Recent discoveries have shed new light onto immunoglobulin M (IgM), an ancient antibody class preserved throughout evolution in all vertebrates. First, IgM – long thought to be a perfect pentamer – was shown to be asymmetric, resembling a quasi-hexamer missing one monomer and containing a gap. Second, this gap allows IgM to serve as carrier of a specific host protein, apoptosis inhibitor of macrophages (AIM), which is released to promote removal of dead-cell debris, cancer cells, or pathogens. Third, recombinant IgM delivered mucosally by passive immunization gave proof-of-concept that this antibody class can prevent mucosal simian-human immunodeficiency virus transmission in non-human primates. Finally, IgM’s role in adaptive immunity goes beyond being only a first defender to respond to pathogen invasion, as long-lived IgM plasma cells have been observed predominantly residing in the spleen. In fact, IgM produced by such cells contained somatic hypermutations and was linked to protection against lethal influenza virus challenge in murine models. Importantly, such long-lived IgM plasma cells had been induced by immunization 1 year before challenge. Together, new data on IgM function raise the possibility that vaccine strategies aimed at preventing virus acquisition could include this ancient weapon. Immunoglobulin M (IgM) is the first responder to foreign invaders – including viral pathogens that cause major pandemics. It is the only antibody class that exists in all vertebrate animals . Its monBecause IgM is the first antibody response in viral infections, this Ig class has important value for diagnosis. IgM’s pentameric structure prevents passage across the placenta. Consequently, viral infections of the fetus or newborn are recognized by IgM responses against the background of transplacentally transferred maternal IgG.IgM’s multimeric structure is well suited to bind viral surface proteins. The high avidity may also allow IgM to better tolerate mutations in viral targets – an important consideration for viral pathogens with high mutation rates. IgM is also a potent complement activator. However, despite IgM’s unique characteristics, its role in the prevention and treatment of viral infections remains understudied. The goal of this review is to give an overview of recent data regarding IgM structure, function, and IgM’s role in acute and longer-lasting antiviral host defenses against virus acquisition.Monomeric IgM consists of two heavy (μ) and two light (L) chains, like monomers of all other antibody classes. The μ chain constant region contains four domains (Cμ1-Cμ2-Cμ3-Cμ4) and a C-terminal tailpiece . The Cμ2Multiple IgM monomers assemble through interchain disulfide bridge formation between cysteines in the Cμ2, Cμ3, and the tailpiece to form polymeric IgM. In the plasma of humans and mice, the pentameric form is the most abundant IgM version. It contains five monomers and an additional small protein, the joining (J) chain, which bridges the cysteine residues within the tailpiece of two neighboring IgM monomers , 9.The most widely accepted structure of the IgM pentamer is a symmetrical pentagonal structure based upon negative-stain electron microscopy (EM) , 11. In Due to its multimeric structure, IgM has a high valency compared to other immunoglobulins. This characteristic allows IgM to compensate for low affinity to a degree and to bind target antigens with high avidity. Multimeric IgM also binds antigens with repeated epitopes efficiently and results in agglutination of bacteria, red blood cells, and viruses , 16.The presence of the J chain allows pentameric IgM to interact with the pIgR expressed on the basolateral surface of epithelial cells. This interaction leads to the formation of an IgM-pIgR complex that is transported across the cell to the luminal side where pIgR is proteolytically cleaved. A pIgR fragment, termed secretory component (SC), remains bound to the pentameric IgM giving rise to secretory IgM. Thus, the pIgR-mediated transport across the epithelial layer grants IgM access to the mucosal lumen . The intcd5l gene), according to EM images by Hiramoto et al. , AIM’s presence affects the interaction of the resulting IgM-ICs with Fcα/μR expressed on the surface of follicular dendritic cells (FDCs). As a result, internalization of the IgM-ICs is slowed, thus prolonging their surface retention. This facilitates antigen presentation to B cells and stimulates secondary antibody responses .After binding to a surface-exposed antigen, multimeric IgM shifts its conformation to a staple-like arrangement . During in vitro. This process could facilitate the processing and presentation of antigens to T-helper cells for the induction of immune response against T-dependent antigens  and Toso/Fas apoptotic inhibitory molecule 3 (FAIM3) . It is e+ B1 subset of B cells (There are two types of secreted IgM (sIgM), natural (innate) and adaptive IgM; the latter is also termed “immune” or “induced” IgM. Even without apparent antigenic stimulation, circulating antibodies are present in human cord blood  and in t B cells  in the s B cells –50. B1 c B cells –53. Natu–/–), viruses disseminated much faster than in wild-type (WT) mice during early-stage infection. Natural IgM trapped virions in the spleen and reduced blood-borne viral trafficking to non-lymphatic organs, such as the kidney. When serum from naïve WT mice that contained predominantly natural IgM was passively transferred to sIgM–/– mice, the recipients were protected against lethal VSV challenge. Moreover, Ochsenbein et al.  neutralization by direct binding to viral proteins or viral receptors expressed on potential viral target cells, with or without complement; (ii) trapping of virions by aggregation; and (iii) transporting viral antigens as ICs to lymphoid tissues, thus promoting adaptive immune responses. The latter most likely occur through complement activation or Fc receptor interaction.In contrast to natural IgM, adaptive IgM is the first antibody class produced by the body in response to an invading pathogen and is mostly produced by B2 cells in the spleen and lymph nodes. Adaptive and natural IgMs are structurally and functionally similar despite the different producer B cells; the only difference is that natural IgM possesses more flexible antigen-binding sites that provide broader reactivity to various antigens –64. Alth–/– mice that, as mentioned above, do not produce sIgM but can secrete other immunoglobulin classes/isotypes. All of the WT mice survived, in contrast to only 25% of the sIgM–/– mice , which can only produce IgM but none of the other Ig classes due to impaired ability to undergo class-switching and somatic hypermutation, survived E. muris challenge. In contrast, 80% of mice lacking all secreted Ig classes succumbed to the challenge, suggesting that IgM alone can protect against E. muris infection. To determine the duration of IgM-mediated protection, the authors exposed AID-deficient mice to a different, more pathogenic bacterial strain, Ixodes ovatus ehrlichia (IOE). The re-exposure occurred 250 days after the initial E. muris infection, from which all mice had recovered; the majority of them survived IOE rechallenge, while none of the naïve mice did. These data suggest the E. muris-induced, IgM-mediated immunity was long-lasting. To exclude the possibility that the long-lasting IgM responses required bacterial persistence, the authors treated the E. muris infection with an antibiotic. After this, titers of E. muris-specific serum IgM were significantly reduced, whereas the IgM-secreting cell population persisted at only slightly lower numbers. These IgM-secreting cells still protected 75% of the mice against lethal IOE challenge 77 days after the initial E. muris infection. In contrast, none of the naïve mice survived, regardless of prior antibiotic treatment. In this mouse model, E. muris infection functioned akin to a live attenuated vaccine that induced antigen-specific IgM responses that cross-protected against lethal IOE rechallenge. Of note, these E. muris-induced IgM responses persisted for months and did not require the chronic presence of E. muris.According to conventional views, adaptive IgM is produced only during acute infection, is short-lived, and is not associated with hypermutation/affinity maturation. However, Racine et al. , 75 demot1/2 = 145 days; p = 0.82)  .Next, Bohannon et al.  immunizeTaken together, the data summarized in this section suggest that adaptive IgM contributes to the humoral memory and long-term protection against pathogen infections. The findings also imply that vaccination could potentially induce long-term protective IgM responses.Adaptive IgM is the first virus-specific antibody class to emerge after HIV infection – like in any infection. In 1987, Cooper et al.  reportedT0), which was approximately 10 days after virus transmission. The first free plasma anti-HIV antibodies were anti-gp41 IgM detected in 41% of subjects at a median of 13 days  after T0. These responses decreased over 20 to 40 days, while IgG responses rose over the same period  shortened the window period, i.e., the time before HIV infection can be diagnosed serologically. Testing for HIV-specific IgM could give positive readouts at a median of 23 days after infection compared to 31 days by tests measuring only IgG .In vitro, 33C6-IgM neutralized and captured challenge virus particles significantly better than its IgG1 counterpart.Unlike anti-HIV IgG or IgA, anti-HIV IgM has remained relatively understudied other than developing it as a diagnostic tool. Recently, we sought to investigate the potential of anti-HIV envelope IgM in preventing virus acquisition in a non-human primate model. We chose passive immunization as our tool because it restricts protection solely to the passively administered antibody and thus establishes a cause-and-effect relationship between the antibody and protection. We first constructed a recombinant human monoclonal anti-HIV IgM using the variable genes of the high-affinity neutralizing antibody, 33C6-IgG1, which targets the conserved V3 loop crown of HIV gp120 . The resenv. After intrarectal (i.r.) passive immunization, 33C6-IgM prevented viremia in four out of six rhesus monkeys challenged i.r. with a single high-dose of SHIV, while all control animals were highly viremic. The degree of protection was similar after i.r. passive immunization with the IgG1 version of the same mAb. We propose that efficient trapping of infectious virions in the mucosal lumen – i.e., immune exclusion – combined with direct virus neutralization, represent the mechanisms of protection by IgM  that carries HIV-1 n by IgM . To our in vitro data lend support to the protective mechanisms we proposed in our passive immunization study in the rhesus macaque/SHIV model.Subsequently, Devito et al.  isolatedSARS-CoV-2, the cause of the newest pandemic, continues to spread exponentially in many countries. More than 16 million people have been infected worldwide , and manThe IgM field is undergoing a major but quiet revolution. There is more to this ancient antibody class than what textbooks have described. To begin with – the structure of sIgM in mice and humans, long thought to be a perfect pentamer – actually has been described as an asymmetric molecule that resembles a quasi-hexamer missing one monomer, thereby creating a “tooth gap.” This new structural feature allows gapped pentameric IgM to serve as host protein carrier. To date, this interaction seems to be specific and restricted to host protein AIM, which is released as needed to promote the removal of dead-cell debris, cancer cells, or pathogens.Second, IgM has unique features against viral infections, such as high avidity. Some viruses tend to exhibit high mutation rates, leading to the generation of quasispecies and neutralization escape mutants. Neutralizing IgG antibodies may lose their affinity to viral targets as a consequence. However, IgM’s high avidity could compensate for the loss of affinity caused by imperfect matching to mutated target epitopes, as shown for influenza virus . As suchFinally, there is more to IgM’s role in adaptive immunity than being a first responder only. According to commonly held views, IgM has been thought to participate solely in the initial, acute response to viral infections without playing any role in long-term adaptive immunity. However, recent findings in mice demonstrated that antigen-specific, long-lived IgM plasma cells do exist – preferentially in the spleen as shown by adoptive transfer from immunized mice. The half-life of such antigen-specific IgM plasma cells was not much shorter than that of long-lived IgG plasma cells. Most interestingly, long-lived IgM plasma cells contained somatic hypermutations ascribed to AID action. These cells were linked to protection against lethal influenza virus challenge 1 year after immunization in mice, in the absence of GCs and antigen-specific IgG plasma cells.Of note, recent data summarized in this review imply that induction of protective, long-term IgM responses may be possible by active immunization. Vaccines that include long-term antiviral IgM responses may possess advantages over traditional IgG responses against fast-mutating viruses, such as HIV and other RNA viruses known to replicate via error-prone viral RNA-dependent polymerases or to frequently recombine with different viral strains. However, generating such long-term antiviral IgM responses through vaccination needs further study. For use in passive immunization against viral pathogens, IgM may have great potential.Both authors listed have contributed to the writing of this manuscript and approved it for publication.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.


