Makeup is commonly attributed with increasing attractiveness in female faces, but this effect has not been investigated in male faces. We therefore sought to examine whether the positive effect of makeup on attractiveness can be extended to male faces. Twenty men were photographed facing forward, under constant camera and lighting conditions, with neutral expressions, and closed mouths. Each man was photographed twice: once without any cosmetics applied and another time with subtle cosmetics applied by a professional makeup artist. Two hundred participants then rated those 40 images on attractiveness. The male faces were rated as higher in attractiveness when presented wearing makeup, compared to when presented not wearing makeup. This was true for both male and female raters, and whether analyzing the data using a by-participant or a by-face analysis. These results provide the first empirical evidence that makeup increases attractiveness in male faces. Following work on female faces, future research should examine the effect of makeup on several other traits in male faces. The market for male cosmetics products is growing and evolving and this study serves as an initial step in understanding the effect of makeup on the perceptions of male faces. Much research has been devoted to understanding the effect of makeup on perceptions. Several studies using carefully controlled before and after photographs have found that women’s faces with makeup are judged as more attractive by both male and female raters than the same faces without makeup –3. This Makeup is believed to increase attractiveness through its effects on visual features, such as skin homogeneity  and faciThis effect of makeup on attractiveness is well documented for women , 10–14, Given the effects of attractiveness on real-world outcomes  were photographed facing forward, under constant camera and lighting conditions, with neutral expressions, and closed mouths. Nineteen of the men self-reported being White and one man self-reported being Asian.Ethical approval was received from the Franklin and Marshall College Institutional Review Board. Participants were recruited through word-of-mouth, social media, and paper flyers. Upon arrival at the lab, written informed consent was obtained from all the participants allowing the use of their photographs for research purposes. Twenty men ranging in ages from 18 to 36 . One hundred and twenty four of the participants identified as male and 76 identified as female. All participants were located in the United States.Ethical approval was received from the Franklin and Marshall College Institutional Review Board. Participants were recruited online through Amazon’s Mechanical Turk platform and each participant provided informed consent. Two hundred participants completed the survey through the platform  in random order, with no time limit. Participants were also asked for demographic information . Upon completion, participants were paid $0.80 for participation in the study.t (199) = 6.35, p < 0.001, Cohen’s d = 0.34). More specifically, the faces with makeup were rated as being more attractive  than the faces without makeup . When splitting by participant sex, both men (t (123) = 6.06, p < 0.001, Cohen’s d = 0.32) and women (t (75) = 2.74, p < 0.01, Cohen’s d = 0.36) rated the faces with makeup as significantly more attractive than the faces without makeup (Participants showed high inter-rater reliability for the ratings of attractiveness (Cronbach’s α = 0.95). When conducting a by-participant analysis, there was a statistically significant difference between the faces with makeup and the faces without makeup (t makeup .t (19) = 2.95, p < 0.01, Cohen’s d = 0.23). Out of the 20 faces, four were rated as less attractive with makeup and 16 were rated as more attractive with makeup.When conducting a by-face analysis, the difference between the makeup and the no makeup conditions was also statistically significant . The sm2 = 0.33 . Regardl2 = 0.33 , 6, evenOne point to note from our study is that not all of the faces were found to be more attractive with makeup. Previous research has found that identity has an effect size that is 1.36 times larger than the effect size attributed to makeup . In our It would also be interesting to further examine what aspect of the makeup application most greatly influences attractiveness perceptions. In our study, the professional makeup artist was instructed to increase skin homogeneity, decrease facial contrast, and accentuate the bone structure. While we got an overall effect of makeup, we are not able to dissociate which of these factors was the most important. For example, perhaps skin homogeneity is responsible for the entire positive effect, or maybe it is a combination of all three factors.Lastly, this study only looked at perceptions of attractiveness in male faces. However, there is vast amount of research examining the effects of makeup on several other traits in female faces. For instance, likeability , leadersIn conclusion, several studies have found that makeup increases attractiveness in female faces –3. Here,


Most systematic reviews of interventions focus on potential benefits. Common methods and assumptions that are appropriate for assessing benefits can be inappropriate for harms. This paper provides a primer on researching harms, particularly in systematic reviews.Commentary describing challenges with assessing harm.Investigators should be familiar with various terminologies used to describe, classify, and group harms. Published reports of clinical trials include limited information about harms, so systematic reviewers should not depend on these studies and journal articles to reach conclusions about harms. Visualizations might improve communication of multiple dimensions of harms such as severity, relatedness, and timing.The terminology, classification, detection, collection, and reporting of harms create unique challenges that take time, expertise, and resources to navigate in both primary studies and evidence syntheses. Systematic reviewers might reach incorrect conclusions if they focus on evidence about harms found in published reports of randomized trials of a particular health problem. Systematic reviews could be improved through better identification and reporting of harms in primary studies and through better training and uptake of appropriate methods for synthesizing evidence about harms. Incomplete knowledge concerning intervention harms threatens public health. Systematic reviews of randomized controlled trials (RCTs) are often thought to provide the most reliable evidence of benefits and harms. Systematic reviews, however, focus largely on potential benefits and typically include trials designed to assess potential benefits. Methods and assumptions that are appropriate for assessing benefits in trials and reviews may be inappropriate for assessing harms. Furthermore, reviews based on publicly available information might be limited because reports often include only a subset of harms, which may be biased.The sources referenced in this commentary have been collected from regulatory and methods guidance documents, textbooks, non-systematic searching and snowball sampling of journal articles, communication with experts in the field, and discussion among the authors. This paper is the first in a series of three articles  dedicate1.When patients “weigh the balance of benefits and harms”, they hope that potential positive effects of an intervention will outweigh any negative effects, such that the intervention would be worth taking. “Harms” There are some important distinctions among terms used to describe harms. 2.Binary or dichotomous harms are often reported as counts or frequencies of events. In regulatory sciences, harms may be collected as events and mapped to standardized terminology before they are analyzed. Examples of standardized terminologies include Common Terminology Criteria for Adverse Events (CTCAE) and Medical Dictionary for Regulatory Activities (MedDRA).-15 The CPatients and clinicians might use multiple terms to describe a single event. For example, an investigator seeking to evaluate whether an intervention makes people drowsy would want to combine similar terms used by patients and clinicians to describe the same feeling.-19 MedDRSpecific events might be related biologically or physiologically, so investigators might want to conduct broader evaluations of harms that share common characteristics.-19 For e2.1.Related harms might occur at several different levels of severity. For example, allergic reactions can be mild to life-threatening or deadly. The Common Terminology Criteria for Adverse Events (CTCAE) defines five grades of severity .14] “Se “Se14] “In addition to classifying severity, harms are often classified according to how likely they are to be caused by the intervention. Relatedness is challenging to determine and the approach is not standardized. The chal3.Harms can be detected in trials, observational studies, and in clinical care. Different sources of evidence for harms have different strengths and weaknesses with regard to the harms signal that they identify and the certainty of the evidence that they provide for the relationships between the harms and interventions.3.1.Before a new drug or biologic can be approved for marketing, regulators such as the FDA may require that several studies be conducted, including RCTs. Trials done before marketing approval typically evaluate a drug’s benefits and look for common and clinically important harms. RCTs can3.2.There are also many different sources of observational data on harms . Compared with RCTs, observational studies face greater challenges stemming from selection bias and information bias. That is, the types of interventions people take for particular health problems might affect the harms that occur for a given intervention in a given population. Despite these limitations, observational studies may be able to achieve larger sample sizes, use less restrictive eligibility criteria , include greater representation of people belonging to minority and underserved populations, and observe longer periods of exposure. These features make observational studies especially useful for detecting unexpected, rare, or long-term harms.,29,31-33Spontaneous reporting systems  can be valuable for detecting rare or long-term harms of drugs after they have been approved for marketing.