Kinetics of serum O-glycosylated M-hepatitis B surface antigen with hepatocellular carcinoma history and nucleos(t)ide analogue therapy in hepatitis B patients.

A newly developed O-glycosylated M-hepatitis B surface antigen (HBsAgGi) measurement system can detect hepatitis B surface antigen (HBsAg) associated with infectious particles. We investigated the association of HBsAgGi levels with clinical parameters and a history of hepatocellular carcinoma (HCC) development in a cross-sectional cohort analysis (Study 1) as well as the quantitative changes in HBsAgGi during nucleos(t)ide analogue (NA) therapy in a longitudinal cohort analysis (Study 2). A total of 124 patients with genotype C chronic HBV infection were analysed in Study 1 to evaluate correlations of HBsAgGi with conventional HBV markers and HCC history. Among those, 36 patients receiving NA therapy were enrolled in Study 2 for quantitative comparisons between pre-treatment baseline and 48 weeks of NA therapy. In Study 1, serum HBsAgGi was significantly associated with HBsAg (r = .5857, p < .00001) and weakly but significantly correlated with HBV DNA (r = .2936, p = .001). Although HBsAgGi (p = .111) was comparable between HCC history (+) group and HCC history (-) group, the HBsAgGi/HBsAg ratio (p = .011) was significantly higher in HCC history (+) patients. In Study 2, HBsAgGi was significantly decreased after 48 weeks of NA therapy (p < .001). HBsAg findings were similar (p = .005) along with an HBV DNA reduction (p < .001). In the baseline hepatitis B e antigen (HBeAg) (+) subgroup, HBsAgGi decreased significantly between baseline and 48 weeks of NA (p = .005), while HBsAg was comparable (p = .051). Low HBsAg and high HBsAgGi were associated with a history of HCC development. HBsAgGi decreased significantly by 48-week NA therapy.

2-Step PLT16-AST44 method: Simplified liver fibrosis detection system in patients with non-alcoholic fatty liver disease.

AIM: Accurate detection of the hepatic fibrosis stage is essential to estimate the outcome of patients with non-alcoholic fatty liver disease (NAFLD). Many formulas, biomarkers, and imaging tests are being developed to predict advanced liver fibrosis without performing a liver biopsy. However, these tests do not have high efficiency in detecting early-stage hepatic fibrosis. Therefore, we aimed to detect the presence of hepatic fibrosis (≥F1) merely by using only standard clinical markers. METHODS: A total of 436 patients with NAFLD who underwent liver biopsy were retrospectively enrolled as the discovery cohort (316 patients) and the validation cohort (120 patients). Liver biopsy and laboratory data were matched to extract simple parameters for identifying ≥F1. RESULTS: We developed a novel simplified ≥F1 detecting system, designated as 2-Step PLT16-AST44 method, where (1) PLT of 16 × 104 /µl or less, or (2) PLT greater than 16 × 104 /µl and AST greater than 44 U/L is determined as having ≥F1 fibrosis. The 2-Step PLT16-AST44 method had a sensitivity of 68%, a specificity of 90%, a positive predictive value (PPV) of 97%, a negative predictive value (NPV) of 40%, and an accuracy of 72% to detect ≥F1 fibrosis in the discovery cohort. Validation studies further supported these results. Despite its simplicity, the 2-Step PLT16-AST44 method's power to detect ≥F1 fibrosis in total NAFLD patients was comparable to hyaluronic acid, type 4 collagen 7S, FIB-4, and APRI. CONCLUSIONS: We propose the 2-Step PLT16-AST44 method as a simple and beneficial early-stage hepatic fibrosis detection system.

Quantitative and qualitative lipid improvement with chronic hepatitis C virus eradication using direct-acting antivirals.

