An allometric pharmacokinetic model and minimum effective analgesic concentration of fentanyl in patients undergoing major abdominal surgery.

BACKGROUND: We aimed to characterise the population pharmacokinetics of fentanyl in adults and to determine the minimum effective concentration (MEC) and minimum effective analgesic concentration (MEAC) of i.v. fentanyl in patients after major abdominal open surgery. METHODS: In the pharmacokinetic study, subjects received an intravenous bolus of fentanyl 100 µg during operation, and arterial blood was sampled at pre-set intervals. In addition, data from previously published fentanyl pharmacokinetic studies were incorporated to build a pharmacokinetic model. In the MEAC study, subjects were asked to rate their pain every 10 min using a VAS (0=no pain, 10=most severe pain) in the PACU. The first blood sample was obtained when wound pain was rated as ≥3 at rest or ≥5 during compression. Then, fentanyl 50 µg was administered every 10 min until the pain intensity had decreased to <3 at rest and <5 during compression, at which point the second blood was sampled and the first MEAC of fentanyl was measured. The same procedure was repeated to obtain a third sample (MEC) and a fourth sample (second MEAC). RESULTS: In the population pharmacokinetic study (n=95), the plasma concentration of fentanyl over time was well-described by the three-compartment mammillary model using an allometric expression. The V1, V2, V3, Cl, Q1, and Q2 of a 70 kg subject were 10.1, 26.5, 206 L, 0.704, 2.38, and 1.49 L min-1, respectively. In the MEAC study (n=30), the median (inter-quartile range) MEC and MEAC were 0.72 (0.58-1.05) ng ml-1, and 0.99 (0.76-1.28) ng ml-1, respectively. CONCLUSION: These results provide a scientific basis for the use of fentanyl for acute postoperative pain management in surgical patients. CLINICAL TRIAL REGISTRATION: KCT0003273 (http://cris.nih.go.kr).

Evaluation of Surgical Pleth Index and Analgesia Nociception Index as surrogate pain measures in conscious postoperative patients: an observational study.

We evaluated the performance of the Surgical Plethysmographic Index (SPI) and the Analgesia Nociception Index (ANI) as surrogate pain measures and determined their respective cut-off values for detecting pain in conscious postoperative patients. In total, 192 patients after elective surgery were enrolled. Baseline SPI and ANI data were acquired for 10 min in the operating room prior to surgery when the patients rated their pain as 0 on the numerical rating scale (NRS). Upon arrival in the post-anaesthesia care unit (PACU) after surgery, SPI and ANI data were recorded for 10 min. The means of the recorded data at OR and PACU were defined as the values representing baseline and postoperative pain, respectively. SPI and ANI data obtained from 189 patients were analysed, who were anesthetized with propofol (n = 149) or sevoflurane (n = 40). Remifentanil was continuously infused intraoperatively in all patients. The values of SPI and ANI were significantly different in conscious patients without (NRS = 0) and with pain (NRS > 0). The areas under the receiver operating curves for SPI and ANI were 0.73 (P < 0.0001) and 0.67 (P < 0.0001), respectively. The cut-off values for SPI and ANI in predicting postoperative pain were 44 (sensitivity: 84%, specificity: 53%) and 63 (sensitivity: 52%, specificity: 82%), respectively, which are different from those suggested by their respective manufacturers for use in intraoperative state under general anaesthesia. The cut-off values of SPI and ANI for detecting pain were similar regardless of the type of anesthesia.

Population-based volume kinetics of Ringer's lactate solution in patients undergoing open gastrectomy.

