In this study, we evaluated the status of and attitudes toward COVID-19 vaccination of healthcare workers in two major hospital systems (academic and private) in Southern California. Responses were collected via an anonymous and voluntary survey from a total of 2491 participants, including nurses, physicians, other allied health professionals, and administrators. Among the 2491 participants that had been offered the vaccine at the time of the study, 2103 (84%) were vaccinated. The bulk of the participants were middle-aged college-educated White (73%), non-Hispanic women (77%), and nursing was the most represented medical occupation (35%). Political affiliation, education level, and income were shown to be significant factors associated with vaccination status. Our data suggest that the current allocation of healthcare workers into dichotomous groups such as “anti-vaccine vs. pro-vaccine” may be inadequate in accurately tailoring vaccine uptake interventions. We found that healthcare workers that have yet to receive the COVID-19 vaccine likely belong to one of four categories: the misinformed, the undecided, the uninformed, or the unconcerned. This diversity in vaccine hesitancy among healthcare workers highlights the importance of targeted intervention to increase vaccine confidence. Regardless of governmental vaccine mandates, addressing the root causes contributing to vaccine hesitancy continues to be of utmost importance. COVID-19, caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and reached a global pandemic level by March 2020 . HealthcVaccine hesitancy, the leading threat to global health , is the Experts suggest that given our global connectivity, addressing the threat of COVID-19 is highly dependent on increasing vaccination rates everywhere to reach herd immunity levels . HCWs arIn the present study, we sought to determine what proportions of HCWs in Southern California were accepting of, hesitant about, or resistant to a COVID-19 vaccine. Additionally, we sought to profile HCWs who are hesitant about or resistant to a COVID-19 vaccine by identifying key sociodemographic, occupational, political factors and specific beliefs that distinguish them from those who accept a COVID-19 vaccine. While this cross-sectional exploratory study had no explicit hypotheses, our approach was guided by several assumptions: (1) most HCWs would accept vaccination, (2) HCWs job characteristics including direct patient interaction would facilitate vaccine acceptance, (3) sociodemographic characteristics of HCWs such as gender, race, age, and educational attainment will be associated with intention to take vaccine, (4) vaccine hesitancy among HCWs will be influenced by several factors, including insufficient knowledge about the vaccines, misinformation from social media, political affiliation, and previous COVID-19 infection. Understanding the characteristics that predict vaccine hesitancy among HCW subgroups will enable health administrators to apply and evaluate tailored interventions.We conducted an online, cross-sectional survey of HCWs at two large hospital systems (academic and private) in Southern California. The study protocol was reviewed by the respective IRBs of each participating institution and deemed exempt. Both hospitals started vaccinating their HCWs against COVID-19 on 17 December 2020. Recruitment occurred relatively early after vaccination distribution; between 5 and 26 February 2021 at the academic hospital, and 3 and 17 April 2021 at the private hospital. We distributed the online Qualtrics survey via institution-wide email listservs, with 8848 recipients at the academic hospital and 3062 recipients at the private hospital. Listserv recipients included physicians, nurses, advanced practice providers, pharmacists, other allied health professionals, administrators, and nonclinical ancillary staff. All employees of both hospital systems were invited to participate in the study. The initial invitation email to complete the survey was followed by two email reminders to encourage participation. The survey was anonymous and voluntary. A working group developed a survey to understand HCWs’ knowledge, attitudes, and perceptions about the COVID-19 vaccination. The survey was based on previous questionnaires conducted in the context of the 2009 H1N1 flu pandemic. It was pilot-tested with 7 healthcare professionals and revised to ensure readability and understandability. The final survey included exclusively forced-choice questions to avoid missing data. The overall survey response rate was 20.9%  with a dropout rate of 17%. This modest response rate can be attributed to the spike in COVID-19 infections and hospitalizations in Southern California, coinciding with the dates when the study survey was administered. The increased workload and burnout among HCWs presented a barrier to survey completion.The survey instrument was composed of five parts: (1) Demographics: including age, gender, race, ethnicity, education level, self-reported history of chronic illness, income level, household size, and political party affiliation; (2) Clinical characteristics: including position within the healthcare field, clinical work setting, medical specialty, frequency of contact with COVID-19 patients, and self-reported history of flu vaccination; (3) COVID-19-related misinformation: including belief in a synthetic origin of the virus, belief in COVID-19 being a hoax, and belief that COVID-19’s impact on the healthcare system is exaggerated; (4) COVID-19 knowledge: understanding that COVID-19 is more deadly and contagious than seasonal flu, estimated COVID-19 mortality for self and an average American, understanding of COVID-19 vaccine effectiveness; (5) COVID-19’s impact: COVID-19’s financial impact and whether someone close had a severe illness or died due to COVID-19. We derived the primary outcome, COVID-19 vaccine behavior/hesitancy, from two questions—(a) receipt of any dose of a COVID-19 vaccine and (b) in case of a negative answer, intention to receive a COVID-19 vaccine within the next six months. We considered answers “unsure”, “probably not”, and “definitely not” indicative of COVID-19 vaccine hesitancy. The 37-item survey took, on average, 15 min to complete.All data were exported into SPSS 26.0 for analysis. Data were first reviewed at a univariate and descriptive statistics level, and the ability of the data to conform to the assumptions of the planned analysis . AnalyseRealizing that there is a continuum between total acceptance and complete refusal of vaccinations, we conducted clustering analysis to further describe groups of currently unvaccinated HCWs holding varying degrees of indecision about vaccination. We used the kernel k-means method (Kernlab R package) and the kernel function of the radial basis (Gaussian kernel) to perform a cluster analysis. This method, representing a more generalized k-means approach to cluster analysis, is well-suited for linear and nonlinear separable inputs because the data type is usually unknown. Kernel k-means cannot determine the number of clusters. Therefore, we used the variance ratio criterion (VRC) to determine the number of clusters. VRC was selected due to its excellent performance against other internal criteria to determine the number of clusters . The litOverall, 2491 respondents answered the survey between 5 February and 17 April 2021. The HCWs participating in the survey were significantly impacted by the pandemic. Sixty-one percent reported having at least intermittent contact with COVID-19 patients, with 28% having frequent contact. Thirteen percent reported being diagnosed with COVID-19, and 42% said that someone close to them had suffered severe disability or died from COVID-19. Forty-seven percent of participants stated that the pandemic had negatively affected them financially. There was diversity in beliefs about the virus and the vaccine. Twenty-three percent considered seasonal influenza as more contagious than COVID-19, 32.6% overestimated the mortality associated with COVID-19, while 11.5% underestimated its severity. A sizeable fraction of the sample held conspiratorial beliefs about COVID-19, with 38% of the sample believing the virus is or could be manmade, 15% suggesting the impact of COVID-19 is overblown, and 6% not rejecting the notion that the pandemic is a hoax. A large majority (80.2%) identified vaccine efficacy to be 90% or greater.COVID-19 vaccination rate at the time of survey administration was high, with 2103 (84.4%) reporting having received it. Vaccine uptake was highest among physicians (96.2%) and lowest among respiratory therapists (70.3%), while 78.6% of nurses were vaccinated. Among the 391 unvaccinated HCWs at the time of the survey, 87 (3.5%) were willing to receive the vaccine, leaving 304 HCW (12.2%) whom we classified as vaccine hesitant. Additional sample characteristics can be found in p < 0.001) and achieved an overall correct classification rate of 88.6%. The Cox and Snell R2 was moderate at 0.33. This reflects the overall characteristics of the model in that the model was strong in predicting whether a participant was vaccinated (correct classification = 97.9%) but had a lower success rate at predicting the not vaccinated group (correct classification = 43.3%) and was fairly weak in predicting the hesitant group (correct classification = 26.6%). Thus, our model effectively predicted the likelihood of a participant being vaccinated. We used a Chi-square test to determine the likelihood of vaccination for all participants. The model’s overall fit was excellent  were slightly older and leaned Republican. They strongly opposed the COVID-19 vaccine, refusing to receive and/or recommend the COVID-19 vaccine. This group underestimated both the COVID-19 vaccine’s efficacy and COVID-19 mortality. They consider seasonal flu as more contagious and deadly than the COVID-19 virus. Finally, members of this cluster were more likely to believe several COVID-19 conspiracies . As seen in Respondents grouped in cluster 1 “misinformed” (cluster 2 “uninformed” (n = 94) tended to be less educated (60% lacking undergraduate degree), were more likely Hispanic/Latinx (47%), and worked in outpatient areas (33%) as allied health providers (60%). This cluster is the second least willing to receive the COVID-19 vaccine. This group underestimated the impact of the pandemic and the efficacy of the vaccine. They were primarily unsure about comparisons between COVID-19 and seasonal influenza. Unlike members of cluster 1, this group is less impacted by disinformation but lacks access to reliable and easy-to-understand vaccine information.Members of Cluster 3 “undecided” (n = 86) members were more open to receiving the COVID-19 vaccine, with half of the respondents unsure about vaccine receipt. Members of this cluster were predominantly White nurses and respiratory therapists working in an ICU. They understood the personal risk of exposure to the virus and knew the severity of COVID-19 disease, correctly assuming it is deadlier than seasonal flu. Participants in this cluster strongly leaned Republican. Cluster 4 “unconcerned” (n = 86) members were younger and racially diverse. This cluster is the most educated and leaned Democrat. Members of this cluster had an accurate knowledge of the vaccine’s efficacy and the lowest support of COVID-19 conspiracies. While hesitating to receive the vaccine themselves, respondents in this cluster were willing to