Immunoglobulin M (IgM) mesangial deposition in pediatric minimal change disease (MCD) has been reported to be associated with steroid dependence and poor renal outcomes. However, the evidence linking the impacts of IgM mesangial deposition to the treatment prognosis in adult-onset MCD is still elusive.In this retrospective cohort study, 37 adult patients with MCD received kidney biopsies from January 2010 to May 2020. Immunofluorescence microscopy was performed and the patients dichotomized according to IgM mesangial deposition . We analyzed the clinical features, the dosage of immunosuppressive agents, and the response to treatment for 2 years between the two groups.p = 0.029). Furthermore, multivariate linear regression revealed that the positivity of IgM mesangial deposition is independently associated with the frequency of relapses .Analysis of the clinical symptoms, the dosage of immunosuppressive treatment, and the time to remission revealed no statistical difference between the groups. However, compared to the negative IgM group, the frequency of relapses was significantly higher in the positive IgM group during the two-year follow-up period (the negative IgM group 0.25 episodes/year; the positive IgM group 0.75 episodes/year, Our findings indicated that adult-onset MCD patients with IgM mesangial deposition have a high risk of relapses. Therefore, intensive monitoring of disease activity should be considered in MCD adults with IgM mesangial deposition.The online version contains supplementary material available at 10.1186/s12882-021-02234-z. Minimal change disease (MCD) is the absence of glomerular changes, tubular injury, interstitial fibrosis, or sclerosis under light microscopy. The immunofluorescence (IF) analysis typically showed negative staining, but some reveal immunoglobulin M (IgM) positive . IgM is A previous study indicated that MCD children with a positive IgM mesangial deposition were more likely to evolve into focal segmental glomerulosclerosis (FSGS) . IgM depA positive IgM mesangial deposition has been reported to be associated with steroid dependence and worse renal outcomes in the pediatric population , 5, 9. OHelsinki Declaration. The need for informed consent was waived because of the retrospective nature of the study. We enrolled patients who were diagnosed as MCD by renal biopsy at the Taipei Veterans General Hospital from January 2010 to May 2020. Eighty-one samples were purely MCD, without significant tubulointerstitial, glomerular, and vascular lesions. As shown in Fig. The protocol of this study was approved by the Institutional Review Board of Taipei Veterans General Hospital, Taipei, Taiwan. The protocol conformed with the ethical guidelines of the Nephrotic syndrome is characterized by overt proteinuria (more than 3.5 g/ 24 h), hypoalbuminemia (less than 2.5 g/dL), hyperlipidemia, and edema. Hematuria is defined by urinalysis (urine red blood cell (RBC) more than 3/high-power field (HPF)). We collected serial urine protein to creatinine ratio (UPCR), urine RBC, serum albumin, serum immunoglobulin, lipid profiles, blood pressure, and serum creatinine. The estimated glomerular filtration rate (eGFR) was estimated according to the Chronic Kidney Disease Epidemiology Collaboration formula . IgM mesImmunosuppressive therapy was initiated after biopsy, and the majority of them used prednisolone. Adjuvant immunosuppressants included cyclosporine (CsA), mycophenolic acid (MPA), and cyclophosphamide (CYC). Because this was a retrospective study, the decision on dosages and types of immunosuppressive agents was at the discretion of each attending nephrologist.Fisher’s exact test was used for comparisons of categorical variables as appropriate. Continuous variables were compared by Student’s t-test. For linear regression analysis, the frequency of relapses was set as the dependent variable, and IgM-associated variables were used as independent variables. In subgroup analysis, patients were divided into two subgroups according to their mean value to examine the subgroup difference. The reference line in Fig. p-value of less than 0.05 was considered to be statistically significant.Chi-square analysis or n = 3; 25%). Because only a small amount of tissue was examined by EM, a negative finding of mesangial deposits on EM may just be a result of limited glomeruli being examined. The larger amount of glomeruli on IF may reduce sampling bias and the possibility of a false-negative result.Clinical characteristics of 37 patients with MCD are listed in Table A summary of immunosuppressive treatment is listed in Supplementary Table p = 1.000). The frequency of relapses was significantly different between these two groups  during 2 years.Response to therapy is presented in Table p = 0.006; and prednisolone daily dose, regression coefficient B 1.660, 95% CI 0.07–3.25, p = 0.041) by univariate analysis. Further multivariate linear regression analysis disclosed that only IgM deposition was independently associated with the frequency of relapses .Univariate and multivariate linear regression analysis of the frequency of relapses were presented in Tables 2; mean cholesterol level was 405 mg/dL) and clinical definition . Figure 2), hypercholesterolemia group (cholesterol ≧ 405 mg/dL), hypoalbuminemia group , proteinuria groups (both UPCR < 3.5 g/g and UPCR ≧ 3.5 g/g), and no microscopic hematuria group (urine RBC < 3 /HPF).We divided patients into two subgroups according to mean  is defined by its immunohistologic features as IgA nephropathy, i.e., the diffuse mesangial deposition of IgM . In our Previous studies showed a significantly higher mean serum IgM level in children with positive IgM deposition , 16. In Intriguingly, our study found the frequency of relapses was more common in men with IgM deposition by our subgroup analysis. In an MCD study composed of both children and adults, gender was not a determinant of renal function progression . Neverth2, serum albumin < 2.5 g/dL group, serum cholesterol ≧ 405 mg/dL, the presence of nephrotic-range proteinuria, and the absence of microscopic hematuria. In accordance with our study, a previous study also demonstrated that microscopic hematuria was a favorable sign [In previous studies, children with IgM deposition were more likely to have hypertension , 18 and ble sign .There are no randomized controlled trials on the treatment of MCD with IgM deposition. Corticosteroids constitute the mainstay of therapy in MCD. The prevalence of steroid resistance in idiopathic nephrotic syndrome patients with positive IgM deposition was inconclusive, varying from 0 to 52% . SeveralAdjuvant immunosuppressive therapy includes CsA, CYC, MPA, and levamisole, etc. . SeveralThere were some limitations to this study. First, the optimal evaluation of the subgroup analysis was hindered due to the small sample size. Second, the follow-up period was relatively short, meaning further evaluation of renal function was halted. Third, constraints of retrospective study apply, indicating that the decision on dosages and types of immunosuppressive agents was at the discretion of each attending nephrologist. However, it should be noted that the average dosages of immunosuppressive agents were similar between patients with and without IgM mesangial deposition, as shown in Supplementary Table Our findings indicated that newly diagnosed MCD adults with IgM mesangial deposition were more likely to experience disease relapses than those without. This effect was even more apparent in patients with older age, male, hypoalbuminemia, lower eGFR, presence of nephrotic-range proteinuria, or the absence of microscopic hematuria. Previous pediatric studies recommended that for this particular group of patients, the immunosuppressive agents may be tapered slowly with close follow-up or might be benefited from combined immunosuppressive therapy than prednisolone alone , 23, 26.Additional file 1: Supplementary Table 1. Dosage of immunosuppressive agents in adult-onset minimal change disease.