AIM: Direct-acting antivirals have revolutionized hepatitis C virus (HCV) therapy by providing a high sustained virological response (SVR) rate and subsequent favorable lipid increases. Proprotein convertase subtilisin-kexin like-9 (PCSK9) plays an important role in regulating quantitative lipid levels. This study examined the interactions between quantitative PCSK9 and lipid changes, as well as qualitative lipid changes in terms of lectin-like oxidized low-density lipoprotein (LDL) receptor-1 ligand containing apolipoprotein B (LAB) and high-density lipoprotein (HDL) cholesterol uptake capacity (HDL-CUC). METHODS: Patients with chronic HCV infection (N = 231) who achieved an SVR by direct-acting antivirals without lipid-lowering therapy were included for comparisons of PCSK9, LAB, HDL-CUC, and other clinical indices between pretreatment and SVR12 time points. RESULTS: LDL (LDL) cholesterol and HDL cholesterol levels were quantitatively increased at SVR12, along with higher PCSK9 (all p < 0.0001). PCSK9 was significantly correlated with LDL cholesterol (r = 0.244, p = 0.0003) and apolipoprotein B (r = 0.222, p = 0.0009) at SVR12. Regarding qualitative LDL changes, LAB was significantly decreased and LAB/LDL cholesterol and LAB/apolipoprotein B proportions were improved at SVR12 (all p < 0.0001). In terms of qualitative HDL changes, HDL-CUC was significantly ameliorated, along with HDL-CUC/HDL cholesterol, HDL-CUC/ apolipoprotein A1, and HDL-CUC/ apolipoprotein A2 at SVR12 (all p < 0.0001). CONCLUSIONS: HCV eradication by direct-acting antivirals may produce quantitative lipid profile changes, along with PCSK9 production recovery in addition to qualitative lipid improvement, which possibly confers the additional secondary benefits of atherosclerosis improvement and cardiovascular disease event reduction.

The ursodeoxycholic acid response score predicts pathological features in primary biliary cholangitis.

AIM: The ursodeoxycholic acid response score (URS) can predict the biochemical response to 12 months of ursodeoxycholic acid (UDCA) treatment in patients with primary biliary cholangitis (PBC). We investigated the relationship between the URS and the histopathological features before and after UDCA treatment. METHODS: Patients with PBC (n = 126) were examined for the association between the probability of response (POR) to UDCA based on the URS formulas and clinicopathological features. Furthermore, 30 patients were examined for the association between the POR and pathological changes. RESULTS: The POR area under the receiver operating characteristic curve (AUROC) for predicting the biochemical response to UDCA was 0.861. The PORs of stage 1 in the Nakanuma system and grade 0 in the CK7 grading in hepatocytes were significantly higher than those of stage 3 and grade 3, respectively. The AUROCs for the prediction of stage ≥2, stage ≥3 and stage 4 in the Nakanuma system at pretreatment were 0.592, 0.710 and 0.817, respectively. The AUROCs for the prediction of grade ≥1, grade ≥2 and grade 3 in the CK7 hepatocyte grading were 0.741, 0.824 and 0.970, respectively. Furthermore, the AUROC for predicting the histological stage progression after UDCA treatment in the Scheuer classification and the Nakanuma system were 0.712 and 0.799, respectively. CONCLUSIONS: The URS not only predicts the biochemical response, but also reflects the Nakanuma system and the CK7 hepatocyte grading at pretreatment. This scoring system can identify an inadequate histological response to UDCA treatment in the Scheuer classification and the Nakanuma system.

aMAP score prediction of hepatocellular carcinoma occurrence and incidence-free rate after a sustained virologic response in chronic hepatitis C.