recommend it to others  were vaccinated, and 304 (12%) were vaccine-hesitant. Vaccination and hesitancy rates varied by age, ethnicity, professional roles, work setting, political affiliation, attitudes toward influenza vaccination, and knowledge of both COVID-19 severity and vaccine efficacy. Furthermore, HCWs who believe that the media has exaggerated the severity of the pandemic perceived the risk of vaccination to be greater than the risk of infection. Our findings parallel those of other studies 4% were v,23,24,25We found that vaccine hesitancy was associated with older age and higher education. In additional analyses, age and education positively correlated with political affiliation (Republican) and occupation (nurse), respectively. Highly educated nurses were more hesitant to accept vaccination, often citing concerns in open-ended comments over unrealized side-effects of the vaccination, including its potential impact on fertility and pregnancy. HCWs who were leaning Republican tended to be older and more hesitant of COVID-19 vaccination. This underscores the politicized nature of the pandemic and the potential of one’s political affiliation to have a more substantial influence on vaccination decisions than age and susceptibility to the virus . This fiTrust is the essential factor in gaining acceptance of the COVID-19 vaccine. Alongside the public, HCWs have been exposed to conspiracy theories such as claims that the government intentionally created COVID-19 or that health organizations have exaggerated its lethality for financial or political purposes. These conspiratorial beliefs were strongly associated with vaccine refusal and were not limited to HCWs with lower education. Cognitive biases can be an underlying cause of conspiratorial beliefs even among educated HCWs. For instance, the availability heuristic may skew their perception of vaccination safety, while confirmation bias may strengthen their vaccine hesitancy through selective exposure to evidence ,33. HCWsWe found four distinct clusters among vaccine-hesitant HCWs, suggesting that the dichotomous “anti-vaccine vs. pro-vaccine” separation of HCWs may not be adequate in informing interventions. Cluster 1 members (misinformed) are dominated by vaccine-related myths and skeptical attitudes toward vaccine effectiveness. This cluster is the highest on the vaccine hesitancy continuum but also the smallest. Building trust within this group may be challenging and require strategies that utilize direct peer-to-peer communication [nication . For insnication . A stronnication . These nnication  or storinication . Vaccinenication . Cluster 2 (uninformed) appears to be the sub-group of HCWs with the greatest need for accurate and easy-to-understand vaccine information. An educational campaign providing evidence-based information on the safety and effectiveness of the vaccination, with contents addressing their concerns, could further COVID-19 vaccine acceptance in this group [is group . Such anMembers of cluster 3 (undecided) are the closest to acceptance on the vaccine hesitancy continuum. Their hesitancy may be attributed to partisan group identity. Several communication strategies can be effective in reaching this group. One is to highlight the non-partisan nature of vaccination decisions and endorsement of the COVID-19 vaccine from various political figures [ figures . It is i figures . Leverag figures  and HPV  figures . Additio figures  and WHO  figures  have relcluster 4 (unconcerned) are willing to recommend vaccination to others but have not been vaccinated themselves (yet). Their hesitancy may stem from under-estimating personal risks. Interventions rooted in behavioral economics (nudges) may increase vaccination rates in this group [Finally, is group . For insis group . Pre-schis group . Additiois group .While our study has many strengths, including recruitment from both a public and private hospital system, and across a broad range of HCWs, in this constantly evolving response to the pandemic, it is limited by its “point in time”, to a time when vaccinations were fairly new. Our response rate of 20.9% introduces nonresponse bias and may not be fully representative of the HCWs population at the two hospital systems or generalized to other hospital systems. However, this response rate mirrors other surveys on the same topic systematically reviewed by Li et al. . As therHCWs have a strong influence on patient and public perceptions of the COVID-19 vaccines, and therefore are one of the most valuable assets in disease prevention. Unvaccinated HCWs are less likely to recommend the vaccine to others ,8. Vacci


Post-operative cognitive dysfunction (POCD) can be a serious surgical complication, and patients undergoing cardiac procedures are at particular risk for POCD. This study examined the effect of blocking neuroinflammation on behavioral and neurogenic deficits produced in a rat model of cardiopulmonary bypass (CPB). Minocycline, a drug with established anti-inflammatory activity, or saline was administered daily for 30 days post-CPB. Treatment with minocycline reduced the number of activated microglia/macrophages observed in the dentate gyrus of the hippocampus at 6 months post-CPB, consistent with an anti-inflammatory action in this CPB model. Behavioral testing was conducted at 6 months post-CPB utilizing a win-shift task on an 8-arm radial maze. Minocycline-treated animals performed significantly better than saline-treated animals on this task after CPB. In addition, the CPB-induced reduction in adult neurogenesis was attenuated in the minocycline-treated animals. Together, these findings indicate that suppressing neuroinflammation during the early post-surgical phase can limit long-term deficits in both behavioral and neurogenic outcomes after CPB. Post-operative cognitive dysfunction (POCD) can complicate surgical outcomes, and is a particular risk for cardiac patients e.g., . The patThe prevalence of POCD is highest during the early post-operative period, and most experimental studies have investigated POCD during the first few post-operative weeks e.g., . Howevern = 6/group). The CPB procedure entails a 60-min period of bypass, and was performed as described previously  were randomized to two groups: (1) CPB + Saline or (2) CPB + Minocycline, , dehydrated and cleared through ascending concentrations of ethanol and xylenes, and coverslipped using Permount. Tissue was examined using a Zeiss Axioplan 2 microscope. Stereological cell counting was performed using optical dissector principles by two investigators blinded to the identity of the sections. Eight sections (30 μm thickness) from the septal  hippocampus were systematically sampled from each brain. ED1-positive or DCX-positive cells were identified and counted in the granular and infragranular layers of both the internal and external leaves (blades) of the dentate gyrus in each section. An average value for the eight sections was calculated for each animal. These analyses focused on the dentate gyrus in the hippocampus because changes in neurogenesis in the hippocampus are associated with alterations in learning and memory. The analyses did not include assessment of the subventricular zone (SVZ) because a sustained presence of activated microglia/macrophages was not observed in the SVZ after CPB.Animals were euthanized after behavioral testing for immunohistochemical assessments of activated microglia/macrophages and immature neurons. Rats were deeply anesthetized with isoflurane and transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) pH 7.4. Brains were removed, post-fixed for 2 days, cryoprotected in 30% sucrose, and cryostat sectioned at 30 μm in the coronal plane. Free-floating serial sections at the level of the septal  hippocampus were rinsed 3× for 10 min in PB prior to blocking for 60 min in hybridization/blocking buffer containing 5% normal serum of the species in which the secondary antibody was raised, 1% BSA, and 0.3% Triton X-100 in PB. Adjacent serial sections were incubated in the following primary antibodies in hyb/blocking buffer overnight at 4°C at the indicated dilutions: mouse monoclonal anti-CD68 antibody  and a rabbit polyclonal anti-Doublecortin (DCX) antibody . Sections were rinsed in PB, exposed to appropriate biotinylated secondary antibodies  in hyb/blocking buffer for 2 h at room temperature, rinsed, and incubated in avidin-biotin complex  2 h. Sections were developed using diaminobenzidine with NiSOMinocycline treatment during the first month post-CPB reduced the amount of activated microglia/macrophages observed in the hippocampus at 6 months post-CPB, a finding that is consistent with the known anti-inflammatory actions of this compound . The numTreatment with minocycline attenuated persistent post-operative cognitive deficits. Performance on the 8-arm, win-shift task at 6 months after surgery was significantly better in the CPB + Minocycline group as compared to the CPB + Saline group .Surgical and medical advances have greatly improved post-operative outcomes in recent decades. Nevertheless, the risk of POCD remains a concern, particularly in the context of cardiac surgery. Increasing evidence suggests that neuroinflammation plays an important role in POCD . AlthougThe present study strengthens this concept by demonstrating that post-CPB treatment with an anti-neuroinflammatory drug: (1) diminishes the activation of microglia, (2) attenuates the suppression of adult neurogenesis, and (3) limits cognitive deficits. Notably, these findings were obtained at 6 months post-surgery using a model of CPB in which substantial neuronal injury does not occur. These findings serve to further implicate neuroinflammation in POCD and, at least in this model, tend to rule out a role for surgically induced neurodegeneration.It is important to consider the design features of any study with respect to potential complicating factors: (a) The minocycline treatment paradigm utilized in this study [adopted from Microglia can play various roles in the physiology and pathophysiology of the central nervous system (CNS), depending on their state of activation. Moreover, beneficial as well as detrimental influences have been defined in the context of CNS disorders . The curIn summary, the current study provides evidence that post-operative treatment with an anti-neuroinflammatory drug can improve cellular and functional outcomes in a rodent model of CPB. Treatment with minocycline during the first post-operative month reduced microglial activation, limited neurogenesis suppression, and improved behavioral deficits when tested at 6 months post-CPB. These findings therefore provide additional support for a role for neuroinflammation in long-term POCD. In addition, the results are consistent with the concept that activated microglia and suppressed neurogenesis contribute to POCD. Finally, the long-term survival model of CPB utilized herein could prove valuable for assessing the behavioral, neuroinflammatory, and neurogenic effects of therapeutic candidates.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 the University of Virginia ACUC.YW: surgeon, manuscript preparation, and data analysis. MM: behavioral testing. TL: surgeon. CW: behavioral study design. RC and MA: histology. IK: study design. KL: overall study design, data analysis, and manuscript preparation. 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.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.


