预测核苷(酸)类似物停药后慢性乙型肝炎复发的生化标记筛查及其临床应用

为探寻核苷(酸)类似物停药后慢性乙型肝炎复发的相关因素及可能血清标记,收集经核苷(酸)类似物治疗的乙型肝炎病毒e抗原(hepatitis B virus e antigen,HBeAg)阳性或阴性慢性乙型肝炎患者,根据治疗应答对其能否停药进行临床评估,分析达到停药标准患者的临床资料,包括性别,年龄,疗程,HBeAg水平,基线丙氨酸氨基转移酶(alanine aminotransferase,ALT)、HBV DNA,停药时的肝硬度值、HBV表面抗原(HBV surface antigen,HBsAg)水平、白细胞介素21(interleukin 21,IL-21)水平、外周血CD4~+IL-21~+T细胞频数。结果显示,共47例患者符合停药标准,其中HBeAg阳性26例,HBeAg阴性21例;平均年龄(42.45±14.08)岁。抗病毒疗程平均为(4.60±1.61)年。抗病毒治疗初始ALT值为(174.38±200.84)U/L;基线HBV DNA为(5.87±1.06)log拷贝/mL;停药时HBsAg精确定量平均为(1 104.82±1 328.11)IU/mL,IL-21平均为(729.82±427.39)pg/mL,CD4~+IL-21~+T细胞频数平均为(0.79±0.49)%,肝硬度测定F1 27例、F2 16例、F3 4例、F4 0例。共27例患者复发,1年内累积复发率为57.45%。Logistic回归单因素分析提示,慢性乙型肝炎患者停用核苷(酸)类似物后复发与服药总疗程及停药时IL-21水平、CD4~+IL-21~+T细胞频数、HBsAg水平有关;Logistic回归多因素分析则提示,停药时HBsAg和IL-21水平与复发有关。因此,停药时HBsAg和IL-21水平可作为慢性乙型肝炎患者停用核苷(酸)类似物后复发的预测因素。     

Predictive indicators for the therapeutic effect of OK-432 in patients with chronic active type B hepatitis

Twenty-two patients with chronic type B hepatitis were treated with OK-432. Immunological parameters were serially measured to find predictive indicators for the seroconversion from hepatitis B envelope antigen(HBe Ag) to anti-HBe. In patients who achieved the disappearance of HBe Ag associated with or without the appearance of anti-HBe, the numbers of CD8⁺DR⁺ and CD4⁺DR⁺T cells in peripheral blood increased gradually during OK-432 therapy and then reduced subsequently to the seroconversion from HBe Ag positive to anti-HBe positive. Increases of DR-positive T cells in numbers were significantly correlated with increased amounts of IFN- produced in response toin vitro OK-432 stimulation.In vitro OK-432-stimulated IFN- production and the increase of CD8⁺DR⁺T cells in number in peripheral blood could be proposed as predictive indicators for the disappearance of HBe Ag.     

Molecular Basis for the High Degree of Antigenic Cross-Reactivity between Hepatitis B Virus Capsids (HBcAg) and Dimeric Capsid-Related Protein (HBeAg): Insights into the Enigmatic Nature of the e-Antigen

The hepatitis B virus core gene codes for two closely related antigens: a 21-kDa protein that forms dimers that assemble as multimegadalton capsids, and a 17-kDa protein that also forms dimers but that do not assemble. The proteins, respectively referred to as core antigen (HBcAg) and e-antigen (HBeAg), share a sequence of 149 residues but have different amino- and carboxy-termini. Their structural and serological relationship has long been unclear. With insights gained from recent structural studies on immune complexes of the capsids, the relationship was reassessed using recombinant forms of the antigens and a panel of monoclonal antibodies (mAbs) commonly believed to discriminate between core and e-antigen. Surface plasmon resonance (SPR) was used to measure the affinities, in contrast to previous studies that used more error-prone and less sensitive plate-type assays. Four of the six mAbs did not discriminate between core and e-antigen, nor did they discriminate between e-antigen and dimers of dissociated core antigen capsids. One mAb (3120) was specific for assembled capsids and one (e6) was specific for unassembled dimers. Epitope valency of the e-antigen was also studied, using a sandwich SPR assay where e-antigen was captured with one mAb and probed with a second. The e-antigen is often considered to be a monomeric protein on the basis of monovalent reactivity with antibody pairs specific for either an α or β epitope (in a prior nomenclature for e-antigen specificity). This model, however, is incorrect, because recombinant e-antigen is a stable dimer and its apparent monovalency is due to steric blockage. This was proven by the formation of a 2:1 Fab e6-e-antigen complex. These results suggest new approaches for the isolation of the authentic e-antigen, its biological assay, and its stabilization as an immune complex for structural studies.     