In order to maintain stable blood pressure and heart rate during surgery, anesthesiologists need to administer the appropriate amount of fluid with appropriate fluid type to the patient, then quantifying how fluid is distributed and eliminated from the body is useful for establishing a fluid administration strategy. In this study we characterized the volume kinetics of Ringer's lactate solution in patients undergoing open gastrectomy. When propofol and remifentanil reached a pseudosteady state at the target concentration and blood pressure was stabilized following surgical stimulation, enrolled patients were administered 1000 mL of Ringer's lactate solution for 20 min, followed by continuous infusion at a rate of 6 mL/kg/h until the time of the last blood collection for volume kinetic analysis. Arterial blood samples were collected to measure the hemoglobin concentration at different time points. The change in hemoglobin-derived plasma dilution induced by the administration of Ringer's lactate solution was evaluated by nonlinear mixed effects modeling. Three hundred and twenty-three plasma dilution data points from 27 patients were used to determine the pharmacokinetic characteristics of Ringer's lactate solution. A two-volume model best described the pharmacokinetics of Ringer's lactate solution. The mean arterial pressure (MAP) and body weight (WT) were significant covariates for the elimination clearance (kr) and central volume of distribution at baseline (Vc0), respectively. The parameter estimates were as follows: kr (mL/min) = 124 + (MAP/70)14.2, Vc0 (mL) = 0.95 + 3440 × (WT/63), Vt0 (mL) = 2730, and kt (mL/min) = 181. A higher MAP was associated with a greater elimination clearance and, consequently, less water accumulation in the interstitium. As body weight increases, volume expansion in the blood vessels increases.

Predictive performance of a new pharmacokinetic model for propofol in underweight patients during target-controlled infusion.

BACKGROUND: In a previous study, the modified Marsh and Schnider models respectively showed negatively- and positively-biased predictions in underweight patients. To overcome this drawback, we developed a new pharmacokinetic propofol model-the Choi model-for use in underweight patients. In the present study, we evaluated the predictive performance of the Choi model. METHODS: Twenty underweight patients undergoing elective surgery received propofol via TCI using the Choi model. The target effect-site concentrations (Ces) of propofol were 2.5, 3, 3.5, 4, 4.5, and 2 µg/mL. Arterial blood samples were obtained at least 10 minutes after achieving pseudo-steady-state. Predicted propofol concentrations with the modified Marsh, Schnider, and Eleveld pharmacokinetic models were obtained by simulation (Asan pump, version 2.1.3; Bionet Co. Ltd., Seoul, Korea). The predictive performance of each model was assessed by calculation of four parameters: inaccuracy, divergence, bias, and wobble. RESULTS: A total of 119 plasma samples were used to determine the predictive performance of the Choi model. Our evaluation showed that the pooled median (95% CI) bias and inaccuracy were 4.0 (-4.2 to 12.2) and 23.9 (17.6-30.3), respectively. The pooled biases and inaccuracies of the modified Marsh, Schnider, and Eleveld models were clinically acceptable. However, the modified Marsh and Eleveld models consistently produced negatively biased predictions in underweight patients. In particular, the Schnider model showed greater inaccuracy at a target Ce ≥ 3 µg/mL. CONCLUSION: The new propofol pharmacokinetic model (the Choi model) developed for underweight patient showed adequate performance for clinical use.

Effects of depth of neuromuscular block on postoperative pain during laparoscopic gastrectomy: A randomised controlled trial.

BACKGROUND: Evidence on whether the use of deep neuromuscular block (NMB) influences postoperative pain after laparoscopic surgery is limited, and existing studies have shown conflicting results. We studied the effect of the depth of NMB during laparoscopic gastrectomy on postoperative pain. OBJECTIVE: The aim of this study was to evaluate the effect of depth of NMB during laparoscopic gastrectomy on postoperative pain by allocating patients randomly to either deep or moderate NMB with a standard-pressure pneumoperitoneum. DESIGN: A randomised, controlled, double-blind study. SETTING: A university-affiliated hospital. PARTICIPANTS: One hundred patients. INTERVENTIONS: Patients were allocated randomly to receive either deep (posttetanic count 1 to 2) or moderate (train-of-four count 1 to 2) levels of NMB. Following surgery, the patients were asked to rate their pain every 10 min using a visual analogue scale (VAS) (0 = no pain, 10 = most severe pain) in the postanaesthesia care unit (PACU). Patients received intravenous oxycodone, 2 mg every 10 min, until the pain intensity (VAS) had decreased to less than 3 at rest and less than 5 on wound compression, at which point the minimum effective analgesia dose (MEAD) of oxycodone was determined. MAIN OUTCOME MEASURES: The primary endpoint was the MEAD of oxycodone. Secondary endpoints included area under the curve of VAS for wound pain, VAS scores for wound and shoulder pain at 6 and 24 h after the end of surgery, rescue analgesics, a five-point surgical rating scale, Rhodes index of nausea vomiting retching at 6 and 24 h after the end of surgery and duration of pneumoperitoneum. RESULTS: The median value for the MEAD of oxycodone was 8 mg in both groups. Area under the curves of VAS over time were similar in both groups. Variables associated with postoperative pain including mean VAS at PACU and frequency of rescue analgesics in the ward did not differ significantly between the two groups. The duration of pneumoperitoneum was a significant variable in determining the MEAD of oxycodone (linear regression, R = 0.07, P = 0.008). The number of patients who reached the acceptable surgical score was not significantly different between the two groups. However, the moderate NMB group did have a significantly higher proportion of cases that required additional muscle relaxants (P < 0.001). CONCLUSION: Deep, compared with moderate, NMB did not significantly reduce the MEAD of oxycodone administered in the PACU. The duration of pneumoperitoneum was positively correlated with the MEAD. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03266419.