My Journey Ahead, to provide information and strategies for managing symptom-focused concerns, which may be easily accessed from the patient’s home. The purpose of this study was to evaluate patient acceptability and satisfaction with the My Journey Ahead program. In Phase 1, five patients with head and neck squamous cell carcinoma (HNSCC) reviewed the web-based program and provided initial feedback, which informed program modifications. In Phase 2, 55 patients were recruited to evaluate the program. Patient assessments were obtained prior to and after use of the web-based program, and included measures of psychological distress, self-efficacy in coping with cancer-related issues, and satisfaction with the website. Among the 55 patients enrolled, 44 logged in and viewed the web-based program. Participants reported high levels of satisfaction with the information received, and indicated that the website was interesting and easy to use. Older age and higher levels of self-efficacy in coping were each associated with higher levels of satisfaction with the website. In summary, the web-based program was well-received by patients, the majority of whom found it to be informative and useful. An easy-to-use web-based program, particularly for older patients who may have difficulty locating reliable evidence-based information on the internet, may be helpful in addressing survivors’ needs in symptom management and coping with cancer.Patients undergoing radiation treatment for head and neck cancer experience significant side-effects that can impact a wide range of daily activities. Patients often report receiving insufficient information during and after treatment, which could impede rehabilitation efforts; they may also encounter practical and logistical barriers to receipt of supportive care. Thus, we developed a web-based program, https://clinicaltrials.gov/, NCT02442336 Patients undergoing treatment for head and neck squamous cell carcinoma (HNSCC) face numerous physical, psychological, interpersonal, and practical challenges as a result of their diagnosis and treatment. Despite advances in diagnosis and management, treating HNSCC often entails considerable functional impairment including not only acute, but chronic long-term changes that impact swallowing, eating, periodontal health and oral care, speech, and social functioning –3. As a HNSCC patients often report a desire for more information during and immediately after their treatment , and 73%My Journey Ahead to provide information and strategies for managing symptom-focused concerns faced by HNSCC patients who were treated with radiation. In this paper, we describe patient acceptability of and satisfaction with My Journey Ahead.Several interventions have targeted HNSCC patient needs , 11, butWebsite content was informed by our prior work  and guidProgram content was developed by healthcare specialists including a radiation oncologist, speech-language pathologist, palliative care and pain management physician, physical therapist, and a clinical psychologist. Website programming and creation of content  was produced by Triad Interactive, Inc. After the initial website content was developed, five other healthcare professionals who treat this patient population reviewed the information for accuracy and readability.The program included an Introduction section to explain the use of the website, followed by four specific units:Unit 1: What is head and neck cancer? This unit provided information about HNSCC, including informational text, brief videos of healthcare professionals describing radiation therapy, and animations to illustrate anatomy and physiology.Unit 2: Potential changes in swallowing and oral care. This unit described potential changes in swallowing that may occur. Proper oral care and other potential side effects of radiation therapy  were also discussed. Brief videos and animations presented strategies for eating and swallowing, including personal experiences described by survivors.Unit 3: Potential changes in speech and social interactions. This unit covered possible temporary or permanent changes in speech and the role of a speech language pathologist. Physical exercises that involve mouth and tongue movement were demonstrated using brief videos. Strategies that other HNSCC survivors have used to facilitate social interactions and effective communication were presented.Unit 4: Coping with cancer. This unit addressed psychosocial challenges and described how other HNSCC survivors have met these challenges. Behavioral strategies, social skills training, and journaling and relaxation exercises were demonstrated using worksheets, brief videos, or audio clips.Five patients who had completed radiation therapy within the past 12 months were recruited to review website content and provide feedback on usability, using a modified version of the guidelines suggested by Usability.gov . Each paParticipants indicated that Unit 1 was not needed because all patients were already undergoing (or had completed) treatment. Therefore, participants believed that the information provided in this unit was not helpful or useful at this point in their cancer experience. Participants requested that we expand the content in Unit 2 pertaining to swallowing and oral care. As a result, the information in this unit was divided into separate units on mouth/swallowing concerns and oral health. With respect to the original Units 3 and 4, participants liked the content provided, but they requested additional examples and exercises. Finally, several participants noted that the program did not contain any information pertaining to physical therapy, nutrition or healthy eating, and pain management. Therefore, the revised program website contained an Introduction section, followed by seven units  and a concluding section entitled “Looking Ahead to the Future” see Figure 1We recruited a separate sample of patients to evaluate the revised website. Eligible participants included any patient with squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx who was currently undergoing or had completed either definitive or adjuvant radiation therapy within the past 12 months. Exclusion criteria included: 1) inability to read and/or communicate in English; 2) diagnosis of a head and neck cancer of non-squamous histology ; 3) blindness or severity of visual impairment that precluded one’s ability to view images/text; and 4) inability to provide informed consent.Eligible patients were identified by collaborating clinicians and approached by a study coordinator who described the study procedures and obtained written informed consent. Following consent, study participants completed a baseline assessment and were then provided with a unique login ID and password for accessing the website. Participants could view the program at home, or in a private clinic space if they did not have Internet access or a web-enabled device. Participants who had not logged into the program after one week were contacted by the study coordinator who offered assistance and answered any questions. After 3 reminder calls, participants who had not logged in to the program were considered to have passively dropped out of the study. Participants who viewed the program were contacted approximately two weeks after their initial login to complete the follow-up assessment.Study assessments were collected at two time points: (1) at study entry and (2) two weeks after viewing the web-based program.Participant characteristics including age, gender, race and ethnicity, education, annual household income, and marital status were assessed at baseline. Disease and treatment-related variables including: tumor site, disease stage, time since diagnosis, treatment(s) received, and time since treatment end were extracted from the electronic medical record by research staff.At both baseline and post-program assessment, self-efficacy for coping with cancer-related stressors was assessed using the brief version of the Cancer Behavior Inventory (CBI-B) , 21, a 1Participant distress was measured at baseline and post-program using the Brief Symptom Inventory-18 [BSI-18 ], an 18-In the post-program assessment, participants were asked to evaluate their level of satisfaction with the program website and the information provided on a scale from 1 to 5, with higher ratings reflecting greater satisfaction. In addition, participants rated the usefulness of each unit on a scale ranging from “1 = Not useful” to “5 = Extremely useful”. Finally, participants provided overall ratings of the website (from “1 = Poor” to “5 = Excellent”) and how informative it was (“1 = Not informative” to “5 = Extremely informative”). An open-ended question was included at the end to allow participants to provide additional comments as desired.The primary objective of this study was to characterize participant usage of and satisfaction with the web-based program. Descriptive statistics were used to characterize the participant sample, program usage, and levels of satisfaction with the program. Indicators of acceptability included: (1) A high degree of satisfaction with the information presented ; (2) high reported ease of use ; and (3) Overall rating .To provide information relevant to the further development of the program, we also explored whether patient-level factors  were associated with participant ratings or time spent exploring the website. Additionally, we explored potential changes in participant self-efficacy or distress after viewing the web-based program. Pearson’s correlations or simple linear regressions were used for hypothesis testing. The study was powered to have 80% power to detect correlations of 0.40 with 45 participants. This assumed a 5% Type I error rate (two-sided).The CONSORT diagram is presented in Participant characteristics are reported in Greater than half of participants (55%) had undergone surgery and radiation therapy and 31% received chemoradiation treatment. Time since diagnosis was, on average, five months (SD = 2.23 months). The majority of participants (82%) had recently completed radiation prior to study enrollment , but 18% of the sample was still in treatment at the time of their participation. There were no differences in age, gender, marital status, education level, or income between participants who had completed treatment and those who were currently in treatment. In addition, no differences in self-efficacy in coping, global distress, program usage, or program evaluation ratings were observed between participants who had completed treatment and those still in treatment. Therefore, subsequent analyses are reported using the entire sample.p = 0.02. Non-users also reported slightly higher levels of global distress  compared with program users , F=3.57, p = 0.06, and had slightly lower levels of self-efficacy in coping  compared with program users , F=3.13, p = 0.08. Finally, non-users were more recently diagnosed  compared with program users , F= 4.92, p = 0.03.Eleven of the 55 consented participants (20%) did not login to the website at all. Analyses indicated no differences in age, gender, marital status, education level, or income between those who visited the program website and those who did not (i.e. non-users). However, non-users reported higher baseline levels of cancer-specific distress  compared with program users , F=5.51, Among the 44 participants who visited the website, 13 logged in once and 15 logged in twice. The remaining 16 participants visited the website between 3 and 11 times. Among the 44 participants who visited the website, 27 (61%) viewed the Introduction and all seven units. Fifteen participants (34%) viewed between two and six units, and two participants viewed only the Introduction, but no additional content.On average, participants spent a total of 68.5 min  on the website, of which 62 min (SD = 56 min) were spent viewing the content contained in Units 1–7. The average length of time spent in each unit is presented in Overall, participant feedback was positive Table 3.With respect to usefulness, the program units that were most highly rated were Oral Health (mean rating = 4.29) and Coping with Cancer (mean rating = 4.19). The unit on Speech concerns received the lowest rating (mean rating = 3.72). When asked to rate the overall website on a scale of 1 = Poor to 5 = Excellent, participants’ mean rating was 4.42.r = 0.48, p < 0.01). Higher baseline levels of self-efficacy in coping were associated with greater satisfaction  and higher overall ratings of the website program . In contrast, higher baseline levels of cancer-specific distress were associated with lower overall ratings . Higher baseline levels of global distress were also associated with lower program ratings  and with lower ratings of the information received from the website .Satisfaction with the program was associated with certain patient characteristics. Older age was associated with higher levels of satisfaction , more minutes spent logged in to the program website , and a greater number of units viewed  were each correlated with higher levels of satisfaction.Participant satisfaction with the program was positively associated with program usage. Specifically, a greater number of program logins  to post-program . Similarly, global distress remained fairly stable from baseline  to post-program .Participants’ qualitative feedback in response to the open-ended item indicated that the information presented was reaffirming, and many patients noted that they especially valued the unit on coping:“It helps to know that others have experienced the same difficulties and challenges, which is somewhat normalizing, and seeing examples of patients who have overcome many of these same challenges increases the likelihood of having a positive attitude when faced with rehabilitation and the hard work that comes with it.” - ID #1023“It is nice to know how other people deal with stress and that it was normal.” - ID #1002“It helped me to accept my limitations.” - ID # 1001“[I] Do like the overall presentation and addressing an issue that does not get discussed.” – ID #1051Participants had mixed responses to the unit on Speech Problems. While some participants felt that this unit was not relevant to them, others reported that it was helpful and provided unique perspectives.