Clustering out-of-hospital cardiac arrest patients with non-shockable rhythm by machine learning latent class analysis.

Aim: We aimed to identify subphenotypes among patients with out-of-hospital cardiac arrest (OHCA) with initial non-shockable rhythm by applying machine learning latent class analysis and examining the associations between subphenotypes and neurological outcomes. Methods: This study was a retrospective analysis within a multi-institutional prospective observational cohort study of OHCA patients in Osaka, Japan (the CRITICAL study). The data of adult OHCA patients with medical causes and initial non-shockable rhythm presenting with OHCA between 2012 and 2016 were included in machine learning latent class analysis models, which identified subphenotypes, and patients who presented in 2017 were included in a dataset validating the subphenotypes. We investigated associations between subphenotypes and 30-day neurological outcomes. Results: Among the 12,594 patients in the CRITICAL study database, 4,849 were included in the dataset used to classify subphenotypes (median age: 75 years, 60.2% male), and 1,465 were included in the validation dataset (median age: 76 years, 59.0% male). Latent class analysis identified four subphenotypes. Odds ratios and 95% confidence intervals for a favorable 30-day neurological outcome among patients with these subphenotypes, using group 4 for comparison, were as follows; group 1, 0.01 (0.001-0.046); group 2, 0.097 (0.051-0.171); and group 3, 0.175 (0.073-0.358). Associations between subphenotypes and 30-day neurological outcomes were validated using the validation dataset. Conclusion: We identified four subphenotypes of OHCA patients with initial non-shockable rhythm. These patient subgroups presented with different characteristics associated with 30-day survival and neurological outcomes.

Clinical Phenotyping of Out-of-Hospital Cardiac Arrest Patients With Shockable Rhythm　- Machine Learning-Based Unsupervised Cluster Analysis.

BACKGROUND: The hypothesis of this study is that latent class analysis could identify the subphenotypes of out-of-hospital cardiac arrest (OHCA) patients associated with the outcomes and allow us to explore heterogeneity in the effects of extracorporeal cardiopulmonary resuscitation (ECPR).Methods and Results:This study was a retrospective analysis of a multicenter prospective observational study (CRITICAL study) of OHCA patients. It included adult OHCA patients with initial shockable rhythm. Patients from 2012 to 2016 (development dataset) were included in the latent class analysis, and those from 2017 (validation dataset) were included for evaluation. The association between subphenotypes and outcomes was investigated. Further, the heterogeneity of the association between ECPR implementation and outcomes was explored. In the study results, a total of 920 patients were included for latent class analysis. Three subphenotypes (Groups 1, 2, and 3) were identified, mainly characterized by the distribution of partial pressure of O2(PO2), partial pressure of CO2(PCO2) value of blood gas assessment, cardiac rhythm on hospital arrival, and estimated glomerular filtration rate. The 30-day survival outcomes were varied across the groups: 15.7% in Group 1; 30.7% in Group 2; and 85.9% in Group 3. Further, the association between ECPR and 30-day survival outcomes by subphenotype groups in the development dataset was as varied. These results were validated using the validation dataset. CONCLUSIONS: The latent class analysis identified 3 subphenotypes with different survival outcomes and potential heterogeneity in the effects of ECPR.

Trends in In-Hospital Advanced Management and Survival of Out-of-Hospital Cardiac Arrest Among Adults From 2013 to 2017　- A Multicenter, Prospective Registry in Osaka, Japan.

BACKGROUND: The aim of our study was to investigate in detail the temporal trends in in-hospital characteristics, actual management, and survival, including neurological status, among adult out-of-hospital cardiac arrest (OHCA) patients in recent years.Methods and Results:From the prospective database of the Comprehensive Registry of Intensive Care for OHCA Survival (CRITICAL) study in Osaka, Japan, we enrolled all OHCA patients aged ≥18 years for whom resuscitation was attempted, and who were transported to participating hospitals between the years 2013 and 2017. The primary outcome measure was 1-month survival with favorable neurological outcome after OHCA. Temporal trends in in-hospital management and favorable neurological outcome among adult OHCA patients were assessed. Of the 11,924 patients in the database, we included a total of 10,228 adult patients from 16 hospitals. As for in-hospital advanced treatments, extracorporeal cardiopulmonary resuscitation (ECPR) use increased from 2.4% in 2013 to 4.3% in 2017 (P for trend <0.001). However, the proportion of adult OHCA patients with favorable neurological outcome did not change during the study period (from 5.7% in 2013 to 4.4% in 2017, adjusted odds ratio (OR) for 1-year increment: 0.98 (95% confidence interval: 0.94-1.23)). CONCLUSIONS: In this target population, in-hospital management such as ECPR increased slightly between 2013 and 2017, but 1-month survival with favorable neurological outcome after adult OHCA did not improve significantly.

