RV/TLC observed in this Chlamydia pneumoniae IgA + /IgG + asthmatics is a consequence of the older age of this subgroup. We indeed recently demonstrated that elderly asthmatics had higher RV/TLC and lower FEV1/FVC [Chlamydia pneumoniae and measure of air trapping while there was a negative correlation with FEV1/FVC, post-BD FEV1 and sGaw.In our population, IgA + /IgG + patients with asthma had lower FEVFEV1/FVC . We founChlamydia pneumoniae infection had a faster decline in FEV1 [Chlamydia pneumoniae. This might be due to remodeling induced by chronic infection with this intra-cellular bacterium. We are the first to report a decreased conductance in IgA + /IgG + patients with asthma, an indirect sign of increased bronchial hyperresponsiveness.Ten Brincke et al.  previous in FEV1 . We foun in FEV1  in a popChlamydia pneumoniae.Even if we found an increased fibrinogen level in the IgA + /IgG + asthmatics, this level remained within the normal range. This may reflect the low grade chronic inflammation induced by the persistence of intracellular Chlamydia pneumoniae seropositive asthmatics, patients with a history of Mycoplasma pneumoniae infection were not frequent in our study and did not exhibit specific features. This is in line with the findings of Ansarin et al. who reported no difference in asthma control and lung function tests between asthmatics with and without evidence of chronic Mycoplasma pneumoniae infection [As opposed to nfection .Chlamydia pneumoniae IgA + /IgG + asthmatics has led to consider treatment of atypical infections as an aspect of the management of asthma patients. Recent studies have evaluated the benefits of using macrolides in asthma and have shown improvement in symptoms and reduction of exacerbations [The more frequent type-1 inflammation observed in rbations . In patirbations  and asthrbations  have beeChlamydia pneumoniae lung infections could indeed explain the effect of macrolides in asthma [Chlamydia pneumoniae and Mycoplasma pneumoniae but this was not confirmed by other groups [Macrolide mechanisms of action in asthma are thought to be directly anti-inflammatory or indirectly anti-inflammatory with an anti-microbial against chronic atypical infections . A pre-en asthma . Kraft en asthma  reportedr groups , 44.Chlamydia pneumoniae received macrolides after the first visit at the Asthma Clinic for neutrophilic asthma insufficiently controlled with ICS/LABA treatment. Patients came back 9 to 12 months later for a follow-up visit. We found a significant decrease in blood neutrophils and a significant reduction in sputum neutrophils after treatment with macrolides. Previous trials have found an improvement in FEV1 value after antibiotic treatment. Like the study of Gibson et al.[Chlamydia pneumoniae IgA + /IgG + and sputum neutrophils ≥ 76% with macrolides may allow a normalization of bronchial neutrophilic inflammation. Concerns about adverse effects of macrolides however include development of antibiotic resistance.In our study, 14 patients IgA and IgG positive against on et al., we did on et al., 45. In Chlamydia pneumoniae and Mycoplasma pneumoniae have been inferred serologically without isolation of organism. Chlamydia pneumoniae and Mycoplasma pneumoniae are however difficult to culture. We used IgA and IgG measurement for identification of chronic infection without the detection of Chlamydia pneumoniae mRNA within tissue and cell types by PCR. Direct methods such as mRNA detection can establish the presence of the pathogen and perhaps its viability. The sensitivity is however limited due to low copy numbers or sampling problems due to deep tissue intracellular location for this species. Therefore seroreactivity is valuable to differentiate acute from chronic infection.A potential limitation of the present study is that infection with C. pneumoniae have more severe disease with increased airway obstruction, higher doses of ICS, more signs of air trapping and less type-2 inflammation. As opposed to Chlamydia pneumoniae seropositive asthmatics, patients with a history of Mycoplasma pneumoniae infection did not exhibit specific features. Treatment of Chlamydia pneumoniae seropositive neutrophilic asthmatics with macrolides significantly decrease sputum and blood neutrophils.Chlamydia serologic test is more often positive than Mycoplasma serology in asthmatics. Patients with elevated levels of IgA and IgG against


Cancer development is mediated by oxidative stress and inflammation, which may correlate with metabolic disorders. The aim of this study was to evaluate antioxidant vitamins status and metabolic parameters in patients with oral cancer according to tumor-node-metastasis (TNM) stages.A total of 194 patients with oral cancer were enrolled in this study. The patients were stratified for four groups according to cancer stages and that the statistics are comparisons across these groups. The levels of antioxidant vitamins , metabolic parameters, oxidative stress, antioxidant enzymes activity, and inflammatory markers were measured.p = 0.03) activity and higher inflammatory markers levels  than patients in the other stages. In addition, the level of β-carotene was negatively associated with waist circumference, and ubiquinone was positively associated with the level of high-density lipoprotein cholesterol (p < 0.05). Higher β-carotene and ubiquinone levels were negatively associated with hypertriglyceridemia and the risk of metabolic syndrome (p < 0.05).More than half of the subjects had high blood pressure, central obesity, hyperglycemia, and hyperlipidemia regardless of TNM stage. With regard to antioxidant vitamins status, 46 and 94% of patients had β-carotene and ubiquinone deficiency, respectively. Patients in T3 and T4 stages had significantly lower antioxidant enzyme , supplementation with antioxidant vitamins such as ubiquinone or β-carotene could be preferentially applied. Current research has stated that tumor progression is related to changes in energy metabolism, resistance to apoptosis, and increased production of reactive oxygen species (ROS) , 2. HighAntioxidants play a critical role in cancer prevention by reducing oxidative stress and chronic inflammation . It is wThis is a cross-sectional study. Patients who were over 20 years old and under 80 years old who were treated at a single medical center and who were diagnosed with oral cancer by stomatologist met the study entry criteria. The exclusion criteria included the following: 1) liver (hepatitis B or C), kidney or gastrointestinal diseases; 2) the use of antioxidant supplements; and 3) pregnant or lactating women. The study was approved by the Institutional Review Board of Taichung Veterans