-38 ExampIn contrast to FAERS and VAERS, which are passive surveillance systems that can be mined for potential drug-harms pairs of interest, Sentinel uses active surveillance of electronic healthcare and administrative claims data. Sentinel is a new program–launched as a pilot in 2008 and fully implemented in 2014–utilizing many databases and surveillance systems. It applies standardized formatting to allow FDA to monitor potential harms of interventions post approval.,44,45 Al4.Unlike benefit outcomes which are typically prespecified in studies, it can be challenging to anticipate every harm that may occur. Harms may be collected systematically  or non-systematically .-54 See A5.Harms data that have been collected in studies may be insufficient for analyses if they do not include relevant dimensions such as date of onset, duration and severity of the event, and concomitant medications. Additionally, the lack of use of standardized terminology when collecting harms, as described in “When we looked at that data, it actually showed an increase in harm amongst those who got the active treatment, and we ditched it because we weren’t expecting it and we were concerned that the presentation of these data would have an impact on people’s understanding of the study findings.” Improving the reporting of harms in trials is an ongoing challenge that remains unsolved, despite the recommendations of the CONSORT-Harms extension being 16 years old.5.1.Most reports about interventions–from journal articles to FDA prescribing information–do not enumerate all harms that are recorded in trials and observational studies. Instead,The extent to which harms are underreported using various selection criteria can be examined by comparing different public sources for trials with the individual participant data: the more restrictive the selection criteria, the less of the overall picture consumers, clinicians, and systematic reviews have when they do not have access to the full, unpublished, data.,68,69 In6.The limitations of harms assessment, analysis, and reporting in individual studies affect evidence syntheses and clinical practice guidelines. The evidence provided by a systematic review can only be as good as the evidence in the included studies. If the limitations of available data are not considered in the review process, then a review might reach incorrect conclusions concerning harms and the trade-offs between benefits and harms. Although the current paradigm for conducting systematic reviews of interventions recommends that harms be assessed so that there can be a balanced discussion of potential benefits and harms, most reviews are not designed to assess harms rigorously,11,78-807.Institute of Medicine’s Finding What Works in Health Care: Standards for Systematic Reviews and the first edition of the Cochrane Handbook for Systematic Reviews of Interventions, both published in 2011.[Based on the early assessments of how systematic reviews synthesized harms, guidance has been developed for searching for evidence, performing analysis, and reporting results. A 2008 report by the Agency for Health-care Research and Quality was foll in 2011.,88 These in 2011.,89 The 2 in 2011. AlthoughImplementing these recommendations requires a substantial investment, both in time and resources. For example, the time required to obtain and make use of unpublished data can be prohibitive for reviewers because harmonizing IPD is labor intensive and because important details may be buried in thousands of pages of reports. Addition8.Assessing harms is a demanding process that requires much work and careful thought, above and beyond that required to synthesize evidence of efficacy or effectiveness. The terminology, classification, detection, collection, and reporting of harms create unique challenges that take time, expertise, and resources to yield valid summaries of harms. Investigators of primary studies should be mindful of these issues as their implementation can greatly impact subsequent evidence synthesis. To avoid tokenism and incorrect conclusions for harms, systematic reviewers should carefully consider how to handle each of these aspects if they plan to draw conclusions about trade-offs between benefits and harms. More systematic reviews are needed that focus exclusively on harms, include appropriate study designs, populations, and data sources, and that apply valid methods for synthesis.Appendix


Most (70.4%) cases were hospital-acquired, and 51.9% of patients were immunocompromised. Corynebacterium striatum pneumonia in South Korea during 2014–2019. As part of an ongoing observational study of severe pneumonia among adult patients, we identified 27 severe C. striatum pneumonia cases. Most (70.4%) cases were hospital-acquired, and 51.9% of patients were immunocompromised. C. striatum cases among patients with severe hospital-acquired pneumonia (HAP) increased from 1.0% (2/200) during 2014–2015 to 5.4% (10/185) during 2018–2019, but methicillin-resistant Staphylococcus aureus (MRSA) infections among severe HAP cases decreased from 12.0% to 2.7% during the same timeframe. During 2018–2019, C. striatum was responsible for 13.3% of severe HAP cases from which bacterial pathogens were identified. The 90-day mortality rates were similarly high in the C. striatum and MRSA groups. C. striatum was a major cause of severe HAP and had high mortality rates. This pathogen is emerging as a possible cause for severe pneumonia, especially among immunocompromised patients.We investigated the proportion and characteristics of severe  Corynebacterium striatum is a nonlipophilic, fermentative coryneform bacterium that commonly occupies the normal flora of the skin and oropharynx  is a major cause of severe hospital-acquired pneumonia (HAP), and the clinical characteristics and outcomes of severe MRSA pneumonia are well-documented. Therefore, comparing C. striatum and MRSA pneumonia could clarify the clinical characteristics of C. striatum pneumonia for clinicians. We investigated the proportion, clinical characteristics, and outcomes of severe C. striatum pneumonia in adults and compared those aspects with those for severe MRSA pneumonia.Methicillin-resistant >16 years of age) patients at Asan Medical Center, a 2,700-bed tertiary referral center in Seoul, South Korea. Since March 2010, we have prospectively identified all adult patients admitted to the 28-bed medical intensive care unit (ICU) who were clinically suspected of having severe pneumonia and monitored them until hospital discharge , which waived the need for informed consent due to the observational nature of the study.This study is part of an ongoing prospective observational study on severe pneumonia in critically ill adult (We defined and categorized pneumonia as previously stated (http://www.synergyinno.com). When coryneform gram-positive bacilli were isolated, we identified and performed antimicrobial susceptibility testing for specimens that were urea positive or from the ICU (>104 CFU/mL (https://www.biomerieux.com) system to identify coryneform gram-positive rods. In September 2015, our facility began using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . We determined antimicrobial susceptibility profiles by ETEST (bioMérieux-Vitek) with MHF medium . We used the Clinical and Laboratory Standards Institute M45 guideline for interpreting susceptibility test results  and considered p<0.05 statistically significant.We compared patient demographics, underlying diseases and conditions, and clinical and laboratory parameters between the C. striatum pneumonia and 103 had severe MRSA pneumonia . Most (70.4%) patients in the C. striatum group had HAP, 14.8% had healthcare-associated pneumonia (HCAP), 11.1% had ventilator-associated pneumonia, and 3.7% had CAP. HAP was significantly more common in the C. striatum group than the MRSA group ; HCAP was more common in the MRSA group , albeit without statistical significance.During the study period, we identified a total of 1,740 patients with severe pneumonia. Among them, 27 had severe neumonia . The medC. striatum HAP increased significantly, from 1.0% (2/200) in 2014–2015 to 5.4% (10/185) in 2018–2019 (p<0.001). Among 75 HAP cases from which bacterial pathogens were identified in 2018–2019, C. striatum was responsible for 13.3% (10/75) of cases, which was the fourth most common pathogen, after Acinetobacter baumannii (30.7%), Klebsiella pneumoniae (21.3%), and Pseudomonas aeruginosa (14.7%).We identified bacterial pathogens in 565 patients who had severe HAP during 2014–2019 . The proC. striatum group and 37 (35.9%) patients in the MRSA group (p = 0.25) . Fungal co-infection, which included 4 Aspergillus species and 1 Pneumocystis jirovecii, was only found in the C. striatum group .We identified co-infection pathogens in 13 (48.1%) patients in the  = 0.25) . Co-infeC. striatum group . The proportion of patients with septic shock at the time of ICU admission was significantly higher in the MRSA group . However, the proportion of mechanical ventilation, acute physiology and chronic health evaluation (APACHE II) score, and sequential organ failure assessment (SOFA) score at the time of ICU admission were similar between the 2 groups. Peripheral leukocyte counts, platelet counts, and serum C-reactive protein levels also were similar between the 2 groups, but serum procalcitonin level was significantly higher in the MRSA group than the C. striatum group .Dyspnea, fever, sputum, and cough were the most common signs and symptoms in both groups . Fever t5 CFU/mL in 8/10 quantitative cultures. Of the 17 semiquantitative culture specimens, 12 specimens were grade many (4+), 1 was grade moderate (3+), 1 grade few (2+), and 3 were grade rare (1+)  of specimens. Among 27 cases, 10 were quantitative cultures and 17 were semiquantitative cultures. Bacterial counts were >10are (1+)  Table. AC. striatum and MRSA group showed no statistically significant differences: 30-day mortality , 60-day , and 90-day  (C. striatum group than in the MRSA group (52.4%), albeit without statistical significance (p = 0.09). Mortality rates were similar for C. striatum and MRSA in subgroups regardless of the patient’s immune status. We noted no statistically significant differences in the median length of ICU stay between the C. striatum and MRSA group, both 14 days (p = 0.33), nor in the length of hospital stay after ICU admission, 30 days for the C. striatum versus 29 days for the MRSA group (p = 0.48).The mortality rates between the  = 0.42) . In-hospC. striatum pneumonia compared with severe MRSA pneumonia. Although the proportion of severe MRSA HAP greatly decreased during 2014–2019, the proportion of severe C. striatum pneumonia sharply increased and surpassed that