AIMS: Hepatocellular carcinoma (HCC) can still occur in hepatitis C virus (HCV) patients who have achieved a sustained virologic response (SVR), which remains an important clinical issue in the direct-acting antivirals era. The current study investigated the clinical utility of the aMAP score (consisting of age, male, albumin-bilirubin, and platelets) for predicting HCC occurrence in HCV patients achieving an SVR by direct-acting antivirals. METHODS: A total of 1113 HCV patients without HCC history, all of whom achieved an SVR, were enrolled for clinical comparisons. RESULTS: Hepatocellular carcinoma was recorded in 50 patients during a median follow-up period of 3.7 years. The aMAP score was significantly higher in the HCC occurrence group than in the HCC-free group (53 vs. 47, p < 0.001). According to risk stratification based on aMAP score, the cumulative incidence of HCC occurrence for the low-, medium-, and high-risk groups was 0.14%, 4.49%, and 9.89%, respectively, at 1 year and 1.56%, 6.87%, and 16.17%, respectively, at 3 years (low vs. medium, low vs. high, and medium vs. high: all p < 0.01). Cox proportional hazard analysis confirmed aMAP ≥ 50 (hazard ratio [HR]: 2.78, p = 0.014), age≥ 70 years (HR: 2.41, p = 0.028), ALT ≥ 17 U/L (HR: 2.14, p < 0.001), and AFP ≥ 10 ng/mL (HR: 2.89, p = 0.005) as independent risk factors of HCC occurrence. Interestingly, all but one patient (99.5%) with aMAP less than 40 was HCC-free following an SVR. CONCLUSION: The aMAP score could have clinical utility for predicting HCC occurrence in HCV patients achieving an SVR.

Successful treatment by glecaprevir/pibrentasvir followed by hepatoprotective therapy of acute chronic hepatitis exacerbation caused by daratumumab-based regimen for multiple myeloma: Case report and review of the literature.

Hepatitis C virus (HCV) exacerbation is relatively rare as compared with hepatitis B virus reactivation in patients treated with immunosuppressive or anticancer drugs. We herein present the first reported case of acute exacerbation of chronic hepatitis in a patient with HCV persistent infection caused by combination treatment with daratumumab (DARA), bortezomib, and dexamethasone (DVd therapy). A 79-year-old woman diagnosed as having chronic HCV infection 11 years prior without successful viral elimination was referred to our hospital for the treatment of acute liver injury. Multiple myeloma (MM; IgG-κ type) was diagnosed two years before referral and subjected to several treatments. She had commenced DVd therapy four months prior to admission. Since her liver enzymes did not normalize with drug discontinuation and hepatoprotective therapy, we suspected HCV exacerbation and began direct-acting antiviral (DAA) treatment with glecaprevir/pibrentasvir (GLE/PIB). Soon afterwards, her liver enzymes normalized, and she achieved a sustained virological response after 8 weeks of treatment. Clinicians should bear in mind HCV exacerbation when encountering chronic HCV with acute liver injury under MM treatment including a DARA-based regimen. In such cases, DAA therapy is an option when other urgent treatments are needed.

Primary Hepatic Extranodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in a Patient with Chronic Hepatitis B Virus Infection: Case Report and Summary of the Literature.

BACKGROUND: The incidence of extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is low, at 7-8% of all non-Hodgkin lymphoma cases. The most common site of MALT lymphoma occurrence is the stomach. Primary hepatic extranodal marginal zone lymphoma of MALT is classified as a type of non-gastric MALT lymphoma and is considered extremely rare, with no consensus on imaging study findings or treatment due to a limited number of reports. We herein describe a rare case of primary hepatic extranodal marginal zone lymphoma of MALT with underlying hepatitis B infection (HBV) and present useful diagnostic findings of various imaging modalities, including contrast-enhanced ultrasonography (CEUS) with Sonazoid. CASE PRESENTATION: A 66-year-old woman was diagnosed as being a non-active carrier of HBV at 51 years of age at the time of total hysterectomy and bilateral adnexectomy for uterine cervical cancer. She was admitted to our hospital following the incidental detection of two focal liver lesions on computed tomography. The lesions were considered malignant based on clinical and other radiologic imaging findings. Her CEUS results of hypo-enhancement in the portal and late phases were consistent with those of previously reported cases of hepatic extranodal marginal zone lymphoma of MALT, and histological liver biopsy findings were compatible with the diagnosis. CONCLUSIONS: Primary hepatic extranodal marginal zone lymphoma of MALT is a rare condition that can appear in HBV carriers. Characteristic CEUS findings may help in disease diagnosis. Clinicians should bear primary hepatic extranodal marginal zone lymphoma of MALT in mind when encountering patients with focal liver lesions which exhibit image findings different from those of typical hepatocellular carcinoma.