“I liked the visual and audio example of a patient who obviously worked hard and found a way to communicate on his terms.” - ID #1009“It gave insight [into] what other people deal with.” - ID #1055“[It emphasized] to me the importance of patience in dealing with verbal deficiencies in social settings.” - ID #1058Several participants mentioned viewing the program website with a family member or caregiver. The program was perceived to be helpful to others, not just patients.“The information I already knew, [so] didn’t find it that useful. Personally [it] may help other family members to understand better.” - ID #1069“We both feel that this site has a lot of potential to help others.” - ID #1060Finally, one participant noted that he has not attended any support groups nor interacted with other patients, so the program was helpful for seeing how other patients have managed their cancer experience.“I like knowing that you all care enough to have created the program at all. Not everyone has someone to be supportive.” - ID # 1009Participant comments also provide guidance for how to improve program content and delivery in future versions. For example, a number of participants noted that this information would have been helpful to have earlier in the process, such as during treatment. Several noted that they would like more information on healthy eating, food choices, and swallowing problems. One commented that since individuals may have different needs, the ability to personalize the program would be useful. Finally, one participant would have liked to see slightly younger patients in some of the videos.My Journey Ahead, to provide information and strategies for managing concerns commonly experienced by HNSCC patients treated with radiation therapy. Participant ratings indicated that the program achieved its acceptability goals. Specifically, participants reported a high degree of satisfaction  with the information presented, and they found the program to be interesting and of value. Most participants (70%) visited the program more than once, and 61% viewed all of the units. Participants spent the most time viewing the units on coping, nutrition, and pain. Overall feedback was positive, and participants found the web-based platform informative and easy to understand.We developed a web-based program, However, assessment of the program varied by patient characteristics. Specifically, older patients reported greater satisfaction with the program and were more likely to find the information presented to be of value. It is possible that younger patients are more facile with the internet, and thus, more able to find the information that they need , 26; wheCompared to the patients who used the program, those who did not log on (non-users) reported slightly higher levels of psychological distress and lower levels of self-efficacy in coping at study entry. Non-users were also more recently diagnosed, compared to program users. Interestingly, patients who engaged with the program indicated that having access to it earlier in their cancer experience would have been helpful. Patients may benefit from having access to this program when they are in the midst of undergoing challenging treatment regimens, but it is also a time when patients may feel least inclined or able to engage in such activities. A potential compromise may be to offer a specific unit each week, rather than providing all program content at once, which could feel intimidating and overwhelming to patients. Since each unit could viewed in a shorter time frame , this would help minimize patient fatigue and burden. Alternatively, patients may benefit from having access to the website earlier in their cancer treatment or before their radiation treatment begins.Levels of distress and self-efficacy in coping did not change from pre to post-program. Because participants spent, on average, only one hour viewing the program, this was not likely to result in any significant impact on those endpoints. Most patients viewed the program in its entirety in either one or two sessions. It may be more optimal to design future iterations of the program so that the information and activities are distributed over time. For example, patients might be instructed to view one unit per week and provided with activities and exercises that they could practice or utilize each day during this period. Incorporating multiple opportunities for patients to apply the skills and tips suggested earlier in their treatment may result in greater benefit in self-efficacy over time.Patients also provided helpful feedback regarding aspects of the program that could be enhanced. Some patients noted that they would like more information on eating, due to the considerable challenges they face with regard to eating and swallowing. One patient noted that since needs are individualized, any ability to personalize the program would be useful, and this may be especially true for younger patients. Similar to other programs developed previously, HNSCC patients are eager to have access to high-quality, evidence-based information to facilitate their recovery. For example, an educational telehealth intervention (utilizing a device attached to the user’s phone line) that was designed to promote symptom management was well-accepted and regularly utilized by HNSCC patients . Badr anWe acknowledge several limitations of the present study. First, this was a single-arm study with a relatively modest sample size. Although there was no control group, the primary objective was to develop and evaluate the acceptability of a web-based program designed to provide information and strategies for managing symptom-focused concerns faced by HNSCC patients who were treated with radiation therapy. Patient responses indicated that the program was well-received and provided helpful information. We also gained useful insight regarding how to enhance program content and delivery. Second, due to the focus on program use and acceptability, other patient assessments were limited in order to reduce participant burden. As a result, only measures of psychological distress and self-efficacy in coping were collected at both study entry and follow-up. Subsequent larger studies should include additional patient-reported outcomes , a longer follow-up time point, and measures of healthcare utilization. Finally, there may be key differences between those patients who viewed the program and those who did not. Our data suggest that patients who did not login to the website had higher levels of distress and lower levels of self-efficacy. Thus, identifying approaches for helping distressed patients obtain information and supportive services to address their needs represents an important element that should be incorporated in the next phase of this research. For example, patients who do not login to the program may benefit from referrals to other healthcare professionals, such as social workers or psychologists, who are trained to manage psychological distress and related issues. This approach is consistent with evidence-based stepped care programs, which have been demonstrated to be effective in reducing distress and improving quality of life among HNSCC patients .My Journey Ahead can serve as an informative resource for HNSCC patients who are undergoing or have recently completed radiation treatment. Further work should evaluate My Journey Ahead in a larger trial with a control group in order to explore potential effects of the program on patient self-efficacy in managing symptom-related concerns. Future studies should also incorporate appropriate strategies to address psychological distress in order to help patients thrive after treatment. Offering an easy-to-use web-based program, particularly for older patients who may have difficulty locating reliable evidence-based information on the internet, may increase information access and help address selected patient concerns.In conclusion, our findings suggest that The de-identified data reported in this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Fox Chase Cancer Center Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.CYF: conceptualization, methodology, formal analysis, investigation, resources, data curation, writing—original draft, writing—review and editing, supervision, project administration, and funding acquisition. TJG and BE: methodology, investigation, resources, and writing—review and editing. BLE: formal analysis, writing—original draft and writing—review and editing. JRB and JAR: validation and writing—review and editing. MC and JGB: resources and writing—review and editing. MLL: investigation, resources, and data curation. SLM: methodology and resources. CM: methodology, software, investigation, resources, data curation, visualization, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.This research was supported by the National Institutes of Health grants R41 CA144100 and P30 CA006927.CM is employed by the company Triad Interactive, 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.


This cross-sectional study examines US states’ compliance with both Patient Protection and Affordable Care Act demonstration program annual reporting and Medicaid guidelines for Section 1115 experimental program evaluations. In 2015, these programs accounted for more than $100 billion in federal expenditures. To date, 43 states have secured 55 active waivers to test various Medicaid program changes, including work requirements, benefit restrictions, or managed long-term services and support systems.4 If none was identified, we also searched state Medicaid websites. We determined the overall percentage of program-years with publicly available annual reports for both 2011-2013 and 2016-2018, and compared percentages with χ2 testing.Using publicly available CMS administrative records, we conducted a 2-part cross-sectional study of state Medicaid 1115 waiver demonstration programs. In the first part, we used CMS administrative records to identify all demonstration programs active during 2011-2013 and 2016-2018. These years correspond to before ACA reporting requirements and after CMS reporting guidance, respectively. In any given year, we excluded programs in their first year of approval because insufficient time had elapsed for required reporting. Among remaining programs, we identified the presence or absence of a publicly available annual report in each year by searching the CMS.gov website, on which the agency is required by law to publish annual reports and evaluations.5: executive summary, background, hypothesis, methods, results, conclusions, interpretations and policy implications, and lessons learned and recommendations. For each component, we used descriptive statistics to summarize the total percentage of evaluation reports with each component present. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline reporting guideline for cross-sectional studies.The second part focused on the 2016-2018 publicly available annual reports. We determined a report to be a program evaluation according to whether it was identified among the CMS administrative records for a respective state as an “evaluation.” By reviewing each evaluation, we determined the presence or absence of the following report components outlined in the CMS guidanceP < .001)  were publicly available. From 2016-2018, we identified 36 active demonstration programs across 24 states. Based on years of program activity, a maximum of 93 annual reports from these programs would be expected; 54 annual reports (58%) were publicly available ( < .001) .From among publicly available annual reports between 2016 and 2018, we identified 20 that were program evaluations, representing 36 demonstration programs (24 states). The median number of components reported per program evaluation was 5 . The most commonly reported program evaluation components were results  and general background information , whereas interpretations and policy implications and lessons learned and recommendations were least commonly reported . None of the 11 evaluations that were submitted to CMS for renewal reported interpretations and policy implications.Despite an increase in the public availability of annual reports for Section 1115 demonstration programs after the ACA and CMS’s evaluation guidance, more than 40% were not publicly available. Moreover, when demonstration program evaluations were made publicly available, they consistently failed to report key evaluation components.There are limitations to consider, including our inability to account for special terms and conditions negotiated between CMS and individual states that may dictate requirements for reporting frequency. In addition, we did not contact federal or state officials when annual reports or evaluations were not identified. However, given the importance of Medicaid Section 1115 waivers to pilot innovative models of health care delivery, and the amount of federal funding involved, enforcement of existing CMS rules to ensure accountability is needed, particularly for program renewals. Availability of program reports and evaluations allows the broader public to better understand the effect of these programs on Medicaid beneficiaries.