Population pharmacokinetics and analgesic potency of oxycodone.

AIMS: This prospective study aimed to characterize the population pharmacokinetics of intravenous oxycodone and to determine the minimum effective concentration (MEC) and minimum effective analgesic concentration (MEAC) of oxycodone for major open intra-abdominal surgery. METHODS: In the pharmacokinetic study, patients were administered intravenous oxycodone (0.1 mg kg-1 ), and arterial blood was sampled at pre-set intervals. In the analgesic-potency study, patients were administered intravenous oxycodone (0.1 mg kg-1 ) 30 min before the end of the surgery, were placed in the postoperative anaesthesia care unit (PACU), and were asked to rate their pain every 10 min using a visual analogue scale (0 = no pain, 10 = most severe pain). On the first occasion that wound pain at rest and during compression was rated as ≥3 or ≥5, respectively, the first blood sample was obtained to determine the MEC. A second blood sample was obtained after titration with 2 mg of oxycodone to yield wound pain <3 at rest and <5 during wound compression, and MEAC was determined. MEC and MEAC were determined again in each patient. RESULTS: In the population pharmacokinetic study (n = 54), oxycodone plasma concentration over time was well described by a three-compartment mammillary model. Lean body mass and age were significant covariates for the volume of distribution and metabolic clearance of the pharmacokinetic model of oxycodone, respectively. The analgesic-potency study (n = 50) showed that the median (95% CI) MEC and MEAC were 31.5 (19.2-42.8) and 74.1 (29.2-128.3) ng ml-1 (first measurements) and 63.4 (15.6-120.1) and 76.1 (32.9-132.7) ng ml-1 (second measurements), respectively. CONCLUSIONS: In major intra-abdominal open surgery, the MEAC and analgesic potency of oxycodone were 75 ng ml-1 and 60 ng ml-1 , respectively.

Response surface modelling of the pharmacodynamic interaction between propofol and remifentanil in patients undergoing anaesthesia.

This study describes the pharmacodynamic interaction between propofol and remifentanil. Sixty patients who were scheduled for elective surgery under general anaesthesia (30 males/30 females) were enrolled. Patients were randomly allocated to receive one of 15 combinations of drug levels. Baseline electroencephalograms (EEGs) were recorded for 5 minutes prior to administering the drugs. Patients received a target-controlled infusion at one of four predefined doses of propofol (high, 3 µg/mL; medium, 1.5 µg/mL; low, 0.5 µg/mL; or no drug) and of remifentanil (high, 6 or 8 ng/mL; medium, 4 ng/mL; low, 2 ng/mL; or no drug). The occurrence of muscle rigidity, apnoea, and loss of consciousness (LOC) was monitored, and EEGs were recorded during the drug administration phase. Electroencephalographic approximate entropy (ApEn) and temporal linear mode complexity (TLMC) parameters at baseline and under steady state conditions were calculated off-line. Response surfaces were developed to map the interaction between propofol and remifentanil to the probability of occurrence for quantal responses (muscle rigidity, apnoea, LOC) and ApEn and TLMC measurements. Model parameters were estimated using non-linear mixed effects modelling. The response surface revealed infra-additive and synergistic effects for muscle rigidity and apnoea, respectively. The effects of the combined drugs on LOC and EEG parameters (eg, ApEn and TLMC) were additive. The C50 estimates of remifentanil (ng/mL) and propofol (µg/mL) were 9.11 and 130 000 for muscle rigidity, 8.99 and 6.26 for apnoea, 13.9 and 3.04 for LOC, 23.4 and 10.4 for ApEn, and 14.8 and 6.51 for TLMC, respectively. The probability of occurrence for muscle rigidity declined when propofol was combined with remifentanil.

