Intensified antimicrobial treatment with higher rifampicin doses may improve outcome of tuberculous meningitis, but the desirable exposure and necessary dose are unknown. Our objective was to characterize the relationship between rifampicin exposures and mortality in order to identify optimal dosing for tuberculous meningitis.An individual patient meta-analysis was performed on data from 3 Indonesian randomized controlled phase 2 trials comparing oral rifampicin 450 mg (~10 mg/kg) to intensified regimens including 750–1350 mg orally, or a 600-mg intravenous infusion. Pharmacokinetic data from plasma and cerebrospinal fluid (CSF) were analyzed with nonlinear mixed-effects modeling. Six-month survival was described with parametric time-to-event models.Pharmacokinetic analyses included 133 individuals . The final model featured 2 disposition compartments, saturable clearance, and autoinduction. Rifampicin CSF concentrations were described by a partition coefficient  and half-life for distribution plasma to CSF . Higher CSF protein concentration increased the partition coefficient. Survival of 148 individuals  was well described by an exponentially declining hazard, with lower age, higher baseline Glasgow Coma Scale score, and higher individual rifampicin plasma exposure reducing the hazard. Simulations predicted an increase in 6-month survival from approximately 50% to approximately 70% upon increasing the oral rifampicin dose from 10 to 30 mg/kg, and predicted that even higher doses would further improve survival.Higher rifampicin exposure substantially decreased the risk of death, and the maximal effect was not reached within the studied range. We suggest a rifampicin dose of at least 30 mg/kg to be investigated in phase 3 clinical trials. Through an individual participant meta-analysis, we characterized the relationship between rifampicin exposure and tuberculous meningitis mortality. Higher rifampicin exposure substantially decreased the risk of death, and doses of at least 30 mg/kg should be moved forward to phase 3 clinical trials. Tuberculous meningitis (TBM) affects 100 000 individuals worldwide annually and has been called the worst possible form of tuberculosis (TB) . OutcomeThe standard dose of rifampicin (10 mg/kg) was selected decades ago, partly based on cost considerations not relevant today . A growimax) function for concentration-effect relationships. Our objective was to characterize the population pharmacokinetics of high-dose rifampicin in plasma and CSF and to evaluate the relationship between individual exposures and mortality. In addition, we performed an exposure–safety analysis relating individual rifampicin levels to occurrence of adverse events. Together this will help to identify the appropriate rifampicin dose to attain desirable exposures, improving the survival of patients with TBM.In this work we conducted a joint analysis using advanced modeling methodology, pooling the data from all 3 trials. For the mortality data, we conducted a time-to-event analysis using parametric hazard models, which yield higher statistical power compared to the conventionally used Cox regression  and alloData originated from 3 randomized phase 2 trials comparing oral rifampicin 450 mg (approximately 10 mg/kg in this patient population) to intensified 14- or 30-day regimens including 750 mg (17 mg/kg), 900 mg 20 mg/kg), or 1350 mg (30 mg/kg) orally, or a 600-mg (13 mg/kg) intravenous infusion (1.5 hours), next to other first-line TB drugs and adjunctive dexamethasone. All 3 studies were conducted in Bandung, Indonesia, and included adult patients with definite , probable, or possible TBM. Details on study design and procedures can be found in the Supplementary Materials and the original publications . In brie mg/kg, oData management, plotting, and postprocessing of results were performed in R , partially using the Xpose package . The modeling and simulations were performed in NONMEM 7.4 , aided by PsN  and Pirana  .Nonlinear mixed-effects methodology was utilized to describe the pharmacokinetic data. Previously published rifampicin population models were used as a starting point and further refined on the current data , 26. PotA total of 148 patients from the 3 studies were available for the joint analysis. The patients were relatively young, 55% were male, and 95% had grade 2 or 3 TBM. Further demographic information is summarized in 0-24h) and peak concentration (Cmax) in plasma and CSF at day 2 ± 1. Typical exposures were imputed based on the model, patient characteristics, and the given dose for the 15 patients missing observed pharmacokinetic data. A histogram depicting the individual exposures and median per dose level can be seen in Based on our pharmacokinetic model, the oral bioavailability of rifampicin was estimated at 77.6% . Rifampicin clearance was found to decrease with increasing plasma concentrations following Michaelis Menten kinetics and the intrinsic clearance was 47.9%  higher after day 4 due to the phenomenon of autoinduction. Volume of distribution was estimated to be 19.3%  lower at the later sampling occasion compared to the first. Concentrations in CSF were directly related to the plasma concentrations through the means of a penetration coefficient  and a half-life for distribution . A correlation between the penetration coefficient and individual protein concentrations in CSF was identified. The relation was described through a linear function with log-transformed protein concentration leading to a 63%  increase in the penetration coefficient with each 10-fold change in protein levels. A full description of the final pharmacokinetic model is available in the Supplementary Materials, including P = .009) and baseline GCS score (P = .0003) were found to significantly affect the hazard, whereas sex, human immunodeficiency virus (HIV) infection, body weight, and CSF neutrophil or protein concentrations did not . Out of the individual exposure metrics, rifampicin plasma AUC0-24h was a better predictor for survival than plasma Cmax or CSF AUC0-24h. An Emax equation allowing for 100% decrease in the hazard was selected to describe the relationship as no maximal effect could be characterized. The plasma AUC0-24h at day 2 ± 1 corresponding to a 50% decrease in the hazard was estimated to 171 mg/L × hour . The bootstrap evaluation confirmed the magnitude but also the relatively large uncertainty in the estimate of this parameter . Further details on the evaluated exposure-response relationships are included in An exponentially declining base hazard model described the survival data the best; P = .47 and P = 1, respectively).The risk of occurrence of at least 1 adverse event for each patient was 65%  in the first 2 studies and 83%  in the last study. The risk of occurrence of at least 1 serious adverse event (grade 3 and 4) was 23%  and not scientifically different between studies. There was no significant effect of rifampicin exposure on either of these risks (Different doses between 450 mg (~10 mg/kg) and 1800 mg (~40 mg/kg) were evaluated for probability of attaining early rifampicin exposures higher than 171 mg/L × hour  or 300 mg/L × hour , as summarized in In this individual patient meta-analysis including data from 3 phase 2 TBM trials investigating intensified rifampicin treatment, we found 3 main factors affecting the chance of survival up to 6 months after start of treatment: disease severity at start of study (quantified by GCS score), age, and rifampicin plasma exposure level. An increase in dose from 450 mg (10 mg/kg) to 1350 mg (30 mg/kg) orally is predicted to increase survival at 6 months from 50% to 70%, and higher doses would further reduce mortality. Based on our analysis an oral dose of 1800 mg is likely to attain desired exposures in the vast majority of patients.Previous analyses of one of the trials included in this work also found improved survival with higher rifampicin dose or exposure , 26, 28,Our analysis is in line with previous analyses that have shown that more severe disease and old age increase mortality . HIV infThe pharmacokinetic analysis showed that only a small proportion of rifampicin penetrated to the CSF (around 5%), that CSF concentrations were directly linked to plasma concentrations, and that distribution to CSF did not saturate within the studied range of rifampicin exposures. The interindividual variability in the penetration was 36% . We found that higher levels of proteins in the CSF were linked to higher rifampicin concentrations. This is probably not a causal relationship, but may reflect more leaky barriers that let both proteins and rifampicin through. In healthy individuals CSF is nearly protein-free ; consequAnother common hypothesis in meningitis treatment is that drug concentrations in CSF should be more closely linked to outcome than plasma levels since the target site is in the brain and CSF is closer to the brain than plasma. This was not supported in our analysis where plasma exposure was found to be a better predictor of survival than CSF exposure. This could be due to different reasons, for example, that the actual individual CSF exposure was not well estimated based on single point concentration measurements or that CSF concentrations are not always a good marker for brain exposure .Our analysis has a number of limitations to be considered. First, data were combined from 3 separate studies with slightly different inclusion and exclusion criteria. The length of the intensified treatment varied between the studies (14 or 30 days); this was evaluated as a covariate in the survival model but not found to be significant. Furthermore, not all patients had confirmed TBM (56%) and this proportion varied between studies. All 3 studies were conducted in Indonesia, potentially limiting the global applicability of the results. Included patients were allowed to have had up to 3 days of rifampicin-containing therapy before the start of the study. The exact number of prestudy treatment days was not recorded and could therefore not be adjusted for. This also mandated a somewhat simplistic way of modeling the autoinduction of rifampicin clearance with a stepwise change over time rather than a more plausible gradual change as earlier described , 43. TotIn conclusion, by combining clinical trial data and employing a model-based analysis, we can support that higher doses of rifampicin improve survival in persons with TBM. Besides intensified treatment using high doses of rifampicin, other aspects of care are relevant for outcome of TBM treatment, including timely diagnosis and treatment initiation, better supportive care, and possibly, host-directed therapy. Still, increasing the dose of rifampicin may be one of the simplest ways to improve treatment outcomes. Hopefully, definitive evidence for such an intervention will come from a planned double-blind randomized controlled clinical trial that will evaluate rifampicin doses of 1500–1800 mg in 500 patients in Uganda, South Africa, and Indonesia (ISRCTN registry number 15668392). The meta-analysis presented herein was part of the work paving the road for this unique phase 3 trial.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciz1071_suppl_Supplementary_MaterialClick here for additional data file.