KCNA5, encoding the Kv1.5 α-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K+ current (IKur). Both loss-of-function and gain-of-function AF-related mutations in KCNA5 are known, but their effects on action potentials (APs) of human cardiomyocytes have been poorly studied. Here, we assessed the effects of wild-type and mutant IKur on APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We found that atrial-like hiPSC-CMs, generated by a retinoic acid-based differentiation protocol, have APs with faster repolarization compared to ventricular-like hiPSC-CMs, resulting in shorter APs with a lower AP plateau. Native IKur, measured as current sensitive to 50 μM 4-aminopyridine, was 1.88 ± 0.49  and 0.26 ± 0.26 pA/pF (n = 17) in atrial- and ventricular-like hiPSC-CMs, respectively. In both atrial- and ventricular-like hiPSC-CMs, IKur blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual IKur, with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native IKur was blocked. Injection of IKur with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD20) of atrial-like hiPSC-CMs. At IKur densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At IKur densities > 100%, simulating gain-of-function mutations, APD20 was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in IKur. In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in IKur, mimicking loss-of-function mutations, has a stronger effect on the AP of hiPSC-CMs than an increase, mimicking gain-of-function mutations, whereas in ventricular-like hiPSC-CMs such increase results in AP shortening, causing their AP morphology to become more atrial-like. Effects of native IKur modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual IKur injection because IKur density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.Atrial fibrillation (AF) is the most common cardiac arrhythmia. About 5–15% of AF patients have a mutation in a cardiac gene, including mutations in  KCNA5 are associated with AF, although rare (KCNA5 encodes the pore-forming α-subunit Kv1.5 of the channel carrying the ultrarapid delayed rectifier K+ current (IKur)   is around 1–2% . Mutatiough rare . KCNA5 et (IKur) . In the he atria , whereasr tissue  and expralso low . Accordils (APs) .KCNA5 have been identified in patients with AF (KCNA5 are supposed to increase susceptibility to AF by prolonging the AP duration (APD) of atrial myocytes, which may eventually result in early afterdepolarizations (EADs)  . Indeed, of EADs . EADs asutations .in silico studies, which demonstrated that increased IKur density, representing gain-of-function mutations, resulted in a shortened APD and arrhythmogenesis in human atrial tissue  of the atrial AP, facilitating re-entry wavelets in the atria . This hyl tissue .in silico studies are instrumental in determining the potential effect of both loss-of-function and gain-of-function mutations in KCNA5, detailed electrophysiological studies of the KCNA5 mutations in human cardiomyocytes are limited. Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) have become a highly suitable tool to study cardiac ion channelopathies and their electrophysiology  on Matrigel coated coverslips in BPEL medium . Data acquisition and analysis were performed with custom software. Signals were low-pass filtered with a cut-off frequency of 2 kHz and digitized at 40 and 5 kHz for AP and IKur recordings, respectively. Cell membrane capacitance  was calculated by dividing the time constant of the decay of capacitive transient when hyperpolarized by 5 mV from −40 mV in voltage clamp by series resistance. Cm of atrial- and ventricular-like hiPSC-CMs was 16.4 ± 2.3 pF , and 19.2 ± 2.5 pF (n = 27), respectively . Patch pipettes with a resistance of ≈2.0 MΩ were pulled from borosilicate glass (Harvard Apparatus) and filled with solution containing (in mM): 125 K-gluconate, 20 KCl, 5 NaCl, 0.44 Amphotericin-B, 10 HEPES; pH set to 7.2 (KOH). Cells were superfused with modified Tyrode’s solution containing (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.5 D-glucose, 5 HEPES; pH set to 7.4 (NaOH). All potentials were corrected for the estimated liquid junction potential of −15 mV , AP amplitude , AP duration at 20, 50, and 90% repolarization , and AP plateau amplitude , derived from the membrane potential (Vm) at 50 ms after the time of dV/dtmax.APs were elicited at 1 Hz by 3-ms, ≈1.2 × threshold current pulses through the patch pipette. The AP parameters analyzed were resting membrane potential , maximum upstroke velocity (dV/dtKur was activated by 200-ms voltage clamp steps from −50 to +50 mV. A 50-ms prepulse to 0 mV was applied to activate and inactivate remaining transient membrane currents. Series resistance was compensated by ≥ 80%. IKur was measured as the current sensitive to 50 μM 4-aminopyridine (4-AP) .Native Ie (4-AP) , and was+ current (IK1) is not necessarily low in hiPSC-CMs  and the associated injected current (bottom panels), which consists of this IK1 and a short inward stimulus current. A virtual Kir2.1-based IK1, characteristic for human ventricular myocytes , gKur (in nS/pF), V (in mV), EK (in mV), and t (in s) denote the ultrarapid delayed rectifier outward K+ current, its fully activated conductance, the membrane potential, the K+ reversal potential, and the time, respectively. The steady-state values of aur and iur are denoted by aur,∞ and iur,∞, respectively, and the associated time constants by τaur (in s) and τiur (in s), respectively. As in the models by Kur. Of note, Kur density in human atrial myocytes.In these equations, the dimensionless Hodgkin and Huxley-type activation and inactivation gating variables, ranging between 0 and 1, are denoted by aKur density of atrial- and ventricular-like hiPSC-CMs was compared with an independent samples t-test. Two-way repeated measures ANOVA followed by the Student–Newman–Keuls post hoc test was used for comparing AP parameters of atrial- and ventricular-like hiPSC-CMs in absence or presence of 4-AP. One-way repeated measures ANOVA followed by the Student–Newman–Keuls post hoc test was used for comparing the effect of injecting virtual IKur at various densities into atrial- and ventricular-like hiPSC-CMs. P < 0.05 was considered statistically significant.Data are presented as mean ± SEM. Statistical analysis was carried out with SigmaStat 3.5 software . Native IK1 was injected into the cells, based on the approach of 20, APD50, and APD90. The ventricular-like APs have a prominent plateau phase at relatively positive potentials, in contrast with the atrial-like hiPSC-CMs that show a less prominent plateau phase at less positive potentials, if any plateau at all. Consequently, APPlatA was significantly smaller in atrial-like hiPSC-CMs and thus appeared a strong tool to distinguish between atrial-like and ventricular-like hiPSC-CMs. APA and dV/dtmax did not differ between the atrial- and ventricular-like hiPSC-CMs, but RMP was less negative in ventricular-like hiPSC-CMs.APs were recorded from single atrial- and ventricular-like hiPSC-CMs that showed spontaneous beating upon visual inspection, clearly indicating a healthy and myocardial status. APs were elicited at 1 Hz and virtual IKur. K1 and the 3-ms stimulus current applied at 10 ms, are displayed in the bottom panels of Kur blockade resulted in a significantly increased APD90 and APPlatA, while other AP parameters were unaffected  and 0.26 ± 0.26 (n = 17) pA/pF, respectively  was present to ensure that any native IKur was blocked . The injected current, which now consists of IK1, 0% or 100% IKur, and a short stimulus current, is shown in the middle panels of Kur in Kur. Injection of IKur shortened the AP of both atrial- and ventricular-like hiPSC-CMs, while the AP plateau was suppressed .ppressed , 6. dV/d of IKur . The smaKur is a repolarizing current that is already activated during the 3-ms stimulus and stays active until repolarization reaches −40 to −50 mV and the black traces ‘fuse’ with the red and blue traces of the action potentials without IKur . At these negative potentials, IKur becomes small because of both deactivation—rather than inactivation, which is much slower—and diminishing driving forces. The maximum IKur during the atrial-like AP is slightly larger compared to the ventricular-like AP because in this particular example the atrial-like AP reaches a higher peak than the ventricular-like AP, which results in a larger driving force for IKur.The phase plane plots of KCNA5, resulting in a decrease in IKur. Therefore, we decreased the fully activated conductance of the virtual IKur conductance to 75, 50, 25, and 12.5% of its control value. 20, APD50, and APD90 significantly increased upon a reduction in IKur . Therefore, we decided to block native IKur and use dynamic clamp to study the effects of IKur, using a virtual IKur with characteristics, including its density, based on observations made in freshly isolated human atrial myocytes.Maximum sustained native IKur density of our atrial- and ventricular-like hiPSC-CMs, it was not surprising that blockade of IKur by 4-AP had only minor effects on AP parameters. When native IKur was replaced with virtual IKur with 100% density, similar to IKur density in freshly isolated human atrial myocytes, more pronounced effects on AP parameters were observed. In atrial-like hiPSC-CMs, APD20 shortened substantially, whereas APD50 shortened only moderately and APD90 even less so. In ventricular-like hiPSC-CMs, on the other hand, not only APD20, but also APD50 and APD90 shortened substantially upon injection of virtual IKur. The more pronounced effect on APD in ventricular-like hiPSC-CMs is likely related to the longer and more positive AP plateau potentials leading to more functional consequences of IKur. The observed decrease in APD20 was accompanied by a lowering of the AP plateau in both atrial- and ventricular-like hiPSC-CMs.Due to the relatively low native IKur to AP repolarization increases with increasing pacing rate  activation kinetics. Accordingly, rate-dependent changes in IKur were largely determined by changes in action potential morphology. In computer simulations with the Kur formulation, we made similar observations (data not shown). We aim to test the rate dependence of the effects of IKur on AP repolarization in future experiments on hiPSC-CMs.We only performed experiments at a pacing rate of 1 Hz and not at higher pacing rates. Therefore, we were unable to confirm that the relative contribution of Iing rate . AguilarKur density from 100% to 12.5–75% of its control value, resulted in prolongation of the AP and raise of its plateau, in line with the differences in AP parameters that were observed between 0 and 100% IKur. KCNA5 in hiPSC-CMs, representing a complete loss-of-function, may result in the development of EADs, which, however, were not observed in the present study, likely due to our higher pacing frequency. At IKur densities > 100%, simulating gain-of-function mutations, effects on AP parameters were somewhat less pronounced. In both atrial- and ventricular-like hiPSC-CMs, APD20 was only significantly decreased upon an increase in IKur density to 200%. A significant lowering of the AP plateau, together with AP shortening, was only observed in ventricular-like hiPSC-CMs.In both atrial- and