Tanzania is one of the countries confronting a multidrug-resistant tuberculosis (MDR-TB) epidemic.Research studies on drug susceptibility testing (DST) for second-line TB drugs given to Tanzanian MDR-TB patients has demonstrated mycobacterial resistance to important MDR-TB drugs, such as ethionamide, ofloxacin, amikacin, kanamycin, and pyrazinamide. Likewise, pharmacokinetic studies have shown a high frequency of patients with circulating serum drug levels below the expected ranges, especially for levofloxacin and kanamycin – key drugs in MDR-TB treatment that also affect ex-vivo plasma drug activity.inhA and/or katG genotypic results to optimize MDR-TB treatment. Quantitative drug susceptibility can guide the selection of options for second-line anti-TB drugs. The TB drug assay, an alternative biomarker for therapeutic drug monitoring, can identify patients who have extensively drug-resistant TB or are exposed to suboptimal serum drug levels of, specifically, levofloxacin and kanamycin.We suggest using molecular diagnostic assays, such as the GenoType MTBDRplus test, and  The empirical MDR-TB regimen recommended by the World Health Organization (WHO) consists of pyrazinamide – which belongs to the first-line TB regimen, or group 1 TB drugs – and at least 4 other second-line TB drugs.2 The second-line drugs are categorized in different groups, and the proposed regimen includes at least 1 drug from each group: 1 from the fluoroquinolone class, also called group 2 ; 1 from the injectable agents or group 3 ; and 2 from group 4 . Of the group 4 drugs, the order of preference is ethionamide, followed by cycloserine and para-aminosalicylic acid. These drugs are added until 4 effective drugs are established.2 If 4 drugs are not established or the efficacy of the combination is doubtful, 2 group 5 drugs  are selected – 2 of these group 5 drugs are counted as a single effective drug to strengthen the regimen.3 The recommended treatment duration is at least 20 months, with a minimum duration of 8 months for the injectable agent.2Multidrug-resistant tuberculosis (MDR-TB) is a public health crisis requiring novel approaches to diagnosis and treatment.8 One of the studies sought to describe the application of second-line drug susceptibility testing (DST) using minimum inhibitory concentration (MIC) results and compare those results with the empirical regimen used during a patient's treatment with a second-line regimen.5 MIC allows categorization of isolates near the resistance breakpoint, ‘borderline susceptible’, that may be labelled as ‘susceptible’ by conventional testing but are subject to clinical resistance with poor drug absorption, altered metabolism, or inhibited protein binding. In this study, we found the majority of patients had at least 1 medication that could have been modified with the application of MIC guidance. The most common medication modification in the MDR-TB regimen was changing ethionamide to para-aminosalicylic acid, which could have happened for more than 50% of patients.5 Although ethionamide is considered tuberculocidal at higher concentrations, a different study showed that only 22% of subjects had pharmacodynamic indices (serum concentration 2 hours post medication [C2hr]/MIC) ratios more than 2.6 While ethionamide may have a less predictable time to peak concentration than the other oral agents tested–which may result in an underestimation of the measured C2hr in some subjects–alternative second-line agents, such as para-aminosalicylic acid, may provide a more reliable benefit in the subset of patients with reduced drug exposure and borderline MICs. Additionally, research has shown that 22% of MTB isolates on kanamycin had borderline or resistant MICs, while amikacin retained full susceptibility and, therefore, required substitution within the aminoglycoside class.5 WHO recommends to substitute aminoglycosides in case of resistance into polypeptides, such as capreomycin, in group 3.3We have conducted several MDR-TB studies in Tanzania, aimed at improving treatment through an individualized approach.8 Moreover, 52% of our MDRTB patients had circulating levofloxacin serum drug levels below the expected range when the levofloxacin was given as a 750 mg daily dose.6 Recent studies of levofloxacin demonstrate that the best pharmacokinetic properties may be achieved at a dose of 1000 mg daily.9 Such optimization may be applicable in our setting, given that no subject in this study was on doses of levofloxacin as high as 1000 mg. The C2hr of levofloxacin for all subjects was below the median maximum serum concentration (Cmax) of 15.5 lg/mL.6 Correlating with what we observed, that a high proportion of MDR-TB isolates had borderline susceptibility on ofloxacin in MIC plates, these findings suggest a value to conducting clinical trials to evaluate levofloxacin at a dose of 1000 mg.Fortunately, in all of the studies we have conducted, resistance to the fluoroquinolone class was low, ranging from 5% to 15%; however, 45% of patients had isolates of borderline susceptibility.rpoB), isoniazid (inhA or katG), ethambutol (embB), pyrazinamide (pncA), ofloxacin (gyrA), amikacin (rrs or eis), and ethionamide (inhA).5 The results were compared with MICs, which showed a good correlation to the drugs tested. The few discrepancies showed resistance with MICs but were genotypically wild on the known regions conferring resistance. Although pyrazinamide MIC assays were not conducted, the pncA mutation was common, meaning that the suspected pyrazinamide resistance could have impact on treatment outcome.10We also looked further into the resistance patterns of MDR-TB isolates using genotypic methods in the following mutations: rifampicin , which identifies inhA and/or katG. The research suggests that a mutation in the inhA region alone can exclude ethionamide from the MDR-TB empirical regimen, while adding high-dose isoniazid and paraaminosalicylic acid to the regimen. While an exclusive mutation on katG includes ethionamide, a mutation of both inhA and katG excludes ethionamide and adds para-aminosalicylic acid only. Further research is required to determine the empiric choice of ethionamide or para-aminosalicylic acid while processing for MIC testing.Our results suggest that in Tanzania, rrs or eis mutations noted in the isolates, and the borderline susceptibility to ofloxacin, the new GenoType MTBDRsl assay  for these targets may be of less value. Instead, MIC testing would allow for selection within the class of aminoglycosides and support the use of high-dose levofloxacin.Given the low proportion of isolates with resistance to injectable agents, the lack of We, therefore, believe that quantitative susceptibility methods would prove as useful and cost-effective as MDRTB programmes that individualize management based on second-line drug susceptibility. This approach can be made even more cost effective by developing in-house laboratory platforms with the capacity to perform TB cultures.7 The TDA assay uses a patient's plasma or serum collected during TB treatment and the patient's own MTB isolate and measures time to detection in liquid culture. Following extensive in-vitro studies, the TDA assay revealed that it predominantly measures the concentration-dependent activity of the aminoglycoside and fluoroquinolone components of the standard MDR-TB regimen.6 The study further demonstrated the inactivity of pyrazinamide at the pH of the media used and the comparatively low concentration/MIC achievable for ethionamide and cycloserine. As such, in a patient on a standard MDR-TB regimen in Tanzania, a TDA value approaching 1.0 may be considered to have little plasma-killing potential. If this phenomenon occurs in the aminoglycoside or fluoroquinolone, it is similar to having XDR-TB, which indicates higher mortality and overall treatment failure. While the TDA assay cannot assess drug activity at the site of infection, subjects from Tanzania who had a faster time-to-sputum culture conversion were more likely to have high TDA values.7 However, it is important to note that plasma drug activity was not exclusively predictive of sputum culture conversion and the TDA assay could not discriminate the relative contribution of individual drugs in the MDR-TB regimen, particularly at the highest TDA values. This may be a consequence of the range of C2hr observed in subjects on the MDR-TB regimen and the unknown target concentration/MIC for a drug such as cycloserine that is not entirely concentration dependent or tuberculocidal in action.Our findings on quantitative MICs in MDR-TB patients led to research work on MDR-TB drug concentrations relative to MICs, particularly when compared to the TB Drug Activity (TDA) assay.11 The study found that modifications of the overnight-pooled sputum collection method improved the time to detection in the MGIT system among culture-positive samples. This is important not only for identifying susceptibility in phenotypic methods, but also for microbio-logical monitoring of patients during the course of MDR-TB treatment. We, thus, recommend improving sputum collection for efficient monitoring of microbiological responses by use of the overnight-pooled method.Several limitations were recognized in these studies: critical concentrations for second-line medication still remain a subject of debate and not all regions of mutations or MTB mechanisms for conferring drug resistance have been identified. Despite these limitations, we believe we have established options and evidence for optimization of MDR-TB management in Tanzania. With the scale up of rapid molecular diagnostics – specifically, GeneXpert and GenoType MTBDRplus – in the country, we expect more MDR-TB cases will be diagnosed and effectively treated than have been in the past. Those who need special attention, for instance those harbouring extensively drug-resistant TB , drug reactions, or delayed culture conversion will continue to be treated at the National Centre of Excellence for MDR-TB management. Previously, we compared the overnight-pooled method to the current standard spot technique