Association between serum lactate level during cardiopulmonary resuscitation and survival in adult out-of-hospital cardiac arrest: a multicenter cohort study.

We aimed to investigate the association between serum lactate levels during cardiopulmonary resuscitation (CPR) and survival in patients with out-of-hospital cardiac arrest (OHCA). From the database of a multicenter registry on OHCA patients, we included adult nontraumatic OHCA patients transported to the hospital with ongoing CPR. Based on the serum lactate levels during CPR, the patients were divided into four quartiles: Q1 (≤ 10.6 mEq/L), Q2 (10.6-14.1 mEq/L), Q3 (14.1-18.0 mEq/L), and Q4 (> 18.0 mEq/L). The primary outcome was 1-month survival. Among 5226 eligible patients, the Q1 group had the highest 1-month survival (5.6% [74/1311]), followed by Q2 (3.6% [47/1316]), Q3 (1.7% [22/1292]), and Q4 (1.0% [13/1307]) groups. In the multivariable logistic regression analysis, the adjusted odds ratio of Q4 compared with Q1 for 1-month survival was 0.24 (95% CI 0.13-0.46). 1-month survival decreased in a stepwise manner as the quartiles increased (p for trend < 0.001). In subgroup analysis, there was an interaction between initial rhythm and survival (p for interaction < 0.001); 1-month survival of patients with a non-shockable rhythm decreased when the lactate levels increased (p for trend < 0.001), but not in patients with a shockable rhythm (p for trend = 0.72). In conclusion, high serum lactate level during CPR was associated with poor 1-month survival in OHCA patients, especially in patients with non-shockable rhythm.

Development and Validation of a Clinical Score to Predict Neurological Outcomes in Patients With Out-of-Hospital Cardiac Arrest Treated With Extracorporeal Cardiopulmonary Resuscitation.

Importance: Extracorporeal cardiopulmonary resuscitation (ECPR) is expected to improve the neurological outcomes of patients with refractory cardiac arrest; however, it is invasive, expensive, and requires substantial human resources. The ability to predict neurological outcomes would assist in patient selection for ECPR. Objective: To develop and validate a prediction model for neurological outcomes of patients with out-of-hospital cardiac arrest with shockable rhythm treated with ECPR. Design, Setting, and Participants: This prognostic study analyzed data from the Japanese Association for Acute Medicine Out-of-Hospital Cardiac Arrest registry, a multi-institutional nationwide cohort study that included 87 emergency departments in Japan. All adult patients with out-of-hospital cardiac arrest and shockable rhythm who were treated with ECPR between June 2014 and December 2017 were included. Patients were randomly assigned to the development and validation cohorts based on the institutions. The analysis was conducted between November 2019 and August 2020. Exposures: Age (<65 years), time from call to hospital arrival (≤25 minutes), initial cardiac rhythm on hospital arrival (shockable), and initial pH value (≥7.0). Main Outcomes and Measures: The primary outcome was 1-month survival with favorable neurological outcome, defined by Cerebral Performance Category 1 or 2. In the development cohort, a simple scoring system was developed to predict this outcome using a logistic regression model. The diagnostic ability and calibration of the scoring system were assessed in the validation cohort. Results: A total of 916 patients were included, 458 in the development cohort (median [interquartile range {IQR}] age, 61 [47-69] years, 377 [82.3%] men) and 458 in the validation cohort (median [IQR] age, 60 [49-68] years; 393 [85.8%] men). The cohorts had the same proportion of favorable neurological outcome (57 patients [12.4%]). The prediction scoring system was developed, attributing a score of 1 for each clinical predictor. Patients were divided into 4 groups, corresponding to their scores on the prediction model, as follows: very low probability (score 0), low probability (score 1), middle probability (score 2), and high probability (score 3-4) of good neurological outcome. The mean predicted probabilities in the groups stratified by score were as follows: very low, 1.6% (95% CI, 1.6%-1.6%); low, 4.4% (95% CI, 4.2%-4.6%); middle, 12.5% (95% CI, 12.1%-12.8%); and high, 30.8% (95% CI, 29.1%-32.5%). In the validation cohort, the C statistic of the scoring system was 0.724 (95% CI, 0.652-0.786). The predicted probability was evaluated as well calibrated to the observed favorable outcome in both cohorts by visual assessment of the calibration plot. Conclusions and Relevance: In this study, the scoring system had good discrimination and calibration performance to predict favorable neurological outcomes of patients with out-of-hospital cardiac arrest and shockable rhythm who were treated with ECPR.