ventricular-like hiPSC-CMs, simulation of loss-of-function mutations through lowering of the virtual IKur density at +50 mV was small in our atrial-like hiPSC-CMs , it was still significantly larger than in our ventricular-like hiPSC-CMs . Within our atrial-like hiPSC-CM population we noted cells lacking IKur  in atrial-like hiPSC-CMs, which was significantly larger than that of ventricular-like hiPSC-CMs . To distinguish between the two types of hiPSC-CMs based on IKur densities would require further investigation, although the available data suggest a trend of a significantly larger IKur density in atrial-like hiPSC-CMs.Although the sustained native Iing IKur , althouging IKur , with thiPSC-CMs . In the max and APA were not only different under control conditions, but also upon blockade of IKur by 4-AP, indicating that the two types of hiPSC-CMs are not only different in their level of Kv1.5 expression, as determined by IKur density, and suggesting that differences in membrane currents other than IKur also contribute to the observed differences in AP parameters. This result is in line with previous findings by both KCNA5 or IKur blockade by 4-AP in atrial-like hiPSC-CMs did not result in completely ventricular-like APs. These findings are, however, to some extent at odds with those by Kur is the major determinant of atrial action potential morphology. Conversely, they observed that injection of a virtual IKur in ventricular-like hiPSC-CMs, employing the dynamic clamp technique using oocytes expressing a cloned Kv1.5 current, resulted in APs similar to those of atrial-like hiPSC-CMs. Apart from the studies by Kur in hiPSC-CMs are limited and the electrophysiology of IKur in atrial- and ventricular-like hiPSC-CMs remains largely unknown.Of note, all AP parameters of our atrial- and ventricular-like hiPSC-CMs except dV/dtKur density and changes in AP parameters, IKur has now been quantified in both atrial- and ventricular-like hiPSC-CMs. Thus, the present study provides additional data toward a complete characterization of individual membrane currents in hiPSC-CMs. Moreover, our study illustrates the potentials of dynamic clamp experiments on hiPSC-CMs, allowing manipulation of characteristics of the injected current in real time, thus facilitating direct, systematic, and efficient testing of changes in those characteristics. In the context of studying drug effects, including effects of anti-AF drugs, dynamic clamp may prove useful in the identification of potential drug targets and in testing model-based hypotheses (Kur based on specific loss- or gain-of-function mutations in KCNA5 can be utilized to assess the cellular effects of these mutations as well as effects of dedicated pharmacological treatment through modulation of IKur. Ultimately, this may lead to mutation-specific treatment of AF.Apart from demonstrating a link between altered Ipotheses . For insThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SH designed and performed the experiments, analyzed the data, and drafted the manuscript. HD cultured the hiPSC line and developed the procedures to generate atrial-like and ventricular-like hiPSC-CMs. LB prepared the hiPSC-CMs used for electrophysiology in the present study. AV and RW designed the study, interpreted the data, and drafted, edited, and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Significant improvements were also achieved for degree of appetite and vitality indices (P < 0.000–0.001). However, incidences of fever were not reduced. These findings indicate that the program effectively improved nutritional status, oral health, vitality, and appetite. However, these effects did not sufficiently remain once the program was finished, thus suggesting the need for a continuous intervention.This study evaluated the effectiveness of a life-enhancement program designed to focus on dining conditions in welfare facilities for seniors living in Japan. Effectiveness was specifically evaluated based on whether improvements were achieved in (1) nutritional status, (2) oral health, (3) frequency of fever, and (4) vitality of appetite across three sites. As part of a comprehensive-care initiative that began with dining support, the program consisted of two main components: (1) a 3-month intensive program comprised of (a) collective experiential learning for residents and staff  and (b) a tailor-made individual program for residents followed by (2) a 3-month continuation program. Participants included 168 individuals  from a total of three facilities (average age was 85.9 [60–104] years). Results showed that the intensive program significantly improved nutritional status ( The hypothesis is that improving oral condition and eating environment will promote appetite to increase nutritional intake from vitalization and reduced fever incidents.We then examined its effects over the course of a (1) 3-month intensive intervention and (2) subsequent 3-month continuation program. Finally, we measured the interventional effects 6 months after the end of the continuation program .This study clarified the effects of a dining-focused life-enhancement program based on the four following research questions:Did the program improve nutritional status among participants?Did the program improve oral health among participants?Did the program improve vitality and appetite among participants?Did the program reduce the frequency of fever among participants?This was a quasi-experimental before and after study. It was conducted between February 2014 and March 2015.We developed a Dining-focused Life Enhancement Program based on our previous conceptual analysis of the dining experience . Figure The program was developed following a pilot study , which sCollective experiential learning sessions were held to help participants improve their knowledge levels and skills of Dining Preparations and Dining Support while stimulating the motivation to improve and promote the continuation of improvement activities by providing opportunities for mutual support. Group learning sessions were separately held for residents and staff members . Each group completed one session per month during the 3-month intensive intervention period.e.g., green tea, sweet red-bean soup, juice, and some carbonated drink). Residents then considered what constituted a desirable menu. Following this, a discussion was held with families and staff members to help fulfill these wishes. During the second session, residents participated in various activities designed to improve their oral function, thus enabling them to eat items from their desired menus. Specific activities included oral care, swallowing exercises, and therapeutic recreation . During the third session, residents participated in games designed to promote correct eating posture and movement. This further promoted the ability to eat food items from their desired menus.Resident learning sessions lasted approximately 30 minutes each, which were designed to increase their desire to eat and appetite rather than improving their ability to do so. At each session, we divided residents into small groups of four or five and assigned one or two nurses/staff members to each group, while an expert in dementia nursing worked as a session facilitator. During the first session, residents discussed memorable food items and confirmed what tastes they most enjoyed while looking at photographs and taking drinks Improving the resident’s oral functionImproving dining-assistance skillsReviewing the dining environmentImproving the resident’s general health conditionRegulating the resident’s life rhythmItems were jointly selected with residents and staff members after displaying them on a whiteboard to increase visibility. The researchers and staff members held additional discussions about the direction of care when difficult cases were encountered during the intensive intervention. This ensured that each care program respected individual needs and preferences.A survey was conducted 3 months after beginning the intensive intervention. Based on this survey, strengthening items were chosen from the list above that would be implemented during the next 3 months. This also involved discussions with residents and staff members. All planned activities were written on the whiteboard.A nonprobability convenience sampling technique was used to recruit participants from three senior welfare facilities in Japan. Specifically, this included elderly residents (60 years of age and older) and staff members who provided them with care. All participants were given oral and written explanations of this study’s procedures. Further, written consent was obtained prior to their participation. However, some residents had difficulty making the decision to participate by themselves. The researchers not only attempted to directly obtain consent in these cases but also accepted written proxy consent from their families when necessary. Approval to conduct research involving human subjects was received from the research ethics committee of the College of Nursing Art and Science, University of Hyogo (KYOUIN24) on March 12, 2013.We asked staff members to record information about the type of care they provided to residents based on what they derived during the learned sessions. Staff members were interviewed to assess this information at three time points .Residents were surveyed to collect demographic information, including gender, age, medical history, and care levels. Based on standards established by the MHLW, care needs were classified into five levels  . An indiWe collected data at four points, including the beginning of the program (baseline), 3 months after the program began (month 3), the end of the program (month 6), and 6 months after the end of the program (month 12) .Body mass index (BMI) measurements were determined for each participant using their heights (m) and bodyweights (kg) ([body weight]/[height]2). This was used as an index for nutritional status.Each participant was also assessed for daily caloric intake by multiplying the daily calorie contents of their meals by the rate of intake (reflected by weight). Mean daily calorie intake was thus determined for each of the three days just prior to measurement date. Finally, daily water intake was determined using records from the nursing station. Mean daily water intake was thus calculated for each of the 3 days just prior to measurement date.A dentist examined each participant to determine oral condition, including dental plaque , tongue The vitality index (VI) was developed to assess QOL for older people with dementia . Vitality was specifically expressed on a scale ranging from 0 to 10, with higher scores indicating higher levels of willingness.Participants also rated their appetites by selecting one of four options, including 1 = no appetite, 2 = uneven appetite, 3 = moderate appetite, and 4 = good appetite.Resident records were checked for the presence/absence of fever  and frequency was counted during the previous 3 months before measurement. Body temperatures were measured at fixed times each day, with median temperatures from the previous week (seven-day period) used as averages for each resident.P < 0.05. All statistical analyses were conducted using PASW Statistics v17 . Finally, interventional programs were