of severe MRSA pneumonia. C. striatum pneumonia was more commonly associated with immunocompromise, viral co-infection, and fungal co-infection. Mortality rates between the C. striatum and MRSA groups were comparable.We investigated the proportion and characteristics of severe C. striatum pneumonia greatly increased and that C. striatum emerged as one of the most common pathogens in patients with severe HAP. Strengthened infection control measures during the study period might have contributed to the decline of severe MRSA pneumonia . Some studies suggest that serum procalcitonin can be used as a marker for bacterial infection and to differentiate bacterial from viral infection or noninfectious causes of inflammation (S. aureus bacteremia group of patients was higher (1.18 ng/mL) than that of a coagulase-negative staphylococci bacteremia group (0.21–0.31 ng/mL)  involved might have affected mortality rates, but we were unable to effectively evaluate each effect because of the small number of patients in each subgroup.Mortality rates were similarly high in both groups, but septic shock at the time of initial clinical manifestation was less common in the C. striatum because co-infection with other pathogens was common among the patient cohort. Finally, we included all C. striatum isolates from sputum, endotracheal aspirate, and bronchoalveolar lavage, but the cultures were mostly semiquantitative, and some of the C. striatum isolates might have been nonpathogenic colonizers. A 2020 study from the United States reported that normal respiratory flora appears to have caused one quarter of CAP cases (The first limitation of our study is that we used a single-center design and our results might not be replicable in other centers or hospital systems. In addition, as we mentioned previously, we were not able to effectively evaluate the sole contribution of C. striatum was associated with severe HAP. Patients with severe C. striatum pneumonia showed similar clinical and laboratory features as patients with severe MRSA pneumonia, and both infections were associated with high mortality rates. Further investigations could clarify incidence, clinical characteristics, and outcomes of severe C. striatum pneumonia in critically ill adults and determine whether infections are due to colonization, or community- or healthcare-acquired infections. Clinicians should be aware of this emerging pathogen as a possible cause for severe pneumonia, especially among immunocompromised patients.In conclusion, we found Corynebacterium striatum in adults, Seoul, South Korea, 2014–2019.Additional information on severe pneumonia caused by


Cerebrospinal fluid (CSF) is an essential and critical component of the central nervous system (CNS). According to the concept of the “third circulation” originally proposed by Cushing, CSF is mainly produced by the choroid plexus and subsequently leaves the cerebral ventricles via the foramen of Magendie and Luschka. CSF then fills the subarachnoid space from whence it disperses to all parts of the CNS, including the forebrain and spinal cord. CSF provides buoyancy to the submerged brain, thus protecting it against mechanical injury. CSF is also transported via the glymphatic pathway to reach deep interstitial brain regions along perivascular channels; this CSF clearance pathway promotes transport of energy metabolites and signaling molecules, and the clearance of metabolic waste. In particular, CSF is now intensively studied as a carrier for the removal of proteins implicated in neurodegeneration, such as amyloid-β and tau. Despite this key function of CSF, there is little information about its production rate, the factors controlling CSF production, and the impact of diseases on CSF flux. Therefore, we consider it to be a matter of paramount importance to quantify better the rate of CSF production, thereby obtaining a better understanding of CSF dynamics. To this end, we now review the existing methods developed to measure CSF production, including invasive, noninvasive, direct, and indirect methods, and MRI-based techniques. Depending on the methodology, estimates of CSF production rates in a given species can extend over a ten-fold range. Throughout this review, we interrogate the technical details of CSF measurement methods and discuss the consequences of minor experimental modifications on estimates of production rate. Our aim is to highlight the gaps in our knowledge and inspire the development of more accurate, reproducible, and less invasive techniques for quantitation of CSF production. The past decade has seen burgeoning interest in the classical topic of cerebrospinal fluid (CSF) production and transport. The recent findings that CSF transport mediates the export from the brain of proteins implicated in neurodegenerative diseases has inspired a range of clinical and preclinical studies –7. FurthHippokrátēs ho Kôios; c. 460–370 BC) first recognized hydrocephalus in animals and humans . T. T121]. The Time-SLIP technique can be used to assess CSF movements in normal physiological and in pathological conditions, with labeling of a selected volume of CSF in any part of the brain and spinal cord. For example, on one application of Time-SLIP served to test whether the two CSF cistern are connected . The inv1 decay of CSF (∼ 4300 ms) to build up the labeled signal for a longer time. By extending the TE , CSF-derived signal can be isolated from other perfusion or faster/turbulent flow compartments within the brain tissue  returns a total BCSFB-labelled water delivery rate to the lateral ventricles of 2.7 µL/min in mouse brain [Arterial spin labeling (ASL) is a non-invasive MRI technique that entails magnetically uses arterial blood water as an endogenous tracer to map and quantify cerebral blood flow –129. These brain . Unsurprse brain .Fig. 12N+-K+-2Cl− cotransporter 1 (NKCC1) expressed by choroid plexus endothelial cells acts as a buffer of K+ and thereby of active water uptake, during both neurodevelopment and in the adult brain [Notwithstanding these results, there are some concerns about the accuracy and the generalizability of the ASL-MRI technique: (1) The rate of labelled water passing from the CP to the ventricles reflects the permeability of water across the BCSFB, not CSF production per se; (2) the observations are quantitative for net rates of water delivery from arterial blood to ventricular CSF, but not in terms of volume. As such, the BCSFB-ASL result represents a correlate of CSF secretion and indicates the expected direction of the water exchange between the two compartments . As an ilt brain , 142. BClt brain . The autlt brain . This dilt brain , might alt brain . FurtherThe study of CSF spans several millennia of recorded history Fig. . The recAs discussed in this review, many different methods have been developed to measure CSF production in different species Table , dating One unexplained observation is that the indirect method consistently indicates a 30% higher CSF production compared to the direct method in dogs , 148, anAlternatively, the contribution of extra-choroidal CSF production might be larger than is generally acknowledged, as suggested by several lines of evidence , 153. InDue to its invasiveness, use of the indirect perfusion method in humans has been limited to intraoperative studies in patients with CNS neoplasms such as gliomas or meningeal leukemia , 76. MRIFultz et al. demonstrate that the sleeping brain exhibits waves of CSF flow on a macroscopic scale, and that these CSF dynamics are interlinked with neural and hemodynamic rhythms . Thus, sSartoretti et al. used the PC-MRI technique to study the influence of age and sex on CSF flow dynamics parameters at the level of the intercollicular sulcus within the cerebral aqueduct . Their sClassical theory of the “third circulation” holds that the CSF is mainly produced by the CP of the brain and subsequently leaves the ventricles via the foramen of Magendie and Luschka . However2O, MW 18 Da), such that its transport may not capture all aspects of CSF flow [217O provided evidence for a much more rapid CNS distribution of the radioactive water than for the heavier MR contrast agent Gd-DTPA . However, we noted that H217O is not confined to the CSF compartment, but can freely pass the BBB and enter the blood pool [217O PET study, muscle tissue served as a control to correct for BBB efflux. However, blood flow in resting muscle is much lower than in brain tissue in anesthetized non-moving animals. Ringstad and colleagues used intrathecal administration of gadobutrol as a tracer in patient with iNPH to study CSF and glymphatic function [More recently, several imaging studies have used gadobutrol, a hydrophilic MRI contrast agent, as a CSF tracer to visualize the movement of CSF , 190. StCSF flow . In factood pool . In the function , 193. A +/K+-ATPase inhibitor, suppresses CSF production by 40–100% [− transport inhibitor DIDS was also reported to reduce CSF production rate [Numerous studies have used pharmacological manipulations to study the mechanisms of CSF production in experimental studies. These studies have shown that treatment with ouabain, a Na 40–100% , 195, wh 40–100% . The Cl−ion rate . In inflion rate . Overallion rate . ASL-MRIion rate –129. Theion rate , 198. Byion rate . Thus, tThere is burgeoning interest in the physiological regulation of CSF production and in how aging and diseases may affect CSF production. We have described in detail the many methods that have been developed to measure the production of the CSF, giving particular attention to their various limitation. Within a given species, the methods give a wide range of quantitative results Table . An idea


