Tsyn-Seq: a T-cell Synapse-Based Antigen Identification Platform.

Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.

Microbiome alteration via fecal microbiota transplantation is effective for refractory immune checkpoint inhibitor-induced colitis.

Immune checkpoint inhibitors (ICIs) target advanced malignancies with high efficacy but also predispose patients to immune-related adverse events like immune-mediated colitis (IMC). Given the association between gut bacteria with response to ICI therapy and subsequent IMC, fecal microbiota transplantation (FMT) represents a feasible way to manipulate microbial composition in patients, with a potential benefit for IMC. Here, we present a large case series of 12 patients with refractory IMC who underwent FMT from healthy donors as salvage therapy. All 12 patients had grade 3 or 4 ICI-related diarrhea or colitis that failed to respond to standard first-line (corticosteroids) and second-line immunosuppression (infliximab or vedolizumab). Ten patients (83%) achieved symptom improvement after FMT, and three patients (25%) required repeat FMT, two of whom had no subsequent response. At the end of the study, 92% achieved IMC clinical remission. 16S rRNA sequencing of patient stool samples revealed that compositional differences between FMT donors and patients with IMC before FMT were associated with a complete response after FMT. Comparison of pre- and post-FMT stool samples in patients with complete responses showed significant increases in alpha diversity and increases in the abundances of Collinsella and Bifidobacterium, which were depleted in FMT responders before FMT. Histologically evaluable complete response patients also had decreases in select immune cells , including CD8+ T cells, in the colon after FMT when compared with non-complete response patients (n = 4). This study validates FMT as an effective treatment strategy for IMC and gives insights into the microbial signatures that may play a critical role in FMT response.

A non-antibiotic-disrupted gut microbiome is associated with clinical responses to CD19-CAR-T cell cancer immunotherapy.

Increasing evidence suggests that the gut microbiome may modulate the efficacy of cancer immunotherapy. In a B cell lymphoma patient cohort from five centers in Germany and the United States (Germany, n = 66; United States, n = 106; total, n = 172), we demonstrate that wide-spectrum antibiotics treatment ('high-risk antibiotics') prior to CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is associated with adverse outcomes, but this effect is likely to be confounded by an increased pretreatment tumor burden and systemic inflammation in patients pretreated with high-risk antibiotics. To resolve this confounding effect and gain insights into antibiotics-masked microbiome signals impacting CAR-T efficacy, we focused on the high-risk antibiotics non-exposed patient population. Indeed, in these patients, significant correlations were noted between pre-CAR-T infusion Bifidobacterium longum and microbiome-encoded peptidoglycan biosynthesis, and CAR-T treatment-associated 6-month survival or lymphoma progression. Furthermore, predictive pre-CAR-T treatment microbiome-based machine learning algorithms trained on the high-risk antibiotics non-exposed German cohort and validated by the respective US cohort robustly segregated long-term responders from non-responders. Bacteroides, Ruminococcus, Eubacterium and Akkermansia were most important in determining CAR-T responsiveness, with Akkermansia also being associated with pre-infusion peripheral T cell levels in these patients. Collectively, we identify conserved microbiome features across clinical and geographical variations, which may enable cross-cohort microbiome-based predictions of outcomes in CAR-T cell immunotherapy.

Bacteroides ovatus alleviates dysbiotic microbiota-induced intestinal graft-versus-host disease.

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

Diet-derived metabolites and mucus link the gut microbiome to fever after cytotoxic cancer treatment.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

Long non-coding RNA MIR4300HG polymorphisms are associated with postoperative nausea and vomiting: a genome-wide association study.

BACKGROUND: Genetic factors such as single-nucleotide polymorphisms (SNPs) play a key role in the development of postoperative nausea and vomiting (PONV). However, previous findings are not widely applicable to different populations because of population-specific genetic variation. We developed a Japanese-specific DNA microarray for high-throughput genotyping. The aim of the current study was to identify SNPs associated with PONV on a genome-wide scale using this microarray in a sample of Japanese surgical patients. METHODS: Associations between 659,636 SNPs and the incidence of PONV 24 h after surgery in a limited sample of 24 female patients were assessed using the microarray. After imputation of genotypes at 24,330,529 SNPs, 78 SNPs were found to be associated with the incidence of PONV. We chose 4 of the 78 SNPs to focus on by in silico functional annotation. Finally, we genotyped these 4 candidate SNPs in 255 patients using real-time PCR to verify association with the incidence of PONV. RESULTS: The T > C variant of rs11232965 in the long non-coding RNA MIR4300HG was significantly associated with reduced incidence of PONV among genotypes and between alleles (p = 0.01 and 0.007). CONCLUSIONS: We identified a novel SNP (rs11232965) in the long non-coding RNA MIR4300HG that is associated with PONV. The rs11232965-SNP variant (T > C) is protective against the incidence of PONV. TRIAL REGISTRATION: This study was registered at the UMIN Clinical Trials Registry (Identifier: UMIN000022903 , date of registration: June 27, 2016, retrospectively registered.