qualitatively assessed based on individual care records.As mentioned above, quantitative data were obtained at four time periods for the purpose of comparison. Variables with normal distributions based on the Kolmogorov–Smirnov test were subjected to the repeated measures ANOVA test, while variables with nonparametric distributions were subjected to the Friedman test. All statistically significant differences were subjected to multiple comparisons via the Bonferroni post hoc test. Here, statistical significance was set to e.g., 136 [81.0%] were diagnosed with dementia, 95 [56.5%] had hypertension, and 83 [49.4%] had cerebrovascular disease)  from three senior welfare facilities . Data obdisease) . Particie.g., salivary-gland stimulation massage, tongue massage, and oral motor exercises) and oral hygiene care .Baseline data showed that 87 (51.8%) residents had insufficient chewing/swallowing functions for eating solid foods, while all residents had substantial dental and/or tongue plaque. The first intervention session therefore included components designed to improve oral function and hygiene. Care records showed that staff members responded by providing residents with oral function care . After the third session, care records showed that staff members began to review and regulate life rhythms, improving more general conditions for residents. This included assisting residents when getting out of bed, ambulating with self-propelled wheelchairs, increasing opportunities for walking, creating comfortable bedroom environments to promote sleep, and providing support to ensure better excretion control.Care records showed that staff members started to provide oral care at the first, then gradually made efforts to enhance the dining experience. Specifically, this included arrangements designed to improve comfort levels in the dining areas, place-setting the dining tables in appealing ways, and creating enjoyable atmospheres. Further, records showed that various types of related care were provided  when compared to baseline measurements, but returned to preintervention levels when measured 6 months after the program ended (month 12). Further, water intake significantly increased by months 3 and 6 when compared to baseline measurements but decreased by 6 months after the program ended (month 12) when compared to the level of month 3. Finally, BMI had significantly increased by month 3 when compared to baseline measurements but returned to preintervention levels by 6 months after the program ended (month 12).Postintervention results showed significant reductions in dental plaque, tongue coating, and tongue movement.While postintervention VI and the degree of appetite indicators significantly increased from baseline, residents tended to return to preintervention levels based on final follow-ups.No significant changes were observed for fever frequency.Resident demographic data for age, gender, and care needs were very similar to those revealed for elderly subjects based on a national survey in Japan . In bothResults showed that the program significantly improved nutritional status, oral health, appetite, and VI scores among residents. However, fever frequency did not change.Oral health care improvements were proposed for the intensive program since all residents exhibited problems in this area. Such improvements also promoted dining habits and conditions by increasing their ability to eat. This indicates that oral function can be improved and maintained by keeping the oral environment clean. Further, our previous study found that oral hygiene was easy to implement since residents and caregivers noticed the issue . For thaDuring the second proposal session for the intensive program, researchers suggested that staff members review the dining environment and devise ways to help residents to enjoy their dining experiences. During the third proposal session, researchers suggested that staff members attempt to regulate life rhythms and improve general health conditions for residents. Care activities were thus extended to all aspects of life. This appeared to contribute to the improvements seen in VI scores, which are used to assess QOL for older persons with dementia and related to life prognoses .As mentioned above, however, there were no significant effects on fever frequency. This is likely because there were already very few incidents both at baseline and during intervention.Professional caregivers tend to focus on routine tasks and safety rather than fulfilling individual needs and preferences . In suchIn this study, interrelationship-based care training was considered effective for teaching staff about the importance of recognizing residents as “dinner guests” while promoting person-centered care rather than approaching the issue solely from the caregiver perspective. These types of positive interactions between residents and staff members may increase vitality among residents. This also includes making efforts to share information. For instance, residents and staff members may use a whiteboard to write jointly decided goals.Excluding oral health, the interventional effects tended to decrease based on 6-month postinterventional follow-up results. While several previous interventional studies have examined dining assistance , few of This study’s effects on oral cleaning were maintained postintervention, which was similar to the results of a previous study targeting community-dwelling elderly persons . This isSince there is no control group for the study and randomization, this study has limitations to conclude a causal association between interventions and outcomes.The program implemented in this study was not focused on treating individuals in the terminal phase. As such, future studies should develop tailor-made programs to address this issue. There was also a tendency for the interventional effects to decrease postintervention. It is thus necessary for future studies to consider additional measures and strategies that can help residents maintain positive interventional effects.This study implemented a comprehensive life-enhancement program that focused on dining conditions for residents of a senior welfare facility in Japan. The program produced notable improvements to their oral health, interest in food, vitality, and nutritional status, but did not have any significant effects on fever frequency. Excluding the persistent improvements to oral health, however, these positive effects tended to disappear postintervention. This indicates the importance of ensuring that welfare facilities provide residents with continual daily support for these matters. This especially includes those with decreased functional abilities, who require additional support when attempting to maintain good health and proper nutrition.We would like to thank the following people for their cooperation and support during this study: nursing care station residents and their families, staff members, and study collaborators.The authors declared no potential conflicts of interest concerning the research, authorship, or publication of this article.This research was funded by a Grant-in-Aid for Scientific Research between 2012 and 2014 (Grant-in-Aid for Scientific Research (B)) .


PRAME and KIT were significantly upregulated in TSQCC . We then examined PRAME expression in 17 TSQCCs and 116 thymomas via immunohistochemistry. All 17 (100%) TSQCCs displayed diffuse and strong PRAME expression, whereas eight of 116 (6.8%) thymomas displayed focal and weak expression (p < 0.0001). KIT and CD5 were positive in 17 (100%) and 16 (94.1%) TSQCCs, respectively, whereas one (0.9%) type B3 thymoma showed double positivity for KIT and CD5. The KIT-/CD5-positive type B3 thymoma was negative for PRAME. Thus, combinatorial evaluation of PRAME with KIT and CD5 may facilitate a more precise diagnosis of TSQCC.Thymic squamous cell carcinoma (TSQCC), accounting for 70–80% of thymic carcinoma cases, is distinct from thymoma. However, differential diagnosis for type B3 thymoma is sometimes challenging, even with established markers for TSQCC, including KIT and CD5, which are expressed in ~ 80% of TSQCCs and ~ 3% of thymomas. Novel TSQCC-specific markers would facilitate precise diagnosis and optimal treatment. Herein, we found that preferentially expressed antigen in melanoma (PRAME) may be a novel TSQCC-specific diagnostic marker. We comprehensively profiled 770 immune-related mRNAs in 10 patients with TSQCC and two healthy controls, showing that  According to the Japanese Association for Research of the Thymus database, most (~ 87%) thymic epithelial tumours are comprised of thymoma, followed by thymic carcinoma (~ 11%) and neuroendocrine tumour (~ 2%)4. Thymic squamous cell carcinoma (TSQCC), accounting for 70–80% of thymic carcinoma cases, shows clinical and pathological distinctions from thymoma. TSQCC shows an aggressive clinical course, developing nodal and distant metastasis with pathological features of squamous cell carcinoma lacking immature T lymphocytes. In contrast, thymoma shows relatively slow progression with local invasion, exhibiting organotypic (thymus-like) features. Therefore, histopathological diagnosis of TSQCC is usually straightforward, but differential diagnosis from type B3 thymoma is challenging in some cases2.Thymic epithelial tumours are rare malignancies, but are the most frequent type of anterior mediastinal malignancy, with an incidence rate of 1–7 individuals/million population/year8. Briefly, KIT and CD5 are positive in ~ 80% of TSQCCs, whereas ~ 3% of thymomas show positivity for KIT or CD54. Other markers, such as glucose transporter 1 and MUC1, have been proposed as TSQCC markers (positive in ~ 90% of cases); however, the positive rate in type B3 thymoma is relatively high 9. Identification of a novel marker specific to TSQCC will facilitate precise diagnosis and lead to optimal management of patients with thymic epithelial malignancy.KIT  and CD5 are established diagnostic markers for TSQCC and are almost always negative in thymoma, with some exceptionsAccordingly, in this study, we identified preferentially expressed antigen in melanoma (PRAME), a type of cancer-testis antigen, as a novel diagnostic marker of TSQCC for the first time, based on comprehensive mRNA expression analysis and immunohistochemical validation.The median ages of patients with TSQCC and thymoma were 64 years (range 46–80 years) and 62 years (range 24–86 years), respectively. There were seven women (41.1%) in the TSQCC group and 65 women (57.0%) in the thymoma group. Among the 116 patients with thymoma, 12 patients had type A thymomas, 34 had type AB thymomas, 23 had type B1 thymomas, 28 had type B2 thymomas, 17 had type B3 thymomas, and two had micronodular thymomas. No patients received neoadjuvant chemotherapy, radiotherapy, or chemoradiotherapy before surgical resection of TSQCC or thymoma.PRAME and KIT showed significant upregulation in TSQCC .Differential gene expression between TSQCC and normal thymus tissues is summarised in the volcano plot shown in Fig. p < 0.0001, each). Positivity for PRAME was significantly frequent in TSQCCs compared with that in all types  of thymoma . Representative images of PRAME immunohistochemistry staining are shown in Fig. p = 1.00; type B2, p = 