A case report of pancreatic panniculitis due to acute pancreatitis with intraductal papillary mucinous neoplasm.

BACKGROUND: Pancreatic panniculitis is a rare skin manifestation in pancreatic disease patients that most frequently develops on the lower legs. We report the unique case of a 68-year-old man who suffered from pancreatic panniculitis on his trunk associated with acute pancreatitis due to an intraductal papillary mucinous neoplasm. CASE PRESENTATION: A 68-year-old man complained of a 2-day history of a tender subcutaneous nodule on his trunk. Laboratory tests and abdominal contrast computed tomography were consistent with acute pancreatitis due to an intraductal papillary mucinous neoplasm. A skin biopsy of the nodule histologically displayed lobular panniculitis with characteristic "ghost cells", which indicated pancreatic panniculitis. CONCLUSIONS: In order to avoid a missed or delayed diagnosis, clinicians should bear in mind that pancreatic panniculitis can be the first manifestation of pancreatic disease when encountering subcutaneous nodules on the trunk.

Clinical utility of FibroScan as a non-invasive diagnostic test for primary biliary cholangitis.

BACKGROUND AND AIM: Primary biliary cholangitis (PBC) is a chronic, slowly progressive, autoimmune liver disease. Some PBC patients display disease progression regardless of medical treatment. Therefore, it is important to accurately diagnose the clinical stage of PBC. This study investigated clinical merits of vibration-controlled transient elastography using FibroScan for assessing disease stage in PBC. METHODS: A total of 74 treatment-naïve PBC patients (84% female, median age: 64 years), 69 of whom having undergone histological assessment and five clinically diagnosed as at the cirrhosis stage, were enrolled for clinical comparisons of liver stiffness measurement (LSM) with other established indices. RESULTS: The number of patients with Nakanuma stages 1, 2, 3, and 4 was 18, 33, 17, and 6, respectively. The median LSM values for Nakanuma stages 1, 2, 3, and 4 were 5.05, 5.90, 8.90, and 23.70 kPa, respectively, and correlated significantly with disease progression based on Nakanuma's classification (r = 0.501, P < 0.001). LSM was also significantly related to other non-invasive serological markers (Mac-2 binding protein glycosylation isomer: r = 0.606, FIB-4 index: r = 0.493, and aspartate aminotransferase-to-platelet ratio index: r = 0.577; all P < 0.001). The areas under the receiver operating characteristic curve for diagnosing Nakanuma stage ≥ 2, stage ≥ 3, and stage 4 were 0.744, 0.763, and 0.907, respectively. A combination of LSM ≥ 7.0 kPa and Mac-2 binding protein glycosylation isomer ≥ 1.00 cut-off index could predict late-stage PBC (i.e. moderate to advanced disease progression) with a sensitivity of 0.58, specificity of 0.82, and accuracy of 0.74. CONCLUSIONS: Liver stiffness measurement using FibroScan provided simple, accurate, and non-invasive assessment of disease stage in PBC patients.

Quantitative evaluation of atrophic acne scars using 3D image analysis with reflected LED light.

BACKGROUND: Methods for objective evaluation of acne scars have not been established yet. In this study, the capability of three-dimensional image analysis of acne scarring was examined. METHODS: Two dermatologists evaluated the severity and counted the number of atrophic acne scars in a defined evaluation area of each cheek (3.5 cm × 3.5 cm) of 22 subjects (age, 21-38 years). Images of the evaluation area were obtained with an Antera 3D® (Miravex Limited, Ireland) camera three times, and three parameters (affected area, volume, and max depth) were measured. Three different filters (small, medium, and large), which limit measurement targets based on the diameters of concavities, were used for measurement. The relationships between each parameter and the evaluation results of scars by dermatologists were analyzed using Spearman's correlation coefficients. RESULTS: The correlations between the evaluation results of scars by dermatologists and each parameter measured were the highest when the large filter was used. The correlation coefficients between the severity of scars by dermatologists and each of affected area, volume, and max depth were 0.736, 0.728, and 0.722, respectively, and those between scar counts and each of affected area and volume were 0.783 and 0.770, respectively. The correlations, scatter plots, and regression lines among three measurements of parameters suggested high repeatability. CONCLUSIONS: Three-dimensional image analysis has the capability to evaluate changes in the shape of scars before and after treatment quantitatively.