Intraoperative auditory evoked potential recordings are more reliable at signal detection from different sensor sites on the forehead compared to bispectral index.

Bispectral index (BIS) and auditory evoked potential (AEP) monitoring require the attachment of forehead sensors, posing difficulties when the surgical field involves the forehead. This study analyzed the relationship between BIS values and AEP indices from different sites on the head to establish alternative sensor locations for AEP recording. Thirty patients scheduled for elective surgery under sevoflurane anesthesia were randomly assigned to the forehead, nose or mandible groups (n = 10 patients per group). AEP sensors were placed at the assigned position for each group and BIS sensors were placed on the forehead. BIS value and AEP index were simultaneously recorded from induction until emergence from general anesthesia. Relationships between BIS values and AEP indices were analyzed using a regression method and compared between groups using Pearson's correlation coefficients. Square regression models better expressed the relationships than linear models in all groups. The z-transformed coefficient in the forehead group was the same as the nose group (p = 0.24) and significantly different in the mandible group (p = 0.0046). These findings suggest that AEPs can be accurately recorded from sensors placed on the nose. Nasal AEP might be useful for monitoring electrical activity in the brain during surgeries involving the forehead.

The influence of depth of anesthesia on motor evoked potential response during awake craniotomy.

OBJECTIVE Motor evoked potentials (MEPs) are a critical indicator for monitoring motor function during neurological surgery. In this study, the influence of depth of anesthesia on MEP response was assessed. METHODS Twenty-eight patients with brain tumors who underwent awake craniotomy were included in this study. From a state of deep anesthesia until the awake state, MEP amplitude and latency were measured using 5-train electrical bipolar stimulations on the same site of the precentral gyrus each minute during the surgery. The depth of anesthesia was evaluated using the bispectral index (BIS). BIS levels were classified into 7 stages: < 40, and from 40 to 100 in groups of 10 each. MEP amplitude and latency of each stage were compared. The deviation of the MEP measurements, which was defined as a fluctuation from the average in every BIS stage, was also considered. RESULTS A total of 865 MEP waves in 28 cases were evaluated in this study. MEP amplitude was increased and latency was decreased in accordance with the increases in BIS level. The average MEP amplitudes in the > 90 BIS level was approximately 10 times higher than those in the < 40 BIS level. Furthermore, the average MEP latencies in the > 90 BIS level were 1.5-3.1 msec shorter than those in the < 60 BIS level. The deviation of measured MEP amplitudes in the > 90 BIS level was significantly stabilized in comparison with that in the < 60 BIS level. CONCLUSIONS MEP amplitude and latency were closely correlated with depth of anesthesia. In addition, the deviation in MEP amplitude was also correlated with depth of anesthesia, which was smaller during awake surgery (high BIS level) than during deep anesthesia. Therefore, MEP measurement would be more reliable in the awake state than under deep anesthesia.

Effect of sevoflurane anesthesia on the comprehensive mRNA expression profile of the mouse hippocampus.

Postoperative nausea and vomiting (PONV) is a common complication after general anesthesia. Recent studies suggested that the hippocampus is involved in PONV. Hypothesising that hippocampal dopaminergic neurons are related to PONV, we examined the comprehensive mRNA profile of the hippocampus, using a sevoflurane-treated mouse model to confirm this. This study was conducted after approval from our institutional animal ethics committee, the Animal Research Center of Sapporo Medical University School of Medicine (project number: 12-033). Eight mice were assigned to two groups: a naïve group and a sevoflurane group (Sev group). In the Sev group, four mice were anesthetised with 3.5% sevoflurane for 1 hour. Subsequently, mRNA was isolated from their hippocampal cells and RNA sequencing was performed on an Illumina HiSeq 2500 platform. Mapping of the quality-controlled, filtered paired-end reads to mouse genomes and quantification of the expression levels of each gene were performed using R software. The Rtn4rl2 gene that encodes the Nogo receptor was the most up-regulated gene in the present study. The expression levels of dopamine receptor genes and the tachykinin gene were increased by sevoflurane exposure, while the genes related to serotonin receptors were not altered by sevoflurane exposure. The expression levels of LIM-homeodomain-related genes were highly down-regulated by sevoflurane. These findings suggest that sevoflurane exposure induces dopaminergic stimulation of hippocampal neurons and triggers PONV, while neuronal inflammation caused by LIM-homeodomain-related genes is down-regulated by sevoflurane.