Population pharmacokinetics and prophylactic anti-emetic efficacy of ramosetron in surgical patients.

AIMS: This study characterized the pharmacokinetics of ramosetron and compared prophylactic anti-emetic efficacy with that of ondansetron in a large population. METHODS: Fifty-eight patients consented to the pharmacokinetic analysis and were assigned randomly to receive 0.3, 0.45 or 0.6 mg ramosetron after induction of anaesthesia. Blood samples were acquired at preset intervals. Non-compartmental and population pharmacokinetic analyses were performed. In total, 1102 patients consented to the evaluation of prophylactic anti-emetic efficacy and were allocated randomly to receive 0.3 mg ramosetron or 4 mg ondansetron at the end of surgery. An additional 16 mg ondansetron were mixed in the intravenous patient-controlled analgesia pump of the ondansetron group. Post-operative nausea and vomiting (PONV) were evaluated 6, 24 and 48 h post-operatively using the Rhodes index of nausea, vomiting and retching (RINVR). Administration of rescue anti-emetics and adverse events were evaluated. RESULTS: The pharmacokinetic parameter estimates were V1 (l) = 5.12, V2 (l) = 108, CL (l⋅min(-1) ) = 0.08 + (59⋅age(-1) ) × 0.09, Q (l⋅min(-1) ) = 1.42. The incidences of PONV in the ramosetron and ondansetron groups were 77 (13.9%) and 113 (20.6%) and 44 (7.9%) and 66 (12.0%) at 24 and 48 h post-operatively, respectively (P = 0.004, 0.030). RINVR was significantly lower in the ramosetron than the ondansetron group 24 and 48 h post-operatively (P = 0.003, 0.025). Use of rescue anti-emetics and incidence of adverse events were comparable. CONCLUSIONS: A two compartment mammillary model was used to describe ramosetron pharmacokinetics. Prophylactic anti-emetic efficacy of ramosetron was significantly better 24 and 48 h post-operatively than that of ondansetron, particularly when the Apfel score was ≥ 3.

Comparison of Needle Insertion and Guidewire Placement Techniques During Internal Jugular Vein Catheterization: The Thin-Wall Introducer Needle Technique Versus the Cannula-Over-Needle Technique.

OBJECTIVES: For needle insertion and guidewire placement during central venous catheterization, a thin-wall introducer needle technique and a cannula-over-needle technique have been used. This study compared these two techniques regarding the success rates and complications during internal jugular vein catheterization. DESIGN: Prospective, randomized, controlled study. SETTING: A university-affiliated hospital. PATIENTS: Two hundred sixty-six patients scheduled for thoracic surgery, gynecologic surgery, or major abdominal surgery, who required central venous catheterization. INTERVENTIONS: Patients were randomly assigned to either the thin-wall introducer needle group (n = 134) or the cannula-over-needle group (n = 132). Central venous catheterization was performed on the right internal jugular vein under assistance with real-time ultrasonography. Needle insertion and guidewire placement were performed using a thin-wall introducer needle technique in the thin-wall introducer needle group and a cannula-over-needle technique in the cannula-over-needle group. MEASUREMENTS AND MAIN RESULTS: The guidewire placement on the first skin puncture was regarded as a successful guidewire insertion on the first attempt. The number of puncture attempts for internal jugular vein catheterization was recorded. Internal jugular vein was assessed by ultrasonography to identify complications. The rate of successful guidewire insertion on the first attempt was higher in the thin-wall introducer needle group compared with the cannula-over-needle group (87.3% vs 77.3%; p = 0.037). There were fewer puncture attempts in the thin-wall introducer needle group than in the cannula-over-needle group (1.1 ± 0.4 vs 1.3 ± 0.6; p = 0.026). There was no significant difference in complications of internal jugular vein catheterization between the two groups. CONCLUSIONS: The thin-wall introducer needle technique showed a superior success rate for first attempt of needle and guidewire insertion and required fewer puncture attempts during internal jugular vein catheterization.