Electron transfer pathways in a multiheme cytochrome MtrF.

In MtrF, an outer-membrane multiheme cytochrome, the 10 heme groups are arranged in heme binding domains II and IV along the pseudo-C2 axis, forming the electron transfer (ET) pathways. Previous reports based on molecular dynamics simulations showed that the redox potential (Em) values for the heme pairs located in symmetrical positions in domains II and IV were similar, forming bidirectional ET pathways [Breuer M, Zarzycki P, Blumberger J, Rosso KM (2012) J Am Chem Soc 134(24):9868-9871]. Here, we present the Em values of the 10 hemes in MtrF, solving the linear Poisson-Boltzmann equation and considering the protonation states of all titratable residues and heme propionic groups. In contrast to previous studies, the Em values indicated that the ET is more likely to be downhill from domain IV to II because of localization of acidic residues in domain IV. Reduction of hemes in MtrF lowered the Em values, resulting in switching to alternative downhill ET pathways that extended to the flavin binding sites. These findings present an explanation of how MtrF serves as an electron donor to extracellular substrates.

Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87.

Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPß chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.

Biochemical and plasma lipid responses to pemafibrate in patients with primary biliary cholangitis.

AIM: Fibrate addition to ursodeoxycholic acid (UDCA) therapy has been shown to improve both liver biochemistry and long-term prognosis in primary biliary cholangitis (PBC) patients showing an incomplete biochemical response to UDCA alone. We herein describe the clinical outcome of seven cases of PBC that received the new selective peroxisome proliferator-activated receptor α modulator, pemafibrate, in combination with UDCA therapy to investigate the biochemical and plasma lipid responses to the drug. METHODS: Of 124 initially enrolled PBC patients, 12 treated with UDCA alone and seven receiving UDCA plus bezafibrate showed alkaline phosphatase (ALP) levels above the upper limit of normal (330 U/L). Ultimately, seven patients with PBC and dyslipidemia who had agreed to biweekly visits at our hospital for UDCA plus pemafibrate combination therapy were retrospectively analyzed. RESULTS: In the four cases that switched from bezafibrate to pemafibrate, ALP became significantly decreased (0.031) and γ-glutamyltransferase tended to decrease (0.063) over the 3 months following pemafibrate addition. Two patients showed a greater than 50% reduction in ALP. No remarkable differences were observed for plasma lipid levels, alanine aminotransferase, aspartate aminotransferase, or the liver fibrosis marker Mac-2 binding protein glycosylation isomer between these time points. No adverse drug reactions were recorded. CONCLUSIONS: Pemafibrate might be another option for PBC patients with an incomplete response to UDCA therapy.

Supramolecular Complex of Methyl-ß-cyclodextrin with Adamantane-Grafted Hyaluronic Acid as a Novel Antitumor Agent.

Methyl-ß-cyclodextrin (M-ß-CyD) exhibits cytotoxic activity, and has the potentials as an antitumor agent. However, a tumor-selectivity of M-ß-CyD is low, leading to low antitumor activity and the adverse effects. Meanwhile, hyaluronic acid (HA) is known as a promising tumor targeting ligand, because various cancer cells overexpress CD44, a HA-binding glycoprotein. In the present study, to develop a tumor-selective delivery system for M-ß-CyD, we designed a supramolecular complex of M-ß-CyD with adamantane-grafted HA (Ad-HA/M-ß-CyD) and evaluated it as a tumor-selective antitumor agent. M-ß-CyD formed a stable complex with Ad-HA (Kc>104 M-1). In addition, Ad-HA/M-ß-CyD formed slightly a negative-charged nanoparticle with ca. 140 nm of a particle size, indicating the favorable physicochemical properties for antitumor agents. Ad-HA/M-ß-CyD showed the superior cytotoxic activity via CD44-mediated endosomal pathways in HCT116 cells (CD44(+)), a human colon cancer cell line. In addition, cytotoxic activity of Ad-HA/M-ß-CyD was induced by apoptosis. These results suggest that Ad-HA/M-ß-CyD has the potentials as a tumor-selective supramolecular antitumor agent.