Vaccination Status and Antibody Titers against Rubella and Measles among Japanese Female College Students Majoring in Childcare between 2015 and 2018.

In 2014, for the protection of medical workers against measles and rubella infection, the Japanese Society for Infection Prevention and Control (JSIPC) recommended either maintaining antibody titers of seroprotective range or two-dose vaccination. JSIPC defined antibody titers into 3 ranges: seroprotective as expected prevention of infection, seronegative as under detection levels, and seropositive as antibody titers ranged between seronegative and seroprotective. This study aimed to explore the association between the number of vaccine doses received and the antibody titers against measles and rubella among Japanese college students majoring in childcare. A total of 841 female students with no history of measles or rubella were serologically screened at the time of college admission between 2015 and 2018. All 841 students had been vaccinated against measles; 738 (87.8%) received two doses of the measles vaccine and 103 (12.2%) received one dose. Likewise, 839 students, except for two, had been vaccinated against rubella; 719 (85.7%) received two doses of the rubella vaccine and 120 (14.3%) received one dose. We thus found that 107 students (12.7%) were seropositive for measles-specific IgG and 731 (86.9%) attained seroprotective titers. By contrast, in case of rubella-specific IgG, only 462 students (55.1%) attained seroprotective titers, and 371 students (44.1%) were seropositive. The two students without receiving rubella vaccination were classified as seronegative. In conclusion, despite that > 85% of students surveyed had received two doses of measles and rubella vaccines, a substantial number of students remain susceptible to measles and especially rubella at the time of college admission.

Meningococcemia in Adults: A Review of the Literature.

We mainly refer to the acute setting of meningococcemia. Meningococcemia is an infection caused by Neisseria meningitidis, which has 13 clinically significant serogroups that are distinguishable by the structure of their capsular polysaccharides. N. meningitidis, also called meningococcus, is a Gram-negative, aerobic, diplococcus bacterium. The various consequences of severe meningococcal sepsis include hypotension, disseminated intravascular coagulation (DIC), multiple organ failure, and osteonecrosis due to DIC. The gold standard for the identification of meningococcal infection is the bacteriologic isolation of N. meningitidis from body fluids such as blood, cerebrospinal fluid (CSF), synovial fluid, and pleural fluid. Blood, CSF, and skin biopsy cultures are used for diagnosis. Meningococcal infection is a medical emergency that requires antibiotic therapy and intensive supportive care. Management of the systemic circulation, respiration, and intracranial pressure is vital for improving the prognosis, which has dramatically improved since the wide availability of antibiotics. This review of the literature provides an overview of current concepts on meningococcemia due to N. meningitidis infection.

[Examination of Patients with Malignant Obstruction Treated with Chemotherapy after Ileo/Coleostomy].

Management for obstructive cancer of the colon diverges into many ways. The aim of this study was to evaluate the treatment course of patients with malignant obstruction after ileo/coleostomy. Thirty-six patients with malignant obstruction who underwent ileo/coleostomy in our hospital from May 2012 to January 2016were enrolled in the study. Clinical outcomes were the period before treatment initiation, chemotherapy, radiotherapy, primary lesion resection, and death, and these were retrospectively analyzed. Although 9 stomal complications occurred, no case experienced a delayed treatment start. However, patients with perioperative complications, sepsis due to the tumor, pneumonia, cerebral infractions, and ileus needed a long recovery period before treatment initiation. Patients who need ileo/coleostomy must be considered for performance status and ways to decrease perioperative complications to prevent stomal complications from chemo/radiotherapy.

[A Case of an Elderly Patient with Unresectable Gastric Cancer Treated by Paclitaxel and Ramucirumab].