for quality, quantity, and time to MTB detection by culture using the BACTEC mycobacterial growth indicator tubes (MGIT) system  for pulmonary TB-suspected patients.Examination of second-line DST conducted with Tanzanian MDR-TB patients showed resistance to the important MDRTB drugs, such as ethionamide, ofloxacin, amikacin, kanamycin, and pyrazinamide. Likewise, pharmacokinetic studies on existing MDR-TB regimens showed a high frequency of patients with circulating drug levels below the expected ranges, especially for levofloxacin and kanamycin – 2 key drugs in MDR-TB treatment that also affect ex-vivo plasma drug activity.inhA or katG results to determine if ethionamide, para-aminosalicylic acid, or high-dose isoniazid should be added to the empirical regimen. Then, quantitative MIC results for second-line DST should be determined within 2 months of initiation of treatment and used to alter the regimen, if needed. Monthly overnight-pooled sputum should be collected to monitor the time-to-culture conversion. Blood should be taken at weeks 2 to 4 for the TDA assay or alternate therapeutic drug-monitoring (TDM) test. In combination with other standard clinical factors, quantitative second-line drug MIC and TDA/TDM results should be used to tailor the appropriate regimen.To optimize MDR-TB management in Tanzania and similar settings at all levels, we propose the following: patients diagnosed with MDR-TB by either the GenoType MTB/RIF or GenoType MTBDRplus rapid molecular diagnostic test shall submit pretreatment overnight-pooled sputum for culture to establish baseline mycobacteriology for culture-based DST. Next, the empirical second-line (MDR-TB) regimen will be started, but the regimen will be based on


To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon  Coronavirus disease 2019 (COVID-19) has led to a global-scale pandemic creating an unprecedented burden on human health and public health processes . AlthougPlatelets are anucleated megakaryocyte-derived blood cells that play a prominent role in hemostasis and thrombus formation . Beyond Despite the importance of platelets in thrombotic events in COVID-19 patients, how heightened platelet activation is linked to clinical features of disease and the associated underlying mechanisms remain poorly understood. These gaps in our understanding of platelet function and dysfunction during SARS-CoV-2 infection limit our ability to identify patients at risk of thromboembolic events and to treat vascular complications of COVID-19 including clots. Moreover, identifying the inflammatory effector molecules and pathways that underlie the activation and dysregulation of platelets in COVID-19 could reveal novel opportunities for therapeutic intervention. To address these questions, we examined platelets and the platelet-activating potential of plasma from severely ill, hospitalized COVID-19 patients. These studies revealed an increase in basal expression of the activation marker P-selectin on platelets from severe COVID-19 patients coupled with poor response to Thrombin Receptor Activation Peptide (TRAP) stimulation, indicating loss of functional reserve, compared to platelets from convalescent and healthy donors. COVID-19 patient plasma robustly activated healthy platelets from control donors and this platelet activating potential was highest prior to the precipitation of a thrombotic event. Correlation of platelet activation induced by COVID-19 plasma with clinical features collected during patient hospitalization revealed significant associations with ferritin levels and key measures of clinical outcome including kidney and liver injury, and APACHE III scores. Moreover, proteomic analysis identified central mediators of inflammation and cardiovascular homeostasis correlating with a platelet hyperactivated state consistent with a role for platelets linking inflammatory events to thrombotic pathology. Finally, we identified a key role for Fc receptor and complement signaling in platelet activation in COVID-19 because blockade of signaling through the FcγRIIa-Syk and the C5a-C5aR axis using antibody blockade, depletion of immunoglobulin from COVID-19 plasma or the FDA approved drug fostamatinib blocked activation of healthy platelets by COVID-19 plasma. Thus, these studies identify a platelet hyperactive state associated with severe SARS-CoV-2 infection, define the underlying mechanisms, and have direct therapeutic implications for the prevention and treatment of thrombotic complications in patients with COVID-19.COVID-19 is associated with heightened activation of platelets both at baseline and after pharmacologic stimulation in response to low doses of agonists , 13. We ex vivo or after stimulation with TRAP that activates platelets via the thrombin receptor (ex vivo TRAP stimulation.CD62P (P-selectin) is bound to the membrane of α-granules within the cytoplasm of platelets, transported to the plasma membrane rapidly after activation and can be used as a marker of platelet activation. To evaluate the activation status of platelets from COVID-19 patients in our cohort, we assayed surface expression of CD62P directly A causal link between cardiovascular disease and COVID-19 outcomes, including thrombotic complications, has been proposed previously –29 but hex vivo analysis of platelets from COVID-19 patients, treatment of healthy platelets using plasma from COVID-19 patients did not induce higher CD62P expression than plasma from recovered or healthy donors . Following response to an extracellular stimulus, platelets degranulate and during this process translocate the granule membrane bound protein CD63 to their plasma membrane, thereby allowing CD63 surface expression to be used as a marker of platelet activation. We also quantified Fcγ receptor IIa , the only Fcγ receptor on platelets, and C3aR to evaluate changes in these receptors that could underlie potential effects of immune complexes and/or anaphylatoxins on platelet activation. The control platelets were pre-gated as CD42b+ (GPIba+) single cells  cutoff of 0.05] that is used to monitor liver injury. Of the markers of inflammation and cellular stress, ferritin had the strongest positive correlation with the ability of plasma to activate platelets . There was a statistically significant positive correlation between ferritin and CD63 or CD32 that also met our FDR cutoff  and other correlations reached nominal p value, but not FDR significance  using the O-link platform. This analysis interrogated 274 analytes in the blood with a focus on cardiovascular, inflammatory and organ damage related processes. We examined which of these circulating inflammatory mediators correlated with the ability of COVID-19 plasma to induce CD63 expression on platelets from healthy donors. Indeed, the concentration of numerous proteins in circulation correlated with the induction of CD63 on platelets Figure 3). Thus, we hypothesized that Fc receptor signaling, IL-6 signaling and/or the signaling by the anaphylatoxins C3a and C5a, might be causally involved in platelet activation by COVID-19 plasma. To test this hypothesis, we examined the effect of blocking each of these pathways on the ability of plasma from COVID-19 patients to activate platelets  strands localized to the region of light exposure perfused with hematoporphyrin. We have previously described this approach in studies of platelet activation in the prothrombotic disorder of heparin-induced thrombocytopenia . Plasma Platelets act as a cellular connection between the immune system and hemostasis, integrating signals from different immune cell subsets, soluble inflammatory mediators and the complement cascade. As a result, platelets have the capacity to link signals from exuberant immune responses to thrombotic complications. Severe COVID-19 results in loss of immune homeostasis either due to a suboptimal, albeit persistent, immune response to SARS-CoV-2 or due to immune system hyperactivation that is disproportionate to what is necessary for efficient virus elimination . The datCardiovascular disease risk factors, including hyperlipidemia, hypertension and diabetes, have been recognized early in the pandemic as important determinants of COVID-19 outcomes –29. Our in vitro platelet activation induced by COVID-19 plasma. Not only do these data identify a potential mechanism of platelet activation in COVID-19 via antibody and/or immune complex mediated platelet Fc receptor signaling, but they also point to a therapeutic intervention opportunity in COVID-19 patients, especially those with a high risk for thrombotic complications. Indeed, fostamatinib is currently being tested in a clinical trial in hospitalized COVID-19 patients . A preliminary report posted by the manufacturer of fostamatinib suggested that Syk inhibition might protect against COVID-19 induced hypoxia. Although speculative at this stage, this information might indicate that Syk is required for the formation of pulmonary microthrombi reported in patients with COVID-19 induced hypoxia . Health care workers were recruited at the Hospital of the University of Pennsylvania and received both a PCR test to assess for active infection and serologic testing for antibodies against SARS-CoV-2; all individuals included in this study were serologically convalescent and PCR negative . Healthy donors were recruited through word of mouth at the University of Pennsylvania and were adults with no prior diagnosis of or recent symptoms consistent with COVID-19. The sample size for all groups and experimental conditions is shown below:Experiments conducted on fresh platelet samples: COVID-19 inpatient (n=38), COVID-19 convalescent (n=23) and healthy donors (n=7)Experiments conducted using plasma samples: COVID-19 inpatient (n=63), COVID-19 convalescent (n=20) and healthy donors (n=9).Peripheral blood was collected from all subjects. For inpatient cases, clinical data were abstracted from the electronic medical record into standardized case report forms. APACHE III scoring was based on data collected in the first 24 hours of ICU admission or the first 24 hours of hospital