Novel bone morphogenetic protein signaling through Smad2 and Smad3 to regulate cancer progression and development.

The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and cellular homeostasis, with aberrant BMP signaling resulting in a broad spectrum of human disease. We report that BMPs unexpectedly signal through the canonical transforming growth factor ß (TGF-ß)-responsive Smad2 and Smad3. BMP-induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP-binding TGF-ß superfamily receptors, activin receptor-like kinase (ALK)3/6, and the Smad2/3 phosphorylating receptors ALK5/7. BMP signaling through Smad2 mediates, in part, dorsoventral axis patterning in zebrafish embryos, whereas BMP signaling through Smad3 facilitates cancer cell invasion. Consistent with increased BMP-mediated Smad2/3 signaling during cancer progression, Smad1/5 and Smad 2/3 signaling converge in human cancer specimens. Thus, the signaling mechanisms used by BMPs and TGF-ß superfamily receptors are broader than previously appreciated.

Comparison of the endotracheal tube cuff pressure between a tapered- versus a cylindrical-shaped cuff after changing from the supine to the lateral flank position.

PURPOSE: Positional change can displace an endotracheal tube (ETT) and change the ETT cuff pressure in a tracheally intubated patient. Endotracheal tubes with different cuff shapes may lead to different cuff pressures after positional change. We hypothesized that the intracuff pressure in the TaperGuard™ ETT with a tapered-shaped cuff would be higher than that in the conventional ETT with a cylindrical-shaped cuff after a change from the supine to the lateral flank position. METHODS: Fifty-eight patients scheduled for open urological procedures in the lateral flank position were randomly allocated to receive either a TaperGuard ETT (group T) or conventional ETT (group C). The ETT cuff pressure was initially set at 20 cm H2O in the supine position and was measured after the change to the lateral flank position. The distance from the ETT tip to the carina was measured in both the supine and the lateral flank positions. RESULTS: Two patients, one from each group, were excluded from the data analysis. The mean (SD) ETT cuff pressure was significantly higher in group T (n = 28) than in group C (n = 28) after the change in position [31 (7) cm H2O vs 25 (4) cm H2O, respectively; mean difference, 6 cm; 95% confidence intervals [CI], 3 to 9; P < 0.001]. The mean (SD) proximal migration of the ETT tip was comparable between the two groups [8 (18) mm vs 4 (14) mm, respectively; P = 0.367]. CONCLUSIONS: After the change from the supine to the lateral flank position, the ETT cuff pressure was significantly higher in the TaperGuard ETT than in the conventional ETT, although the extent of cephalad displacement of the ETT was comparable between the two groups. This trial was registered at Clinicaltrials.gov: NCT02165319.

Increased tracheal cuff pressure during insertion of a transoesophageal echocardiography probe: A prospective, observational study.

BACKGROUND: Excessive tracheal cuff pressure reduces tracheal mucosal blood flow and increases tracheal morbidity. Inserting a transoesophageal echocardiography (TOE) probe has been shown to increase tracheal cuff pressure. OBJECTIVE: To evaluate the effect of inserting a TOE probe on tracheal cuff pressure and compare the effect in patients who received a single-lumen endotracheal tube (SLT) with those who received a double-lumen endotracheal tube (DLT). DESIGN: A prospective, observational study. SETTING: Single-centre trial, study period from October 2013 to January 2014. PATIENTS: Forty-four adult patients scheduled for elective cardiothoracic surgery requiring intraoperative TOE monitoring. INTERVENTIONS: After tracheal intubation with a SLT (n = 22) or DLT (n = 22), the tracheal cuff was inflated to 18 mmHg (25 cmH2O) with air. Tracheal cuff pressure was monitored continuously for 5 min after inserting the TOE probe. MAIN OUTCOME MEASURES: The primary endpoint was steady-state tracheal cuff pressure after insertion of the TOE probe. RESULTS: Median (interquartile range, IQR) tracheal cuff pressure stabilised at 3 (2 to 3) min in the SLT group and at 2 (1 to 3) min in the DLT group. Steady-state cuff pressure was significantly higher in the DLT group than that in the SLT group [36.7 (31.3 to 44.1) vs. 31.3 (29.6 to 35.7) cmH2O; (P = 0.03)]. Steady-state cuff pressure more than 40 cmH2O was observed in two patients (18.2%) in the SLT group and nine patients (40.9%) in the DLT group (P = 0.02). CONCLUSION: Insertion of a TOE probe increased tracheal cuff pressure in both the SLT and DLT groups. The increase in cuff pressure was greater in patients who received a DLT. Frequent measurement and adjustment of cuff pressure should be emphasised particularly when TOE is used in patients receiving a DLT. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02034643.