The evaluation of biofunctional sperm parameters can explain some cases of idiopathic male infertility. Among these, sperm DNA fragmentation (fDNA) is the most studied biofunctional sperm parameter. Mitochondrial membrane potential (MMP) correlates positively with sperm motility, the evaluation of sperm apoptosis by flow cytometry allows us to identify a population of spermatozoa not recognizable at the optical microscopy and finally, lipid peroxidation (LP) and mitochondrial superoxide levels measurements are rational oxidative stress indices. Male age seems to affect sperm concentration and sperm fDNA. For these reasons, this study was undertaken to evaluate the correlation, if any, between male age and biofunctional sperm parameters evaluating their possible impact on fDNA. To accomplish this, MMP, degree of chromatin compactness, sperm apoptosis/vitality, fDNA, LP, and mitochondrial superoxide levels were evaluated by flow cytometry in a cohort of 874 men. A significant negative correlation was found between age and the percentage of alive spermatozoa . The percentage of spermatozoa with low MMP (L-MMP) correlated positively with the percentage of spermatozoa with abnormal chromatin compactness . Spermatozoa with abnormal chromatin compactness and L-MMP correlated negatively with the percentage of alive spermatozoa  and positively with spermatozoa with PS externalization . The percentage of alive spermatozoa correlated negatively with both the percentage of spermatozoa with PS externalization  and of the spermatozoa with fDNA . Spermatozoa with PS externalization correlated positively with the percentage of spermatozoa with fDNA . Spermatozoa with LP correlated positively with the percentage of spermatozoa with increased mitochondrial superoxide  In conclusion, these findings in a large number of men suggest that age, mitochondrial damage, and alteration of chromatin compactness could activate the apoptotic cascade which could result in an increased fDNA rate. Biofunctional sperm parameters can alter conventional sperm parameters and can explain some cases of idiopathic male infertility .Sperm DNA fragmentation (fDNA) is the most studied biofunctional sperm parameter to ensure normal fertilization, embryo development, and implantation . PhysiolAmong the causes of fDNA, some are reversible and the improvement of this parameter could be fundamental not only for natural pregnancies but also for patients requiring assisted reproductive techniques (ART) for their infertile condition. A recently published meta-analysis showed a negative impact of fDNA on ICSI outcomes . Other sMitochondrial membrane potential (MMP) evaluation is another important marker of sperm mitochondrial function that correlates positively with sperm motility . The evaHowever, many of the studies so far cited do not consider the age of the male patients. This aspect is of fundamental relevance because couples are trying to conceive at an increasingly advanced age than in the past. The correlation between age and sperm fDNA has been well studied, however, some aspects remain controversial. Male age seems to affect significantly sperm parameters, such as sperm concentration and sperm fDNA . Men oldTherefore, this study aimed to analyze the correlation between male age and biofunctional sperm parameters and to evaluate, for the first time, their possible impact that can result in fDNA. To accomplish this, biofunctional sperm parameters  were evaluated in a large cohort of men aged 18 to 55 years to investigate possible intracellular pathways that may alter sperm fDNA.The study was conducted on 874 unselected men, aged between 18 and 55 years (35.1 ± 8.2 years). This study was approved by the Ethics Committee of the University Teaching Hospital of “Policlinico-Vittorio Emanuele”, University of Catania, . All methods were performed by following the more relevant guidelines , 18 and Each man enrolled in this study underwent to semen analysis. Semen samples were collected by masturbation after 3–5 days of sexual abstinence. After 30 min of liquefaction at 37°C, they were washed with PBS and immediately acquired by flow cytometry for the evaluation of biofunctional sperm parameters. The latter included: mitochondrial membrane potential (MMP) evaluation, degree of chromatin compactness, sperm apoptosis/vitality evaluation, fDNA assessment, lipid peroxidation (LP), and mitochondrial superoxide levels determination, as previously reported , 7.Cytofluorimetric analysis was performed using flow cytometer CytoFLEX  equipped with two argon lasers and six total fluorescence channels (four 488 nm and two 638 nm). We used the FL1 detectors for green (525 nm), FL2 for orange (585 nm), and FL3 for red (620 nm) fluorescence; 100,000 events (low velocity) were measured for each sample and analyzed by the software CytExpert 1.2.6/ml of spermatozoa was incubated with JC-1 in the dark, for 10 min, at a temperature of 37°C. At the end of the incubation period, the cells were washed in PBS and analyzed. JC-1 penetrates selectively in mitochondria existing in monomeric form, emitting at 527 nm (giving a green fluorescence for cells with low MMP (L-MMP), while after excitation at 490 nm and based on the membrane potential, JC-1 forms aggregates emitting at 590 nm . Negative control was obtained omitting fluorescence from the reaction mixture, while the addition of carbonyl cyanide 3-chlorophenylhydrazone can be used to confirm that the JC-1 response is sensitive to changes in membrane potential.MMP was evaluated using the lipophilic probe 5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethyl-benzimidazolylcarbocyanine iodide . An aliquot containing 1x106 spermatozoa was incubated with LPR DNA-Prep Reagent containing 0.1% potassium cyanate, 0.1% NaN3, non-ionic detergents, saline, and stabilizers  for cell membrane permeabilization, in the dark, at room temperature for 10 min and then further incubated with Stain DNA-Prep Reagent containing 50 µg/ml of propidium iodide (PI) (<0.5%), RNase A (4 KUnitz/ml), <0.1% NaN3, saline, and stabilizers  in the dark at room temperature. Flow cytometry analysis was performed after 30 min, by FL3 detector: the greater the fluorescence emitted, the greater the binding points inside the double helix indicate a worse chromatin compactness.An aliquot of 1x10The signal of early apoptosis is the phosphatidylserine (PS) externalization on the outer cell surface. The assessment of PS externalization was performed using annexin V, the protein that binds selectively to PS in the presence of calcium ions, FITC-labeled. When the cell membrane is also damaged the intracellular passage of the propidium iodide (PI) takes place. Therefore, marking simultaneously the cells with annexin V and PI, we distinguished three different cell populations: viable cells, cells in early apoptosis, and cells in late apoptosis. Staining with annexin V and PI was obtained using a commercially available kit .6/ml was suspended in 0.5 ml of buffer containing 10 µl of annexin V-FITC and 20 µl of PI and incubated for 10 min in the dark. After incubation, the sample was analysed immediately by the detectors FL-1 (FITC) and FL3 (PI).An aliquot containing 0.5 × 106) with 1 mg/ml of deoxyribonuclease I, not containing RNAse, at 37°C for 60 min before staining. The reading was performed by flow cytometry using the FL1 detector.The evaluation of fDNA was performed by the TUNEL method. This uses the TdT , an enzyme that polymerizes, at the level of DNA breaks, modified nucleotides conjugated to a fluorochrome. The TUNEL assay was performed by using a commercially available kit . To obtain a negative control, TdT was omitted from the reaction mixture; the positive control was obtained pretreating spermatozoa (about 0.5 × 10The probe BODIPY (581/591) C11 was used for LP evaluation by flow cytometry. It is incorporated into cell membranes and responds to the attack of free oxygen radicals changing its spectrum of emission from red to green. This displacement of the emission is shown by the flow cytometer, which provides an estimate of the degree of peroxidation.6 of spermatozoa were incubated with 5 mM of the probe for 30 min in a final volume of 1 ml. After washing with PBS, flow cytometry analysis was conducted using the FL1 and FL2 detectors  was used. A p-value lower than 0.05 was considered statistically significant.The median and interquartile range of each biofunctional sperm parameter evaluated in the 874 men enrolled in this study are reported in A significantly negative correlation was found between the age of the men and the percentage of alive spermatozoa   showed that type 1 DM damages the mitochondrial function and impairs sperm motility but has no effects on fDNA, whereas type 2 DM with its inflammatory conditions and the increased oxidative stress leads to reduced sperm viability and an increase in the rate of sperm fDNA rate . Other sOur study showed that age is associated with a decreased percentage of viable spermatozoa. The latter correlated negatively with fDNA; this is supported by other studies that have reported similar observations without however investigating its mechanism , 27. MurThe effects of aging on the decline of conventional sperm parameters  over time seem to be related to an alteration of steroidogenesis and increased oxidative stress . AdvanceThe present study did not show a correlation between age and increased oxidative stress indices . We used BODIPY (581/591) C11 as a sensitive marker of membrane LP by flow cytometry, regardless of whether it is activated by superoxide anion, or any other type of oxygen free radical. The positive correlation between LP and mitochondrial superoxide confirms the sensitivity of this assay. We found a statistically significant improvement in LP with advancing age. This finding seems to be in contrast with data reported elsewhere . HoweverFinally, the results of this study add an important element in the pathophysiology of DNA damage. The advancing age of the male patient correlating with the increased apoptosis rate, which damages later the other biofunctional sperm parameters , could increase in fDNA. Thus, the results of this study suggest evaluating fDNA to evaluate sperm fertilizing capability, especially in those couples undergoing ART cycles, as also suggested by specific guidelines . We suggIn conclusion, the results of this study suggest that it is very important to take into account the contemporary evaluation of the biofunctional sperm parameters described. This to allow us to grasp subclinical elements and nuances that would go unnoticed through the conventional seminal fluid analysis or the evaluation of single biofunctional sperm parameter. Adult men should maintain a good quality of the conventional and biofunctional sperm parameters even if they do not have reproductive aims as a test of adequate functionality of the hypothalamic-pituitary-testicular axis and therefore of general health.rosita.condorelli@unict.itThe datasets presented in this article are not readily available because of patient confidentiality and participant privacy. Requests to access the datasets should be directed to Rosita A. Condorelli: The studies involving human participants were reviewed and approved by Division of Endocrinology “Policlinico G. Rodolico” Teaching Hospital–Catania University. The patients/participants provided their written informed consent to participate in this study.RC and SV: conceptualization and draft preparation. FB, AA, and LM: methods. AA, RC and AC: revision of the text. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pre- and postoperative laryngofiberoscopy was performed to confirm vocal cord function. The proposed technique was successfully used in all patients, and typical EMG signals were effectively detected. No significant change in EMG signals before and after tumor resection was noted, and a normal vocal cord movement was ensured in all patients with postoperative laryngofiberoscopy. IONM helped localize the position of the RLN and facilitated the safe resection of the parathyroid tumor during MIP. The novel transcutaneous EMG recording method proposed in this study was feasible, convenient, reliable, and inexpensive.Intraoperative neuromonitoring (IONM) facilitates recurrent laryngeal nerve (RLN) protection in thyroid and parathyroid surgeries. This study aimed to investigate a novel transcutaneous electromyography (EMG) recording method for IONM of the RLN during minimally invasive parathyroidectomy (MIP). Twenty patients with primary hyperparathyroidism undergoing MIP were enrolled. Two paired needle electrodes were percutaneously inserted into the bilateral laminas of thyroid cartilage for monitoring the vagus nerve and RLN. A standardized IONM procedure (V In recent decades, intraoperative neuromonitoring (IONM) has been increasingly applied in thyroid surgery; it is effective in RLN protection2. The application of IONM has seldom been reported in parathyroid surgery, although it has been considered to be equally important as thyroid surgery3.Recurrent laryngeal nerve (RLN) injury is one of the most common complications in thyroid surgery. Unilateral RLN injury can result in hoarseness, exerting a great impact on singers, teachers, and other professional voice users. Bilateral RLN injuries can lead to airway compromise, and emergent tracheostomy may be required. Furthermore, it has been reported as one of the most common causes of medicolegal litigation4. Surgery is a favorable regimen for treating PHPT. Also, minimally invasive parathyroidectomy (MIP) has become highly popular among patients with a single parathyroid adenoma because of its minimal invasion and appropriate cosmetic features5. Clear identification of the RLN may be difficult in a narrow surgical field during MIP, which is potentially associated with a high risk of RLN injury due to the close anatomical relationship between the enlarged parathyroid and RLN. Therefore, locating and mapping the RLN are of great significance before tumor resection using IONM during MIP6.The primary hyperparathyroidism (PHPT) can be caused by adenoma (85%), hyperplasia (15%), and carcinoma (1%–2%). The hypersecretion of parathyroid hormone (PTH) leads to persistent hypercalcemia and is clinically manifested as weakness in limbs, bone pain, recurrent urinary calculi, dry mouth, dyspepsia, and constipation9. Chiang first compared two different EMG recording methods between an EMG tube and thyroid cartilage (TC) electrodes and concluded that TC electrodes obtained higher and more stable EMG signals and fewer false results during the IONM of the RLN10. However, the trans-thyroid-cartilage neuromonitoring method becomes difficult when only a small skin incision is needed for surgery. This study aimed to investigate a novel transcutaneous EMG recording method for the IONM of the RLN during MIP.Nowadays, the endotracheal tube–based nerve monitoring method  is widely accepted as a standard for IONM of the RLN. However, several limitations have been reported during the clinical use of EMG tubesP < 0.0001), but no significant difference was noted in the serum phosphorus level (P = 0.0860). A total of 20 parathyroid tumors (8 superior parathyroids and 12 inferior parathyroids) were resected, and all were diagnosed as parathyroid adenomas by the pathological examination.A total of 20 patients successfully underwent MIP, including 9 men and 11 women, with a mean age of 48 years . The preoperative serum level of calcium, phosphorus, and PTH was 10.80 ± 0.68 mg/dL, 2.67 ± 0.59 mg/dL, and 172.66 ± 118.44 pg/mL, respectively. On the next day after the surgery, the serum level of calcium, phosphorus, and PTH was 8.88 ± 0.48 mg/dL, 3.04 ± 0.71 mg/dL, and 13.96 ± 13.39 pg/mL, respectively. A significant difference in the serum levels of calcium and PTH was noted before and after the surgery . No significant change in the VHI-10 score was observed before and after the surgery . In the process of needle electrode placement, TC electrodes were inserted easily in 14 patients. The needle electrodes were inserted with great difficulty in six patients owing to the calcified TC, and the initial EMG amplitudes were less than 500 μV  verifying proper electrode–cord position and readjusting EMG tube is troublesome and time-consuming for anesthesiologists; (2) displacement of the EMG tube during surgical maneuvers on the thyroid lobe or trachea often occurs and may cause unstable EMG signals; (3) use of the EMG tube is difficult where IONM is unexpectedly required during the surgery; and (4) cost of the EMG tube is relatively high.Previous studies reported the use of various EMG recording methods during the IONM of RLN, including intramuscular vocal cord electrodes placed endoscopically or through the cricothyroid membrane29  and MIP. The present clinical study reported a transcutaneous EMG recording method for the IONM of the RLN during MIP and verified its feasibility preliminarily. It was speculated that this method was worth further clinical research in MIT.Several clinical and animal studies have reported an alternative trans-thyroid-cartilage EMG recording method as feasible, stable, and convenientWhen the RLN is stimulated, the innervated internal laryngeal muscle (except the cricothyroid muscle) can be induced to generate depolarization. Meanwhile, the EMG signal can diffuse into the skin through the TC and decline rapidly. For patients with TC calcification, the tip of the needle electrode is hardly inserted into the periosteum, which may be just under the skin.The RLN should be dry, as the fluid or blood on the tissue can weaken the EMG signal, which could explain why the EMG amplitude of RLN in patient 3 decreased by 40% (from 1487 μV to 876 μV), while the EMG amplitude of VN increased from 1480 μV to 1808 μV.TC calcification was evaluated in this study by evaluating the resistance of the insertion of the needle electrode. In some cases with high resistance, the needle electrode might not reach the periosteum to avoid the injury on the TC. Hence, it is better to use computed tomography (CT) to evaluate TC calcification.The EMG amplitudes in six patients with TC calcification were less than 500 μV (490 μV). Besides the learning curve, other possible reasons were also speculated:In all patients, the IONM setup was completed within a few minutes, and no complication of subcutaneous or laryngeal hematoma caused by percutaneous insertion of recording electrodes into the TC occurred. In addition, the cost of using IONM technology is also an issue worth consideration. The cost of using an EMG tube in the hospital under study is about $750 each time, and the routine use is restricted by the medical insurance policy. However, the cost of using needle electrodes is only $100, which most patients can afford.The recording needles were only 12 mm in length and too short to be inserted into the TC in patients with an obese neck. However, EMG signals were still well recorded.Inserting needle electrodes into a severely calcified TC could be difficult. Also, relatively lower EMG amplitudes (less than 500 µV) were noted in four of six patients with a severely calcified TC.It was an invasive method, despite no complications, such as hemorrhage and TC injury in the present study. Further studies with a large number of cases should be conducted to verify the secure use of the proposed method.The sample size was relatively small, and no control group was used in this study. Hence, further clinical studies are needed to verify the findings.The novel transcutaneous EMG recording method proposed in this study showed reliable, feasible, and cheap. However, it still had some limitations:IONM helps localize the position of the RLN and facilitate safe resection of parathyroid tumors during MIP. It also increases surgeons’ confidence and comfort through ensuring the functional integrity of RLN after parathyroid resection. The novel transcutaneous EMG recording method proposed in this study is a simple, reliable, and inexpensive way for recording the IONM of the RLN during MIP.This study was conceived as a pilot study. Participants were recruited from the Department of Thyroid and Parathyroid Surgery, Peking University Shenzhen Hospital. The study was approved by the ethics committee of the Peking University Shenzhen Hospital (2019-025) and conducted according to the guidelines of the Declaration of Helsinki on biomedical research involving human participants. All participants provided written informed consent.From May 2016 to December 2017, patients with PHPT admitted to the Department of Thyroid and Parathyroid Surgery  were enrolled in this study. The inclusion criterion was as follows: a single parathyroid adenoma on the unilateral neck after imageological examinations . The exclusion criteria were as follows: multiple parathyroid adenomas, multiple endocrine adenoma type 1, suspected parathyroid carcinoma, tumors located in the thoracic cavity, need to undergo thyroid surgery simultaneously, and preoperative vocal cord palsy.The electronic fiber laryngoscope and Voice Handicap Index 10 (VHI-10) scores were evaluated preoperatively. The electronic fiber laryngoscope was rechecked on the first day after the surgery. If the paralysis of vocal cords occurred, the monitoring was performed in months 1, 3, and 6 after the surgery, and it was considered as permanent vocal cord paralysis not recovered in 6 months. The VHI-10 score was rechecked on the third day after the surgery. All the aforementioned data were recorded in detail.All patients underwent general anesthesia induced with lidocaine (1 mg/kg), propofol (4.0 μg/L), sufentanil (0.5 μg/kg), rocuronium bromide (0.3 mg/kg), and target-controlled infusion (TCI). After performing assisted ventilation for 5 min, regular endotracheal tubes were inserted assisted with a visible laryngoscope. The anesthesia was maintained as follows: propofol (3.5–4.0 μg/L) for TCI continuous intravenous pumping and remifentanil [0.1 μg/(kg · min)] for continuous intravenous pumping.After successful anesthetic intubation, the shoulders were cushioned with pillows to achieve cervical hyperextension. Palpation was performed to determine the outline of the TC. Then, two paired needle electrodes  were obliquely inserted about 1 cm from the midline of the thyroid cartilage into the left and right TC laminas  was used to stimulate the vagus nerve surface (without dissecting the carotid sheath), and the V2 and V2 signals were obtained. Then, subcutaneous tissues and skin were sutured without placing a drainage tube.After resecting the parathyroid tumor and submitting it for the frozen-section examination, the blood was drawn subsequently from the same side of the internal jugular vein after 10 min of tumor resection for intraoperative parathyroid hormone (IOPTH) analysis. If the IOPTH level decreased by more than 50% compared with that obtained preoperatively, the surgery was ended. After the complete hemostasis of the wound, the RLN function was routinely reconfirmed by stimulating the RLN and the vagus nerve. The RP value ≤ 0.05 was considered statistically significant.Microsoft Office Excel 2007 was used for recording data related to the sex and age of patients, position of the parathyroid tumor, hardness of the TC, laryngofiberoscopic examination, and pathological results. Moreover, the serum calcium, phosphorus, and PTH levels, amplitude, and latency of EMG before and after resection were expressed as mean ± standard deviation. GraphPad Prism 7.0 software was used to compare differences among all parameters before and after the surgery. A Supplementary information.