Efficacy and Safety of a Lidocaine and Ropivacaine Mixture for Scalp Nerve Block and Local Infiltration Anesthesia in Patients Undergoing Awake Craniotomy.

BACKGROUND: Mixtures of various local anesthetics, such as lidocaine and ropivacaine, have been widely used. However, their efficacy and safety for scalp nerve blocks and local infiltration during awake craniotomy have not been fully elucidated. METHODS: We prospectively investigated 53 patients who underwent awake craniotomy. Scalp block was performed for the blockade of the supraorbital, supratrochlear, zygomaticotemporal, auriculotemporal, greater occipital, and lesser occipital nerves with a mixture containing equal volumes of 2% lidocaine and 0.75% ropivacaine, including 5 µg/mL of epinephrine. Infiltration anesthesia was applied at the site of skin incision using the same mixture. The study outcomes included changes in heart rate and blood pressure after head pinning and skin incision, and incidence of severe pain on emergence from anesthesia. Total doses and plasma concentrations of lidocaine and ropivacaine were measured at different time points after performing the block. RESULTS: The heart rate and blood pressure after head pinning were marginally, but significantly, increased when compared with baseline values. There were no significant differences in heart rate and blood pressure before and after the skin incision. Nineteen percent of the patients (10/53) complained of incisional pain at emergence from anesthesia. The highest observed blood concentrations of lidocaine and ropivacaine were 1.9±0.9 and 1.1±0.4 µg/mL, respectively. No acute anesthetic toxicity symptom was observed. CONCLUSIONS: Scalp block with a mixture of lidocaine and ropivacaine seems to provide effective and safe anesthetic management in patients undergoing awake craniotomy.

Recovery of postoperative cognitive function in elderly patients after a long duration of desflurane anesthesia: a pilot study.

Postoperative cognitive dysfunction (POCD) increases morbidity and mortality. The mechanisms underlying POCD remain elusive; however, systemic responses induced by anesthesia and surgery might trigger neuroinflammation and POCD. Desflurane is a preferable volatile anesthetic agent for elderly patients because it facilitates shorter recovery from general anesthesia. The aim of this study was to determine whether quality of emergence and cognitive function in elderly patients undergoing a long duration desflurane anesthesia are better than those in the case of sevoflurane anesthesia. Forty-two patients who were older than 65 years of age and scheduled for surgery of more than 4 h in duration were enrolled in this study. Patients were randomly assigned to a desflurane anesthesia group (D group) and sevoflurane anesthesia group (S group). General anesthesia was maintained with 3.5 % desflurane (D group) and 1.0 % sevoflurane (S group). The Mini-Mental State Examination (MMSE) was used for assessing cognitive function 24 h before and after surgery. Postoperative MMSE score in the D group was significantly improved compared to that in the preoperative period. In conclusion, elderly patients undergoing desflurane anesthesia have significantly better quality of emergence and may have better cognitive function than those in elderly patients undergoing sevoflurane anesthesia.

Association of µ-opioid receptor gene (OPRM1) haplotypes with postoperative nausea and vomiting.

Genetic variants, such as single-nucleotide polymorphisms (SNPs), of the µ-opioid receptor gene (OPRM1) might be associated with individual differences in opioid sensitivity, as well as with the incidence and severity of postoperative nausea and vomiting (PONV). The goal of the present study was to determine, in a cohort of Japanese surgical patients, genotypes and haplotypes of several SNPs in the OPRM1 gene, and their association with PONV during the early (first 24 h) postoperative period. We examined the incidence and severity of PONV, during the first 24 h after surgery, in 85 Japanese patients receiving intravenous patient-controlled analgesia fentanyl analgesia for postoperative pain control. Eight tag SNPs of the OPRM1 gene (rs1799971, A/G; rs510769, G/A; rs4870266, G/A; rs3798683, G/A; rs1323042, A/C; rs609623, C/T; rs9397685, A/G; and rs644261, C/G) were selected based on their minor allele frequency (>10%) and linkage disequilibrium strength (<80%), and genotyped for haplotype analysis and determination of associations with PONV. Only one out of eight investigated SNPs, rs9397685, in the intronic part of the OPRM1 gene was associated with differences in the occurrence and severity of PONV. We also found four common haplotypes with a frequency of >10% in the investigated patients, including GGGAACAC (33%), AGGGACAC (19%), GGGAACGC (12%), and AGAGACAC (10%). The severity of PONV in carriers of the GGGAACGC haplotype was significantly lower than in the carriers of the other haplotypes (P < 0.05). One intronic SNP, rs9397685, and haplotypes constructed from eight SNPs within the OPRM1 gene locus might be involved in the severity of PONV associated with general anesthesia and opioid administration. This novel finding, if validated and verified in larger and additional ethnic cohorts, might contribute to better knowledge of the contribution of the OPRM1 gene to PONV.