An 80-year-old man was admitted to our hospital with appetite loss in December 2014. Gastroduodenal scope, abdominal computed tomography(CT), and laparoscopy revealed type 4 advanced gastric cancer(poorly differentiated adenocarcinoma) with multiple lymph node(LN)involvement and multiple peritoneal metastasis. S-1(80mg/body)was administrated between January 2015 and September 2015 in the outpatient clinic. A partial response was obtained, but a gastric tumor, ascites, and LN re-growth were observed. Since October 2015, paclitaxel(PTX)(70mg/m2; day 1, 8, and 15)and ramucir- umab(RAM)(8mg/kg; day 1 and 15)have been administered. After 2 courses, bi-weekly PTX plus RAM were continued for grade 3 neutropenia and grade 2 anorexia. The tumor and LNs partially responded, and the ascites disappeared. With this dosage and administration schedule, the partial response(PR)was maintained for approximately 8 months without any severe adverse reactions. This successful case might indicate that it is important for elderly patients with gastric cancer that progressed with prior chemotherapy regimens to consider appropriate reduction of the PTX dosage, schedule, and continuation of RAM.

Sebaceous carcinoma of the parotid gland: a case report.

BACKGROUND: Primary sebaceous carcinoma of the parotid gland is extremely rare, and because of its rarity, clinicopathological characteristics and histogenesis are not fully understood. METHODS: Here, we report a patient who presented with a left infra-auricular painless mass. We present the histological features and discuss possible optimal treatments based on previous literature. RESULTS: The mass was suspected to be a myoepithelial tumor or possibly a pleomorphic adenoma. Initially, the mass was resected with preservation of the facial nerve, but this caused facial palsy. Because the histological examination showed a sebaceous carcinoma and a part of the mass could be remaining on the facial nerve, additional surgery was performed, and the facial nerve was reconstructed with cervical nerve. Follow-up after 7 months showed no sign of recurrence of metastasis. CONCLUSION: We encountered a rare sebaceous carcinoma of the parotid gland. Additional surgery was performed because preoperative diagnosis was difficult.

[A Case of Invasive Ductal Carcinoma in the Fat Replacement of the Pancreatic Body and Tail].

A 56 year-old woman with obesity (BMI3 2) and diabetes mellitus was diagnosed with right renal cell carcinoma. She underwent right nephrectomy 1 year ago. Seven months after surgery, CT revealed a rapidly growing mass near the spleen. The mass showed slight accumulation of FDG (SUVmax=2.4) on PET-CT. Since the lesion grew rapidly and was not enhanced in the early phase of enhanced CT, we diagnosed pancreatic cancer. Distal pancreatectomy and splenectomy were performed. The final pathological diagnosis was invasive ductal carcinoma in the fat replacement of the pancreatic body and tail. Postoperatively, the patient had no complications such as pancreatic fistula or aggravation of glucose intolerance. She received postoperative chemotherapy with gemcitabine. Since she developed pulmonary artery thrombosis, postoperative chemotherapy was interrupted after 8 courses. Thirty-two months after the surgery, she was still living without any recurrence. Acinar cells were absent in the fat replacement of the pancreas, but the pancreatic duct cells were still present. There was carcinoma in situ in the main pancreatic duct surrounding chronic inflammation. Fat replacement itself could be potentially precursor of the pancreatic cancer.

Seroprevalence of measles- and mumps-specific immunoglobulin G among Japanese healthcare students increased during 2007-2012.

We evaluated the seroprevalence of vaccine-preventable infectious diseases among Japanese healthcare students to create immunization guidelines. Between 2007 and 2012, a total of 1746 Japanese medical, nursing, and paramedical students were serologically screened for measles, mumps, rubella, varicella, and hepatitis B virus (HBV) antibodies at the time of admission. In 2007, the seroprevalence of measles and mumps was 52.7% and 65.6%, respectively. The seroprevalence of measles dramatically increased to 96.6% in 2009 and was then sustained at >90%. The seroprevalence of mumps gradually increased to >80.0% between 2010 and 2012. The seroprevalence of rubella remained at >90% except in 2008 (85.6%), and the seroprevalence of varicella was sustained at >92% throughout 2007-2012. The seroprevalence of HBV antibody remained at <7% during 2007-2012. Although the seroprevalence of vaccine-preventable infectious diseases among Japanese healthcare students increased during the 2007-2012 study period, a substantial number of students were susceptible to vaccine-preventable infectious diseases. Therefore, we propose targeted immunization of Japanese healthcare students using serological screening prior to clinical training.