Mutagenesis Study of the Cytochrome c Subunit Responsible for the Direct Electron Transfer-Type Catalytic Activity of FAD-Dependent Glucose Dehydrogenase.

The FAD-dependent glucose dehydrogenase from Burkholderia cepacia (FADGDH) is a hetero-oligomeric enzyme that is capable of direct electron transfer (DET) with an electrode. The cytochrome c (cyt c) subunit, which possesses three hemes (heme 1, heme 2, and heme 3, from the N-terminal sequence), is known to enable DET; however, details of the electron transfer pathway remain unknown. A mutagenesis investigation of the heme axial ligands was carried out to elucidate the electron transfer pathway to the electron mediators and/or the electrode. The sixth axial ligand for each of the three heme irons, Met109, Met263, and Met386 were substituted with His. The catalytic activities of the wild-type (WT) and mutant enzymes were compared by investigating their dye-mediated dehydrogenase activities and their DET abilities toward the electrode. The results suggested that (1) heme 1 with Met109 as an axial ligand is mainly responsible for the electron transfer with electron acceptors in the solution, but not for the DET with the electrode; (2) heme 2 with Met263 is responsible for the DET-type reaction with the electrode; and (3) heme 3 with Met386 seemed to be the electron acceptor from the catalytic subunit. From these results, two electron transfer pathways were proposed depending on the electron acceptors. Electrons are transferred from the catalytic subunit to heme 3, then to heme 2, to heme 1 and, finally, to electron acceptors in solution. However, if the enzyme complex is immobilized on the electrode and is used as electron acceptors, electrons are passed to the electrode from heme 2.

[A Case of Intussusception in the Jejunum Due to Small Intestinal Adenocarcinoma].

The patient was a woman in her 60's with an 11-month history ofpersistent epigastralgia and abdominal distension, without abnormal findings on upper endoscopy, abdominal ultrasonography, and abdominal computed tomography in other hospitals. She presented to our hospital with a complaint off requent vomiting; abdominal CT indicated intussusception in the jejunum due to a small intestinal tumor, and laparoscopic exploration and partial jejunectomy were performed. The histopathological diagnosis was tub1>tub2>pap, pT4(SE), pN1, pPM0, pDM0, pStage III A. She was treated with oral chemotherapy( S-1)and developed no recurrence 7 months after surgery. Laparoscopic exploration was useful to detect intussusception in the jejunum due to small intestinal adenocarcinoma.

Specific roles of each TCR hemichain in generating functional chain-centric TCR.

TCRα- and ß-chains cooperatively recognize peptide-MHC complexes. It has been shown that a "chain-centric" TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain-centric HLA-A*02:01(A2)/MART127-35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127-35. Although the generated A2/MART127-35-specific T cells used various TRBV genes, TRBV27 predominated with >10(2) highly diverse and unique clonotypic CDR3ß sequences. T cells individually reconstituted with various A2/MART127-35 TRBV27 TCRß genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127-35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance.

CDR3ß sequence motifs regulate autoreactivity of human invariant NKT cell receptors.