Eucalyptus blakelyi and yellow box, E. melliodora, occurred in similar proportions irrespective of vegetation density; (2) E. blakelyi had the largest number of stems and basal area, but while E. melliodora had fewer stems, it had a similar basal area to E. blakelyi. E. melliodora also showed fewer signs of dieback than E. blakelyi with between 40–50% trees in good condition compared to 2% for the latter species; (3) woody debris loads were low compared to other studies in woodland, but there were levels of heterogeneity indicating ‘natural’ accumulation was occurring; (4) tree basal area and woody debris loads had a 1:1 relationship across all sites and vegetation densities. Overall, our study indicated that ecosystem recovery was taking place (i.e. with many young trees), but there were fewer large trees that are known to supply most woody debris. Our findings highlight the slow accumulation of this critical resource because the volumes were lower than expected. Based on our results, we recommend: (1) aiming for approximately a 50:50 ratio of yellow box to Blakely's red gum basal area in woodland restoration projects; (2) to accelerate the recovery of woodland structure, addition of woody debris should be added at a minimum ratio of 1:1 to standing basal area  (3) managing for both volume and heterogeneity of woody debris loads; (4) preserving large diameter trees to harness proportionally higher woody debris and litter inputs.Simplification of stand structure of forests and woodlands through human-induced modification is a serious threat to biodiversity. Restoring lost habitat complexity and heterogeneity, such as woody debris, requires an understanding of the relationships between different elements that contribute to stand structure. In this study, we examine the structure and composition of a critically endangered box-gum grassy woodland in south-eastern Australia and relationships with woody debris loads. We found that: (1) despite modification by humans and differing susceptibility to dieback, the two dominant tree species, Blakeley’s red gum,  The structure and composition of vegetation is critically important for maintaining biodiversity, ecological processes and ecosystem services , 2. For Studying the structure and composition of a woodland provides information about its past, and its potential future , 17. ManEucalyptus melliodora) and Blakely’s red gum (E. blakelyi) (ACT Government 2004a). Together with white box , these woodlands once covered a vast area of south-eastern Australia prior to European settlement [The maintenance, restoration and spatial expansion of woodlands through active management is particularly important where an ecosystem type is highly modified and greatly reduced in extent. A good example is the box-gum grassy woodlands in south eastern Australia, which occur in an internationally recognised endangered ecoregion . Box-gumttlement , 20. Howttlement –22. As attlement , with fettlement . Consequttlement , 26.A key effect of the process of modification of woodlands by humans is stand structural ‘simplification’ . This is a result of: (1) removal of woody debris for firewood, ‘tidying up’ and prescribed burns to manage fuel loads, (2) removal and prevention of regeneration of shrubs and trees, (3) clearing and thinning of trees , 27. SimTo restore highly modified or destroyed woodlands requires an understanding of current woodland structure and composition and relationships between key elements. Through better understanding of current, simplified, woodland states we can plan and implement interventions that aim to reverse this process , 29.In this study, we examined relationships between measured vegetation structure and woody debris loads in box-gum grassy woodlands in the northern part of the Australian Capital Territory (ACT) in two adjacent reserves with different management histories. Our aim was to answer three questions:What are the structural and compositional characteristics of woodlands, and how do proportions of dominant species vary?What are the predicted volumes of woody debris across vegetation density classes?What is the relationship between woodland stand basal area and woody debris loads?We make recommendations for future research and conservation in box-gum grassy woodlands specifically, and that also have broader relevance to woodlands generally.The study area was in north-eastern ACT, and comprised two adjacent nature reserves—Mulligans Flat and Goorooyarroo Nature Reserves see . Togethewww.mfgowoodlandexperiment.org.au) [The reserves are the location of a major designed ecological restoration experiment, “The Mulligans Flat–Goorooyarroo Woodland Experiment” (.org.au) , 29. TheOur multi-level experiment consists of 24 polygons, with four 1 hectare sites per polygon High tree cover, low shrub cover (HTLS)Low tree cover, low shrub cover (LTLS)Low tree cover, high shrub cover (LTHS)These classifications describe the vegetation structure (i.e. density) of each polygon, with ‘High Tree’ and ‘High Shrub’ meaning ‘dense’ structure, and ‘Low Tree’ and ‘Low Shrub’ meaning ‘open’ structure.Woody debris and tree inventory data were collected at each of the 96 1 ha sites . SurveysAll woody debris (>2cm diameter) was measured on each transect, always commencing at the same relative side of the site, moving in the same direction. Woody debris was assessed using a continuous line intercept procedure , 32, recWoody debris volume was calculated using the line intercept method , 32 by aWoody vegetation was measured within the 10 m wide ‘bands between the tape transects used for woody debris measurement  being reserve, polygon and site. An appropriate statistical modelling framework for assessing the effects of ‘reserve’, ‘vegetation’ and their interaction, and other relationships, was a general linear mixed model with random effects for polygon and site  and number of stems (between 25:75 and 30:80) for yellow box versus Blakely’s red gum, the proportion did not change significantly with reserve or vegetation classification.3  to 2.28 m3  in Mulligans Flat and from 7.54 m3  to 1.93 m3  in Goorooyarroo.Predicted volume of woody debris (derived by back-transformation) varied between reserves and vegetation class . The preThere was no evidence of any variation in heterogeneity of pattern of woody debris loads within sites (i.e. between bands–2 to 6.05 m2 (a 1% increase), woody debris would be predicted to increase from 3.11 m3 to 3.15 m3 (a 1% increase) tonnes per ha. Although this relationship is strong and statistically significant, prediction of woody debris at specific levels of basal area is imprecise due to the high variability around the relationship between the two variables.Fine woody debris (2–10 cm diameter) and coarse woody debris (>10 cm diameter) were analysed separately, but the result was similar to that for both combined (termed “woody debris”). There was a strong and highly significant  and species associations (e.g. beetles) that each species supports. In this study, the proportions of yellow box basal area and stems to Blakely's red gum was the same in both reserves and across all vegetation classes. This is important information, as each species contributes different ecological values to the woodland environment. For example, McElhinny et al. (2010) found that the amount of litter input from yellow box and Blakely's red gum was not significantly different. However, they did find that Blakely's red gum produced significantly higher loads of leaf and other kinds of litter whereas yellow box had significantly more fine twig litter. These litter differences affect beetle assemblages and functional groups, and a proportion of the beetle assemblages associated with both species are mutually exclusive . Yellow ucophaea . Large Bainsonii  and provainsonii . TherefoBlakely's red gum had a larger basal area and a greater number of stems than any other species. This supports the observation that the species can regenerate vigorously given suitable conditions. Stands of yellow box, the next most abundant tree species in our area, were characterised by considerably fewer stems, but a similar basal area because individuals of the species had a larger mean DBH. Yellow box also was considerably healthier (i.e. less dieback) than Blakely's red gum. These factors, and the consistent proportion of the two species across reserves and vegetation classes  than those published for box-gum grassy woodlands elsewhere [3 per ha (+/- 2.6) for yellow box communities, and Killey et al. (2010) predicted 7.0 m3 per ha . The estimates in this study are also less than those estimated CWD volumes in [3 to 247 m3 per ha, mean 34 m3 for both reserves). The relatively low volumes found in these woodlands reflects the DBH (i.e. age) profile of the trees. Killey et al. (2010) studied yellow box in Goorooyarroo and found that yellow box trees that were 100cm DBH were 10 times more likely to produce CWD than a 50cm DBH tree, and produced 10 times more CWD volume. Most trees measured in this study were under 40 cm DBH, with few over 50 cm DBH. So while this DBH profile indicates a woodland ecosystem in recovery (i.e. many young trees), and the heterogeneity of the woody debris (see below) indicate ‘natural’ falls, the low volumes highlight the slow rate of accumulation of this critical resource. as part of the recovery process. This is an issue that potentially acts as a barrier to persistence of biodiversity and its recovery [This study adds to the growing body of work on woody debris loads in box-gum grassy and similar woodlands , 45, 46.lsewhere , 48. Thelumes in . Results also indicate the level of ‘natural’ variability in woody debris patterns, and indicate that this should be replicated if woody debris is being added to a site.2 basal area per hectare (see above) could constitute 7.6 trees at 100 cm DBH, or 30.5 trees at 50 cm DBH.There was a strong 1:1 relationship between overall basal area of a site and woody debris volumes. This means that managers of areas of box-gum grassy woodlands from which woody debris has been removed can use the basal area of a site to determine what the minimum woody debris volume should be. An example of such a situation could be where a paddock with only scattered trees is to be restored to functional box-gum woodland. Logically, if there are more, larger trees per ha, basal area can be maintained in a woodland but that woodland can be open (i.e. fewer stems) and still produce the same amount of woody debris and litter. For example, 5.99 mPrior to European settlement, there would have been more, large eucalypts in box-gum grassy woodlands . More, lIn light of our findings, management of box-gum grassy woodlands should consider the following:aim for approximately a 50:50 ratio of yellow box to Blakely's red gum basal area in woodland restoration projects;2 requires a minimum volume of 3.11 m3), and higher volumes if possible to enhance biodiversity [add deadwood to accelerate the recovery of woodland structure, and should be, at a minimum, added proportionally 1:1 to standing basal area  harness proportionally higher woody debris and litter inputs, (b) create more open woodland while maintaining these inputs. This could be achieved by encouraging tree regeneration or planting in woodlands with low tree and low shrub cover and/or maintaining woodland structural and compositional heterogeneity i.e. have a range of all four broad vegetation classifications.The field sampling approach and resulting data collected for this study provide the basis for tracking the effects of differing woodland structures and composition on biodiversity and ecological processes. In future, it will be important to model how tree populations change through time, and in relation to existing vegetation structure and experimental treatments, and how this affects biodiversity and ecosystem processes. The relative recruitment and survival of small stems of yellow box and Blakely's red gum, and the associated cascading ecosystem effects on biodiversity will be important information for managers in deciding whether or not to intervene with management actions. Although the characteristics such as woody debris volume and heterogeneity within these reserves suggest a ‘natural’ or undisturbed woodland, the paucity of larger trees reflecting a recovering ecosystem rather than a ‘pristine’ benchmark site. The recovery of key structures in these box-gum grassy woodlands has probably been taking place for a longer period than the formal designation of the reserves. Our results highlight the slow accumulation of woody debris, even after vegetation recovery has been underway for some time. This supports the argument for the addition of woody debris in such woodlands in Australia, and also may have applicability to other woodlands elsewhere.