To evaluate the effects of manganese superoxide dismutase (Mn-SOD) from thermophilic bacterium HB27 (name as Tt-SOD) on chemical cystitis.Control and experimental rats were infused by intravesical saline or hydrochloric acid (HCl) on the first day of the experiments. Saline, sodium hyaluronate (SH) or Tt-SOD were infused intravesically once a day for three consequent days. On the fifth day, the rats were weighted and sacrificed following a pain threshold test. The bladder was harvested for histological and biochemical analyses.mRNA expression of inflammatory factors in tissues, and increase perineal mechanical pain threshold and serum MDA and ROS levels in HCl-induced chemical cystitis. Furthermore, Tt-SOD alleviated inflammation and oxidative stress by the negative regulation of the NF-κB p65 and p38 MAPK signaling pathway.Tt-SOD could reduce the bladder index, infiltration of inflammatory cells in tissues, serum inflammatory factors and SOD levels, Intravesical instillation of Tt-SOD provides protective effects against HCl-induced cystitis. Chemotherapy is recognized as the most common treatment for patients with bladder cancer, and chemical cystitis is the most common complication following chemotherapy. Such treatment of chemical cystitis is often tedious, complex, cumbersome, and time consuming. If required, additional surgical procedures are performed: bladder augmentation or ureteral replantation. This caused distress, depression, and in some instances lead to patients refusing further chemotherapy, thereby compromising their treatment plan. Chemical cystitis is mainly caused by harmful chemical substance or metabolite that enters bladder, which is characterized by bladder mucosal inflammation and damage. Bladder mucosa consists of the urothelium, basement membrane and underlying lamina propria. It is located in a very thin layer of bladder transitional epithelial tissue and plays an important role in defending against the invasion of harmful factors . BladderROS is mainly generated by mitochondria, mostly from inflammatory cells, such as neutrophils . In addiIn the present study, a Mn-SOD was used from Thermus thermophilus HB27 (denoted as Tt-SOD), which has a precise active center structure, thermodynamic stability and kinetic inertia. For example, Tt-SOD exhibits superior stability and activity under high-temperature conditions, high acidity and alkalinity and several types of proteases, such as pepsin and trypsin. In addition, Tt-SOD overcomes the shortcomings of existing related products, which have a high application value. A previous study has demonstrated that inflammatory bowel disease (IBD) is attenuated by treatment with Tt-SOD, which is possibly due to decreased SOD activity and attenuated neutrophil infiltration in IBD animal models . The preThe following reagents were purchased from the indicated companies: Tt-SOD; Sodium hyaluronate (SH) ; IL-1β, IL-6 and TNF-α ELISA Kits ; SOD and MDA Kits ; Total RNA Kit , and a reverse transcription polymerase chain reaction (RT-PCR) kit ; ROS Fluorescent Probe -DHE Kit ; Anti-phos-p65 (Ser536) antibody(3033), Anti-p65 antibody (8242), Anti-p38 antibody (6390), Anti-phos-p38(Thr180/Tyr182) (4511), Anti-IκBα antibody(4812) .The experiments were carried out in female Sprague Dawley rats (200–250 g). The animals were kept in the laboratory and maintained at 25 ± 3 °C, with a 12 h light and 12 h darkness cycle. They were provided free access to food and water.This method refers to the previously published protocol of Konkol et al. . The aniThe rats were randomly assigned to one of the following treatment groups:N = 6): Rat bladders were treated only with saline once a day for 4 consecutive days. Saline (0.25 ml) was injected into the rat bladder for 30 min on days 2, 3 and 4. The pain threshold test was performed on day 5.Group 1 : Rat bladders were washed and emptied with saline three times before and after drug administration. The tissues were infused with HCl  for 90 s to induce chemical cystitis by a sterile polyethylene catheter (PE10) on day 1. Then, saline (0.25 ml) was injected into the rat bladder for 30 min on days 2, 3 and 4. Pain threshold test was performed on day 5.Group 2 : Rat bladders were washed and emptied with saline three times before and after drug administration. The tissues were infused with HCl  for 90 s to induce chemical cystitis by a sterile polyethylene catheter (PE10) on day 1. Then, SH  was injected into the rat bladder for 30 min on days 2, 3 and 4. Pain threshold test was performed on day 5.Group 3 : Rat bladders were treated with washed and emptied with saline three times before and after drug administration. The tissues were infused with HCl  for 90 s to induce chemical cystitis by a sterile polyethylene catheter (PE10) on day 1. Tt-SOD  was injected into the rat bladder for 30 min on days 2, 3 and 4. The pain threshold test was performed on day 5.Group 4 : Only the dosing concentration of Tt-SOD was different and the other operations were the same as group 4.Group 5 : Only the dosing concentration of Tt-SOD was different and the other operations were the same as group 4.Group 6  by ELISA according to the manufacturer's instructions and previous studies.Prior to sacrificing, 3 ml blood samples were collected from the heart of each animal under general anesthesia. Subsequently, the blood was centrifuged at 1000 × Body and bladder weight (wet weight) was weighed on the date of euthanasia and the bladder index was calculated as the proportion of bladder weight (mg) to body weight (g)*100% .The bladder samples were washed repeatedly with sterile normal saline to remove excess blood. Following removal of excess water with paper towels, the samples were weighed and sterile normal saline (0.9 ml/100 mg) was added to prepare the tissue homogenate by a tissue homogenizer. The homogenates were centrifuged at 5000 r.p.m. at 4 °C for 10 min and the supernatants were collected to measure the expression of MDA and SOD activity by Elisa kit .Bladder tissue was fixed in 4% paraformaldehyde fixative solution in the presence of 30% sucrose gradient dehydration solution for 48 h and subsequently used to produce frozen sections and paraffin sections.Dihydroethidium (DHE) staining was performed as previously described . In geneH&Estaining was applied to the paraffin sections according to routine protocols. All of the paraffin sections were assessed by a pathologist with experience in bladder pathology. The method of assessment mainly refers to the previous article of Bae WJ et al.  It is diTNF-α, IL-1β, and IL-6). The sequences of the primers  are listed in Table target gene—CTreference gene △△CT = △CTtarget sample—△CTcontrol, where the relative expression level of the target genes = 2−△△CT, and the relative expression of the control group was 20 = 1.Total RNA was extracted and reverse-transcribed using kits as previously described ; quantitFor WB analysis, we followed the methodology described previously . The folN represented the number of animals. One-way ANOVA and Tukey’s test were used to evaluate the statistical significance of the differences between groups. p values of p < 0.05 (*) and p < 0.001(**) were considered for significant differences.Statistical analysis was performed using the SPSS software. All data were expressed as mean ± SD. p < 0.01), while that of the SH and Tt-SOD  groups were significantly higher than those of the HCl group (p < 0.05) , while the Tt-SOD  and SH groups was decreased significantly compared with that of the HCl group; the body weight was not significantly different among the different groups (P > 0.05); the bladder index was increased in the HCl group compared with that of the Sham group (P < 0.01), while the Tt-SOD  and SH groups was decreased significantly compared with that of the HCl group (p < 0.05).The bladder index of each rat was calculated. As shown in Fig. p < 0.01) . The increased inflammatory cell infiltration, edema and urothelial exfoliation were identified on a bladder mucosa in the HCl group Fig. a. The SH01) Fig. b.Fig. 2TWe assessed the therapeutic effects of Tt-SOD on HCl-induced cystitis by detecting the serum levels of certain inflammatory factors  by specific Elisa kits. HCl treatment caused a significantly increased in the level of TNF-α, IL-1β and IL-6 as compared with control group. The SH and Tt-SOD  groups indicated a significant decrease in the level of TNF-α, IL-1β and IL-6 as compared with the HCl group . HCl treatment caused a significantly increased in the mRNA level of TNF-α, IL-1β and IL-6 as compared with Sham group. The SH and Tt-SOD  groups indicated a significant decrease in the mRNA level of TNF-α, IL-1β and IL-6 as compared with the HCl group , while it was significantly decreased in the SH and Tt-SOD groups at all doses compared to those of the HCl group (p < 0.01)  Fig. a, b.Fig.p < 0.01), while the SH and Tt-SOD  groups exhibited a significant increase in SOD levels compared to those of the HCl group (p < 0.05). As shown in Fig. p < 0.01), while the SH and the Tt-SOD groups  exhibited a significant decrease of MDA levels compared to those of the HCl group (p < 0.01). However, there was no difference identified between the Tt-SOD group (100 U/ml) and HCl group (p > 0.05).As shown in Fig. p < 0.01). Compared with those in the HCl group, P65 and P38 phosphorylation levels were significantly decreased, and I-κBα levels significantly increased (p < 0.05), in the SH group and Tt-SOD groups .To determine the therapeutic mechanism of Tt-SOD on chemical cystitis, we examined the expression of phosphorylated p65, p65, phosphorylated p38, p38, and I-κBα proteins in the bladder tissue. As shown in Fig. Cystitis in humans is characterized by the symptoms of urinary frequency, urgency and pain at voiding . This diVarious experiments have shown that SOD plays an important role in various biological functions, such as ROS scavenging –34. KanaHyaluronic acid (HA) is an acidic mucopolysaccharide that is involved in inflammation, angiogenesis, fibrosis, cancer, oxidative stress, and other processes. SH is an extremely stable form of HA, which is often used as a protective agent for the bladder mucosa in cystitis. Therefore, SH was used as the positive control . When exThe present study suggests, for the first time, that treatment with Tt-SOD have anti-inflammatory and decrease antioxidant stress in the rat model of chemical cystitis induced by hydrochloric acid. NF-κB p65 and p38 MAPK signaling pathways play key roles in the ability of Tt-SOD to regulate inflammatory responses and oxidative stress injury in the rat model of chemical cystitis. These results suggest that Tt-SOD is useful for treating chemical cystitis. The detail mechanisms of Tt-SOD in chemical cystitis will be further investigated in the future, and our further work is to observe the therapeutic effect of Tt-SOD in chemical cystitis model caused by bladder cancer.In this study, the HCl-induced model of chemical cystitis reduced inflammation and promoted the repair of bladder mucosa injury in rats after Tt-SOD treatment, which may be related to the amelioration of oxidative stress and inflammation by Tt-SOD treatment. Therefore, further experimental and clinical studies are needed to provide more data to support the role of Tt-SOD in the treatment of chemical cystitis, and Tt-SOD may shows promised for used as an adjuvant drug to reduce the occurrence of chemical cystitis during intravesical chemotherapy for bladder cancer in future.