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid ligands presented by monomorphic CD1d. Human iNKT T cell receptor (TCR) is largely composed of invariant Vα24 (Vα24i) TCRα chain and semi-variant Vß11 TCRß chain, where complementarity-determining region (CDR)3ß is the sole variable region. One of the characteristic features of iNKT cells is that they retain autoreactivity even after the thymic selection. However, the molecular features of human iNKT TCR CDR3ß sequences that regulate autoreactivity remain unknown. Since the numbers of iNKT cells with detectable autoreactivity in peripheral blood is limited, we introduced the Vα24i gene into peripheral T cells and generated a de novo human iNKT TCR repertoire. By stimulating the transfected T cells with artificial antigen presenting cells (aAPCs) presenting self-ligands, we enriched strongly autoreactive iNKT TCRs and isolated a large panel of human iNKT TCRs with a broad range autoreactivity. From this panel of unique iNKT TCRs, we deciphered three CDR3ß sequence motifs frequently encoded by strongly-autoreactive iNKT TCRs: a VD region with 2 or more acidic amino acids, usage of the Jß2-5 allele, and a CDR3ß region of 13 amino acids in length. iNKT TCRs encoding 2 or 3 sequence motifs also exhibit higher autoreactivity than those encoding 0 or 1 motifs. These data facilitate our understanding of the molecular basis for human iNKT cell autoreactivity involved in immune responses associated with human disease.

Structure and dynamics of ionic liquids: Trimethylsilylpropyl-substituted cations and bis(sulfonyl)amide anions.

Ionic liquids with cationic organosilicon groups have been shown to have a number of useful properties, including reduced viscosities relative to the homologous cations with hydrocarbon substituents on the cations. We report structural and dynamical properties of four ionic liquids having a trimethylsilylpropyl functional group, including 1-methyl-3-trimethylsilylpropylimidazolium (Si-C3-mim+) cation paired with three anions: bis(fluorosulfonyl)imide (FSI-), bis(trifluoromethanesulfonyl)imide (NTf2-), and bis(pentafluoroethanesulfonyl)imide (BETI-), as well as the analogous N-methyl-N-trimethylsilylpropylpyrrolidinium (Si-C3-pyrr+) cation paired with NTf2-. This choice of ionic liquids permits us to systematically study how increasing the size and hydrophobicity of the anions affects the structural and transport properties of the liquid. Structure factors for the ionic liquids were measured using high energy X-ray diffraction and calculated from molecular dynamics simulations. The liquid structure factors reveal first sharp diffraction peaks (FSDPs) for each of the four ionic liquids studied. Interestingly, the domain size for Si-C3-mim+/NTf2- indicated by the maxima for these peaks is larger than for the more polar ionic liquid with a similar chain length, 1-pentamethyldisiloxymethyl-3-methyl-imidazolium bis(trifluoromethanesulfonyl)imide (SiOSi-mim+/NTf2-). For the series of Si-C3-mim+ ionic liquids, as the size of the anion increases, the position of FSDP indicates that the intermediate range order domains decrease in size, contrary to expectation. Diffusivities for the anions and cations are compared for a series of both hydrocarbon-substituted and silicon-substituted cations. All of the anions show the same scaling with temperature, size, and viscosity, while the cations show two distinct trends-one for hydrocarbon-substituted cations and another for organosilicon-substituted cations, with the latter displaying increased friction.

Expression of a rice glutaredoxin in aleurone layers of developing and mature seeds: subcellular localization and possible functions in antioxidant defense.

MAIN CONCLUSION: A rice glutaredoxin isoform (OsGrxC2;2) with antioxidant capacity is expressed abundantly in seed tissues and is localized to storage vacuoles in aleurone layers in developing and mature seeds. Seed tissues undergo drastic water loss at the late stage of seed development, and thus need to tolerate oxidative injuries associated with desiccation. We previously found a rice glutaredoxin isoform, OsGrxC2;2, as a gene expressed abundantly in developing seeds. Since glutaredoxin is involved in antioxidant defense, in the present study we investigated the subcellular localization and expression profile of OsGrxC2;2 and whether OsGrxC2;2 has a role in the defense against reactive oxygen species. Western blotting and immunohistochemistry revealed that the OsGrxC2;2 protein accumulated at a high level in the embryo and aleurone layers of developing and mature seeds. The OsGrxC2;2 in developing seeds was particularly localized to aleurone grains, which are storage organelles derived from vacuoles. Overexpression of OsGrxC2;2 resulted in an enhanced tolerance to menadione in yeast and methyl viologen in green leaves of transgenic rice plants. These results suggest that OsGrxC2;2 participates in the defense against oxidative stress in developing and mature seeds.