An update on purple urine bag syndrome.

Purple urine bag syndrome is characterized by the urinary drainage bag turning purple in patients on prolonged urinary catheterization, especially those in the bedridden state. It is associated with bacterial urinary tract infections caused by indigo-producing and indirubin-producing bacteria, usually affects women, and is associated with alkaline urine, constipation, and a high bacterial load in the urine. Almost all patients with purple urine bag syndrome are catheterized due to significant disability, and the urinary pH is 7.0 or more. In general, intensive treatment with antibiotics is not recommended. Purple urine bag syndrome per se almost always appears to be asymptomatic and harmless. However, caution is needed, because some cases have been reported to show progression to severe disease states, so further research into the morbidity and mortality of this infection is warranted.

Downregulation of N-methyl-D-aspartate receptor ζ1 subunit (GluN1) gene in inferior colliculus with aging.

Presbycusis is the impairment of auditory function associated with aging, which stems from peripheral cochlear lesions and degeneration of the central auditory process. The effect of age-induced peripheral hearing loss on the central auditory process is not fully understood. C57Bl/6 (C57) mice present accelerated peripheral hearing loss, which is well developed by middle-age and mimics the human presbycusis pattern. The aim of this study was to elucidate the molecular effects of peripheral hearing loss in the inferior colliculus (IC) with age between young and middle-aged C57 mice using cDNA microarray. Glutamate receptor ionotropic NMDA ζ1 (GluN1) exhibited the greatest decrease in the middle-aged group as determined using cDNA microarray and by further assessment using real-time PCR (qPCR). Histological assessment with in situ hybridization of GluN1 showed significantly decreased expression in all IC subdivisions of the middle-aged group. GluN1 is a receptor for excitatory neurotransmission, and significant downregulation of this gene may be subsequent to the decline of afferent input from the cochlea in aging C57 mice. Consequently, using the combination of microarray, qPCR, and in situ hybridization, we showed that the decline of GluN1 in the IC of aging animals might have a key role in the pathogenesis of presbycusis.

Cryoglobulinemia (review).

Cryoglobulins are immunoglobulins that precipitate at low temperatures and redissolve upon rewarming. Cryoglobulinemia refers to the presence of circulating cryoglobulins in serum, and generally leads to a systemic inflammatory syndrome characterized by fatigue, arthralgia, purpura, neuropathy and glomerulonephritis. The disease mainly involves small to medium-sized blood vessels and causes vasculitis due to cryoglobulin-containing immune complexes. Cryoglobulinemia is classified into three types (I, II and III) on the basis of immunoglobulin composition. Predisposing conditions include lymphoproliferative disease, collagen disease and hepatitis C virus (HCV) infection. The diagnosis of cryoglobulinemic syndrome is predominantly based on the laboratory demonstration of serum cryoglobulins. Treatment is often directed towards the underlying disease state. For patients with chronic HCV infection, anti-viral therapy is indicated. Intense immunosuppressive or immunomodulatory therapy, including steroids, plasmapheresis and cytotoxic agents, is reserved for organ-threatening or recalcitrant disease. In this review, we discuss the clinical characteristics of the three types of cryoglobulinemia.

Potent anti-tumor killing activity of the multifunctional Treg cell line HOZOT against human tumors with diverse origins.

The T cell line HOZOT has a unique FOXP3+CD4+ CD8+CD25+ phenotype, exhibits suppressive activity in allogeneic mixed lymphocyte reactions (MLR), and produces IL-10, defining HOZOT as regulatory T cells (Tregs). Interestingly, in addition to possessing a suppressive Treg ability, HOZOT was also found to show cytotoxicity against certain representative human cancer cell types. In order to disclose the range of anti-tumor activity by HOZOT, we screened it by using a panel of twenty human tumor cell lines with different origins. Consequently, HOZOT showed potent cytocidal effects against a wide spectrum of neoplastic cells including carcinomas, sarcomas, mesotheliomas and glioblastomas except for hematopoietic malignancies. Its anti-tumor activity was strong enough with an E:T ratio of 4:1, which is considered to be more effective than that by conventional CTLs. Furthermore, an in vivo representative mouse tumor model by implanting human colon adenocarcinoma cells revealed that adoptive transfer of HOZOT almost completely eradicated disseminated lesions on peritoneum, markedly reduced metastases in lung and liver, and dramatically decreased bloody ascites caused by peritoneal carcinomatosis. Treatment of the tumor model mice by HOZOT with an E:T ratio of 2:1 even indicated the prolongation of their survival, although not reaching obvious statistical significance. In vitro blocking experiments using antibodies and inhibitors suggested that the cytotoxic mechanism of HOZOT against tumors is different from conventional cytotoxic cells such as CTL, NK or NKT cells. Altogether, our studies demonstrated the potent killing activity of HOZOT against a broad range of human malignancies, which indicates that HOZOT is a powerful tool in immunotherapy for advanced stage tumors.