基于临床数据构建慢性乙型肝炎患者肝纤维化的新型预测评分：S-risk score

基于临床数据构建一种预测慢性乙型肝炎肝纤维化的无创诊断模型。

收集2021年1月至2023年7月宁波市医疗中心李惠利医院收治的165例CHB患者病例资料作回顾性分析,根据肝活检病理结果将患者分为无肝纤维化组（S0,n = 22）和肝纤维化组（≥S1,n = 143）。收集患者的血清学指标和临床数据,运用单因素和多因素 logitstic回归分析筛选出独立预测指标并建立模型,同时采用受试者工作特征曲线（ROC）评价模型的预测效能。

单因素分析结果显示,两组患者在白蛋白、谷草转氨酶、甘油三酯、总胆汁酸、胆碱酯酶、凝血酶原时间、 BMI、血清Ⅳ胶原和血清透明质酸等指标中存在差异（P＜0.05）。通过logistic多因素的回归分析构建肝纤维化模型S-risk score = −4.30+0.12×白蛋白+0.02×谷草转氨酶−0.05×碱性磷酸酶+0.29×甘油三酯+0.06×总胆汁酸−0.47×凝血酶原时间+0.20×BMI+0.03×血清Ⅳ胶原测定+0.02×血清透明质酸。该评分下的ROC曲线下的面积为0.866,其预测肝纤维化的准确性明显优于APRI和FIB-4两项评分模型。

我们构建的S-risk score模型对CHB患者肝纤维化有良好的预测能力,其预测准确性均高于APRI和FIB-4两项评分模型。

     

Pegylated interferon alpha-2a (40 kDa) in the treatment of chronic hepatitis B

Chronic hepatitis B virus (HBV) is a serious and life-threatening disease afflicting 350 million of the world's population. So far, current monotherapy with conventional interferon-alpha, lamivudine, and adefovir dipivoxil remains unsatisfactory. In addition, the use of conventional interferon-alpha needs to be administered subcutaneously daily or thrice weekly and is associated with frequent adverse events. Although nucleoside-nucleotide analogs such as lamivudine and adefovir dipivoxil are well tolerated and can normalize serum alanine aminotransaminase rapidly, 1-year therapy with either lamivudine or adefovir dipivoxil results in low hepatitis B e antigen (HBeAg) seroconversion rates. In HBeAg negative patients, most of the patients would relapse after lamivudine has been discontinued. Pegylated interferon alpha-2a, an immunomodulatory agent, is a new drug that has just completed phase III clinical trials for the treatment of both HBeAg positive and HBeAg negative chronic HBV infection. The advantage of pegylated interferon alpha-2a in achieving sustained virological response over nucleoside-nucleotide analogs is particularly obvious in the HBeAg negative group. In both of these phase III studies, sustained off-treatment response is superior to the use of lamivudine. These recent data put pegylated interferon alpha-2a as the first choice of anti-HBV therapy, especially in young and motivated patients with chronic HBV infection.     

Role of the cytomegalovirus (CMV)-antigenemia assay as a predictive and follow-up detection tool for CMV disease in AIDS patients

Forty-two patients were evaluated to determine the value of the CMV antigenemia (CMV-Ag) test as a follow-up marker as well as a prediction marker of CMV disease. Twenty patients were positive for at least one positive CMV-Ag assay and 9 of them developed CMV retinitis. With the threshold value (10 positive cells), sensitivity was 56% and specificity was 94%. The CMV-Ag assay, with the threshold value, produced high specificity, positive predictive value and negative predictive value but relatively poor sensitivity. Eight patients experienced CMV disease relapse a total of 16 times. At relapse, 8 of the 16 times showed negative for CMV-Ag assay; 7 underwent systemic maintenance while 1 underwent local maintenance. It is inferred that the CMV-Ag test is a poor follow-up marker to detect the relapse of CMV disease, particularly in patients undergoing systemic maintenance.     