Extent of tumor load is an important factor in the selection of ovarian cancer patients for cytoreductive surgery (CRS). The Peritoneal Cancer Index (PCI) gives exact information on tumor load but still is not standard in ovarian cancer surgery. The aim of this study was to find a PCI cutoff for incomplete CRS. The secondary aims were to identify reasons for open-close surgery and to compare surgical complications in relation to tumor burden.The study included 167 women with stage III or IV ovarian cancer scheduled for CRS. Possible predictors of incomplete surgery were evaluated with receiver operator curves, and a PCI cutoff was identified. Surgical complications were analyzed by one-way analysis of variance and Chi square tests.n = 19). The PCI predicted incomplete CRS, with an area under the curve of 0.94 . Complete CRS was obtained for 67.2% of the patients with a PCI higher than 24, who experienced an increased rate of complications (p = 0.008). Overall major complications were found in 16.9% of the cases. Only 28.6% of the patients with a PCI higher than 33 achieved complete CRS. The reason for open-close surgery (n = 14) was massive carcinomatosis on the small bowel in all cases.The median PCI score for all the patients was 22 (range 3–37) but 33 (range 25–37) for the patients with incomplete surgery  contains supplementary material, which is available to authorized users. Ovarian cancer, the fourth most common cause of cancer-related death among women, is diagnosed for 240,000 women globally every year.4The mainstay of treatment is cytoreductive surgery (CRS), with the aim of removing all macroscopic tumors because the absence of any residual disease is the most important factor for survival and prognosis.6Currently, the extent of tumor load is estimated by different imaging methods, usually by computed tomography (CT) and magnetic resonance imaging (MRI) or preoperative laparoscopy. However, no universally accepted reference standard exists for the imaging of peritoneal carcinomatosis.7To describe peritoneal carcinomatosis, the Peritoneal Cancer Index (PCI) was introduced by Jacquet and Sugarbaker–15Different cutoffs of total PCI have been applied for the investigation of resectability and survival in ovarian cancer. For these outcomes, most studies have used PCI cutoffs of 10–15.11–If information on tumor load from PCI were adequately estimated preoperatively, it could be used in selecting patients for primary surgery. In an attempt to achieve this, the PCI has been used in the interpretation of images.16Establishment of a PCI cutoff for CRS in ovarian cancer would be of great value for identifying inoperable patients. This primary aim of this study was to find a PCI cutoff for incomplete CRS. The secondary aims were to identify reasons for open-close surgery and to compare surgical complications in relation to tumor burden.This single-center study analyzed patients with ovarian, fallopian tube, or primary peritoneal cancer (later referred to as ovarian cancer) treated at Uppsala University hospital, an ESGO-certified tertiary referral center that performs 70–80 primary ovarian cancer operations per year.The preoperative workup included diagnosis based on histology and/or on cytology and a tumor marker profile suggesting gynecologic cancer. A CT scan of the abdomen, pelvis, and thorax (chest) was an obligatory part of the workup. Patients were selected for neoadjuvant chemotherapy (NACT) on the basis of poor general condition, advanced age, massive ascites, hypoalbuminemia, significant pleural effusion, or other factors indicating massive disease spread .The inclusion criteria specified women who had suspected ovarian cancer with carcinomatosis, recorded data on estimated PCI during surgery, and disease in the range of FIGO stages IIIB to IVB. Women who previously had undergone open-close surgery and received NACT were not included in the study.Data on FIGO stage, histology, patient characteristics, surgery, and surgical complications were collected from the medical records. A certified gynecologic cancer surgeon participated in all the operations. The study was approved by the Swedish Ethical Review Board .Surgery was performed with the intention of achieving complete cytoreduction. At the beginning of the surgery, a PCI score was estimated as follows. For the anatomic distribution, 13 regions were defined. Two transverse planes and two sagittal planes divide the abdominopelvic cavity into nine regions. Regions 9–12 divide the small bowel. Lesion size (LS) refers to the greatest diameter of the tumor implants distributed on the peritoneal surfaces, with LS ranging from 0 (no tumor seen) to 3 (tumor > 5 cm). The PCI score ranges from 0 to 39 (Supplementary Data 1).8At completion of the surgery, the completeness of the cytoreduction score (CCS) was estimated as follows: CC0 , CC1 , CC2 , and CC3 . Complete cytoreduction was defined as CC0 and CC1, whereas CC2 and CC3 were considered to indicate incomplete cytoreduction. If the surgeon did not see any possibility for radical or near radical surgery, no surgery was performed, the abdomen was closed (open-close procedure), and the procedure was classified as incomplete cytoreduction.To evaluate the complexity of operations, the Aletti score was used, which divided the operations into three groups using low, intermediate, or high complexity scores. These scores are based on the number of procedures performed in every operation and their degree of difficulty.19The 30-day postoperative complications were graded with the Clavien–Dindo classification system.20t test, Chi square tests, or one-way analysis of variance (ANOVA). Patient characteristics, divided into groups by completion of CRS, were compared with an independent t test and Chi square tests. A two-sided p value lower than 0.05 was considered statistically significant. The data were analyzed using IBM SPSS statistics 24 .The PCI cutoff values for incomplete CRS were evaluated with a receiver operator characteristic (ROC) curve and other possible predictors of incomplete CRS explored with ROC curves. The predictors tested were age, preoperative albumin levels, body mass index, smoking, American Society of Anesthesiologists [ASA] score, physical status classification system, FIGO stage, treatment, histology, and PCI score. Subgroups identified by PCI cutoff were compared regarding peri- and postoperative complications using an independent The study enrolled 204 women scheduled for primary or interval CRS from 1 January 2014 to 15 October 2018 at Uppsala University Hospital. The study excluded 29 women due to a FIGO stage lower than IIIB or because of histology types other than epithelial ovarian, fallopian tube, or primary peritoneal cancer. Seven women were excluded due to missing registration of PCI . Complete CRS was achieved for 148 patients (88.1%), corresponding to CC0 in 121 cases (82%). Of 19 patients who had incomplete CRS, 14 underwent open-close surgery because of inoperability due to massive carcinomatosis on the small bowel. For the remaining five patients, CRS resulted in CC2-3 despite surgical effort that found carcinomatosis at the end of surgery on the surface of the small bowel or basal pleura or on the main artery of the liver. One patient was judged too weak for completion of the larger surgery because of a preoperative pulmonary embolism.p < 0.001; Table Patients with an incomplete CRS had a higher median PCI of 33 , whereas those with a complete CRS had a median PCI of 19   with a PCI score higher than 24 had a complete CRS. Second, to obtain high specificity for detecting cases of incomplete CRS, a PCI cutoff of 33 was identified, at which the sensitivity and specificity for incomplete CRS cases were respectively 45.0% and 99.3%. Among the patients with a PCI higher than 33, 71.4% had incomplete operations.n = 1), ureter injury (n = 1), and bleeding (n = 2).In Table p = 0.024), with major complications for 27% of the group with a PCI of 25–32 and 42.9% of the group with a PCI of 33–37. The operation time, SCS, and blood loss increased with a higher PCI. The duration of surgery was 1.7 times longer for the group with a PCI of 33–37 than for the group with a PCI of 1–24. The stoma rate was 30.3% for the patients with a PCI lower than 24 compared with 81.1% and 85.7% for the groups with the higher PCI .25Attempts have been made to use PCI to predict operability before opting for CRS. Most studies have focused on imaging, whereas Llueca et al.29Imaging studies have shown that diffusion MRI is superior to CT scan because it provides greater accuracy, whereas CT underestimates small carcinomatosis nodules. However further studies are needed before preoperative patient selection is based on PCI, and our group has an ongoing study on this issue.In conclusion, the PCI proved to be an excellent predictor of incomplete CRS, and more complications were found with a greater tumor burden. The PCI cutoff of 24 or higher was related to incomplete CRS and inoperability, and we suggest that with this PCI cutoff, neoadjuvant chemotherapy can be considered, especially for fragile patients. Our findings support further studies on PCI in preoperative imaging because more accurate knowledge of tumor load could enhance patient selection before surgery. We recommend that the PCI be used as a standard parameter in clinical management of advanced gynecologic cancers and included as a parameter in registers and research.Supplementary material 1 (DOCX 154 kb)Below is the link to the electronic supplementary material.