Malignancies in the urinary tract and the kidney graft are quite common after kidney transplantation. In some selected cases tumours develop from donor-derived tissue.We hypothesised that there is a clinical value to investigate donor/recipient origin in urologic malignancies in renal transplant recipients.In this retrospective study, including patients transplanted between the years 1969 and 2014 at Uppsala University Hospital, Sweden, 11 patients with malignancies in urinary tract and 4 patients with malignancies in kidney transplants were investigated. Donor/recipient origin of tumour tissue was analysed by polymerase chain reaction (PCR) of human leucocyte antigen (HLA) genotypes or by fluorescence in situ hybridization  of sex chromosomes. HLA genotype and sex chromosomes of the tumour were compared to the known HLA genotype and sex chromosomes of recipient and donor.Three of ten cancers in the urinary tract and three of four cancers in the kidney transplants were donor-derived.We suggest that urologic malignancies in renal transplant recipients can be investigated for transplant origin. In addition to conventional therapy the allograft immune response against these tumours can be valuable to treat donor-derived cancers. Development of malignancies after organ transplantation is one of the major risks with lifelong immunosuppressive treatment. To assess the origin, malignant tumours after solid organ transplantation can be divided into de novo tumours, recurrence of earlier malignancies and donor-related malignant tumours. The donor-related tumours develop from donor tissue and can further be divided into donor-transmitted tumours and donor tissue-derived tumours . While dDonor-derived tumours are managed by reduction or cessation of immunosuppression to allow the recipient’s own immune system to be activated. The tumours can be rejected merely by host immune surveillance. Most patients usually need conventional therapy such as surgery, chemotherapy or radiation , 12, 13.We hypothesised that there is a clinical value to investigate donor/recipient origin in urologic malignancies in renal transplant recipients. In this study we investigated whether urologic tumours in renal transplant recipients more frequently may be of donor origin to a greater degree than shown this far.Hence, we investigated all cancers in the renal allograft or cancer of the urinary tract in transplanted patients between the years 1969 and 2014 with regard to tissue origin.Between June 1969 and December 2014, 2835 renal or simultaneous renal and pancreas transplantations were performed in 2437 patients at Uppsala University Hospital, Sweden. Ninety-two patients were lost to follow up, the remaining number of patients were 2345. All recipients received a kidney transplant or kidney and pancreas transplants according to the allocation rules of Scandiatransplant and according to Swedish legislation. None of the transplant donors was from a vulnerable population and all donors and the next of kin provided written informed consent that was freely given. Informed consent was obtained according to the Swedish law  and according to Scandiatransplant allocation rules. The informed consent can, according to Swedish law, be found in a nationwide donor registry, be oral or written, or according to the interpretation of relatives to the donor. This is then signed by the treating physician.Since September 2009 information on post-transplant malignancies have been retrieved by linkage between the Hospital Transplant Database and the Regional Tumour Register in the Uppsala-Örebro region (RTR). This allows for more than 95% detection of post-transplant malignancies . In totaSeventeen patients were included in this partly retrospective study; all patients with malignancy of the urinary tract (n = 13) as well as all patients with malignancy in the renal transplant (n = 4). Clinical data, including patient survival, were collected from medical records. Information on sex, age and HLA genotype of donor and recipient were retrieved from the hospital transplant database at the Department of Transplantation. The primary endpoint was to analyse the donor or recipient origin of the tumour.Archival paraffin blocks of urologic tumour tissue were obtained from the respective regional hospitals of the patients. There was insufficient tumour tissue for further analysis in one patient, and accidently tumour tissue from another patient was not correctly coded; hence these tumours of the urinary tract (n = 2) were excluded from the analyses. Thus, the final number of included patients in the study was 15 .This study was approved by the Regional Ethical Review Board in Uppsala, Sweden (Dnr 2007/032) and approved by the Uppsala Biobank, Sweden (ID: BbA-827-2015-081). Written informed consent was obtained from all living patients prior to inclusion. The use of data and cancer tissue for analysis from deceased patients was approved by the Regional Ethical Review Board.An experienced pathologist re-evaluated the histology of the tumour tissue in the obtained archival paraffin blocks. Representative tumour material from transplantectomy specimens and biopsies from renal transplants and the urinary tract was fixed in 4% phosphate buffered formaldehyde and embedded in paraffin. Tissue sections (4 to 5 μm thick) were processed for routine histology and stained with haematoxylin-eosin, and Sirius red for collagen.HLA genotyping of the tumour was performed when the donor and recipient had different HLA-A, HLA–B or HLA-DRB1. Genomic DNA was extracted from manually dissected tumour tissue by using Recover All Total Nucleic Acid Isolation kit  following the protocol of the manufacturer. The HLA region was amplified by polymerase chain reaction (PCR) using sequence-specific primers  according to a standard protocol . HLA-A, FISH was used to determine donor or recipient origin of tumours in cases where the donor and recipient were HLA identical but had different gender. Representative tumour specimens, 4 μm thick, formalin-fixed and paraffin-embedded were analysed. First the tumour specimen was deparaffinised and incubated in hydrochloric acid (0.2M). After 20 min at room temperature the tumour specimen was rinsed and kept in VP2000 Pretreatment Reagent . In order to separate the DNA strands a protease was added. DNA probes with fluorescent label, AneuVysion probe kit , caused hybridization to the centromeres of the X- and Y-chromosomes. Vectashield mounting medium with 4’,6-diamidino-2-phenylindole  was used to mount the chromosomes. To identify the XX or XY karyotype a fluorescence microscope  was used. The analyses of the images were performed with the ISIS software .All data were presented as medians with ranges. Occurrence of donor-derived tumours were presented as ratios.The characteristics of the patients are summarized in The immunosuppressive protocol was in concordance with the maintenance immunosuppression used at the Department of Surgery, Section of Transplantation Surgery in Uppsala at the time the patients were transplanted (intention to treat). All patients who were transplanted before the year 2000 (n = 11) received a T-cell depleting agent as induction theraphy while all patinets transplanted from the year 2000 and forth (n = 7) received an anti-IL2 receptor antagonist as induction therapy. Maintenance immunosuppression is mentioned in Of the 11 patients with cancer of the urinary tract, 9 patients suffered from cancer of the bladder and 2 patients experienced cancer of the ureter. Four patients were diagnosed with cancer of the renal allografts. The morphological diagnoses of the cancers in the bladder were distributed between urothelial cancer (n = 8) and adenocarcinoma (n = 1), while the cancers of the transplant ureter comprised urothelial cancer (n = 2). The morphological diagnoses of the renal cancers were clear cell carcinoma (n = 1), adenocarcinoma (n = 1) and papillary cancer (n = 2)  Tables. The results of the HLA genotyping and FISH analyses showed that 6 of 14 (43%) examined tumours were of donor origin, 3 of 10 tumours (30%) in the urinary tract and 3 of 4 tumours (75%) in the renal allografts  Tables. Treatment and outcomes are presented in data in This survey showed that three of four investigated cancers of the renal allografts and three of ten examined cancer of the urinary tract originated from the donor. One of the cancers in the renal allograft was identified to have cells/tissue of two tissue types, i.e. cells/tissue of donor and recipient origin. An explanation for mixed tissue origin could be that the tumour is of donor origin and invading lymphocytes of recipient origin.At least 0.2% 6 of 2345 patients) of all transplanted patients in Uppsala and 1% (6 of 531 patients) of all patients with post-transplant malignancies developed donor-derived tumours. No donor-transmitted tumours are included in these numbers. Most current studies comprise only donor-transmitted (0.01–0.05%) and not donor-derived malignancies because the latter has not been systematically investigated , 8. Dono patientsOne of the cancers in the renal allografts originated from the recipient, and three were of donor origin. Boix et al. observed the same phenomenon in a kidney transplant 2009 . They hyCancers of the kidney and urinary tract occur at a 6-fold and 2-fold increased incidence respectively among renal transplanted patients compared to immunocompetent individuals in Sweden . The staDonor-derived tumours compose a unique immunological feature because they occur as a consequence of immunosuppressive treatment after organ or cell transplantation . This ofThe strengths of the study were that all patients at the department of transplantation in Uppsala diagnosed with cancer in the kidney transplant or in the bladder were included. Clinical information was confirmed in the medical records and all analyses were performed at the same department of pathology. The number of patients was however limited and therefore the estimated proportion of donor-derived patients may change in a larger patient material. The proportion of the results might also have been influenced by the prolonged storage time of some tumour specimens; 2 of 15 tumour specimens were indeterminable.In conclusion, we suggest that donor-derived tumours may occur more frequently than previously thought. We suggest that urologic malignancies in renal transplant recipients are investigated for donor/recipient origin. Donor-derived tumours are unique from an immunological point of view and this offers new options for cancer treatment by immunomodulatory drugs in addition to reduced immunosuppression.S1 Checklist(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableTreatment and outcome.(PDF)Click here for additional data file.S5 TableTreatment and outcome.(PDF)Click here for additional data file.S6 TableTreatment and outcome.(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(DOC)Click here for additional data file.