admission for subjects who remained in an inpatient unit. Acute kidney injury (AKI) represented incident acute kidney injury that occurred on days zero – five of ICU admission, defined according to the AKIN creatinine criteria. Eligible patients for this outcome were free of end stage renal disease at baseline . Comorbidities including prior history of clotting (deep vein thrombosis (DVT), pulmonary embolism (PE), cerebrovascular accident (CVA), myocardial infarction (MI), or other thrombus) were collected prospectively based on the EMR. Cardiovascular disease (CVD) risk factors were considered present if patients had any of the following: diabetes, hypertension, hyperlipidemia, peripheral arterial disease, cerebrovascular disease, or known coronary artery disease. In-hospital clotting events were determined by EMR chart review and requiring a documented and date-stamped DVT, PE, CVA, MI, or other thrombus by duplex, echocardiogram, or contrast-enhanced imaging. Anticoagulation (AC) treatment at the time of research blood collection was recorded; “intermediate” dose AC was equivalent to enoxaparin 0.5 mg/kg subcutaneously twice daily in contrast to “prophylactic” dose enoxaparin of 0.5 mg/kg once daily. Clinical laboratory data were collected from the date closest to the date of research blood collection.ex vivo assays, whole blood was diluted 10-fold with Tyrode’s buffer and then used for stimulation assays with TRAP or vehicle in the presence of 50mM CaCl2. TRAP was added at a final concentration of 35uM for 15mins at 37°C. The activation was terminated by adding PBS/4% PFA for 20 mins. For in vitro activation assays of control platelets with COVID-19 plasma, healthy platelets were first isolated. Whole blood collected in citrate tubes was spun at 125g for 15 minutes and the supernatant was collected to isolate PRP (platelet rich plasma). PRP was spun at 330g for 10 minutes to collect pelleted platelets that were resuspended in 1x Tyrode’s Buffer. Platelets were subsequently activated on a 96-well plate with 50uL of sodium heparin isolated plasma derived from COVID-19 patients or healthy volunteers.Peripheral blood was collected into sodium heparin tubes . For directly Neutralizing monoclonal antibodies against C3a , C5a , IL-6  were used for the neutralization assays. Inhibition of activation of FcγRIIa by human IgG/immune complexes was achieved with the use of Human TruStain FcX . IgG depletion from plasma was performed using the Albumin IgG Depletion Spintrap from Millipore-Sigma (cat # GE28-9480-20) based on the manufacturer’s instructions. The active metabolite of fostamatinib R406 was purchased from Selleckchem (cat # S1533), dissolved in DMSO and used at a final concentration of 5μM.Antibodies used for staining of whole blood and platelets:5 platelets were acquired per each sample.Samples were acquired on a 4 laser BD FACS LSR. Standardized SPHERO rainbow beads  were used to track and adjust PMTs over time. UltraComp eBeads  were used for compensation. Up to 1x10Proximity Extension Assays using plasma derived from COVID-19 patients were performed using the commercially available Olink protein biomarker platform. Specifically, three Olink Target 96 panels were used: Olink Target 96 Cardiovascular III, Olink Target 96 Inflammation and Olink Target 96 Organ Damage panel. SARS-CoV2- RBG IgG and IgM measurements were performed by enzyme-linked immunosorbent assays (ELISA) as previously described .6 cells/channel of human umbilical vein endothelial cells  were seeded into the fibronectin  coated channels of a 48-well microfluidic plate  and then injured by flowing a 50ug/mL solution of hematoporphyrin (Sigma-Aldrich) with exposure to blue light using the HXP-120 C light source with 475-nm excitation and 530-nm emission filters as previously described (6 platelets in HBSSCa-Mg-) from healthy donors was labelled with calcein-AM  for 15 mins and then R406, Human TrueStain FcX or anti-C5a were added into the respective tube for a 15-minute incubation. 20µl of either healthy control or COVID-19 patient plasma  was added just prior to being flowed through the channel at 10 dynes/cm2. Platelet accumulation in the injured endothelium field was captured by Zeiss Axio Observer Z1 inverted microscope using Montage Fluxion software and analyzed using ImageJ as described  correction was performed by the Benjamini-Hochberg procedure at the FDR < 0.05 significance threshold unless otherwise specified.Due to the heterogeneity of clinical and flow cytometric data, non-parametric tests of association were preferentially used throughout this study unless otherwise specified. Correlation coefficients between ordered features  were quantified by the Spearman rank correlation coefficient and significance was assessed by the corresponding non-parametric methods (null hypothesis: ρ = 0). Tests of association between mixed continuous versus non-ordered categorical variables were performed by Wilcoxon test (for n = 2 categories) or by Kruskal-Wallis test (for n > 2 categories). All tests were performed two-sided, using a nominal significance threshold of Other statistical analysis was performed using Prism software (GraphPad). Other details, if any, for each experiment are provided within the relevant figure legends.All participants or their surrogates provided informed consent prior to participation in accordance with protocols approved by the regional ethical research boards and the Declaration of Helsinki.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the regional ethical research boards (University of Pennsylvania) and in accordance with protocols approved by the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.SA, CSA, and EW conceived the study. SA, AmS, HG, RG, DM, MA-H, and SG carried out experiments. SA, HG, AB, AG, CA, JW, ZC, YH, AP, OK, JD, AW, CI, DD, IF, AH, LV, JR, and NM were involved in clinical recruitment, sample allocation, processing and acquisition. All authors participated in data analysis and interpretation. SA, AmS, AaS, LR, LZ, MP, CSA, and EW wrote the manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.st Century Scholar Award from the University of Pennsylvania. DM and JG were supported by T32 CA009140. HMG was supported by T32 HL007586-34 from NHLBI. ZC was funded by NIH grant CA234842. DO was funded by NHLBI R38 HL143613 and NCI T32 CA009140. JB was supported by T32 HL007775-19. LAV is funded by a Mentored Clinical Scientist Career Development Award from NIAID/NIH (K08 AI136660). AH was funded by grant CA230157 from the NIH and the Tara Miller Foundation. JG is a Cancer Research Institute-Mark Foundation Fellow. MP and LR were supported by NIH R35 HL150698. CSA was supported by the US Public Health Service grants PO1 HL120846 and PO1 HL40387 from the National Heart, Lung, and Blood Institute (NIH). JG, JW, CA, and EW are supported by the Parker Institute for Cancer Immunotherapy which supports the Cancer Immunology program at the University of Pennsylvania. We thank Jeffrey Lurie and Joel Embiid, Josh Harris, and David Blitzer for philanthropic support. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.This work was supported by NIH AI105343, AI08263, and the Allen Institute for Immunology (EW). The adult COVID-19 cohort was supported by NIH HL137006 and HL137915 (NM) and the UPenn Institute for Immunology. SA was supported by T32 AR076951-01 from NIH, the Allen Institute for Immunology and the Chen Family Research Fund. RG was supported through a Raffensperger 21SH has received consultancy fees from Sanofi Pasteur, Lumen, Novavax, and Merck for work unrelated to this report. AH is a consultant for Immunai. EW is consulting or is an advisor for Merck, Elstar, Janssen, Related Sciences, Synthekine and Surface Oncology. EW is a founder of Surface Oncology and Arsenal Biosciences. EW is an inventor on a patent  submitted by Emory University that covers the use of PD-1 blockade to treat infections and cancer.NM reports funding to her institution from Quantum Leap Healthcare Collaborative, Athersys, Inc., Biomarck, Inc.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. 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Although pig-to-non-human primate (NHP) corneal xenotransplantation has shown long-term graft survival, xenogeneic antigen-related immune responses are still stronger than allogeneic antigen-associated responses. Therefore, there is an unmet need to investigate major rejection pathways in corneal xenotransplantation, even with immunosuppression. This study aimed to identify biomarkers in aqueous humor for predicting rejection and to investigate rejection-related pathways in grafts from NHPs transplanted with porcine corneas following the administration of steroids combined with tacrolimus/rituximab. NHPs who had received corneas from wild-type (WT) or α-1,3-galactosyltransferase gene-knockout (GTKO) pigs were divided into groups with or without rejection according to clinical examinations. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the proteomes of corneal tissues or aqueous humor. The biological functions of differentially expressed proteins (DEPs) were assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathways and protein–protein interaction network analysis. Among the 66 DEPs in aqueous humor, complement proteins  and cholesterol metabolic proteins (APOA1 and APOA2) were related to xenogeneic rejection as biomarkers, and alternative pathways of the complement system seemed to be important in xenogeneic graft rejection. Among the 416 DEPs of the cornea, NF-κB1 and proteosomes  seemed to be related to xenogeneic graft rejection. Additionally, oxidative phosphorylation and leukocyte activation-related pathways are involved in rejection. Overall, our proteomic approach highlights the important role of NF-κB1, proteosomes, oxidative phosphorylation, and leukocyte activation-related inflammation in the cornea and the relevance of complement pathways of the aqueous humor as a predictive biomarker of xenogeneic rejection. Corneal allotransplantation is the standard surgical treatment for medication-resistant corneal blindness . AlthougRecently, LC-MS-based proteomics has evolved to detect low abundant critical proteins in various diseases and allows possible longitudinal monitoring in small-volume samples . LC-MS-b+IFNγ+ T cells and the aqueous complement protein C3a were discovered to predict rejection in corneal xenotransplantation  rejected after corneal transplantation due to bullous keratopathy were obtained during re-corneal transplantation.This study adhered to the ARVO Statement regarding the Use of Animals in Ophthalmic and Vision Research. This study was approved by Seoul National University (SNU) (IACUC: SNU‐151102‐3) and SNU Hospital . Human corneas (n = 20) or GTKO (n = 5) miniature pig corneal xenotransplantation between 2016 and 2018 (We investigated a cohort of 25 rhesus macaques that had undergone full-thickness WT (and 2018 –16. Peneand 2018 . After tn = 5), AH at the nearest 20 weeks after transplantation, all recipients were transplanted with WT porcine cornea; 2) RO group (n = 6), AH at graft scores of 4 or 5, three recipients were transplanted with WT porcine cornea and the other three with the GTKO porcine cornea; and 3) rejection group (n = 7), AH at graft scores of 6, four recipients were transplanted with WT porcine cornea and the other three with the GTKO porcine cornea  survival (control) group (n = 2), in which all recipients were transplanted with WT porcine cornea, and 2) rejection group (n = 4), in which two recipients were transplanted with WT porcine cornea and the other two with the GTKO porcine cornea  were used for proteomics analysis. The six analyzed corneal samples were stratified as follows: 1) survival (control) group  analysis to minimize protein loss during the experiments. Each AH sample was collected and the protein concentration of each sample was measured using the BCA assay . An equal amount of protein of each AH sample (20 μg) was digested into peptides using 8 M urea in 100 mM ammonium bicarbonate  incubated for 20 min at room temperature (RT). Reduction using 10 mM dithiothreitol (Sigma) and alkylation with 30 mM iodoacetamide (Sigma) were held to denature the proteins. Trypsin was used to treat the sample [with a 1:50 (trypsin:sample) ratio] and incubated at 37°C for 12 h. To quench the trypsin activation, 0.4% TFA was added to the sample and the digested peptides were desalted using the C18 spin column (Pierce). The desalted peptides were dried and resuspended with sol A (0.1% formic acid) for the next step—LC-tandem mass spectrometry (MS/MS).m/z) was acquired in a resolution of 70,000 at m/z 200. Automated gain control (AGC) target value was targeted to 1.0 × 106 with a maximum ion injection time of 120 ms. The maximal ion injection time for MS/MS was set to 60 ms with a resolution of 17,500. To acquire various MS/MS features, dynamic exclusion time was set to 30 s.A high-throughput and high-resolution mass spectrometer, Q Exactive Orbitrap Hybrid Mass Spectrometer coupled with the EASY-nLC 1000 system , was adopted to identify as many proteins as possible. Three-hour gradients  were used. Full MS with scan range   . At leasp-value from independent t-test) were enriched and used for further analysis.LFQ intensity was processed with R software (v. 4.0.2). Potential contaminants were removed  and the intensity of the protein left was converted into log2 value and statistically analyzed. Differentially expressed proteins with strict cutoff  method  in water (pH 7.5) for labeling tandem mass tag (TMT) reagents (The cornea is composed of keratocytes and extracellular matrix (ECM) with collagens. Therefore, unlike AH, the cornea was individually cryopulverized using a cryoPREP device  and dissociated in lysis buffer  with sonication using a focused ultrasonicator. Twenty sonication cycles were repeated at 16°C. The homogenate was centrifuged at 16,000) method . An equareagents . A six-pm/z, the fixed first m/z was set to 100 m/z on MS/MS scan. The rest of the values are the same as the AH LC-MS/MS analysis.Since the cornea peptides were labeled with TMT reagents, different MS settings were used. To acquire the reporter ion Macaca mulatta, Sus sucrofa) from the UniProt database (2021.09 released. Reviewed + TrEMBL). In modification, carbamidomethylation on cysteine and TMT six-plex modification of lysine, N-termination as static modification, and oxidation of methionine as a variable modification were used. The 1% FDR for peptide level was adopted. Unlike MaxQuant LFQ analysis, TMT quantitative analysis used the reporter ion intensity to quantify the proteins. The exported reporter ions were normalized and used for statistical analysis. Since TMT quantitative analysis reduced the variation during the experiments, different cutoff values were applied to the cornea proteomics. Differentially expressed proteins with cutoff  were enriched and used for further analysis.Raw files from LC-MS/MS analysis proceeded with postexperiment monoisotopic mass refinement (PEMMR) to increase sensitivity in peptide identification and accuracy by selecting the unique mass class (UMC). MSGF+ was used to find proteins using two species  according to the manufacturer’s protocol. The upper detection limit of C3a concentration was 25 ng/ml.p-value GO terms were enriched and used for constructing a network. The STRING public database was used for the protein-to-protein interaction network model, and the network was built with sorted DEPs and interactome data using Cytoscape  or GraphPad Software .Gene Ontology (GO) search and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping were performed using g:Profiler . Less thytoscape . For mulQuantitative proteomics analysis identified 649 host proteins in the AH samples. In the principal component analysis (PCA), the RO and rejection groups were close to each other and were distinctly different from the survival group (p < 0.05) between the rejected and surviving grafts (p < 0.05) for DEPs between the rejected and surviving groups. The most enriched BP pathways included localization, myeloid leukocyte-mediated immunity, immune system process, and oxidative phosphorylation  affected protein expression in the rejected cornea. After the RO and rejection groups were combined and AH was compared between the groups transplanted with WT and GTKO porcine corneas, 17 proteins showed a more than 2-fold significant difference, including AZGP1, Ig-like domain-containing protein, SAA1, SAA2, and RAA1  , were inIncreased levels of other proteins, such as the acute phase response protein SERPINA3 as well as α-albumin, a protein related to the inflammatory breakdown of the blood–aqueous barrier, were observed (EGK_15801) , 35. TheComparing the results of xenografts with those of allografts, complement or cholesterol-related proteins did not change in aqueous humor of cases with allograft rejection , indicatvia Toll-like receptors (TLRs), tumor necrosis factor receptor (TNFR), T-cell receptor (TCR), B-cell receptor (BCR), or membrane attack complex (MAC) of the complement  and protein disulfide-isomerase  are involved in xenogeneic rejection, with a high betweenness centrality (Nuclear factor-κB (NF-κB), proteasomes, protein disulfide-isomerase, and immunometabolism regulated by ETC coupled with oxidative phosphorylation appear to play important roles in corneal xenogeneic rejection under treatment with conventional immunosuppressive agents. NF-κB signaling contributes to the activation of T or B cells or hampers regulatory T-cell (Treg) activation in allogeneic as well as xenogeneic rejection –43. NF-κmplement , 42. Givmplement , 39, com cascade , 45. Pro cascade –46. Consltration –49, protntrality , 51. Metntrality –54. Protntrality , 55–59. ntrality . In addintrality –63, was In the subgroup AH analysis, we compared the groups transplanted with WT and GTKO porcine corneas. Surprisingly, there were only a few DEPs in either the AH or rejected grafts. Most DEPs in the AH were Ig-like domain-containing proteins. When the GTKO cornea was transplanted, it was expected that the antibody reactions would be mild; however, the expression of Ig-like domain-containing protein was higher in the GTKO group than in the WT group. The GTKO porcine cornea showed decreased αGal antigen expression; however, it may increase the expression levels of non-Gal antigens. Indeed, elevated levels of non-Gal IgG were reported in rejected GTKO porcine corneas after corneal xenotransplantation . In addiBecause the cornea is an avascular organ, the effect of hyperacute rejection involving alpha Gal is expected to be less than that of a solid organ such as the kidney and heart. In addition, GTKO porcine cornea may increase the expression levels of non-Gal antigens because the level of non-Gal antigens from GTKO porcine fibroblasts is much higher than that from WT porcine fibroblasts . Indeed,There is only one study regarding pig-to-NHP solid organ xenotransplantation. In the study of heart xenotransplantation, 14 potential proteins including fibronectin, annexin, and vimentin were identified as targets of IgG antibodies . In lineM. mulatta and S. scrofa lacks reviewed data, unlike human or mouse databases. These sample- and species-associated limitations further restricted the protein identification process. In addition, the cornea is composed of abundant amounts of ECM and collagen matrix, and the major proteins present in the cornea are mostly isoforms of collagen or ECM proteins. Of course, due to the nature of the cornea, mass spectrometry analysis with a higher resolution and novel methods for analyzing more proteins, such as cytokines or interleukins, will be necessary in order to deepen our understanding of the cornea. Due to the lack of samples, it was difficult to quantify each protein using targeted methods to validate our results.This study has several limitations. First, the immunosuppressive regimen was variable among the subjects, which may have affected protein expression. There were no different basic conditions of the recipients with and without immunosuppression, and we performed several NHP studies to find the most