[Introduction of translational research in omics science to clinical anesthesia].

Much progress has been made in omics research following completion of the Human Genome Project. This comprehensive analysis produced a new discipline (i.e., bioinformatics), and its findings contributed to the clinical practice of anesthesiology. Genomes of patients show genetic variations and may predict the sensitivity to anesthetics and analgesics, incidence of adverse effects, and intensity of postsurgical pain. Changes in the transcriptomes of patients may also reflect anesthesia-related expression profiles of various types of neurons in the brain, and information on such changes may contribute to molecular targeted therapy in anesthetized patients. In addition, novel epigenome research may explain why environments change the phenotypes of clinical anesthesia. We currently hypothesize that female gender is associated with DNA methylation in pain-related and vomiting-related gene promoter regions at the genome-wide level and that epigenetic mechanisms are involved in gender differences in anesthesia practice.

The usefulness of an earphone-type infrared tympanic thermometer for intraoperative core temperature monitoring.

BACKGROUND: In this study we sought to determine the usefulness of a novel earphone-type infrared tympanic thermometer (IRT) for core temperature monitoring during surgery. METHODS: Two groups of patients were studied under different surgical conditions. The first group consisted of 18 adult patients (ASA I or II) who had been scheduled for elective surgery under general anesthesia. Before induction of general anesthesia, an earphone-type IRT was inserted into either the left or right ear canal. Tympanic temperature was monitored and recorded along with both rectal and esophageal temperatures during anesthesia. The second group consisted of eight adult patients (ASA II or III) who had been scheduled for cardiac surgery with cardiopulmonary bypass. Similar to the first group, tympanic temperature was measured by the earphone-type IRT and recorded along with the rectal and esophageal temperatures during cooling and rewarming phases of cardiopulmonary bypass. RESULTS: Study 1-The average temperature (+/-2 sd) measured with the IRT was +0.08 degrees C (+/-0.34 degrees C) above the esophageal temperature, and that with the rectal temperature was +0.11 degrees C (+/-0.55 degrees C) above the esophageal temperature. Study 2-The average temperature (+/-2 sd) measured with the IRT was +0.72 degrees C (+/-2.2 degrees C) above the esophageal temperature during cooling and warming phases during cardiac surgery with cardiopulmonary bypass. CONCLUSIONS: The earphone-type IRT might be used in a clinical setting for reliable and continuous core temperature monitoring during an operation.

[Usefulness of an earphone-type infrared tympanic thermometer for intraoperative core temperature monitoring].

BACKGROUND: Thermometers widely used intraoperatively are invasive and non-hygienic. We developed an earphone-type infrared tympanic thermometer and evaluated its usefulness as a core temperature monitor. METHODS: Sixteen adult patients who required nonabdominal surgery under general anesthesia were enrolled in this study. After induction of general anesthesia, thermistor probes were inserted into the rectum and esophagus for measurements of rectal and esophageal temperatures, respectively. An earphone-type infrared tympanic thermometer was inserted into the ear canal on one side. These measured temperatures were monitored and recorded at 1-min intervals. Regression analysis and Bland-Altman analysis were used to compare the data (tympanic/rectal temperatures) with esophageal temperature as a core temperature. RESULTS: Tympanic temperature showed a good correlation with esophageal temperature (r = 0.976, n= 2490, P < 0.001). The mean difference between these temperature monitors was+0.06 degrees C, and 2x standard deviation (SD) was 0.32 degrees C. Rectal temperature also showed a good correlation with esophageal temperature (r = 0.946, P < 0.001). The mean difference between these monitors was+0.22 degrees C, and 2 SD was 0.28. CONCLUSIONS: The earphone-type tympanic thermometer can be used in a clinical setting as a reliable core temperature monitor.