Understanding the early events involved in the induction of immune tolerance to harmless environmental antigens and microbiota compounds could reveal potential targets for allergic disease therapy or prevention. Regulatory T cells (Treg), particularly induced Treg (iTreg), are crucial for the induction and maintenance of tolerance against environmental antigens including allergens. A decrease in the number and/or function of Treg or iTreg could represent an early predictor of allergy development. We analyzed proportional and functional properties of Treg in the cord blood of children of allergic mothers  and healthy mothers . We observed a higher number of induced Treg in the cord blood of females compared to males, suggesting an impaired capacity of male immunity to set up tolerance to allergens, which could contribute to the higher incidence of allergy observed in male infants. The decreased proportion of iTreg in cord blood compared with maternal peripheral blood documents the general immaturity of the neonatal immune system. We observed a positive correlation in the demethylation of the Treg-specific demethylated region (TSDR) and the proportion of Treg in cord blood. Our data suggest that immaturity of the neonatal immune system is more severe in males, predisposing them to increased risk of allergy development. The roots of this dysregulation can be tracked to the prenatal period, when developing fetal immunity is maintained under Th2 bias in order to prevent harmful reactivity against antigenically foreign maternal determinants [Allergic diseases are a highly diverse, multifactorial group of immunological disorders, characterized by unwarranted immune reactivity to and/or failure of tolerance to relatively innocuous exogenous antigens, i.e., allergens. The underlying cause of allergy is a dysregulation of the fine balance among various branches of the immune system which are orchestrated by specialized subpopulations of CD4Early postnatal rebalancing of immune system branches is regulated by many environmental and intrinsic factors, including exposure to microbiota . Central+CD25+/highCD127low cells which express FOXP3 as a lineage-specific transcription factor. The crucial importance of FOXP3+ Treg can be demonstrated by severe syndromes caused by FOXP3 deficiency, which include multiple symptoms of autoimmunity and allergy, both in humans [In humans, Treg are characterized as CD4n humans . MFI of n humans  and exprn humans .Lately, epigenetic regulation of Treg has been proposed as a key mechanism which plays a role both complementary to and independent from that of FOXP3 in inducing and maintaining Treg function and lineage stability . A charain vitro generated iTreg, have been described by various studies as less stable than nTreg, especially under inflammatory conditions [It has been postulated that Treg can be broadly divided into two major subpopulations—natural Treg (nTreg), which are generated during thymic negative selection and which predominantly express T cell receptors (TCR) specific toward autoantigens and play a major role in preventing autoimmune disorders ; and indnditions ,22. Thisnditions ,24,25 annditions  or retinnditions  or withdnditions .Due to the globally high medical, economic and social burden allergic diseases continue to pose, many studies have been and are being conducted to assess the suitability of various immunological parameters as factors useful for prediction of individual risk of allergy development. Identification of such factors could enable clinical testing and early introduction of appropriate preventive measures or at least more timely intervention. Cord blood is a particularly promising biological material for similar studies as it is non-invasively and readily available at the moment of birth. Nevertheless, despite the progress in our understanding of the early etiopathology of allergy development, few reliable predictors of individual risk have been conclusively identified so far. Various factors have been studied without much success, including cord blood IgE ,30, leveIn our study, we aimed to compare proportional and selected functional characteristics of total Treg, nTreg and iTreg as well as the TSDR demethylation status of Treg in the cord blood of children of allergic mothers  and newborns of healthy mothers .The study included healthy (n = 118) and allergic (n = 108) mothers with physiological pregnancies who delivered children via cesarean section at full term in the Institute for the Care of Mother and Child in Prague, Czech Republic. The allergy status of the mother was determined following clinical manifestations of allergy, persisting for at least 24 months: allergy against respiratory and/or food allergens manifested by various individual combinations of symptoms ; monitoring by an allergist; positive skin prick tests or positive specific IgE; and anti-allergic treatment before pregnancy. No difference in pregnancy length was observed between allergic and healthy mothers. The study was approved by the Ethical Committee of the Institute for the Care of Mother and Child  and carried out with signed written informed consent of the mothers.Approximately 15 mL of cord blood was collected via umbilical vein punction into sterile heparinized tubes immediately after birth, as described previously . The monProportional characteristics of Treg were analyzed with flow cytometry using a TregFlowEx kit . Following the manufacturer’s instructions, non-stimulated whole blood samples were stained for cell surface markers CD4  and CD25 , fixated, permeabilized and stained for intracellular markers FOXP3  and Helios .+/Helios− subpopulations is shown in +CD25+FOXP3+ cells  control are shown in + and Helios− cells were gated and relatively quantified from the whole Treg population  and analyzed using FlowJo 7.2.2 .+CD25+CD127low) and target cells  were magnetically isolated from cord blood mononuclear cells using an EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit . Obtained Treg were used for DNA/RNA extraction and for assessing Treg cytokine production and capacity for suppressing production of selected effector cytokines in a functional assay based on coculture of Treg with Tconv.Treg . Total DNA concentration was estimated by spectrophotometric measurement at 260 nm, assuming that 50 μg of DNA per milliliter equals one absorbance unit. Purity of isolated DNA was estimated by the ratio of absorbance at 260 and 280 nm and was in the range of 1.8–2.3. Total RNA concentration was estimated by spectrophotometric measurement at 260 nm, assuming that 40 μg of RNA/ml equals one absorbance unit. RNA purity was likewise assessed by the ratio of absorbance at 260 and 280 nm and was in the range of 1.9–2.2. Isolated nucleic acids were stored in aliquots at −20 °C prior to downstream processing: reverse transcription followed by qPCR expression analysis of selected genes (RNA); or bisulphite modification followed by TSDR demethylation analysis using high-resolution melting (HRM) PCR (DNA).Il10, Tgfb and Il35 (p35 subunit) was determined using TaqMan probes ; actin beta Hs 99999903_m1 was used as an endogenous control . PCR reactions were run, and data were analyzed as described previously [Gene expression of cytokines in Treg isolated from cord blood was performed as described by Hrdy et al. . Brieflyeviously .Bisulphite conversion of Treg genomic DNA was performed using an EpiTect Bisulfite Kit (48)  according to the manufacturer’s instructions. Briefly, 0.5–1 μg DNA was mixed with 85 μL of bisulphite mix and 35 μL of DNA protect buffer and incubated in a thermal cycler programmed according to the manufacturer’s instructions. Converted DNA was stored at −20 °C before HRM-PCR analysis.FOXP3 in bisulphite-converted Treg genomic DNA was analyzed using an EpiTect HRM PCR Kit (100)  according to the manufacturer’s instructions. Briefly, 10 ng of bisulphite-converted DNA was added to 5 μL of EpiTect HRM PCR Master Mix and a mixture of forward and reverse primers at a concentration of 0.75 μM each; the reaction mixture was topped to a total volume of 10 μL per reaction with RNase-free water. The real-time cycler was programmed as follows: 5 min incubation at 95 °C; followed by 45 cycles at 95 °C for 10 s, 55 °C for 30 s, 72 °C for 15 s; and, finally, high-resolution melting, performed from 65 to 95 °C, with a ramp rate of 0.02 °C/s and 25 acquisitions per second. All reactions were run in triplicates in a 384-well plate on LightCycler 480 .Methylation status of the TSDR of FOXP3 intron 1, as described by Baron et al. [The TSDR was considered as CNS2, located in n et al.  and Wiecn et al.  Primer p® 480 Software, version 1.5.0.39 (Roche). Proportion of demethylated TSDR (in %) was then calculated from comparison of area under the curve of the methylated and demethylated DNA-specific melting peaks.Methylated and demethylated bisulphite-converted human control DNA from the Epitect PCR Control DNA  set was mixed in varying ratios for use as a standard curve. Melting peaks specific for methylated products and demethylated products, differing in melting temperature, were detected and characterized using LightCycler6 target cells to stimulate proliferation and cytokine production. After 72 h, culture supernatants were collected, and levels of selected cytokines were analyzed by ELISA.The target cells were cocultured with Treg as described previously . BrieflyThe cell culture supernatants were stored at −20 °C. Levels of IL-10, TGF-β, TNF-α, IL-1β and IL-4 in cell culture supernatants were quantified by ELISA, as described previously .Statistical and graphical analysis was performed using GraphPad Prism 6.0 software . Differences between groups were compared using the unpaired Student’s t-test or standard ANOVA in case of data with normal distribution  and the nonparametric Kruskal–Wallis test for the rest of the data (culture supernatant levels of cytokines). Pearson’s correlation coefficient was calculated for all correlations. Results are presented as column plots showing mean + SEM for most of the data and as scatter plots in the case of all correlations.