appropriate immunosuppressive regimen. Through the studies of pig-to-NHP corneal xenotransplantation using various immunosuppressant regimens , 16, we In this study, we aimed to evaluate the proteomic changes in the corneal tissue, identify AH biomarkers related to graft rejection, and explore the changes in xenogeneic rejection-networking pathways according to donor characteristics (WT and GTKO porcine graft) after corneal xenotransplantation. The complement pathways of the AH can be used as predictive biomarkers in xenogeneic rejection, and alternative pathways are expected to be mainly involved in the complement system. The difference in protein expression according to the donor characteristics was small. Proteins related to platelet activation or thrombosis were downregulated in the GTKO cornea (compared with their expression in WT). Nevertheless, both the GT KO and WT grafts were similarly rejected, suggesting that the cornea was affected by other biological pathways related to rejection even when the GTKO cornea was used. Upon combining the results of proteomic analysis of the AH and cornea, the complement pathway of AH or corneal NFKB/proteasome-related proteins was found to be associated with graft rejection. It is presumed that the combination of immunosuppressive agents used in the study is not sufficient to inhibit these pathways and the additional use of eculizumab (a complement pathway inhibitor) or bortezomib (a proteasome/NF-κB activation inhibitor) can reduce xenogeneic rejection after corneal xenotransplantation. In addition, the use of genetically modified pigs expressing GTKO, CD46, CD55, and/or human A20 that modulate the complement and NF-κB pathway may also reduce rejection in pig-to-non-human primate corneal xenotransplantation.In conclusion, the NFKB/proteasome-related proteins were associated with corneal xenograft rejection even with immunosuppressive agents. Furthermore, the complement activation product C3a was found to be an aqueous biomarker for the progression of xenogeneic rejection in pig-to-NHP corneal xenotransplantation, and xenogeneic rejection-networking pathways were found to be not so different between WT and GTKO corneal grafted NHPs. Further studies with a larger number of experiments are needed to determine and select more accurate markers and to validate our hypotheses.via the PRIDE partner repository with the dataset identifier PXD031320.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium The studies involving human participants were reviewed and approved by Seoul National University Hospital Institutional Review Board for the use of human corneas (No. 1102-092-353). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Seoul National University (SNU) (IACUC: SNU‐151102‐3) and SNU Hospital .MK and KK conceived and designed this study. JO, CY, and JR performed the experiments. JO, CY, KK, and MK analyzed and interpreted the data and prepared the manuscript. All authors contributed to the article and approved the submitted version.This research was carried out with the support from the Korea Healthcare Technology R&D Project, Ministry for Health & Welfare, Republic of Korea (grant number: HI13C0954), and the Gyeonggi-do Regional Research Center Program of Gyeonggi Province [grant number: GRRC-Kyung Hee 2018(B03)].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.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.


Older age and a higher overhydration state were associated with GDF-15/Albumin ratio. The ROC analysis confirmed that the ability of the GDF-15/Albumin ratio to predict mortality was superior to GDF-15 or albumin alone. In conclusion, the GDF-15/Albumin ratio measured at the initial maintenance hemodialysis is an independent prognostic marker of two-year mortality in ESRD patients.It is important to identify risk factors related to mortality in end-stage renal disease (ESRD) patients starting renal replacement therapy. Recently, several studies proposed that growth-differentiation factor-15 (GDF-15) is a possible biomarker for the prognosis of patients on maintenance hemodialysis. Here, we investigated the predictive value of serum GDF-15/Albumin ratio on two-year mortality in ESRD patients initiating maintenance hemodialysis. The study was a single center, retrospective study on ESRD patients starting maintenance hemodialysis with a follow-up of two years. All patients completed laboratory test and bioimpedance spectroscopy prior to the initiation of the first dialysis. The patients were stratified into quartiles according to the quartiles of serum GDF-15/Albumin ratio. Among the 159 patients, the mean age was 61.78 ± 12.52 years and median survival was 20.03 ± 7.73 months. The highest GDF-15/Albumin quartile was significantly more associated with the increased risk of all-cause mortality than other quartiles (unadjusted hazard ratio (HR): 8.468, 95% CI 2.981–24.054,  Chronic kidney disease (CKD) is one of the many increasing global public health issues causing a high socioeconomic burden to health systems . As a reDuring the last decade, the all-cause mortality of ESRD patients on maintenance hemodialysis has gradually decreased  owing toResearchers have attempted to develop biomarkers to predict mortality in ESRD patients. Serum albumin, creatinine and cholesterol were the focus in the 1990s ,15, paraRecently, growth differentiation factor-15 (GDF-15), also known as macrophage inhibitory cytokine-1 (MIC-1) a stress-responsive member of the transforming growth factor-β (TGF-β) cytokine superfamily, has been studied in various chronic diseases as biomarker for disease progression and mortality. GDF-15 is weakly expressed under normal physiologic conditions and upregulated in response to oxidative stress, tissue ischemia, inflammation, and metabolic stress ,20. Its GDF-15 is also known to be elevated in various kidney diseases and recent studies have suggested that GDF-15 is a prognostic marker of disease progression and mortality in CKD patients. In a cohort study of non-dialysis CKD patients, circulating GDF-15 levels were significantly associated with increased risk of progression of CKD . Tuegel Meanwhile, whether GDF-15 levels measured before starting hemodialysis can reflect the survival of ESRD patients during maintenance hemodialysis remains poorly studied. Considering that the premature mortality of ESRD patients occurs more frequently within a year after the initiation of dialysis , it is iIn this study, we investigated the prognostic value of serum GDF-15 at the initiation of hemodialysis for all-cause mortality in ESRD patients and the associations between GDF-15 and clinical variables. We analyzed the role of GDF-15 in combination with serum albumin as a newly designed index, GDF-15/albumin. We assumed that GDF-15/Albumin measured at the initiation of hemodialysis is associated with increased risk of all-cause mortality in patients starting hemodialysis.This study was a single-center retrospective study at the Chungnam National University hospital, Daejeon, Republic of Korea. We reviewed the medical records of the patients who were diagnosed with ESRD, naïve to renal replacement therapy and prescribed to start maintenance hemodialysis by our division in the outpatient-clinic, emergency room, general ward or intensive care unit from May 2014 to August 2019. Nephrologists in our hospital diagnosed ESRD based on clinical features, and laboratory tests including eGFR and ultrasonographic findings of chronic kidney disease . Maintenance hemodialysis was defined as three hemodialysis sessions per week required for the life-long replacement of renal function. All patients were encouraged to undergo full baseline evaluation on the first day of hemodialysis before the initiation of dialysis if possible. The full baseline evaluation included a bioimpedance spectroscopy (BIS) for the assessment of body composition, routine laboratory tests which were necessary for for proper treatment. Also, full evaluation included deposition of additional blood samples for extended laboratory tests requiring written consent for the single center CKD cohort conducted in our hospital. The exclusion criteria were being under age 18 or over age 85, evidence of acute kidney injury or acute kidney disease, a hemodialysis duration less than two weeks, having baseline studies not conducted before the first hemodialysis on the date of initiation, incomplete pre-dialysis bioimpedance spectroscopy results, incomplete laboratory data, or previous history of transplantation. The protocol for this study was reviewed and approved by the Institutional Review Boards of Chungnam National University Hospital (No. 2021-05-068).The body composition and hydration state for patients was assessed with the Body Composition Monitor , a portable bioimpedance spectroscopy device. All measurements were performed by one trained nurse before the start of the first hemodialysis with the patients relaxing in supine position for at least 5 min prior to the measurement. Collected data were overhydration (OH), total body water (TBW), extracellular water (ECW), intracellular water (ICW), lean tissue index (LTI), fat tissue index (FTI), lean tissue mass (LTM), fat mass, body cell mass (BCM) and dry weight. To compare the volume state between patients, the hydration state was normalized to OH/ECW and OH/ECW > 15% was considered overhydrated ,35.The biospecimens and data used for further analysis on GDF-15 were provide by the Biobank of Chungnam National University Hospital, a member of the Korea Biobank Network. Blood sampling to measure the level of GDF-15 was carried out at the first access to previously formed hemodialysis vascular accesses sites .Within six hours of sampling, the blood samples were centrifuged and the serums were carefully stored at −80 °C in the Human Resource Tissue Bank (Biobank) at our hospital. An experienced researcher in the lab who was not privy to the patients’ clinical information performed the measurement of the GDF-15 serum levels twice using an enzyme-linked immunosorbent assay (ELISA) . In this study, the average of