General Hospital, Taiwan. The subjects participated in the research after signing the informed consent form.A questionnaire was used to understand the characteristics, including sex, age, lifestyle habits, and family medical history, of the subjects. The definitions for lifestyle habits as follows: smokers: individuals regularly smoking one or more cigarettes per day; for alcohol use: individuals regularly consuming one or more drink per day; betel nut use: individuals regularly consuming one or more betel nut per day. Exercise: individuals regularly exercising at least 3 times per week. The height, weight, and waist circumference of each subject were measured, and then the body mass index (BMI) was calculated. Central obesity was defined by a waist circumference ≥ 90 cm for male; ≥ 80 cm for female. Blood pressure was measured by an electronic sphygmomanometer, and a systolic blood pressure (SBP) ≥ 130 mmHg or a diastolic blood pressure (DBP) ≥ 85 mmHg was defined as high blood pressure.Subjects who were hospitalized and asked to fast for at least 8 h before surgical therapy, blood samples were collected in vacutainers containing K3-EDTA anticoagulant, sodium fluoride, or in the tube without anticoagulant. Then, these samples were centrifuged for 15 min to separate plasma, red blood cells (RBCs), and serum and stored at − 80 °C until analysis. After tumor resection surgery, the pathologist determined the pathological staging of the tumor in patients with oral cancer.Fasting glucose was analyzed by an automated chemistry analyzer , and a level ≥ 5.55 mmol/L was defined as hyperglycemia. Lipid profiles, including triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were analyzed by an automated chemistry analyzer , and hyperlipidemia was defined by TG ≥ 1.70 mmol/L, LDL-C ≥ 2.59 mmol/L, or HDL-C ≤ 1.04 mmol/L for male and < 1.30 mmol/L for female. High sensitivity C-reactive protein was measured by latex immunoassay. The level of IL-6 was analyzed by an enzyme-linked immunosorbent assay using commercial kits . The definition of high blood pressure, central obesity, hyperglycemia, and hyperlipidemia were according to the guidelines of the Administration of Health Promotion, Ministry of Health and Welfare, Taiwan (2007). The diagnostic criteria for metabolic syndrome in Taiwan are in accordance with the National Cholesterol Education Program Adult Treatment Panel III , International Diabetes Federation , and the American Heart Association/National Heart, Lung, and Blood Institute  criteria –23.The concentration of malondialdehyde was analyzed using the thiobarbituric acid colorimetric method . The antAntioxidant vitamins status was measured by high-performance liquid chromatography (HPLC). The measurement of ubiquinone was performed according to the method used by Littarru et al. . The anap value < 0.05.All statistical tests in the study were conducted using SigmaPlot software . Descriptive statistics are presented as the mean ± standard deviation (median) or percentages. The normality of the distribution of the data was examined using the Shapiro-Wilk test. One-way ANOVA or Kruskal-Wallis test was used to examine the differences in antioxidant vitamins, metabolic parameters, oxidative stress, antioxidant enzymes activity, and inflammation according to the stage of the oral cancer; post hoc tests were further used to compare the differences among the stages. A chi-square test or Fisher’s exact test was used to compare the differences in categorical variables. Spearman’s rank order correlation coefficient was used to determine the correlations between antioxidant vitamins status and metabolic parameters in patients with oral cancer. The logistical regression analyses were used to examine the correlations between antioxidant vitamins and metabolic syndrome . The significance of level was set at a p < 0.05), and slightly higher level of fasting glucose (p = 0.06) than patients in the other stages. Over 60 % of the subjects had current or ever smoking cigarette habits, alcohol use, or betel nut use, and subjects in the T4 stage had the highest proportion of smoking, alcohol consumption, and betel nuts use.The characteristics of the subjects according to TNM stages are shown in Table Figure p = 0.03) activity than the subjects in the other stages. With regard to inflammation status, the subjects in the T4 stage had a significantly higher level of hs-CRP (p < 0.01), and the level of IL-6 was significantly higher in the subjects with T3 and T4 stages than in the subjects in the other stages (p < 0.01).Table The prevalence of antioxidant vitamins deficiency in patients with oral cancer according to TNM stages is shown in Fig. r = 0.17, p < 0.05) and inflammation status . Among the four antioxidant vitamins, β-carotene and ubiquinone status were significantly negatively correlated with the level of TG . In addition, the level of β-carotene and ubiquinone was significantly correlated with waist circumference  and HDL-C level , respectively. Furthermore, we examined the correlations between β-carotene and ubiquinone status and the risk of metabolic syndrome  tended to increase the probability of death [In the present study, we found that more than half of the patients had high blood pressure, central obesity, hyperglycemia, and hyperlipidemia regardless of the TNM stage Fig. . The preof death . In factRecently, a case-control study conducted by Godala et al. revealed that patients with metabolic syndrome had a higher risk of antioxidant vitamins deficiency, particularly deficiencies in vitamin A, C, and E , 32. In Patients with malignancies had a lower β-carotene level, which has been found in previous studies , 40. Thep = 0.02; IL-6, p = 0.01). Unhealthy behaviors, such as betel-nut chewing, smoking or alcohol consumption, may contribute to chronic inflammation, and increase the risk of metabolic syndrome [Inflammation and ROS are both key factors in carcinogenesis . Patientsyndrome , 44 In Tsyndrome  and incrThis study is the first to investigate the antioxidant vitamins status in patients with oral cancer. In this cross-sectional study, we observed that a high proportion of patients with oral cancer had ubiquinone or β-carotene deficiency. Higher ubiquinone or β-carotene status was associated with reduced risk of central obesity, hypertriglyceridemia, and metabolic syndrome. Since patients with oral cancer suffer from high oxidative stress and inflammation, supplementation with antioxidant vitamins such as ubiquinone or β-carotene could be preferentially applied. Further interventional studies addressing metabolic issues are needed to clarify the significance of antioxidant vitamins in patients with oral cancer.