+FOXP3+ Treg proportion and also evaluated the ratio of Helios+ Treg (putative nTreg) and Helios− Treg (putative iTreg). The MFI (median of fluorescence intensity) of FOXP3 and Helios, transcription factors crucial for Treg phenotype stability and subpopulation homeostasis, were also compared between the groups. Furthermore, we compared the same immunological characteristics between male (n = 104) and female (n = 122) children. Later in the study, collection of maternal peripheral blood samples was added to the study setup, in order to assess the interdependence of parameters of maternal and neonatal immune systems in allergic (n = 85) as well as healthy (n = 82) mothers. To further evaluate the functional and developmental maturity of the neonatal immune system, we measured the effect of Treg on the secretion of selected cytokines in a Treg/Tconv cocultivation assay. Gene expression of IL-10, TGF-β and IL-35 (subunit p35) at the mRNA level was analyzed in magnetically isolated Treg by real-time qPCR. Finally, analysis of demethylation of the TSDR of the FOXP3 promoter was performed in a smaller subset of samples.In our study, we compared immunological characteristics of Treg in the cord blood of newborns of allergic mothers  with Treg in the cord blood of children of healthy mothers . Chiefly, we used flow cytometry to analyze the total CD25+ and Helios− subsets, adding up to 100% of CD25+FOXP3+ Treg. Therefore, nTreg, by definition, represent a complementary population to iTreg and reflect iTreg in all relevant characteristics. For the sake of clarity, and since induced Treg hold particular relevance in the context of allergy control, we elected to present graphs showing iTreg in the main body of the article and include the corresponding graphs of nTreg as In our study, nTreg and iTreg were identified as Helios+FOXP3+ Treg (− iTreg (+ nTreg (p = 0.0098); correspondingly, male newborns had significantly higher nTreg (p = 0.0099). No difference was observed among the four groups for total Treg  but not allergic mothers.No statistically significant difference in total CD25P3+ Treg a, Helios as well as nTreg (p < 0.0001) was found in maternal peripheral blood, with iTreg (p < 0.0001) being correspondingly higher in maternal samples. The highly significant differences between cord blood and maternal peripheral blood (p < 0.0001) were also preserved upon subdivision according to maternal allergy status  but not FOXP3 (p = 0.0008) and a significantly lower MFI of Helios (p < 0.0001) than cord blood Treg, although no difference was observed between peripheral blood of allergic and healthy mothers ; this correlation was not significant in children of allergic mothers but was significant and more pronounced in children of healthy mothers (p = 0.0134), and an even stronger significant positive correlation was revealed between the two in male but not female children (p = 0.0045). While there was no correlation between the Helios MFI and total Treg in any of the groups (p < 0.0001). This negative correlation was comparable when the data were analyzed according to maternal allergic status  as well as according to the newborns’ sex . Due to the role of Helios for nTreg characteristics, as well as its use in nTreg identification in our study, it is of little surprise that there is an equally strong and significant positive correlation between the Helios MFI and nTreg in the pooled samples . The interdependence is somewhat stronger in healthy mothers  than in allergic mothers , both shown in p = 0.0061) than in male newborns (p = 0.0238). As nTreg, by definition, represent a complementary population to iTreg, it is not surprising that both correlation coefficients and measures of significance of the correlation of nTreg between cord blood and maternal peripheral blood are comparable with those of iTreg, regardless of whether they are considered in all samples  at a 1:5 Treg/Tconv ratio. Selected relevant cytokines were detected by ELISA in the culture supernatants. Additionally, real-time qPCR analysis of gene expression of regulatory cytokines IL-10, TGF-β and IL-35 (p35 subunit) was performed in mRNA isolated from a magnetically separated population of Treg (CD4+CD25+CD127low).Since Treg functional  properties are more likely to prove informative regarding Treg maturation status and/or the risk for future allergy development, we performed an assay based on cocultivation of magnetically isolated Treg with magnetically isolated non-Treg CD4Although there was no significant difference in IL-10 concentration between the samples with Treg and the samples including only stimulated Tconv, regardless of maternal allergy status, an observable, though non-significant, trend toward lower concentration of this chief regulatory cytokine was visible between samples isolated from the cord blood of children of allergic mothers compared to children of healthy mothers a. RegretIl10, Tgfb and Il35 (p35 subunit) , supporting the role of TSDR demethylation in Treg maturation already in the perinatal period.Considering the importance of epigenetic control of Treg maturation, stability, function and homeostasis, particularly demethylation of the TSDR of the FOXP3 promoter, we performed epigenetic analysis of this locus. Although we found no difference in TSDR demethylation in DNA isolated from cord blood Treg, regardless of maternal allergy status a and theRegulatory T cells play a key role in inducing and maintaining immune tolerance toward innocuous stimuli, including allergens . While iContrary to our previous observations ,47, we dOther potential sources of variability include differences in gating and the method of staining utilized, factors known to strongly influence the variability among the published studies ,54,55,56In our study performed mostly on an urban cohort of probands, we observed a slight but significant correlation between the MFI of FOXP3, the chief Treg lineage-specific factor, and the total Treg proportion in cord blood. This correlation was stronger in the cord blood of children of healthy mothers and particularly in male children but was not significant in children of allergic mothers and female children. In addition, we found a slightly but significantly lower proportion of iTreg, as well as a higher proportion of nTreg, in male children regardless of the allergy status of their mothers. This was also apparent when analyzing the MFI of transcription factor Helios: male newborns exhibited significantly higher values of the Helios MFI in cord blood Treg than female children, while no difference was seen between children of healthy and allergic mothers. The Helios MFI, in fact, showed a consistent, significant positive correlation with the nTreg population in cord blood and, correspondingly, a strong negative correlation with iTreg.+ nTreg and a corresponding higher positivity of Helios reflect a less mature state of immune regulation [Importantly, we observed a significantly larger proportion of iTreg and a lower proportion of nTreg in maternal peripheral blood compared with cord blood, as well as higher values of the Helios MFI in cord blood than in maternal blood. While the peripheral blood of mothers at birth is itself an immunologically highly specific kind of adult blood sample , it undegulation ,47. Thisgulation ,60. CompInterestingly, a higher incidence of various types of allergic disorders has been reported in male children. Studies including meta-analyses have described this phenomenon in bronchial asthma ,62,63,64+ nTreg Helios MFI in cord blood Treg of male newborns, and, indeed, it is the first analysis of sex-dependent differences in Helios-expressing cells in cord blood. More extensive, rigorously conducted confirmatory studies will be needed to evaluate the clinical relevance of this effect, and inclusion of further surface markers such as CD127, CD45RA, CTLA-4, PD-1 and LAG-3 would provide better population identification and more insight into the activation status and biological significance of these cells. It would also be of interest to compare cord blood samples of newborns delivered vaginally with the current cohort of probands who were delivered with cesarean section.Although the magnitude of the difference between nTreg in male and female children is not very high, to the best of our knowledge, our findings present the first report of an increased HeliosInterestingly, while maternal peripheral blood contained significantly fewer Treg than cord blood, the MFI of FOXP3 was higher in maternal blood. Sufficient FOXP3 expression is key for Treg functional efficiency and phenotypic stability ,75,76,77We were also able to uncover correlations between the Treg population and iTreg as well as nTreg subpopulations in cord blood and maternal blood, consistent with the well-known tight inter-regulation of the maternal and fetal immune systems ,82. In p+FOXP3+ Treg described in cord blood may include a population of activated non-Treg CD4+ T cells in the neonatal circulation rather than bona fide Treg. Since epigenetic regulation (particularly TSDR demethylation) is a more reliable marker of stable, lineage-committed Treg than simple FOXP3 positivity [+ T cells [+FOXP3+ T cells could then, in fact, signify a higher number of activated, potentially inflammatory T cells in the higher-risk groups. To this day, the real biological significance of Helios has not been conclusively determined [+ and Helios− cells consistent with the functional role of nTreg and iTreg, respectively [FOXP3 is generally accepted as the chief Treg lineage-specific transcription factor. Nonetheless, it has been shown that FOXP3 expression can transiently be upregulated in activated T cells ,84. As tsitivity ,85,86, Tsitivity ,87, its  T cells ,89. Hightermined , though ectively ,92.While comparison of proportional and phenotypic characteristics of Treg and Treg subpopulations can provide valuable insight into neonatal homeostasis, it is increasingly clear that the functional capacity of these cells is more likely to hold predictive value for later allergy development. This can be assessed both indirectly  as well as methodical differences in Treg identification and analysis may underlie the significant variability observed among the many published studies. Our data instead suggest a slightly decreased maturation status in male children compared to females, a finding consistent with the reported higher risk of male infants to develop various kinds of early-age allergic diseases ,69,70,71The observed correlation of iTreg and nTreg subpopulations between cord blood and maternal peripheral blood supports the generally accepted notion of a tight interconnection of maternal and fetal immune system regulation, which, under physiological conditions, may prime neonatal immunity to react to harmless exogenous antigens in a tolerogenic fashion or, in the case of maternal allergic inflammation, may contribute to the higher risk of allergy development in the children ,115,116.We acknowledge several limitations of our study. The discrepancy between Treg proportional and functional characteristics published in this study and findings reported in our previous studies ,47 as we