The effect of intraoperative dexmedetomidine on postoperative catheter-related bladder discomfort in patients undergoing transurethral bladder tumour resection: A double-blind randomised study.

BACKGROUND: Catheter-related bladder discomfort (CRBD) due to an indwelling urinary catheter causes postoperative distress. Dexmedetomidine is used as an anaesthetic adjuvant during general anaesthesia and has an antimuscarinic effect, which may be beneficial for the prevention and treatment of CRBD. OBJECTIVE: To determine the effect of intraoperative dexmedetomidine administration on the incidence of CRBD. DESIGN: A double-blind, placebo-controlled, randomised study. SETTING: A tertiary care teaching hospital. PATIENTS: One-hundred and nine patients undergoing transurethral bladder tumour excision (TURB). INTERVENTIONS: Patients were randomly allocated to two groups: control group (n = 55) received placebo and dexmedetomidine group (n = 54) received intraoperative dexmedetomidine (1 µg kg(-1) loading dose followed by 0.5 µg kg(-1) h(-1) continuous infusion). MAIN OUTCOME MEASURES: The incidence and severity (mild, moderate, severe) of CRBD assessed at 0, 1, 6 and 24 h postoperatively. RESULTS: The incidence of CRBD was significantly higher in the control group at 0 (78 vs. 50%; P = 0.004), 1 (86 vs. 57%; P = 0.002) and 6 h (82 vs. 63%; P = 0.047) postoperatively. The incidence of moderate to severe CRBD was higher in the control group at 0 (38 vs. 11%; P = 0.002) and 1 h (29 vs. 7%; P = 0.006) postoperatively. The number of patients having CRBD treated with tramadol was higher in the control group (24 vs. 12; P = 0.006). The mean end-tidal desflurane concentration during the surgery was higher in the control group (4.5 vs. 3.9%; P = 0.04). The postoperative pain score (numerical rating scale: 0 to 10) was higher in the control group at 0 (4.6 vs. 2.7; P = 0.002), and 1 h (3.8 vs. 2.7; P = 0.041). The number of patients treated with opioids was higher in the control group (21 vs. 8; P = 0.011). CONCLUSION: Intraoperative dexmedetomidine administration decreased the incidence and severity of early postoperative CRBD as well as intraoperative desflurane and postoperative opioid requirements in patients undergoing TURB. TRIAL REGISTRATION IDENTIFIER: NCT01991223 (www.clinicaltrials.gov).

Efficacy of a newly developed guidewire for selective biliary access.

A clinical efficacy study of 0.025-inch guidewires (GWs) according to mechanical property analysis has not been reported yet. This study was designed to evaluate the clinical efficacy of a newly developed 0.025-inch GW for biliary access according to the basic mechanical property. Commercially available 0.025-inch GWs were in vitro tested based on parameters of mechanical property. Patients with naïve papilla requiring diagnostic or therapeutic ERCP were randomly assigned to an experimental 0.025-inch newly developed GW or a control 0.025-inch GW group. Technical success rate of wire-guided cannulation (WGC), difficult biliary cannulation (DBC), and adverse event rates were measured in this multicenter randomized trial. The technical success rate of primary WGC was 79.1% (151 of 191) in the experimental group and 70.8% (131 of 185) in the control group (95% two-sided confidence interval: 8.25%; p < 0.001; for a noninferiority margin of 15%). The technical success rate including cross-over to each other was also non-inferior. However, the chi-square test showed a statistical difference (81.7% vs. 68.1%; p = 0.002). Median biliary cannulation time was shorter in the experimental group (53 s vs. 77 s; p = 0.047). The rate of DBC was more frequent in the control group (34.6% vs. 50.3% p = 0.002). Multivariate analysis revealed that control group was one of contributing factors for DBC. Overall rate of post-ERCP pancreatitis was not different (4.7% vs. 8.6%; p = 0.125). WGC using a newly developed GW with superior physical performance GW in a bench test showed similar clinical efficacy and the rate of DBC was significantly lower in experimental GW.