Herein, we examined whether H2 has a salutary effect on hypertension in animal models. We subjected 5/6 nephrectomised rats to inhalation of either H2 (1.3% H2 + 21% O2 + 77.7% N2) or control (21% O2 + 79% N2) gas mixture for 1 h per day. H2 significantly suppressed increases in blood pressure after 5/6 nephrectomy. The anti-hypertensive effect of H2 was also confirmed in rats in a stable hypertensive state 3 weeks after nephrectomy. To examine the detailed effects of H2 on hypertension, we used an implanted telemetry system to continuously monitor blood pressure. H2 exerted an anti-hypertensive effect not only during daytime rest, but also during night-time activities. Spectral analysis of blood pressure variability revealed that H2 improved autonomic imbalance, namely by suppressing the overly active sympathetic nervous system and augmenting parasympathetic nervous system activity; these effects co-occurred with the blood pressure-lowering effect. In conclusion, 1-h daily exposure to H2 exerts an anti-hypertensive effect in an animal model of hypertension.A recent clinical study demonstrated that haemodialysis with a dialysate containing hydrogen (H We previously demonstrated in animal models that H2 inhalation is a promising therapeutic option for ischaemia–reperfusion injury in emergency and critical care settings such as acute myocardial infarction, cardiac arrest, and haemorrhagic shock5. We also conducted a proof-of-concept clinical study to validate the efficacy and safety of H2 gas inhalation therapy for patients with acute myocardial infarction and post–cardiac arrest syndrome7. A multicentre, double-blind, randomised controlled trial is currently underway to test the therapeutic effects of H2 on brain damage in patients resuscitated from cardiac arrest to obtain Pharmaceutical Affairs approval from the Ministry of Health, Labour and Welfare8. Apart from inhibiting ischaemia–reperfusion injury, H2 has been shown in animal studies to effectively suppress cytokine storms in a state of overreaction of the immune system. Notably, the Chinese National Health and Medical Commission recommends H2 inhalation in addition to general O2 therapy for treating coronavirus disease 2019 (COVID-19)-associated pneumonia or acute respiratory distress syndrome, and the effect of H2 inhalation in patients with COVID-19 was recently reported10.Molecular hydrogen  and control (control 5/6 Nx) for 4 weeks , although the difference in body weight between the two groups disappeared after 4 weeks  or 5/6 nephrectomy (5/6 Nx). Post–5/6 Nx rats were randomised into two groups: 1-h daily exposure to H2 reduced inflammation associated with the nephrectomy. To test this hypothesis, we examined whether the blood pressure–lowering effect persisted even when H2 inhalation was started 3 weeks after 5/6 Nx. Blood pressure was measured weekly using a tail cuff. Thirty-four rats were subjected to 5/6 Nx; of these, 4 rats whose mean blood pressure did not increase to 105 mmHg or more 3 weeks after 5/6 Nx were excluded from subsequent experiments. The remaining 30 rats were randomly assigned to treatment and control groups   Fig. b. As treant Fig. c.Figure 2 therapy on autonomic nervous system activity, we conducted spectral analysis of blood pressure variability. Spectral components were obtained in normalised units (nu). Low-frequency (LF) power indicates a predominantly sympathetic tone, whereas high-frequency (HF) indicates a mostly parasympathetic tone. Although the increase in LF power and decrease in HF power over time associated with 5/6 Nx showed a tendency to be suppressed by H2 inhalation, the results were not significant when compared as continuous variables over a 4-week time course . A longer treatment period led to greater reductions in blood pressure, heart rate, and LF power and increases in HF power in the 5/6 Nx + H2 group compared to in the control 5/6 Nx group  has been widely accepted as the most likely mechanism of action of H2. Although H2 has been shown to selectively eliminate •OH in a test tube27, whether H2 reduces oxidative stress by eliminating •OH at the cellular and tissue levels remains unclear.The therapeutic potential of H. showed that one brain region, the rostral ventrolateral medulla (RVLM), is important for the activation of sympathetic nerves, with a decrease in nitric oxide activity and increase in the production of ROS in RVLM as its possible mechanisms30. Campese et al. also showed that increased ROS production occurs in the brain during central sympathetic activation, which occurs during kidney injury32. H2 may suppress central sympathetic activity by scavenging ROS in the RVLM, thereby lowering the blood pressure and heart rate.Activation of sympathetic nerves can lead to high blood pressure. Hirooka et al2 treatment reduced the degree of weight loss and promoted weight recovery after nephrectomy. Postoperative weight loss in humans contributes to poor prognosis by increasing the likelihood of complications and weakening the effectiveness of chemotherapy. Our results suggest that if these effects occur in humans, postoperative administration of H2 may shorten the length of hospital stays and improve the prognosis of patients after a variety of surgeries.Although not the focus of our study, it is worth noting that postoperative H33 may not have been completely eliminated when the measurements were taken. Third, further studies are needed to determine the optimum concentration of H2 and optimise the time of inhalation. Fine-tuning of the concentration of H2 required for different therapeutic outcomes will be required. Although concentrations of 1.3–4% have mostly been used in clinical trials relying on H2 inhalation, an increase in capillary blood flow has been observed by nail capillary microscopy even when inhaling much lower concentrations of H2 and after drinking H2-rich water. The amount of H2 required may vary depending on the biological effects being targeted. As this is the first study to investigate the therapeutic effects of H2 on hypertension, we used H2 with a sufficiently high concentration (1.3%) to ensure that any effect was detected. In fact, the maximum H2 concentration allowed under Japanese regulation to be added to a cylinder containing O2 is 1.3%, which is a third of the lower flammability limit of 4%. Our results revealed that at least 1 h of H2 inhalation per day was required to observe haemodynamic changes; however, whether inhalation of H2 for a longer time has a better effect should be further evaluated. Finally, additional studies are required to elucidate the versatility of H2 on blood pressure in other animal models of hypertension.Our study has several limitations. First, insertion of the implanted telemetry system is a stressful procedure for rats and thus may