Cell-in-cell structures formed between human cancer cell lines and the cytotoxic regulatory T-cell line HOZOT.

We previously established a novel cell line, termed HOZOT, derived from umbilical cord blood mononuclear cells that is characterized as a human cytotoxic regulatory T (Treg) cell line with a FOXP3(+)CD4(+)CD8(+)CD25(+) phenotype. Here, we describe a new property of HOZOT cells: they actively penetrate into a variety of human cancer cell lines, but not into normal cell lines, and form apparent cell-in-cell structures. In the process of cell penetration, we observed that HOZOT cells adhered to target cells seemed to first insert their nuclei into the cytoplasm of target cells, distinct from the process of phagocytosis. In addition, blocking experiments showed that major histocompatibility complex class I is one of the target cell recognition molecules for HOZOT cells. Furthermore, we propose that cell-in-cell structures between HOZOT cells and target cancer cells could be one of the cytotoxic mechanisms of HOZOT cells.

[A case report: multiple air embolism after the laryngopharyngoesophagectomy occurred by the cervical infection from postoperative fistula].

Systemic air embolism, a very rare clinical condition, has many causes. We report a case of multiple air embolisms following laryngopharyngoesophagectomy salvage surgery for hypopharyngeal residual cancer after concurrent chemoradiotherapy. Cervical infection arose from a fistula caused by postoperative suture failure in which the 56-year-old man suddenly lost consciousness and went into shock. A few days post operation, an air embolism happened and caused in the brain, pulmonary, myocardiac and cerebral infarction. The man died two months after initial occurrence. We suspect that air entered through the ruptured left internal jugular vein via infection due to aspiration at the injury site. Air embolisms are associated with different medical maneuvers, and it is necessary to recognize that they may become a serious perioperative complication.

Interleukin-8 and RANTES are signature cytokines made by HOZOT, a new type of regulatory T cells.

Distinct cytokine production profiles define the effector functions of both helper and regulatory T cells. Recently, we established novel cytotoxic regulatory T (Treg) cell lines, HOZOT, which have been characterized as IL-10-producing T cells. In this study, we further characterized HOZOT by performing comprehensive analyses of cytokines produced by HOZOTs in order to identify a signature cytokine profile. Using DNA microarrays, we compared the gene expression profiles of HOZOT-4, a representative HOZOT cell line, under three different conditions. Seven genes, including IL-8, IL-10, IL-13, MIP-1alpha, and MIP-1beta, were identified as inducible cytokines when stimulated with stromal cells or anti-CD3/CD28 antibodies. Twelve genes, including IL-2, IL-3, IL-4, IL-22, CCL1, and lymphotactin, were categorized as antibody stimulation-responsive but stromal cell-non-responsive. Three genes, IL-32, RANTES, and CCL23, were constitutively expressed irrespective of stimulation condition. Among these cytokines, we focused on two chemokines, IL-8 and RANTES for further studies, and found that only HOZOT produced both of them at considerable levels whereas other T cell subsets, including Tregs and helper T cells, did not. Kinetic and inhibition experiments revealed contrasting properties for the two chemokines. IL-8 was induced only after stimulation, whereas RANTES mRNA and protein accumulated to high levels even before stimulation. Interestingly, IL-8 mRNA was induced by cycloheximide treatment and RANTES showed reduced mRNA but increased protein expression by antibody stimulation. As a whole, the unique cytokine signature profile consisting of Th1, Th2, and cytolytic T cell cytokines as well as Treg cytokines reflect the multifunctional nature of HOZOT. In particular, the dual production of IL-8 and RANTES by distinct mechanisms is a hallmark of HOZOT.

The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus.

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4(+)CD25(+) naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.