Carnobacterium sp. and an increase in the abundance of operational taxonomic units within the classes Clostridia and Gammaproteobacteria. In contrast, cortisol concentrations in skin-mucus were lower than in the feces, and were not related to any detectable changes in the skin microbiome. Our results demonstrate that stressor-induced cortisol production is associated with disruption of the gut microbiome, which may, in turn, contribute to the adverse effects of stress on fish health. They also highlight the value of using non-invasive fecal samples to monitor stress, including simultaneous determination of cortisol and stress-responsive bacteria.Exposure to environmental stressors can compromise fish health and fitness. Little is known about how stress-induced microbiome disruption may contribute to these adverse health effects, including how cortisol influences fish microbial communities. We exposed juvenile Atlantic salmon to a mild confinement stressor for two weeks. We then measured cortisol in the plasma, skin-mucus, and feces, and characterized the skin and fecal microbiome. Fecal and skin cortisol concentrations increased in fish exposed to confinement stress, and were positively correlated with plasma cortisol. Elevated fecal cortisol was associated with pronounced changes in the diversity and structure of the fecal microbiome. In particular, we identified a marked decline in the lactic acid bacteria  Stress can be broadly defined as a state in which a series of adaptive responses re-establish homeostasis following exposure to a stressor . In fishRecent research has revealed the diverse influence of microbiota and their metabolites on many aspects of host health and fitness, including digestion and nutrient uptake, metabolism and immune development . In mammDisruption of the microbiome is likely to represent an important mechanism by which stress affects fish health, welfare and performance in aquaculture, as well as in the natural environment. There is some evidence that environmental and social stressors disrupt microbial communities associated with the fish gut and skin  however,Prior to the start of the experiment 8 month post hatch Atlantic salmon fry , were housed in stock tanks (80 L) supplied with a recirculating flow of aerated and de-chlorinated tap water, with a temperature of 15 ± 0.5°C and photoperiod of 12L:12D. Water oxygen saturation (>90%), ammonia (<0.02 mg/L), nitrite (<0.01 mg/L), nitrate (<15 mg/L), and pH (7.5 ± 0.2) were maintained within the optimal range for the species. Fish were fed with a commercial feed (Skretting Nutra Parr) at 3% body weight/day.g) and the plasma supernatant was removed and stored at −80°C prior to cortisol analysis. For each fish, a sample of skin-associated mucus for microbiome analysis was collected by swabbing the left side lateral line five times using Epicentre Catch-AllTM Sample Collection Swabs . A sample of skin-associated mucus for cortisol analysis was collected by scraping mucus from a 2 cm2 region of skin on the left-hand side of the fish, above the lateral line between the head and dorsal fin, using a scalpel blade. Fecal samples were collected from each fish by gently pressing along the length of the abdomen and collecting expelled feces, which were then split evenly between samples for cortisol and microbiome analysis. All skin-mucus and fecal samples were directly frozen at −80°C prior to analysis.Experimental fish were assigned at random to the control and confinement-stress treatment groups, with three replicate 20 L tanks per group, each containing 28 fish. Confinement consisted of slowly lowering the water volume in each tank (via draining) from 20 to 5 L for 1 h, and this was repeated every day at the same time (1100 h) for two weeks. All other husbandry conditions were as before. At the end of the experiment, fish were euthanized using Phenoxyethanol (0.5 mg/L), followed by destruction of the brain according to UK Home Office regulations. The fish were measured (fork length), weighed (wet weight), and Fulton’s condition factor was calculated as a measure of body condition. Blood samples were collected from the caudal vein using heparinized capillary tubes, centrifuged , then the supernatant was then diluted in assay buffer (1:20) before cortisol measurement. Skin-mucus samples were suspended in 100 μL 1M Tris–HCl, vortexed for 30 min and centrifuged , then the supernatant was used directly in the assay without dilution.Quantification of cortisol concentration in plasma, skin mucus and fecal samples was performed using the DetectX Cortisol Enzyme Immunoassay Kit , according to the manufacturer’s recommendations. Briefly, plasma samples were pre-treated with dissociation reagent then diluted in assay buffer  before cortisol measurement. Fecal samples were suspended in 100 μL ethanol, vortexed for 30 min, centrifuged  and 1.6× higher than the next highest value in this group (29.1 ng/g).Cortisol concentration was measured in the plasma, feces and skin-mucus for a total of 60 individual fish . We included a greater number of stressed individuals in the analysis in order to encompass variation in cortisol stress responses . Each o16S rRNA amplicon sequencing was performed using the fecal and skin mucus samples for the same 60 individual fish for which cortisol quantification was performed. DNA was extracted from all samples using the PowerSoil DNA Isolation Kit (Qiagen) according to the manufacturer’s instructions, but including an additional 10 min incubation step at 65°C prior to bead beating. Libraries were prepared according to the Illumina Metagenomic Sequencing Library Preparation protocol, targeting the V3–V4 hypervariable region of the universal prokaryote 16S rRNA gene using the primers 341F and 785R. The first PCR reaction consisted of an initial denaturation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, then a final elongation at 72°C for 5 min, using 12.5 ng input genomic DNA, 0.2 μM of primers and KAPA HiFi HotStart ReadyMix (Kapa Biosystems) in a total volume of 25 μL. All products were purified with Agencourt Ampure XP beads (Beckman Coulter) then used as template for the second PCR reaction to add indexed sequencing adaptors to each library . The reaction conditions used were the same as before, but using eight cycles, and a total reaction volume of 50 μL. The final libraries were purified using AMPure XP beads, and the presence of amplicons of the expected size was checked using gel electrophoresis. All libraries were quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific), multiplexed in equimolar concentrations and sequenced on an Illumina MiSeq (2x 300 bp).Raw sequence reads were quality filtered using Trimmomatic  before aAll statistical analysis was performed in R v3.6.1; . FirstlyComparison of microbial community structures (beta diversity) was performed within the Vegan package  using th56r = 0.615, P < 0.001), between plasma and skin cortisol , and between fecal and skin cortisol , but not in blood plasma . There was no association between cortisol and the length, weight or body condition of fish at the end of the experiment .Confinement stress induced a modest elevation in plasma, feces and skin cortisol concentration . There was a significant increase in cortisol in the feces and skin of stressed fish compared to unstressed controls , but positively correlated with Shannon diversity . For the skin, there was no significant effect of confinement stress, skin cortisol, or fish size on skin microbial Chao1 richness of Shannon diversity , although there were significant tank effects for skin Chao1 richness .Fecal cortisol was negatively correlated with fecal Chao1 microbial richness .Microbial community structural diversity was performed based on the Bray-Curtis dissimilarity metric, and visualized using NMDS analysis . For theVagacoccus sp.) was significantly elevated in the confinement stress group independently of cortisol  were from the class Gammaproteobacteria and, notably, two highly abundant OTUs from the family Clostridiaceae were also increased. In contrast, for the skin microbiome, no OTUs were significantly associated with either confinement stress or measured skin cortisol concentration.The effects of confinement stress and cortisol concentration on OTU relative abundance was investigated using DeSeq2. For the fecal microbiome, the abundance of 44 OTUs (27 increased and 17 decreased) were significantly associated with fecal cortisol concentration, but only one OTU  increased cortisol concentration in the skin and feces of juvenile salmon compared to control fish. There was considerable variation in cortisol response among individuals, which is consistent with the existence of low and high cortisol responders . We alsoCarnobacterium sp. with increasing levels of fecal cortisol, including the most abundant OTU in non-stressed fish, together with +10 other OTUs assigned to this genus. Carnobacterium  is a genus of facultatively anaerobic, cold tolerant lactic acid bacteria comprising +12 species  is commonly found in the gut of mammals and fish, but includes a number of opportunistic pathogens. Although relatively little is known about the role that Clostridiaceae play in the fish gut, in mammals an increased abundance of this family has been associated with microbial dysbiosis, intestinal inflammation and gastrointestinal diseases .The animal study was reviewed and approved by Swansea Animal Welfare and Ethical Review Body, Swansea University (number IP-1415-2).TU and DR-B performed the experiment. TU and CG analyzed the data. TU drafted the manuscript. All authors designed the study and contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.