have affected the variability in blood pressure and heart rate in. In addition, environmental factors such as sounds or odours2 therapy lowered blood pressure in a rat model of hypertension and improved the imbalance of the autonomic nervous system caused by hypertension. Additional pre-clinical studies are required before the translational research.In conclusion, our study showed that daily 1-h H2 and control groups was performed randomly. All animal experiments were performed in accordance with ARRIVE guidelines34.Male Lewis rats  were used . Animals were fed standard chow ad libitum with free access to water and were not fasted prior to the experiments. Animals were housed under standardised temperature (22 ± 1 °C) and humidity (55 ± 5%) conditions with a 14-h:10-h light: dark cycle. The rats were allowed to acclimatise to the above-mentioned conditions for a minimum of 1 week prior to the experiments. The study was approved by the Institutional Animal Care and Use Committee . Allocation to the H2 inhalation system that simultaneously allowed gas inhalation and ambulatory blood pressure monitoring. We used an anaesthetic box  for gas administration. The oxygen concentration was measured using a portable complex gas detector  at nine areas in the box   was flowed and H2 and O2 concentrations were measured at nine areas in the box using a GX-2009 detector . We confirmed that the inside of the box was uniformly filled with the mixed gas.We sought to establish a precisely titrated H2 gas (1.3% H2 + 21.0% O2 + 77.7% N2) and control gas (21.0% O2 + 79.0% N2) at a factory . The H2 group and control group inhaled the premixed H2 gas and control gas, respectively. The gas flow rate was 10 L/min and the animals were kept in the box for 1 h at a time. An additional 3 min was set for purging the box . In all experimental protocols, the day on which the gas inhalation was initiated was uniformly defined as day 0. The treatment was repeated every day for 4 weeks.Gas cylinders for injection into the anaesthetic box were filled with H37, with minor modifications. Briefly, the rats were anaesthetised via isoflurane inhalation , and an upper median laparotomy of approximately 4 cm was performed. Surgical procedures were carried out under a microscope; the branches of the left renal artery were identified and then selectively ligated with 7–0 silk sutures as close to the left kidney as possible to grossly infarct approximately 2/3 of the left renal cortex. Subsequently, the right renal artery, renal vein, and ureter were ligated with 4–0 silk sutures and the right kidney was resected. The abdominal wall and skin were closed with 4–0 nylon thread. Sham-operated rats were subjected to the same laparotomy as 5/6 Nx except that surgical interventions to the kidneys was not performed. The anaesthesia time was 30 min, which was uniform for all rats.To induce renal hypertension, 5/6 nephrectomy was performed as previously describedIn the first experiment, arterial blood pressure was measured at the right femoral artery using a pressure transducer  after 4 weeks of gas inhalation under anaesthesia induced by isoflurane inhalation. In the second experiment, blood pressure and heart rate were measured by the tail cuff manometry  method. Blood pressure and heart rate were measured in triplicate, and the median value was used as the representative value for that individual.38. The tip of the transmitter catheter was placed in the abdominal aorta caudally from the renal artery bifurcation. The transmitter body was inserted into a subcutaneous pocket created on the left lower back, and the skin was sutured. All surgical interventions were performed in an aseptic manner.Rats were anaesthetised using isoflurane and the left groin was disinfected with 1% chlorhexidine. An incision of approximately 1.5 cm was made and the left femoral artery was exposed. A telemetric transmitter  catheter was inserted into the left femoral arteryBlood pressure and heart rate were continuously monitored using a telemetry system . Data were recorded for 60 min in a breeding cage. Blood pressure data obtained at 1 and 6 h after stopping inhalation were used for analysis. If this measured value was a significant outlier compared to the other measurements of the same individual on the same day, the data obtained from the hour before inhalation were used instead. Of the recorded data, those acquired within the first 15 min of each measurement period were excluded from analysis. Data retrieved in the remaining 45 min were divided into 5-min blocks. Blood pressure and heart rate display great fluctuations when the rats move; thus, in each 5-min data block, the values in the first 1 min of stable measurement were recorded and averaged. Stable measurement was defined as data without body movement and without any parts missing due to loss of signal; if these conditions were not met, the data were considered as invalid. Within each 5-min block, if the data of the first minute was deemed as invalid, data from the following minute were used. In the rare cases in which all single-minute intervals within the 5-min block were invalid, the data from that block were discarded completely.Arterial blood pressure variability was analysed using a telemetry system software . Frequency domain analysis  was performed using arterial pressure waveform data of the first minute (approximately 300–400 beats) every 5 min. Similar to the method employed for evaluating haemodynamic parameters, in case there was a body movement or any missing data due to loss of signal, the next minute was used, and if all single-minute intervals of the 5-min block were invalid, data from that block were discarded.Renal function was assessed before, one day after nephrectomy, and weekly by using a handheld blood analyser . Daily urine volume was measured weekly using a metabolic cage  and creatinine clearance (mL/min/kg BW) was calculated. The left kidney weight was measured at the end of the experiment after 4 weeks of gas inhalation.t-test, paired t-test, or Mann–Whitney U-test, as appropriate. The Tukey’s multiple comparison test was followed by analysis of variance. The mixed-effect model was used to analyse the repeated measures. All tests were two-tailed, and a P value < 0.05 was considered as statistically significant. All statistical analyses were conducted using GraphPad Prism 8.0 .Descriptive statistics are presented as the mean ± standard error of mean. Comparisons were conducted using analysis of variance, unpaired Supplementary Information