Data collections were repeated during iFL and iHL conditions (2–3/5 AAEP), induced on separate days using a modified horseshoe. Motion asymmetry parameters and continuous joint and pro-retraction angles for each limb were calculated from kinematic data. Normalized average rectified value (ARV) and muscle activation onset, offset and activity duration were calculated from sEMG signals. Mixed model analysis and statistical parametric mapping, respectively, compared discrete and continuous variables between conditions (α= 0.05). Asymmetry parameters reflected the degree of iFL and iHL. Increased ARV occurred across muscles following iFL and iHL, except non-lame side forelimb muscles that significantly decreased following iFL. Significant, limb-specific changes in sEMG ARV, and activation timings reflected changes in joint angles and phasic shifts of the limb movement cycle following iFL and iHL. Muscular adaptations during iFL and iHL are detectable using sEMG and primarily involve increased bilateral activity and phasic activation shifts that reflect known compensatory movement patterns for reducing weightbearing on the lame limb. With further research and development, sEMG may provide a valuable diagnostic aid for quantifying the underlying neuromuscular adaptations to equine lameness, which are undetectable through human observation alone.The relationship between lameness-related adaptations in equine appendicular motion and muscle activation is poorly understood and has not been studied objectively. The aim of this study was to compare muscle activity of selected fore- and hindlimb muscles, and movement of the joints they act on, between baseline and induced forelimb (iFL) and hindlimb (iHL) lameness. Three-dimensional kinematic data and surface electromyography (sEMG) data from the fore-  and hindlimbs  were bilaterally and synchronously collected from clinically non-lame horses ( Pain reduction during lameness is mainly achieved by redistributing load from the painful limb onto the non-lame or less lame limbs, which is manifested as compensatory movement –7 and isSurface electromyography (sEMG) represents an excellent tool for exploring these unanswered questions about equine neuromuscular control. It offers a non-invasive, quantitative means of estimating the degree of muscle activation by recording a summation of motor unit action potentials from electrodes placed on selected superficial muscles . sEMG hasEMG is gaining popularity in equine biomechanics research mainly due to significant advancements in sensor technology, custom wireless transmission protocols, and electromechanically stable interfaces. Collectively, these advances make it possible for researchers to acquire high fidelity sEMG signals from a large animal in motion. Most of the studies to date have investigated muscle activity of selected superficial muscles during normal locomotion –34, and The induction of standardized, mild and temporary lameness, allows the researcher to investigate compensatory mechanisms in animals with a known diagnosis and minimal variation across subjects , 36. ThiEthical approval for this study was obtained from Utrecht University (CCD: AVD108002015307) and the University of Central Lancashire (Reference number: RE/17/08a_b).Eight (n = 8) horses  were used. Horses were part of the university herd, were in regular use for low-level dressage and pleasure riding and were accustomed to being walked and trotted in-hand. Horses were deemed as clinically non-lame  through visual assessments by two qualified veterinarians .sEMG sensors were positioned to record bilaterally from the following superficial appendicular muscles: long head of triceps brachii (triceps), latissimus dorsi (latissimus), superficial gluteal , vertebral head of biceps femoris (biceps) and semitendinosus. These muscles were chosen as they are “prime movers” and thus purported to exhibit adaptive functionality during lameness. Wireless sEMG sensors , with a bipolar parallel bar electrode configuration and a fixed interelectrode distance of 10 mm, were employed. A combination of ultrasonography and previously described anatomical locations and/or sEMG sensor locations –40 were After the sensor sites were determined, the overlying hair was removed using clippers (No. 40 clipper blade), and the skin was thoroughly cleaned using isopropyl alcohol. A small amount of saline solution was applied to each electrode bar to act as an electrolytic solution , 42. SenThree-dimensional (3D) kinematic data were collected using an optical motion capture system of 18 high-speed, infrared cameras , which were secured to the walls of a large indoor hall, where veterinary lameness examinations are conducted. The system was calibrated for each data collection session, producing a large calibration volume ~56 m long and 10 m wide. The optical motion capture system was hardware synchronized to the sEMG system to record the two sets of time series data in one file for further processing. To collect 3D kinematic data, retro-reflective markers  were positioned over anatomical landmarks . Hair waTo simulate a real-world lameness examination, sEMG  and 3D kinematic (200 Hz) data were synchronously collected from in-hand trot trials, conducted on a straight, hard-surfaced, indoor runway during control  and induced lameness  conditions. Data were collected using Qualisys Track Manager  software. Each horse undertook four passes of the straight runway and horses were permitted to trot at their preferred velocity. One handler led the horses during in-hand trot trials. After collecting data for the control (baseline 1) condition, temporary, mild iFL  was induced by qualified veterinarians  using a mechanical, flat headed screw to exert pressure on the sole of the hoof using a modified horseshoe . The vetn = 4) for right and left iFL, in a cross-over design. Following iFL, trot trials were repeated. After a washout period of at least 24 h, the same data collection process was repeated for control (baseline 2) and iHL conditions, where iHL was again randomized to the right (n = 4) or left (n = 4) HL. After each data collection session, the screw/sole pressure was removed. None of the horses showed adverse reactions to the mild, temporary lameness that was induced for the purposes of this study.Horses were randomly divided into two groups  and low-pass filter . Post-processing and analysis of sEMG signals was conducted in Visual3D . Post-processing of sEMG signals included DC-offset removal, followed by the application of a high-pass filter  to attenuate low-frequency noise contamination  . Within-Muscle activity onset and offset events were calculated across strides, in accordance with the double threshold method, described by St. George et al. . Events To complement the discrete variables, continuous sEMG data, in the form of amplitude-normalized sEMG signals across all strides/conditions were prepared for analysis. For each horse, high-pass filtered, and full-wave rectified sEMG signals were enveloped using a Butterworth 4th order, low-pass filter (25 Hz cut-off) and were normalized to an RVC. This was the peak amplitude value of the enveloped signals observed for each muscle location across all strides (excluding detected ARV outlier strides) from the corresponding baseline condition. As the RVC represents a submaximal contraction, it was possible for both normalized ARV and continuous data from the iFL/iHL conditions to exceed 100% of the RVC. To ensure that signals were normalized to a peak value that accurately reflects muscular effort, outliers in peak amplitude data were detected from baseline conditions prior to normalization of continuous signals, using the method employed for ARV data. Any additional outlier strides, detected using peak amplitude data, were then excluded from the sEMG dataset.Kinematic data were tracked in Qualisys Track Manager  and imported into Visual3D and Matlab  software for further analysis. Gait event detection for stride segmentation and the calculation of stride duration, stride speed, and motion asymmetry variables  were conducted in Matlab, with joint time-angle data calculated in Visual3D. Hip Hike was calculated as the difference between the upward movement of the left and right tubera coxae during swing phase . Stride Continuous angle-time data were calculated in Visual3D for the fore- and hindlimb joints affected by the studied muscles, as well as overall fore- and hindlimb pro-retraction angles. For the forelimb, shoulder and elbow joint angles were calculated. For the hindlimb, hip, stifle and tarsal joint angles were calculated. Kinematic data from the fore- and hindlimb markers were interpolated (maximum gap: 10 frames) and filtered using a Butterworth 4th order low-pass filter, with a 10 Hz cut-off frequency, which was determined using Fast Fourier Transform to ensure that 95% of signal content was retained. For each horse, rigid body models of the forelimb and hindlimb were created. Briefly, virtual landmarks were created 2 cm medial to each anatomical landmark, as described by Hobbs et al. , and rigTo increase statistical power, motion asymmetry variables from right iFL and iHL were multiplied by −1 to mirror the indices and thus categorize all data as if they were derived from left fore- and hindlimb inductions only. The remaining variables, including sEMG variables and other data from right iFL and iHL, were also mirrored. Therefore, all results are reported as results of the “lame” side (LS)  and the “non-lame” side (NLS) .Linear mixed models were used to estimate the effect of lameness induction, with iFL and iHL modeled separately. Stride level data for discrete sEMG and kinematic variables were entered into the model from the baseline measurements and the corresponding induced lameness measurement  from each horse. Models were calculated in RStudio  using the package “lme4” (Version 1.1-15). In each model, horse was used as a random effect and the lameness condition as fixed effect. To evaluate the effect of speed on results, additional, separate analyses were conducted, using speed as a random slope to correct for this variable. Model fit was evaluated using q-q plots and boxplots of the residuals. For each model, results are presented as estimated marginal means, standard error (SE) calculated using the package “emmeans” (Version 1.7.1). Significance values were corrected for multiple comparisons using the false discovery rate method .