Synthesis and characterization of a C6 nucleoside analogue for the in vivo imaging of the gene expression of herpes simplex virus type-1 thymidine kinase (HSV1 TK)

The synthesis and biological evaluation of '6-(1,3-dihydroxyisobutyl)thymine' (DHBT; 1), which corresponds to 6-[3-hydroxy-2-(hydroxymethyl)propyl]-5-methylpyrimidine-2,4(1H,3H)-dione, is reported. DHBT (1) was designed as a new substrate for herpes simplex virus type-1 thymidine kinase (HSV1 TK). The compound was found to be exclusively phosphorylated by HSV1 TK, and to exhibit good binding affinity (Ki = 35.3+/-1.3 microM). Cell-proliferation assays with HSV1-TK-transduced human osteosarcoma cells (143B-TK+-HSV1-WT) and with both human-thymidine-kinase-1-negative (143B-TK-) and non-transduced parental (MG-63) cells indicate that 1 is less cytotoxic than the standard drug Ganciclovir. Thus, DHBT (1) represents a promising precursor of a nontoxic reporter probe for the monitoring of HSV1 TK gene expression by means of positron-emission tomography (PET).     

The defective protein level of myosin light chain phosphatase (MLCP) in the isolated saphenous vein, as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM)

We examined the contractile reactivity to 5-hydroxytryptamine (5-HT) in isolated human saphenous vein (SV), as a vascular conduit in coronary artery bypass grafting (CABG), harvested from patients with diabetes mellitus (DM) and non-DM (NDM). Vascular rings of endothelium-denuded SV were used for functional and biochemical experiments. The vasoconstrictions caused by 5-HT were significantly greater (hyperreactivity) in the DM group than in the NDM group. RhoA/ROCK pathway is activated by various G-protein-coupled receptor agonists and consequently induces phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin light chain phosphatase (MLCP), which inhibits MLCP activity. In the resting state of the vessels, total tissue protein levels of 5-HT_2A receptor, 5-HT_1B receptor, RhoA, ROCK1, and ROCK2 did not differ between NDM and DM groups. However, the total protein level of MYPT1 was significantly lower in the DM group than in the NDM group. Furthermore, the ratio of P(Thr⁶⁹⁶)-MYPT1 to total MYPT1 was significantly higher in the DM group than in the NDM group. These results suggest that the hyperreactivity to 5-HT in the SV smooth muscle of patients with DM is due to not only enhanced phosphorylation of MLCP but also defective protein level of MLCP. Thus, we reveal for the first time that the defective protein level of MLCP in the DM group can partially explain the poor patency of SV graft harvested from patients with DM.     

A long-term study on interactions between the Adh and αGpdh allozyme polymorphisms and the chromosomal inversion In(2L)t in a seminatural population of D. melanogaster

The Adh and αGpdh allozyme loci (both located on the second chromosome) showed considerable fluctuations in allele frequencies in a seminatural population of Drosophila melanogaster during 1972–97. Both long-term and short-term fluctuations were observed. The short-term fluctuations occurred within almost all years and comparison of allele frequencies between winters and summers showed significantly higher Adh^S (P < 0.001) and αGpdh^F (P < 0.01) allele frequencies in summers. Frequencies of these alleles were significantly positively correlated with environmental temperature, suggesting the adaptive significance of these allozyme polymorphisms. Frequency changes of the Odh locus (located on the third chromosome) showed no seasonal pattern and were not correlated with environmental temperature. Almost all short-term and long-term increases in Adh^S frequency were accompanied by a corresponding decrease in αGpdh^S frequency (r = –0.82, P < 0.001) and vice versa. Further analysis showed that gametic disequilibria between the Adh and αGpdh loci, which frequently occurred, were due to the presence of inversion In(2L)t located on the same chromosome arm and In(2L)t frequencies were positively correlated with environmental temperature. Gametic disequilibria between Adh and Odh and between Odh and αGpdh were hardly observed. Because In(2L)t is exclusively associated with the Adh^S/αGpdh^F allele combination, the observed correlated response in Adh/αGpdh allele frequencies is (at least partly) explained by hitchhiking effects with In(2L)t. This means that the adaptive value of the allozyme polymorphisms has been overestimated by ignoring In(2L)t polymorphism. Fluctuations in Adh allele frequencies are fully explained by selection on In(2L)t polymorphism, whereas we have shown that αGpdh frequency fluctuations are only partly explained by chromosomal hitchhiking, indicating the presence of selective differences among αGpdh genotypes in relation with temperature and independent of In(2L)t. Frequency fluctuations of αGpdh and In(2L)t are consistent with their latitudinal distributions, assuming that temperature is the main environmental factor varying with latitude that causes directly or indirectly these frequency distributions. However, the results of the tropical greenhouse population show no correlation of Adh (independent of In(2L)t) and Odh allele frequencies with environmental temperature, which may indicate that the latitudinal distribution in allele frequencies for these loci is not the result of selection on the F/S polymorphism in a direct way.     