ICGap, representing the gap between baseline ICP and baseline CVP. The aim of the current study was to explore the relationship between PICGap and ICP responsiveness to PEEP.Mechanical ventilation (MV) with positive end-expiratory pressure (PEEP) is commonly applied in patients with severe traumatic brain injury (sTBI). However, the individual responsiveness of intracranial pressure (ICP) to PEEP varies. Thus, identifying an indicator detecting ICP responsiveness to PEEP is of great significance. As central venous pressure (CVP) could act as an intermediary to transduce pressure from PEEP to ICP, we developed a new indicator, P2) were recorded at the initial (3 cmH2O) and adjusted (15 cmH2O) levels of PEEP. PICGap was assessed as baseline ICP - baseline CVP (when PEEP = 3 cmH2O). The patients were classified into the ICP responder and non-responder groups based on whether ICP increment with PEEP adjusted from 3 cmH2O to 15 cmH2O was greater than 20% of baseline ICP. The above parameters were compared between the two groups, and prediction of ICP responsiveness to PEEP adjustment was evaluated by receiver operating characteristic (ROC) curve analysis.A total of 112 patients with sTBI undergoing MV were enrolled in this prospective cohort study. ICP, CVP, cerebral perfusion pressure (CPP), static compliance of the respiratory system (Cst), and end-tidal carbon dioxide pressure , lower baseline ICP, and higher baseline CVP. ROC curve analysis suggested that PICGap was a stronger predictive indicator of ICP responsiveness to PEEP  compared with baseline ICP and baseline CVP, with favorable sensitivity  and specificity , at a cut off value of 2.5 mmHg.Compared with the non-responder group, the responder group had lower PICGap. In addition, PICGap is a potential predictor of ICP responsiveness to PEEP adjustment in patients with sTBI.The impact of PEEP on ICP depends on the gap between baseline ICP and baseline CVP, i.e. P Mechanical ventilation (MV) with positive end-expiratory pressure (PEEP) is often required in patients with severe traumatic brain injury (sTBI) due to neurologic, airway, and pulmonary dysfunctions , 2. Appl2O range increases ICP (> 10 mmHg)  < 90%) and MV  were initially included. The patients whose hypoxemia could not be corrected even by increasing the fraction of inspired oxygen (FiO2) by more than 60% in combination with suction and intensive airway management were eventually enrolled. Exclusion criteria included brain death, age below 18 or over 80 years, pregnancy in women, hemodynamic instability , pneumothorax, pulmonary bulla, acute myocardial infarction  and neurosurgical intervention.All patients with sTBI  diagnosed via head CT scan, ICP monitoring transducers or catheter, hypoxemia  through an intraparenchimal transducer or a ventricular catheter  that was associated with a closed external ventricular drain in each measurement when applicable. Both central venous and arterial catheters were inserted to measure intra-arterial mean arterial pressure (MAP) and CVP. CPP was maintained at more than 60–65 mmHg. The static compliance of the respiratory system (Cst) recorded from the ventilator was indexed to the predicted body weight of the patient. During the study, end-tidal carbon dioxide pressure (PetCO2)  was maintained at 30–35 mmHg by adjusting tidal volume and the respiratory rate, in order to avoid any effect of CO2 on ICP [The treatment process followed the Guidelines for the Management of sTBI . All pat2 on ICP .2 was set, and PEEP was increased stepwise  every 2 min, which was a recruitment maneuver known as “extended sigh”. ICP, CVP, Cst, PetCO2, and CPP at both levels of PEEP (3 cmH2O and 15 cmH2O) were measured, respectively. After PEEP at 15 cmH2O was maintained for 2 min, baseline ventilator setting was resumed.The stepwise increase of PEEP was set according to a method by Lim et al.  when hypICGap and other measurements  were compared between the responder and non-responder groups, and prediction of ICP responsiveness to PEEP was tested by assessing the area under the receiver operating characteristic (ROC) curve (AUC).Based on our research hypothesis and the specific relationship between CVP and PEEP , 17, PIC2O ; (4) SpO2 declined progressively (PEEP was restored to 0); and (5) suspicion of pneumothorax . An equilibration period (≥ 90s) was entailed to ensure a normalized baseline PetCO2 through tidal volume and respiratory rate modulation, as described by Flexman and colleagues [In this study, treatment was provided in case of: (1) CPP < 60 mmHg (norepinephrine 0.3–1.0 μg/kg/min); (2) ICP > 25 mmHg (PEEP was restored to 0); (3) increase in pressure plateau > 35 cmHperiod ≥ 0s was en2O to 15 cmH2O. Because there is no specified definition for ICP responsiveness, we stipulated that response and non-response referred to increments greater than and less than 20% of baseline ICP, respectively. The patients were then classified into the responder and non-responder groups accordingly.The main outcome was ICP responsiveness to PEEP adjustment from 3 cmH2 and CPP, were expressed as mean ± standard error. One-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used for multiple comparisons. The predictive roles of PICGap other related parameters recorded for ICP responsiveness to PEEP were tested by calculating the AUCs of the ROC curves of ICP over the baseline value at two levels of PEEP (3 and 15 cmH2O). P <  0.05 was considered statistically significant. All statistical analyses were performed with SPSS 20.0 for windows .Categorical variables were presented as number and percentage, and analyzed by Fisher’s exact test. Continuous covariates, including hemodynamic variables ICP, CVP, CPP, Cst, PetCO2, and 112 were entered into the final analysis . Baseline characteristics of the study population in the responder (n = 49) and non-responder (n = 63) groups are shown in Table From May 2016 to May 2019, a total of 174 patients were included initially. Sixty-two patients were excluded for hemodynamic instability neurosurgical interventions before admission of ICU, and hypoxemia corrected by suction and increase of FiOICGap , lower baseline ICP , and higher baseline CVP .Compared with the non-responder group, the responder group had lower P2O increased CVP levels significantly in both groups  , with no change in the non-responder group   among the three parameters. At a cut off value of 2.5 mmHg, PICGap had favorable sensitivity  and specificity  in predicting ICP responsiveness to PEEP. However, baseline ICP had an overtly weaker predictive ability compared with PICGap , and baseline CVP had the weakest ability for predicting ICP responsiveness among the three parameters .As shown in Table ICGap was narrower in the responder group compared with the non-responder group (1.63 ± 1.33 versus 6.55 ± 2.46 mmHg). This indicates that the same increment of CVP (4.39 ± 1.30 versus 4.25 ± 1.58 mmHg) could obliterate PICGap in the responder group, but not in non-responders  is higher than the venous pressure outside the dura (0–5 