*101*1 vector fields  for each signal, condition, and horse. The open source spm1d package (Version M.0.4.1.) was used to conduct the SPM analysis in Matlab (Version 2020b). For both the sEMG and kinematic data, separate analyses were performed to compare signals between baseline measurements and the corresponding induced lameness measurement . For sEMG and kinematic data, paired samples T-tests were performed on forelimb muscles  and joints , and on hindlimb muscles  and joints  together, but separately for the lame side and non-lame side. The two-tailed significance level was set at α = 0.05 and p-values were adjusted for multiple comparisons using the Bonferroni correction.For the statistical analysis of the continuous kinematic and sEMG data , statistical parametric mapping (SPM) was used , 54. TheAppendicular muscle activation and limb movement patterns during trot are presented in p-values and the beginning and end time points for each data cluster that exceeded the critical thresholds are presented in For forelimb muscles , significant decreases in sEMG activity, quantified using ARV, were observed on the NLS, with significant increases observed on the LS . ActivitAn increase in most kinematic asymmetry variables was found for iFL , includiSignificant increases in sEMG ARV were observed bilaterally across all hindlimb muscles  . Muscle An increase in most asymmetry variables was found for iHL , includiThis study combined, for the first time, motion capture and sEMG technology to quantify and compare activity of superficial fore- and hindlimb muscles and associated joint and limb pro-retraction angles during baseline and induced fore- and hindlimb lameness conditions. Findings from this study support the hypotheses that adaptations in muscle activity occur during trotting under iFL and iHL conditions, are identifiable using sEMG, and result in altered temporal and angular kinematics that can characterize the phenomenon in an individual horse when compared to their non-lame condition. Briefly, increases in muscle activation were observed bilaterally across all muscles, except for NLS forelimb muscles during iFL in which activation significantly decreased. Phasic shifts in muscle activation patterns were observed across fore- and hindlimb muscles, which were generally consistent within each limb and reflected phasic shifts/measured changes in the associated limb movement cycle.We investigated the triceps and latissimus, based on their superficial location (for sEMG), their potential to contribute to joint angle changes, and the fact that they represent two of the largest muscles of the proximal forelimb , 56. TheDuring iFL, changes in temporal stride patterns are particularly evident in the LS forelimb, where the shortened suspension phase of the lame diagonal pair –3, 61 siThe NLS triceps and latissimus were the only muscles to exhibit significant decreases in ARV during iFL. These decreases were seemingly achieved through differing control over the amplitude and duration of muscular response to iFL, with the NLS forelimb muscles exhibiting longer activation bursts of lower amplitude and vice versa for the contralateral LS forelimb. Further, activation of NLS triceps and latissimus occurred significantly earlier during iFL at around 35% of the stride cycle, which corresponded to peak protraction, elbow flexion, and shoulder extension angles. As retractors of the forelimb , 55, 56,For iHL, visual analysis of Taken together, our findings from the forelimbs show that, within the lame diagonal pair, significant increases in triceps and latissimus ARV during iFL and iHL are required to stabilize the forelimb against sagittal plane forces, albeit to a seemingly lesser degree during iFL, which we postulate as being related to the greater compensatory role of the head and neck  for unloading the affected limb , 63, 69.The hip extensor musculature, including biceps and semitendinosus as studied here, are specialized for force and power generation during stance , but alsDuring both iFL and iHL, ARV significantly increased across all LS and NLS hindlimb muscles, where increases in amplitude were particularly evident during stance phase . These fBuchner et al.  reportedTo our knowledge, equine hindlimb muscles have not previously been studied during forelimb lameness conditions. Like iHL, we noted significant increases in ARV across all NLS and LS hindlimb muscles during iFL, but with differing phasic activation shifts for delayed NLS activation and precipitated LS activation. Significantly delayed activation across all NLS hindlimb muscles is likely related to the prolonged transition from sound to lame diagonal stance , 61, 69,Given the preliminary nature of this study, there are limitations that must be considered when interpreting our findings. Firstly, an acute lameness induction model was employed to study standardized conditions in a relatively small sample of horses. This transient model served our purpose of exploring the muscular adaptations to lameness and as such is not a limitation. However, the acuteness of the model can be seen as a limitation in the light of the high clinical prevalence of chronic lameness in horses, about which no direct inferences can be made based on the outcome of this study. However, currently no ethically acceptable models of chronic lameness in horses exist and future research employing a larger sample of clinical lameness cases is required as a follow-up to this experimental study. Future studies of this nature should also determine whether the significant changes observed here constitute clinically meaningful changes. In addition, limitations associated with the acquisition and analysis of sEMG data from equine subjects should be considered. sEMG is limited to the evaluation of superficial musculature and only select fore- and hindlimb muscles were studied here, so future studies should evaluate additional muscles. In equine subjects, obtaining a maximum voluntary contraction for normalization purposes is not possible, but normalization to an RVC, as employed here, allowed us to explore the meaningful proportional change in muscle activity between non-lame and induced lameness conditions. Further, normalization of equine sEMG data using an RVC has been shown to improve the sensitivity and accuracy of equine gait analysis . ARV wasThe known effect of speed on kinematic and sEMG variables can be considered a limitation of this study, where overground trot speed was not standardized. We addressed this by presenting results from models with- and without a statistical correction for speed , 73. OnlIn this study, SPM results for continuous group-level sEMG data showed non-significant differences, which contrasts results from the linear mixed model analysis of group-averaged discrete data. This could be considered a limitation, but the observed variation in group-level sEMG data and the tighter control of alpha that is required for SPM , may expUp until now, descriptions of compensatory muscular mechanisms that occur during lameness have been largely based on subjective clinical perception. However, findings from this study offer the first objective evidence for underlying muscular adaptations and the associated movements of the fore- and hindlimbs that occur during acute lameness. It is clear from our results that adaptation mechanisms to unilateral lameness occur across all limbs and are primarily related to increased bilateral muscular activity and phasic shifts in activation patterns, which facilitate and/or adapt to pain avoidance mechanisms to reduce loading on the affected limb. The long-term consequences of these muscular adaptations are not known, but further research should investigate this, as prolonged abnormalities in muscular function can manifest as muscle injury or pain  and may By combining sEMG and motion capture, we identified distinctive differences in appendicular muscle activity that occur during iFL and iHL and this resulted in altered temporal and angular kinematics of the fore- and hindlimbs. The muscular adaptations identified here were primarily related to increased muscular activity, as quantified using ARV, and phasic shifts in activation patterns, which reflected distinctive changes in associated joint angles and phasic shifts of the limb movement cycle. Further studies are required to investigate the clinical meaningfulness of these mechanisms with other lameness causes, in clinical cases, and to better understand the long-term consequences of these muscular adaptations. Still, the successful combination of sEMG and kinematics can be used to further our understanding of the clinical signs and etiology of equine lameness, and with further development, sEMG has the potential to aid in the diagnosis, treatment, and management strategies for lameness.