0.735; type B3, p = 0.569). No significant differences in presentation stage were found between PRAME-positive and PRAME-negative thymomas . Furthermore, metastatic lesions of PRAME-positive thymomas (n = 4) showed no expression of PRAME, in contrast to metastatic lesions of TSQCC (n = 1), which showed strong and diffuse expression of PRAME.The results of immunohistochemical analyses are shown in Table PRAME mRNA expression in TSQCC was higher than that in PRAME-negative thymomas, with a log2 fold change of 3.96 . In contrast, PRAME mRNA expression in TSQCCs tended to be higher than that in PRAME-positive thymomas, although the difference did not reach statistical significance .14, comprehensive mRNA profiling of TSQCC had not been performed previously. However, in this study, we clearly demonstrated the upregulation of PRAME mRNA in TSQCCs compared with that in normal control samples and the overexpression of PRAME protein in TSQCCs compared with that in thymomas and normal control samples. To the best of our knowledge, this is the first report identifying PRAME as a novel diagnostic marker for TSQCC, but not for thymoma.Although several studies have analysed the genomic profiles of TSQCC by focusing on mutational status15. PRAME can bind to the retinoic acid receptor in the presence of retinoic acid, leading to inhibition of retinoic acid receptor signalling and tumour necrosis factor-related apoptosis-inducing ligand expression. This in turn blocks cell differentiation and promotes cell proliferation by downregulating pro-apoptotic genes17. In normal tissues, PRAME expression is regulated at very low levels by DNA methylation, except in the testes15. Notably, overexpression of PRAME has been observed in malignant melanoma (88% of primary lesions and 95% of metastases), lung carcinoma (46% of adenocarcinomas and 78% of squamous cell carcinomas), renal cell carcinoma, neuroblastoma, myxoid liposarcoma, and synovial sarcoma22. Moreover, PRAME expression correlates with higher tumour grade and poorer prognosis in neuroblastoma, urothelial carcinoma, osteosarcoma, head and neck carcinoma, breast cancer, liposarcoma, chronic myeloid leukaemia, and lymphomas23.PRAME, a type of cancer-testis antigen, was first recognised as a tumour-associated antigen through analyses of cytotoxic T-cell clones collected from a patient with metastatic malignant melanoma24, PRAME positivity in TSQCC may reflect the biological distinctions in the differentiation state of the tumour cells compared with thymoma. The underlying mechanisms of PRAME overexpression in TSQCC remain unclear. To date, several mechanisms have been shown to upregulate PRAME in other malignancies, including hypomethylation of 5′-C-phosphate-G-3′ sites in the 3′ sequence of the promoter region and the exon 1b region of PRAME, microRNA-211 downregulation, production of breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue fusion protein, and activation of FMS-like tyrosine kinase 328.In this study, we demonstrated the distinct expression patterns of PRAME in TSQCC and thymoma. Given that PRAME inhibits cell differentiation and is expressed in tumour cells with stem cell characteristicsImportantly, all TSQCCs showed diffuse and strong expression of PRAME, whereas thymomas showed no expression or only focal and weak expression. This indicated that PRAME could be a novel diagnostic marker for TSQCCs. Although some cases of types AB, B2, and B3 thymomas  showed positivity for PRAME, the staining pattern was focal and weak. In addition, PRAME-positive thymomas are always KIT- and CD5-negative. Interestingly, a case of type B3 positive for both KIT and CD5 showed no expression of PRAME. These findings suggested that the use of PRAME in combination with KIT and CD5 may facilitate more precise differential diagnosis between TSQCC and thymoma, specifically for type B3 tumours.31. Notably, overexpression of PRAME has been shown to be associated with resistance to chemotherapy34. Additionally, PRAME may contribute to chemoresistance in TSQCC. The chemosensitivity of thymic carcinoma to classical cytotoxic antitumor agents appears to be heterogenous, varying from unexpected responses to clear nonresponses. Recently, specific therapeutic agents targeting KIT, such as sunitinib and imatinib, have been investigated as potential treatment options; of these, sunitinib appears to be more promising35, whereas imatinib failed to show significant therapeutic effect in a clinical trial36. Accordingly, therapeutic strategies targeting PRAME could be promising for patients with TSQCC because no standard systemic therapies have been established for this unresectable disease. In our study population, no patients with PRAME-positive thymoma developed postoperative recurrence; thus, we could not examine whether PRAME-positivity affected the chemosensitivity of thymomas.From a therapeutic perspective, PRAME has been studied as an attractive target for antigen-specific immunization by adoptive T-cell therapy in some tumours, such as uveal melanoma and non-small cell lung cancer2. Thus, it remains unclear whether overexpression of PRAME is TSQCC-specific or thymic carcinoma-specific. Accordingly, immunohistochemical assessment of other subtypes of thymic carcinoma is urgently needed.There were some limitations to this study. First, the presented results were based on a single-centre study with a small sample size. Thus, the findings of the study may not be generalised, and type II error may be present. Further studies are needed to validate our results in larger study populations. Second, PRAME was studied exclusively in TSQCC but not in other histological types of thymic carcinoma. Although TSQCC is a major subtype of thymic carcinoma accounting for ~ 70% of cases, there are 10 other histological variants according to the 4th edition of the World Health Organization classificationIn conclusion, our results suggested that PRAME may be a novel diagnostic marker differentiating TSQCC from thymoma. Specifically, the use of PRAME in combination with KIT and CD5 will facilitate more precise diagnosis of patients with TSQCC. Further investigations are necessary for optimal diagnosis of thymic epithelial malignancy and to improve our understanding of the underlying biology.37. Information regarding this study, such as the inclusion criteria and the opportunity to opt out, was provided through the institutional website.This study was conducted in accordance with the Declaration of Helsinki and was approved by the Kansai Medical University Hospital Clinical Research Ethics Board . Informed consent was obtained from patients by opt-out methodology owing to the retrospective design of the study, with no risk for the participantsChart review was carried out for all patients (n = 150) who underwent surgery for thymic epithelial malignancy between January 1, 2006 and December 31, 2019. The inclusion criteria for this study were a histopathological diagnosis of TSQCC or thymoma and adequacy of the surgical specimen for analysis. Nineteen of 150 patients were excluded owing to inadequate sample amount. Two patients had synchronous multiple thymomas: each patient had two thymomas with different WHO types. Finally, 17 patients with TSQCCs and 116 with thymomas were included. None of the 17 patients with TSQCC had previously presented with squamous cell carcinoma that originated from any tissue other than the thymus. For controls, three patients with thymic cysts were included, and non-cystic sections of resected specimens  from these patients were used for RNA extraction and immunohistochemistry staining.2.The diagnosis and subclassification of thymoma and the diagnosis of TSQCC were confirmed by two diagnostic pathologists (MI and KT) independently according to the 4th edition of WHO classification38. Briefly, for mRNA extraction, 5-μm-thick sections from formalin-fixed and paraffin-embedded (FFPE) blocks from 10 TSQCCs and two normal controls were cut. RNA was extracted using a NucleoSpin total RNA FFPE kit , including on-column treatment with DNase. A quantitative evaluation of RNA was performed using a Nanodrop 1,000 spectrophotometer . RNA quality was evaluated by measuring the 260/280 nm ratio. We excluded samples in which the total amount of RNA was less than 50 ng/μL or the 260/280 ratio was less than 1.6.We extracted RNA from the archived samples as described in our previous reports2-transformed, and normalised using housekeeping genes selected using the nSolver analysis software version 4.0 . Fold changes, p values, and adjusted p values were determined using the nSolver analysis software version 4.0 with nCounter Advanced Analysis add-on software version 2.0.115. In particular, adjusted p values were determined using the Benjamini–Yekutieli method to control the false discovery rate39.We performed comprehensive mRNA expression profiling of 770 immune-related genes in 10 patients with TSQCC and two normal controls using the nCounter PanCancer Immune Profiling Panel , according to the manufacturer’s instructions. The RNA was hybridised with the probe sets for 16 h at 67 °C, and the samples were then processed using an automated nCounter Sample Prep Station . Cartridges containing immobilised and aligned reporter complexes were subsequently imaged on an nCounter Digital Analyzer  that had been set at a data resolution of 555 fields of view. Reporter counts were determined, log40. Briefly, the most morphologically representative tumour regions were selected using haematoxylin and eosin-stained slides, and two tissue cores (2 mm in diameter) were punched out from the selected regions from the paraffin-embedded blocks for each patient. These tissue cores were then arrayed in a paraffin block. Moreover, two tissue cores from thymic cysts of three patients were also arrayed in a paraffin block.Tissue microarrays were created as previously describedp values of less than 0.05 were considered statistically significant. EZR , a modified version of R , was used for statistical analysis41.Immunohistochemical analyses were performed using autostainers , according to the manufacturers’ instructions. The primary antibodies used in this study were mouse anti-CD5 monoclonal antibody , rabbit anti-CD117 polyclonal antibody , and rabbit anti-PRAME polyclonal antibody . The normal lymph node tissues were used as outer positive controls for CD5, gastrointestinal stromal tumours were used as controls for CD117, and normal testis tissues were used as controls for PRAME. When more than one core from the same patient showed immunoreactivity, we considered it to be a positive case. Differences in the classification rates of immunohistochemistry staining for PRAME between TSQCCs and thymoma or those between TSQCCs and healthy controls were evaluated using Fisher’s exact test. Results with PRAME mRNA levels in TSQCCs (n = 10) and thymomas (n = 29), which were subdivided based on negativity (n = 21) or positivity (n = 8) for PRAME immunohistochemistry in tissue microarray, were analyzed and compared using the nCounter PanCancer Immune Profiling Panel . The methodology used for digital mRNA counts and data analysis in this comparative analysis is the same as that in comprehensive mRNA expression profiling by digital mRNA counts as described above.