the duplicated values of serum GDF-15 were applied for further analysis.Information regarding the patient’s baseline characteristics and comorbidities were collected by reviewing previous medical records. Major comorbidities included coronary artery disease, heart failure, cerebrovascular disease and malignancy. All laboratory tests were performed on the blood samples within 6 h prior to the start of the first hemodialysis. The estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) equation.The primary outcome was all-cause mortality. Deaths were identified by reviewing hospital records on the National Health Insurance Service database. Patients were censored at the last visit or when receiving kidney transplantation during the 24-month follow-up period.p value of <0.05 was regarded as statistically significant and two-tailed test of the hypothesis was assumed to obtain 95% confidential intervals. To compare baseline characteristics, one-way analysis of variance (ANOVA) and Kruskal-Wallis test were used for continuous variables and Chi-square tests or Fisher’s exact test were used for categorial variables. Pearson’s correlation coefficients were calculated to investigate the correlation of the GDF-15/albumin ratio and other clinical parameters. Univariate and multivariate linear regression analysis were performed to test the association of the GDF-15/albumin ratio and clinical parameters. Also, a univariate and multivariate logistic regression analysis were conducted to determine the association between the highest quartile of GDF-15/albumin ratio and continuous variables. Kaplan-Meier analysis was performed to compare the survival time for patients classified by quartiles of GDF-15/albumin ratio, and the Log-rank test was used to compare the difference in survival times. ROC analysis was used to evaluate the predictive value of GDF-15/albumin to estimate all-cause mortality within the first 24-months after the initiation of hemodialysis. Youden’s index was used for selecting the optimal cut point for each variable. All statistical analyses were performed using SPSS version 26 .Data are presented as means ± standard deviatioor median (interquartile range) for continuous variables and number with percentage for categorical variables as indicated. A A total of 2123 patients started maintenance hemodialysis at Chungnam National University Hospital between May 2014 and August 2019. A total of 293 patients underwent baseline evaluation including blood samples and pre-dialysis bioimpedance spectroscopy (BIS) on the same day of the initiation of hemodialysis. A total of 178 patients deposited additional blood samples for further investigation with informed consent. Finally, 159 patients were eligible for analysis and they were divided into four groups according to quartiles of GDF-15/albumin ratio .The baseline characteristics of the study population are listed in n = 12) with the shortest mean survival (16.58 ± 7.79 months). And these patients had lower levels of total protein, albumin and albumin-to-globulin ratio (A/G ratio) with statistical significance. Furthermore, these patients were likely to have lower levels of serum blood urea nitrogen and creatinine and higher levels of eGFR although the differences among quartiles were not statistically significant. Underlying cardiovascular comorbidities did not differ across quartiles.The patients in the highest GDF-15/albumin quartile were older, more overhydrated (higher OH/ECW), and had higher levels of serum GDF-15. Compared to the other three quartiles, the patients in the highest quartile exhibited the largest numbers of mortality (n = 1), septic shock (n = 2), multiorgan failure (n = 2), multiple myeloma (n = 1), chronic myeloid leukemia (n = 1), acute subdural hemorrhage (n = 1). The exact cause of death in remaining nine patients were impossible to assess since the mortality events occurred outside of our hospital and the information from the National Health Insurance Service database lacked the exact cause of death.In this study population, 17 all-cause mortality events occurred during the 24-month follow-up and the causes are as follows; aspiration pneumonia (p < 0.001) than the other three quartiles , OH/ECW , OH , total cholesterol , and eGFR-MDRD  levels were positively correlated with GDF-15/albumin while total protein  levels were negatively correlated with GDF-15/albumin.Correlations with GDF-15/albumin and clinical and biochemical parameters were examined with Pearson’s correlation coefficients. Age  . Also, ip < 0.001) during the first 24-months after starting maintenance hemodialysis : 8.468, 95% CI 2.981–24.054, dialysis . After mp < 0.001), 0.758 for GDF-15 alone , 0.715 for A/G ratio  and 0.638 for OH/ECW . Serum albumin had an AUC of 0.766 . Overall, GDF-15/albumin was a better predictor for all-cause mortality than GDF-15 alone, the overhydration state, A/G ratio or albumin in patients starting maintenance hemodialysis. The optimal cut-off value of GDF-15/albumin was 219.23 ng/g with a sensitivity of 76.5% and a specificity of 79.1%.We performed ROC analysis to examine the predictive accuracy of all-cause mortality of GDF-15/albumin . Accordip-value less than 0.05, which was considered statistically significant in univariable linear regression analysis. For model 2 in multivariable linear regression, we selected age and OH/ECW since these two markers were previously reported to have association with increased mortality in hemodialysis patients. For model 3, we included all variable with statistical significance to compare the independent power in the regression model. As a result, we confirmed that age and OH/ECW were eligible for prediction of GDF-15/Albumin.Here, we demonstrated that an elevated GDF-15/albumin ratio measured at the initiation of hemodialysis is associated with all-cause mortality within two years. The association was independent of all-cause mortality after adjustment for age, diabetes mellitus, heart failure, OH/ECW and eGFR. In addition, the predictive value of the GDF-15/albumin ratio was better than that of GDF-15 alone or other markers such as overhydration status or hypoalbuminemia proven by ROC analysis. In Pearson’s correlation coefficient analysis, GDF-15/Albumin showed positive correlations with age, OH/ECW, OH, total cholesterol and eGFR. For the multivariable linear regression models , we selePrevious studies showed correlations between GDF-15 and various kidney disease. Studies conducted from our hospital showed that GDF-15 is a predictor of the progression of membranous nephropathy  and adveIn terms of hemodialysis, several studies have investigated the role of GDF-15 for predicting mortality. A study by Yilmaz et al. (2015)  revealedOur preliminary data focused on GDF-15 alone also demonstrated that a higher GDF-15 measured at the dialysis initiation was correlated to an increased risk of death during the follow-up period . When thAlbumin is a well-known marker for inflammation as well as nutrition in various chronic diseases. The two previously mentioned studies also revealed that higher GDF-15 levels were associated with hypoalbuminemia ,31 in marel: hydration state/extracellular water) had a high predictive value to predict all-cause mortality in patients on maintenance hemodialysis and ΔHSrel > 15% was strongly related to increased death in the study population [Fluid overload is a well-established risk factor for increased mortality in patients on hemodialysis. The findings from our study support previous studies showing the value of volume overload assessed with whole-body BIS on predicting all-cause mortality in ESRD patients . The relpulation . For patpulation . Exposurpulation .Although the exact correlation between GDF-15 and OH/ECW has not been fully evaluated, serum GDF-15 was reported to be elevated in patients with heart failure ,42. GeneLower BMI in dialysis patients is also associated with higher all-cause mortality while higher BMI seems to have a protective effect on survival, partially explained by the obesity paradox ,45. NoneThe strength of this study is the uniformity of race and the same treatment protocol performed in a single hospital despite retrospective study. In addition, according to our knowledge, this is the first study to show that GDF-15/Albumin ratio is a useful tool for predicting survival in patients starting maintenance hemodialysis. This study may provide inspiration to further survival studies using GDF-15.Our study has several limitations to be acknowledged. First, since this was a retrospective study, some findings may have been exposed to possible confounding factors. Second, the number of subjects in this study was relatively small. This is primarily due to the fact that we were strictly allowing cases to be included only if all baseline studies were fully completed on the same day of dialysis initiation and before the start of the first session. Third, wherever GDF-15/Albumin ratio is associated with cardiovascular disease or heart failure is not fully studied. Only a limited number of patients were evaluated by a cardiologist with previous or on-going evidence of co-existing cardiac comorbidities. Specifically, it was not a routine for patients to undergo coronary angiogram or transthoracic echocardiogram prior to starting maintenance hemodialysis. Therefore, the number of patients with underlying cardiac conditions, which may have an influence on the increase in both GDF-15/Albumin ratio and risk of early mortality, may have been underestimated.In conclusion, elevations in the GDF-15/Albumin ratio at the start of the initial maintenance hemodialysis are associated with a two-year mortality in ESRD patients. GDF-15/Albumin, the newly designed marker, may be helpful to assist physician to predict early mortality and provide chances to search for modifiable factors to prevent early mortality in this population. Further prospective studies are required to determine whether the GDF-15/Albumin ratio at the start of hemodialysis is valuable for predicting long-term survival during life-long renal replacement therapy.