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Utrecht University (CCD: AVD108002015307) and the University of Central Lancashire (Reference Number: RE/17/08a_b).LS, TS, SJH, and FS contributed to conception and design of the study. LS, TS, and FS conducted data collection. LS, SJH, and FS conducted data processing and analysis. FS and IS performed the statistical analysis. LS and TS wrote the first draft of the manuscript. FS and IS wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This study was made possible with support from Morris Animal Foundation (Grant ID: D21EQ-406) and the British Society of Animal Science (BSAS) 2018 Steve Bishop Early Career Award. Open Access publication fees were funded by the University of Central Lancashire.Author SR is employed by Delsys Inc., the manufacturers of the sEMG sensors used in this study. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neutrophil-to-lymphocyte ratio (NLR) is a simple parameter implying the inflammatory status. We aimed to explore the association of brain-dead donor NLR change with delayed graft function (DGF) in kidney transplant recipients.We retrospectively analyzed the data on 102 adult brain-dead donors and their corresponding 199 kidney transplant recipients (2018 − 2021). We calculated ΔNLR by subtracting the NLR before evaluating brain death from the preoperative NLR. Increasing donor NLR was defined as ΔNLR > 0.p = .018), and remained significant  after adjustment of confounders including BMI, hypertension, diabetes, and the occurrence of cardiac arrest. When acute kidney injury (AKI) was included in the multivariable analysis, increasing donor NLR lost its independent correlation with DGF, while AKI remained an independent risk factor of recipient DGF . The area under the curve of combined increasing NLR and AKI in donors (0.873) for predicting DGF was superior to increasing donor NLR  and AKI alone .Forty-four (22%) recipients developed DGF after transplantation. Increasing donor NLR was significantly associated with the development of DGF in recipients (OR 2.8, 95% CI 1.2 − 6.6; Dynamic changes of donor NLR are promising in predicting post-transplant DGF. It will assist clinicians in the early recognition and management of renal graft dysfunction. Validation of this new biomarker in a large study is needed. Kidney transplantation is the therapeutic option for patients with end-stage renal failure. Complications after transplantation influence graft function and even contribute to graft loss, which lay a great social and economic burden on the healthcare system . DelayedThe neutrophil-to-lymphocyte ratio (NLR) is an inexpensive, easy-obtained, widely available parameter to evaluate inflammation status. Previous literature suggested the predictive role of NLR in outcomes of many diseases including stroke, cardiovascular disease, and chronic kidney disease . DynamicOrgans procured from brain-dead donors is the main source used in transplantation . HoweverAdult brain-dead patients who donated their kidneys in our institution between January 2018 and December 2021 were enrolled. Data of brain-dead donors and their corresponding recipients were reviewed from the electronic records. The information of their corresponding recipients was provided from the local Organ Procurement Organization (OPO) and the department of transplantation in our institution.NLR value was defined as the ratio of absolute neutrophil count and absolute lymphocyte count in the peripheral blood. NLR was collected from the blood drawn at two periods: within 24 h before evaluating brain death (aNLR) and within 6 h before organ procurement (pNLR). The dynamic change of NLR (ΔNLR) was calculated by subtracting the NLR before evaluating brain death from the preoperative NLR (ΔNLR = pNLR – aNLR), and increasing donor NLR referred to ΔNLR > 0.via Banff criteria by allograft biopsy [Expanded criteria donors (ECDs) were defined as age ≥ 60 years, or age 50–59 years with two of the following: history of hypertension, serum creatinine level ≥ 133 μmol/l (1.5 mg/dl), or cerebrovascular disease as a cause of death . Accordit biopsy .No prisoners or non-consenting donors were enrolled. The study was performed adhere to the principles of the Declaration of Helsinki. The Ethics Committee of Nanfang Hospital approved this research (NFEC-2021-410). Procurement of kidneys from brain-dead donors was approved by the Human Organ Transplantation and Ethics Committee of local institution. Organs were obtained by the Organ Procurement Organization of the local hospital and allocated by the China Organ Transplant Response System.P value < .05 was considered statistically significant. Quantitative variables were expressed as median and 25th–75th interquartile ranges (IQRs), while categorical variables were expressed as number (percentages). Comparisons were made using Mann–Whitney test for continuous variables. The χ2 test or Fisher exact test was used for categorical variables. To account for the cluster effect of paired kidneys from the same donor, we used generalized estimating equations (GEEs) to perform multivariable analysis [p < .05 by univariate analysis were included. The value of increasing donor NLR in predicting graft DGF was evaluated by receiver operating characteristic (ROC) curve.Statistical analysis was performed on SPSS for Windows . analysis . In the p < .05) (p > .05).During the study period, a total of 102 adult brain-dead donors and their corresponding 199 kidney transplant recipients were included . Both kip < .05) . Recipiep = .655). The median preoperative NLR value in the DGF group  seemed higher over the non-DGF group , but the difference was of no statistical significance (p = .161) (p = .004) (p = .148).The median NLR value within 24 h before evaluating brain death were similar between the DGF  and non-DGF group  . Compari = .004) , while tp = .018). Besides, other risk factors for DGF related to brain-dead donors included higher BMI (p = .018), hypertension (p = .014), diabetes (p = .032), occurrence of cardiac arrest (p = .006), and AKI (p < .001). Multivariate model 1 showed increasing donor NLR remained to be related to posttransplant DGF  after adjustment of BMI, hypertension, diabetes, and the occurrence of cardiac arrest. However, in another model that included donor AKI, increasing donor NLR lost its significant association with the risk of posttransplant DGF , while AKI in brain-dead donors was strongly related to the post-transplant DGF  (Supplementary File 1).Twenty‐two percent of recipients following renal transplantation developed DGF. Univariate and multivariate analysis were performed to analyze associated factors with DGF. In univariate analysis, increasing NLR in donors was significantly associated with DGF  . The resThis study explored the predictive value of dynamic changes of donor NLR in graft DGF. The dynamic changes of donor NLR have a significantly predictable role in the early graft outcome. Although AKI in donors was strongly related to the development of DGF in recipients, the combination with increasing donor NLR would further improve its predictive value.One of the multiple risk factors in relation to the incidence of DGF after kidney transplantation are donor-related factors. A study reported kidneys procured from brain-dead donors were at higher rate of DGF . In our NLR is a simple and inexpensive parameter indicating inflammation state. It integrates general information of three systems: vegetative nervous system, neuroendocrine and immune systems . Some stSupplementary File 2). We speculate that donor AKI may be the most significant factor related to the posttransplant DGF and the role of increasing donor NLR in predicting DGF may be limited owing to the small sample size. Alternatively, there may exist some other unexpected or confounding factors related with graft dysfunction.In our study, despite the NLR value before organ procurement in the DGF group is higher than non-DGF group, this result is of no statistical difference. In univariate analysis, increasing donor NLR was proved to correlate with the risk of post-transplant DGF. When adjusted by BMI, hypertension, diabetes, and occurrence of cardiac arrest, increasing donor NLR was still associated with posttransplant DGF. But this relation lost statistical significance when adjusted by the aforementioned factors and donor AKI. Donor AKI become the only risk factor related to the posttransplant DGF. Notably, recipients in the DGF group all received donations from donors with AKI. We found that increasing donor NLR was independently associated with donor AKI (Additionally, donor BMI was observed related to the occurrence of DGF in this study. Some authors presumed that it might result from the impact of immune system in the overweight/obese donors and the insufficient cooling of organs taken from these donors. Because the perirenal fat is not always removed adequately ,39. We sEarly prediction of graft dysfunction could serve as a warning for clinicians to initiate renal protective measures. Various factors in brain-dead donors have been proposed for predicting the development of DGF in recent years. Similar to some studies ,40–42, dOur study also has limitations. First, it is a small-sized study of a single center which inevitably introduces biases in data collection. The validation from a large multi-institutional study is needed. Second, despite adjustment for some critical factors, there may still exist unmeasured confounders given the observational study design. Nevertheless, it has strength with regard to homogeneity of patient population, donor management, standard immunosuppression, and other postoperative management protocols in a single center.Dynamic changes of NLR in brain-dead donors were promising in predicting the development of DGF in recipients. Furthermore, increasing NLR combining AKI in brain-dead donors had significantly better predictive efficacy and may assist in early recognition and management of renal graft dysfunction. A large and prospective study is warranted to validate this relationship in the future.Click here for additional data file.Click here for additional data file.