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.     

Evidence for the involvement of the RNA-dependent protein kinase (PKR) in the induction of human cytotoxic T lymphocytes against a synthetic peptide of HIV-1 by regulatory RNA

The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms . After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation.To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation of WEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHI-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.     

Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

TMEM126A is a mitochondrial located mRNA (MLR) protein of the mitochondrial inner membrane

Background

Hereditary optic neuropathies (HONs) are a heterogeneous group of disorders that affect retinal ganglion cells (RGCs) and axons that form the optic nerve. Leber's Hereditary Optic Neuropathy and the autosomal dominant optic atrophy related to OPA1 mutations are the most common forms. Nonsyndromic autosomal recessive optic neuropathies are rare and their existence has been long debated. We recently identified the first gene responsible for these conditions, TMEM126A. This gene is highly expressed in retinal cellular compartments enriched in mitochondria and supposed to encode a mitochondrial transmembrane protein of unknown function.

Methods

A specific polyclonal antibody targeting the TMEM126A protein has been generated. Quantitative fluorescent in situ hybridization, cellular fractionation, mitochondrial membrane association study, mitochondrial sub compartmentalization analysis by both proteolysis assays and transmission electron microscopy, and expression analysis of truncated TMEM126A constructs by immunofluorescence confocal microscopy were carried out.

Results

TMEM126A mRNAs are strongly enriched in the vicinity of mitochondria and encode an inner mitochondrial membrane associated cristae protein. Moreover, the second transmembrane domain of TMEM126A is required for its mitochondrial localization.

Conclusions

TMEM126A is a mitochondrial located mRNA (MLR) that may be translated in the mitochondrial surface and the protein is subsequently imported to the inner membrane. These data constitute the first step toward a better understanding of the mechanism of action of TMEM126A in RGCs and support the importance of mitochondrial dysfunction in the pathogenesis of HON.

General significance

Local translation of nuclearly encoded mitochondrial mRNAs might be a mechanism for rapid onsite supply of mitochondrial membrane proteins.     

A complex of Co(II) with 2-hydroxyphenyl-azo-2'-naphthol (HPAN) is far less cytotoxic than the parent compound on A549-lung carcinoma and peripheral blood mononuclear cells: Reasons for reduction in cytotoxicity

Cytotoxic studies using an azo compound HPAN and its Co(II) complex were carried out on non-small lung epithelium carcinoma (A549) cells and peripheral blood mononuclear (PBM) cells. The results obtained suggest that the Co(II) complex is much less toxic toward both cell lines and the decreased toxicity due to the complex was more pronounced with carcinoma A549 cells. An attempt was made to correlate the findings related to cytotoxicity with the interaction of the compounds with DNA using calf thymus DNA as the target. The study was able to conclude that the complex was a relatively weak binder to calf thymus DNA. This information was used to explain the interaction of azo compounds with DNA in peripheral blood mononuclear cells and A549 lung carcinoma cells. It was concluded that the Co(II) complex interacts with DNA to a much lesser extent than HPAN alone. Cyclic voltammetry experiments carried out with HPAN and the Co(II) complex further showed that the presence of the metal ion in the complex prevents reduction of the azo group to such species that are responsible for inducing cytotoxicity. The overall finding was that complex formation with azo compounds might serve as a possible route to curb their toxicities.