mmHg), the changes of extradural venous pressure transfer to the brain circulation might rely on a certain situation . The degCVP could act as a surrogate marker of extradural venous pressure, because the pressure falling on the jugular vein is negligible in the supine position. A preliminary experiment also showed that CVP values were the same as those of jugular bulb pressure. According to the Starling resistor model , once th2O in PEEP caused a more than 4 mmHg rise of CVP in the current study, which was consistent with previous findings [ICGap narrowing by CVP.The relationship between PEEP and CVP has been validated by previous studies. Stepwise PEEP elevation induces an increase of CVP . An incrfindings . Thus, iICGap, the easier it is for CVP after PEEP to exceed baseline ICP; then, the Starling resistor would lose effectiveness as a result of the elimination of venous outflow resistance. As indicated in Table ICGap compared with the non-responder group. Thus, based on the hypothesis that CVP is an intermediary which connects PEEP to ICP, we found that PICGAP, a new indicator, could provide a rational explanation regarding the underlying mechanism, which also accounts for the individual heterogeneity proposed by Yang and colleagues [The lower the Plleagues .ICGap is narrowed to zero. A study by Robba and colleagues investigated the effects of pneumoperitoneum and the Trendelenburg position on ICP in non-brain injured patients (lower ICP), and demonstrated that both increase ICP [Brain compliance is unfavorable in patients with sTBI because of cerebral edema associated with injury. In this case, cerebral venous return impeded by increased CVP after PEEP would contribute to increasing ICP after Pease ICP . There wease ICP .ICGap.Several studies used baseline ICP to predict the responsiveness of ICP to PEEP, and found that patients with lower baseline ICP have a positive response to various PEEP levels , 26. The2 increase [It should be mentioned that ICP responsiveness to PEEP may be influenced by compliance of the respiratory system , 28. Assincrease .2O during lung recruitment was to avoid the harmful effects of high PEEP (> 20 cmH2O), such as worsened hemodynamics and significantly increased ICP. Consequently, no serious complication was recorded in this study (Table 2O for 2 min helped achieve pulmonary bloating [2 to avoid the impact of CO2 retention on ICP [It is known that sTBI patients tend to progress rapidly early after injury; therefore, they were kept in the ICU for 24 h after injury for close monitoring of ICP in this study. To prevent catheter-related infections, the ICP monitoring probe was implanted for no more than 7 days. In case of hypoxemia, increased PEEP was provided for lung recruitment and increased oxygen saturation. The purpose of a PEEP of 15 cmHdy Table . In addih PEEP >  cmH2O, sn on ICP . PEEP in2 at the normal level, and ICP elevation was in a permissible range, indicating likely stable CBF. Furthermore, although PICGap is a dynamic marker, it did not change dramatically in the early stage of TBI in certain patients after neurosurgery, ensuring the predictive value individually.The limitations of this study should be mentioned. First, the sample size was relatively small for a clinical study. In addition, the effect of PEEP on CBF was not evaluated. However, we maintained PetCOICGap, a new indicator, could be a potential predictor of ICP responsiveness to PEEP adjustment in patients with sTBI.The impact of PEEP on ICP depends on the gap between baseline ICP and CVP. PAdditional file 1: Table S1. Hemodynamics and respiratory variables at PEEP of 15 cmH2O between two groups.Additional file 2: Table S2. Comparison of prognosis between two groups.


Supplemental Digital Content is available in the text. P=0.876), despite an initial increase in mass in donors compared with controls at 12 months. Pulse wave velocity, which increased in donors at 12 months, returned to levels not different from controls at 5 years . Change in ambulatory systolic blood pressure was not different in donors compared with controls . We found no evidence that the reduction in estimated glomerular filtration rate after kidney donation was associated with a change in left ventricular mass detectable by magnetic resonance imaging at 5 years.Kidney donation reduces renal function by ≈30% allowing study of the cardiovascular effects of a reduced estimated glomerular filtration rate without comorbidities. We report 5-year results of a longitudinal, parallel-group, blinded end-point study of living kidney donors (n=50) and healthy controls (n=45). The primary end point, left ventricular mass, was measured using cardiac magnetic resonance. Secondary end points, 24-hour ambulatory blood pressure, and pulse wave velocity were measured using validated blood pressure monitors and the SphygmoCor device. Effect sizes were calculated as differences between change from baseline in the donor and control groups. In donors, estimated glomerular filtration rate was 95±15 mL/min per 1.73 m2 at baseline (predonation) and 67±14 mL/min per 1.73 m2 at 5 years. In controls, there was a −1±2 mL/min per 1.73 m2 decline per annum. Change in left ventricular mass at 5 years was not significantly different between donors and controls (mean difference, +0.40 g   To date, most clinical outcome studies from kidney donors have not demonstrated an increase in major cardiovascular events.https://www.clinicaltrials.gov (unique identifier: NCT02973607).The data that support the findings of this study are available from the corresponding author upon reasonable request. Preregistration of the study can be found at 12CRIB-DONOR II (NCT02973607) was a longitudinal, 5-year prospective parallel-group study designed to follow-up kidney donors and healthy controls recruited into the CRIB-DONOR study. All participants who originally consented to take part in the CRIB-DONOR study (NCT01028703) were approached for follow-up between May 2017 and May 2019.Ethical approval was obtained from the West Midlands Solihull Research Ethics Committee (REC 17/WM/0048) and approved by the Health Research Council. All subjects gave informed consent to take part in accordance with the principles set out in the Declaration of Helsinki.13 The methods and protocol have been published previously.13The study was designed to collect data ≈5 years after the original date of enrollment and as far as possible, to use the same methods, equipment, and assays described in the CRIB-DONOR study.Data Supplement, Supplemental Methodology 1).12CMR studies  were performed at 5 years using the same standard steady-state free-precession cine and aortic distensibility imaging protocol as described previously . Three-dimensional tissue tracking for 3-dimensional global circumferential