Comprehensive Analysis of Epstein-Barr Virus LMP2A-Specific CD8+ and CD4+ T Cell Responses Restricted to Each HLA Class I and II Allotype Within an Individual.

Latent membrane protein 2A (LMP2A), a latent Ag commonly expressed in Epstein-Barr virus (EBV)-infected host cells, is a target for adoptive T cell therapy in EBV-associated malignancies. To define whether individual human leukocyte antigen (HLA) allotypes are used preferentially in EBV-specific T lymphocyte responses, LMP2A-specific CD8+ and CD4+ T cell responses in 50 healthy donors were analyzed by ELISPOT assay using artificial Ag-presenting cells expressing a single allotype. CD8+ T cell responses were significantly higher than CD4+ T cell responses. CD8+ T cell responses were ranked from highest to lowest in the order HLA-A, HLA-B, and HLA-C loci, and CD4+ T cell responses were ranked in the order HLA-DR, HLA-DP, and HLA-DQ loci. Among the 32 HLA class I and 56 HLA class II allotypes, 6 HLA-A, 7 HLA-B, 5 HLA-C, 10 HLA-DR, 2 HLA-DQ, and 2 HLA-DP allotypes showed T cell responses higher than 50 spot-forming cells (SFCs)/5×105 CD8+ or CD4+ T cells. Twenty-nine donors (58%) showed a high T cell response to at least one allotype of HLA class I or class II, and 4 donors (8%) had a high response to both HLA class I and class II allotypes. Interestingly, we observed an inverse correlation between the proportion of LMP2A-specific T cell responses and the frequency of HLA class I and II allotypes. These data demonstrate the allele dominance of LMP2A-specific T cell responses among HLA allotypes and their intra-individual dominance in response to only a few allotypes in an individual, which may provide useful information for genetic, pathogenic, and immunotherapeutic approaches to EBV-associated diseases.

Fibromodulin suppresses nuclear factor-kappaB activity by inducing the delayed degradation of IKBA via a JNK-dependent pathway coupled to fibroblast apoptosis.

Fibulin-5 (FBLN5) belongs to the Fibulin family of secreted extracellular matrix proteins, and our laboratory first established FBLN5 as a novel target for TGF-ß in fibroblasts and endothelial cells. To better understand the pathophysiology of FBLN5, we carried out microarray analysis to identify fibroblast genes whose expressions were regulated by FBLN5 and TGF-ß. In doing so, we identified fibromodulin (Fmod) as a novel target gene of FBLN5, and we validated the differential expression of Fmod and 12 other FBLN5-regulated genes by semi-quantitative real time PCR. Fmod belongs to the small leucine-rich family of proteoglycans, which are important constituents of mammalian extracellular matrices. Interestingly, parental 3T3-L1 fibroblasts displayed high levels of nuclear factor-κB (NF-κB) activity, although those engineered to express Fmod constitutively exhibited significantly reduced NF-κB activity, suggesting that Fmod functions to inhibit NF-κB signaling. By monitoring alterations in the activation of NF-κB and the degradation of its inhibitor, IκBα, we demonstrate for the first time that Fmod contributes to the constitutive degradation of IκBα protein in 3T3-L1 fibroblasts. Mechanistically, we observed Fmod to delay the degradation of IκBα by promoting the following: (i) activation of c-Jun N-terminal kinase; (ii) inhibition of calpain and casein kinase 2 activity; and (iii) induction of fibroblast apoptosis. Taken together, our study identified a novel function for Fmod in directing extracellular signaling, particularly the regulation of NF-κB activity and cell survival.

Multiple regulatory roles of the carboxy terminus of Ste2p a yeast GPCR.