strain and global longitudinal strain was performed as previously described with the baseline and 12-month data reanalyzed (TomTec 2D not available for CRIB-DONOR II) to allow comparison.15 Aortic distensibility was assessed using software developed in Matlab, version R2017a .16All LV mass and volume measurements were made at a central CMR core laboratory by 2 independent expert observers (A.M.P. and W.E.M.) blinded to both donor/control status and temporal order . Delineation of trabeculations and papillary muscles was performed using thresholding to determine the endocardial border.17 Quantification was made using full-width half-maximum methodology.18 For assessment of T1, T2, and extracellular volume, the myocardium at the midventricular slice was segmented into American Heart Association segments, and global values were calculated as an average of the valid segments .21Late gadolinium enhancement (LGE) was defined based on definitions described previously.Data Supplement, Supplemental Methodology 3).23Pulse wave analysis, pulse wave velocity, and central BP were measured with the SphygmoCor device  and a high-fidelity micromanometer  as described previously  with an appropriate size cuff on the nondominant arm with the elbow rested on a pillow in the dorsiflex position.26At the end of the study, subjects were fitted with ambulatory BP monitors  set to measure BP every 30 minutes during the day (from 8:00 to 22:00) and every hour at night (from 22:01 to 7:59). An appropriate size cuff was chosen and fitted to the nondominant arm by a trained observer. The validity of a recording and definition of hypertension was in accordance with the European Society of Hypertension guidelines.27 Three measurements were taken 1 cm from the carotid bifurcation, and the mean of both internal carotid arteries was used in final analysis.12Carotid intima-media thickness was measured in real time using ultrasound  using IMT QLAB  software for automated tracking of the wall.28 At 5 years, kidney donors but not controls underwent iGFR assessment. For assessment of isotopic glomerular filtration rate, a total of 1.85 MBq of chromium-51 labeled EDTA was injected into a vein in the antecubital fossa. Venous blood samples were taken at 2, 3, and 4 hours post-injection if the eGFR was >60 mL/min per 1.73 m2, otherwise 2, 4, and 6 hours if <60 mL/min per 1.73 m2. Samples were counted the following day using a Cobra Auto Gamma Counter . The CKD Epidemiology Collaboration 2009 equation was used to calculate eGFR.29Isotopic glomerular filtration rate (iGFR) measurement was determined using clearance of chromium-51 labeled EDTA in accordance with the British Nuclear Medicine Society guidelines.FGF23 (fibroblast growth factor-23) was measured using frozen plasma stored at −80 °C, using the C-terminal kit from Immunotopics . N-terminal pro-B-type natriuretic peptide and high-sensitivity troponin T were measured on frozen serum stored at −80 °C, using the Elecsys Cobas immunoassay (Roche Diagnostics).Data Supplement . Using the effect sizes and variances from the CRIB-DONOR study , recruiting 50 subjects in each group would provide 93% power to detect a difference in LV mass of 7 g with an alpha value of 0.05.30 For 80% power, 34 subjects in each group were required.The primary end point was change in LV mass at 5 years compared with baseline. Exploratory secondary end points included changes in BP, pulse wave analysis, pulse wave velocity, aortic distensibility, biomarkers, and carotid intima-media thickness. For details of a combined BP end point, see the t tests. Between-group difference was analyzed using independent samples t tests to compare within-group change at 5 years between groups and generate the P for the primary end point. Nonparametric data were analyzed in a logged format and then antilogged and displayed as multipliers. Categorical data are displayed as counts and percentages, between-group changes are displayed as relative risks and 95% CIs, and analyses were performed using MedCalc for Windows, version 19.4 . Interactions between each variable and donor/control status were determined by general linear models. Multivariable model analysis was performed using linear regression and incorporating any significant interactions. An interval-censored cox regression was used for analysis of the combined BP end point using the icenReg package in R. T1, T2, and extracellular volume, which were measured at 5 years only, were analyzed using independent samples t tests. Reproducibility was assessed using intraclass correlation coefficients.Statistical analysis was performed using SPSS, version 23 . Continuous variables were assessed graphically using histograms to determine normality. Nonparametric data were log10 transformed and assessed graphically. For continuous data, within-group change from baseline to 12 months and baseline to 5 years was analyzed using paired samples Data Supplement, Supplemental Table S1).Records from all 124 subjects who took part in the original study were reviewed. Of these, 1 had died of bronchial carcinoma, and 3 were not contactable; 120 were approached; 50 kidney donors and 45 healthy controls agreed to participate . Three subjects had contraindications to 3T CMR and had 1.5T scans using the same protocol.Data are presented in Table Data Supplement Supplemental Table S2.There were no deaths or major cardiovascular events in subjects during the study period. For details of all incidental findings during the study, see 2; 5 years, 94±15 mL/min per 1.73 m2.In kidney donors, the mean eGFR was 95±15 mL/min per 1.73 m2 at baseline before donation, 65±13 mL/min per 1.73 m2 at 12 months, and 67±14 mL/min per 1.73 m2 at 5 years. Changes in iGFR  in kidney donors were comparable: baseline, 91±12 mL/min per 1.73 m2; 12 months, 59±11 mL/min per 1.73 m2; 5 years, 64±11 mL/min per 1.73 m2. In controls, there was a mean −1±2 mL/min per 1.73 m2 decline annually in eGFR: baseline, 99±16 mL/min per 1.73 m2; 12 months, 96±15 mL/min per 1.73 mP=0.876) Table At 5 years, change in LV mass in kidney donors was not different from healthy controls, +0.40 g . Carotid intima-media thickness at 5 years was greater in donors versus controls but had not increased significantly from previous values.There were no between-group differences in office BP or heart rate at 5 years Table . CompareAt 12 months, there was an increase from baseline in pulse wave velocity in kidney donors, which was not seen in controls. From 12 months to 5 years, the pulse wave velocity increased in both groups, and by 5 years, the between-group difference was not significantly different . There was no significant influence of sex or LV mass at baseline on change in LV mass at 5 years. Change in ambulatory systolic BP, however, was significantly associated with change in LV mass. None of the other variables were significant when included in a multivariable model with change in day systolic BP.A linear regression analysis was performed to determine variables influencing change in LV mass from baseline to 5 years adjusted for both follow-up time and donor/control status .There was high reproducibility for LV mass assessment. The interclass correlation coefficients (95% CIs) for interstudy, intraobserver, and interobserver variability were 0.99 (0.98–0.99), 0.99 (0.96–0.99), and 0.99 (0.97–0.99), respectively .12 Given this, and the strong associations of LV mass with reduced eGFR in community studies, a reduced difference in eGFR might be expected to be associated with a reduced difference in LV mass.37 Other direct and indirect effects of the nephrectomy surgery on LV mass seem unlikely to explain the 12-month findings. Although donors experience an acute reduction in hemoglobin and a rise in erythropoietin and in C-reactive protein, most of these effects have resolved by 12 months.38 Laparoscopic nephrectomy seldom causes long-term pain and is not known to result in autonomic dysfunction. The prevalence of late anemia in kidney donors has been reported at only 11%; consistent with this, we found no difference in hemoglobin at 12 months in our cohort.39 We did not, however, measure erythropoietin, which has been associated with LV hypertrophy.40In the first CRIB-DONOR study, there was a significant increase in LV mass in kidney donors compared with healthy controls at 12 months.41 In the Framingham study, LV mass was second only to age in its ability to predict cardiovascular morbidity and mortality.42 We acknowledge that a causative relationship cannot be assumed and that a meta-analysis has questioned the validity of using LV mass as a surrogate for total mortality in CKD; this study, however, included patients on dialysis, and many of the studies were of inadequate size and duration and measured LV mass by echocardiography, which has limitations in CKD subjects.43Change in LV mass was chosen as the primary outcome for this study because of the well-recognized association of LV hypertrophy with adverse clinical outcomes and the graded relationship between LV mass and prognosis.26 It is likely that the apparent finding of increased rates of hypertension in donors was a result of surveillance bias.44 This phenomenon has been seen repeatedly in living kidney donor studies.44 Our study was not powered to detect small effects on BP, and as the ambulatory BP values in donors at 5 years were numerically slightly higher than those in controls, we suggest that longer and larger studies of ambulatory BP in kidney donors are still required.The increase in self-reported hypertension in the living kidney donor group at 5 years was not consistent with the use of antihypertensives or associated with a significant increase in mean office or ambulatory BPs compared with the control group. Likewise, we found no significant difference in hypertension prevalence according to the European Society of Hypertension ambulatory BP monitoring criteria or combined end-point analysis.45 Of the donors in our cohort, 36% had a glomerular filtration rate of <60 mL/min per 1.73 m2 at 5 years. The precise threshold at which cardiovascular damage and risk occurs is still a subject under study. Most studies suggest that risk increases at around 60 mL/min per 1.73 m2 although effects at levels of kidney function above this have been reported.46 It is possible that epidemiological studies have attributed increased cardiovascular risk to early-stage CKD as a result of inadequate correction for traditional risk factors or that factors present in early-stage CKD due to renal injury but not loss of functioning nephrons play a role in the causation of cardiovascular disease. Proteinuria which is commonly viewed as a reflection of inflammatory mediated endothelial damage is a frequent occurrence in early-stage CKD but is seldom seen in donors.47 Further long-term studies of cardiovascular disease markers and events in kidney donors are required.This study suggests that a reduction in eGFR of ≈30% as a result of living kidney donation is not inevitably associated with adverse cardiovascular effects including a rise in BP. It is possible that the reduction in eGFR in donors is insufficient to cause cardiovascular damage, but we and others have reported adverse cardiovascular structural and functional findings in subjects with early-stage CKD who have eGFR values similar to our cohort.The major strength of this study is that it was a blinded end-point analysis from a prospective longitudinal study of a donor cohort with an appropriately healthy control group allowing assessment of serial change. We experienced a high return rate for a longitudinal study with 79% from the original cohort.48 Our cohort was predominantly White and, therefore, cannot be generalizable to all kidney donors. It has previously been established that risk is highly likely to be race and age dependent.44 Finally, we recognize that due to the large number of variables analyzed, some significant differences are likely to occur by chance and that our sample size limited our ability to detect small changes in secondary end points.Limitations include potential selection bias due to attrition as a result of the longitudinal design. While attempts were made to minimize changes in techniques and methodology, upgrades to our imaging system meant that the magnetic resonance scanner used at 5 years was 3T rather than 1.5T. Signal-to-noise ratio and artifact increase with increasing field strength and can potentially affect scan quality; however, the field strength itself is not deemed to have a significant influence on mass and volume quantification.49 These results provide reassuring information, suggesting lack of cardiovascular harm and increase in BP at 5 years.In summary, we have found no evidence to suggest kidney donation has an adverse effect on cardiovascular structure and function at 5 years over and above those of aging in the general population. The greatest predictor of a change in LV mass in this cohort is in keeping with those well established in the general population, systolic BP.This research was funded by the British Heart Foundation and performed at the National Institute for Health Research (NIHR)/Wellcome Trust Birmingham Clinical Research Facility. The views are those of the authors and not of the National Health Service, the NIHR, or the Department of Health. We would like to thank Peter Nightingale for his statistical input and support.A.M. Price is supported by a British Heart Foundation Fellowship (FS/16/73/32314). V.M. Stoll is supported by a National Institute for Health Research Clinical Lecturer grant.None.