Signaling and internalization of Ste2p, a model G protein-coupled receptor (GPCR) from the yeast Saccharomyces cerevisiae, are reported to be regulated by phosphorylation status of serine (S) and threonine (T) residues located in the cytoplasmic C-terminus. Although the functional roles of S/T residues located in certain C-terminus regions are relatively well characterized, systemic analyses have not been conducted for all the S/T residues that are spread throughout the C-terminus. A point mutation to alanine was introduced into the S/T residues located within three intracellular loops and the C-terminus individually or in combination. A series of functional assays such as internalization, FUS1-lacZ induction, and growth arrest were conducted in comparison between WT- and mutant Ste2p. The Ste2p in which all S/T residues in the C-terminus were mutated to alanine was more sensitive to α-factor, suggesting that phosphorylation in the C-terminus exerts negative regulatory activities on the Ste2p signaling. C-terminal S/T residues proximal to the seventh transmembrane domain were important for ligand-induced G protein coupling but not for receptor internalization. Sites on the central region of the C-terminus regulated both constitutive and ligand-induced internalization. Residues on the distal part were important for constitutive desensitization and modulated the G protein signaling mediated through the proximal part of the C-terminus. This study demonstrated that the C-terminus contains multiple functional domains with differential and interdependent roles in regulating Ste2p function in which the S/T residues located in each domain play critical roles.

Comprehensive analysis of mycobacterium tuberculosis antigen-specific CD4+ T cell responses restricted by single HLA class II allotype in an individual.

Mycobacterium tuberculosis infection is generally asymptomatic as latent tuberculosis, but it is still known as the world's leading bacterial cause of death. The diagnosis of latent tuberculosis infection relies on the evidence of cellular immunity to mycobacterial antigens. Since the association between HLA class II and tuberculosis infection has been reported in several population groups, a detailed study on the CD4+ T cell response to major tuberculosis antigens is needed. To elucidate which HLA class II allotypes in an individual are preferentially used in tuberculosis, CD4+ T cells specific to TB10.4, Ag85b, ESAT-6, and CFP-10 of Mycobacterium tuberculosis antigens were analyzed comprehensively. A total of 33 healthy donors were analyzed by ex vivo and cultured ELISPOT using panels of artificial antigen-presenting cells expressing a single HLA class II allotype. The CD4+ T cell responses were increased by an average of 39-fold in cultured ELISPOT compared with ex vivo ELISPOT. In ex vivo and cultured ELISPOT, CD4+ T cell responses showed significantly higher by HLA-DR than those of HLA-DQ and HLA-DP locus. In cultured ELISPOT, 9 HLA-DR allotypes, 4 HLA-DQ allotypes, and 3 HLA-DP allotypes showed positive CD4+ T cell responses. Among ten donors with positive CD4+ T cell responses when tested for mixed Mycobacterium tuberculosis antigens, seven donors were positive for only a single allotype, and three were positive for two allotypes in an individual. However, only one allotype was used for a single antigen-specific response when a single tuberculosis antigen was used individually. These results on the distribution of HLA class II allotypes showing high CD4+ T-cell responses to Mycobacterium tuberculosis antigens and the intra-individual allotype dominance will provide valuable information for understanding the immunobiology and immunogenetics of tuberculosis, which can contribute to the development of more effective vaccines.

Role of TGF-ß and the tumor microenvironment during mammary tumorigenesis.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that functions to inhibit mammary tumorigenesis by directly inducing mammary epithelial cells (MECs) to undergo cell cycle arrest or apoptosis, and to secrete a variety of cytokines, growth factors, and extracellular matrix proteins that maintain cell and tissue homeostasis. Genetic and epigenetic events that transpire during mammary tumorigenesis typically inactivate the tumor suppressing activities of TGF-beta and ultimately confer this cytokine with tumor promoting activities, including the ability to stimulate breast cancer invasion, metastasis, angiogenesis, and evasion from the immune system. This dramatic conversion in TGF-beta function is known as the "TGF-beta paradox" and reflects a variety of dynamic alterations that occur not only within the developing mammary carcinoma, but also within the cellular and structural composition of its accompanying tumor microenvironment. Recent studies have begun to elucidate the critical importance of mammary tumor microenvironments in manifesting the TGF-beta paradox and influencing the response of developing mammary carcinomas to TGF-beta. Here we highlight recent findings demonstrating the essential function of tumor microenvironments in regulating the oncogenic activities of TGF-beta and its stimulation of metastatic progression during mammary tumorigenesis.

Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II.

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4+ T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016-2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance.