A previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans

Respiratory infections constitute the most widespread human infectious disease, and a substantial proportion of them are caused by unknown etiological agents. Reoviruses (respiratory enteric orphan viruses) were first isolated from humans in the early 1950s and so named because they were not associated with any known disease. Here, we report a previously unknown reovirus (named "Melaka virus") isolated from a 39-year-old male patient in Melaka, Malaysia, who was suffering from high fever and acute respiratory disease at the time of virus isolation. Two of his family members developed similar symptoms approximately 1 week later and had serological evidence of infection with the same virus. Epidemiological tracing revealed that the family was exposed to a bat in the house approximately 1 week before the onset of the father's clinical symptoms. Genome sequence analysis indicated a close genetic relationship between Melaka virus and Pulau virus, a reovirus isolated in 1999 from fruit bats in Tioman Island, Malaysia. Screening of sera collected from human volunteers on the island revealed that 14 of 109 (13%) were positive for both Pulau and Melaka viruses. This is the first report of an orthoreovirus in association with acute human respiratory diseases. Melaka virus is serologically not related to the different types of mammalian reoviruses that were known to infect humans asymptomatically. These data indicate that bat-borne reoviruses can be transmitted to and cause clinical diseases in humans.     

HCV受体及入胞相关分子的研究进展

HCV是丙型肝炎的致病体,在被感染组织和血清中浓度较低,病毒高度变异及缺乏稳定的体外细胞培养系统及理想的实验动物模型等问题,阻碍了对HCV的进一步研究。近年来各种HCV体外培养系统的建立,为HCV的研究提供了强有力的工具,使人们对HCV的认识提升到新的层次。本文就CD81、低密度脂蛋白受体、清道夫受体B1及claudin—1等受体及入胞相关分子研究进展作一简要综述。     

Phosphorylation of Arl4A/D promotes their binding by the HYPK chaperone for their stable recruitment to the plasma membrane

The Arl4 small GTPases participate in a variety of cellular events, including cytoskeleton remodeling, vesicle trafficking, cell migration, and neuronal development. Whereas small GTPases are typically regulated by their GTPase cycle, Arl4 proteins have been found to act independent of this canonical regulatory mechanism. Here, we show that Arl4A and Arl4D (Arl4A/D) are unstable due to proteasomal degradation, but stimulation of cells by fibronectin (FN) inhibits this degradation to promote Arl4A/D stability. Proteomic analysis reveals that FN stimulation induces phosphorylation at S143 of Arl4A and at S144 of Arl4D. We identify Pak1 as the responsible kinase for these phosphorylations. Moreover, these phosphorylations promote the chaperone protein HYPK to bind Arl4A/D, which stabilizes their recruitment to the plasma membrane to promote cell migration. These findings not only advance a major mechanistic understanding of how Arl4 proteins act in cell migration but also achieve a fundamental understanding of how these small GTPases are modulated by revealing that protein stability, rather than the GTPase cycle, acts as a key regulatory mechanism.

The Arl4 [ADP ribosylation factor (Arf)-like 4 proteins] small GTPases, comprising Arl4A, Arl4C, and Arl4D, are developmentally regulated. In adults, they have restricted tissue expression and have been found to act in a variety of cellular events. Arl4A recruits ELMO-Dock180, a conserved Rac regulator, to membranes for actin remodeling (1) and also directs cell migration through Robo1 and Pak1 effectors (2, 3). Several studies have reported that Arl4C functions in filopodia formation (4), cancer cell invasion (5, 6), and tumorigenesis (7–9). Arl4D has been found to activate Arf6 by recruiting cytohesin-2/ARNO for actin remodeling to promote cell migration (10, 11). How the Arl4 proteins are regulated in these cellular roles has been enigmatic. Small GTPases are typically activated by guanine nucleotide exchange factors and deactivated by GTPase-activating proteins (12, 13). However, neither class of key regulators has been identified for the Arl4 proteins. Consistent with this current state of knowledge, these small GTPases have been found to undergo rapid nucleotide exchange and appear structurally unable to bind GDP (13, 14). Thus, a major goal has been to elucidate how Arl4 proteins are regulated independent of the canonical mechanism that involves the GTPase cycle.The p21-activated kinases (Paks) stand at the hub of several signaling pathways for cell proliferation, migration, and survival (15). In cell migration, Pak1 phosphorylates paxillin at the leading edge of migrating cells for the rapid turnover of focal adhesion (16). Pak1 also phosphorylates LIMK1 and myosin light chain for actin reorganization to promote cell migration (17, 18). Pak activation requires cell adhesion through the interaction between integrins and the extracellular matrix (19). A well-characterized example has been the stimulation of cells by fibronectin (FN), which recruits Pak1 to the plasma membrane, where it then interacts with other molecules to coordinate downstream events (20, 21). In this role, FN acts in a variety of physiologic and pathologic circumstances, with wound healing being an example of the former (22) and cancer cell invasion being an example of the latter (23). With respect to the latter, alternatively spliced forms of FN have been found to act in collective tumor migration and are predictive of adverse outcomes for patients with cancer (24–26).We have recently found that FN promotes the cooperative recruitment of Arl4A and Pak1 to the plasma membrane, which contributes to sustained Pak1 activation needed for cell migration (3). In this study, we find that Arl4A and Arl4D (Arl4A/D) undergo rapid proteasomal degradation. FN stimulation activates Pak1 to phosphorylate these small GTPases, which leads to their binding by the chaperone-like protein HYPK (Huntingtin-interacting protein K). This binding stabilizes the targeting of Arl4A/D to the plasma membrane to promote cell migration.     

Single Nucleotide Variants of the Human TIM-1 IgV Domain with Reduced Ability to Promote Viral Entry into Cells

Human T-cell immunoglobulin mucin 1 (hTIM-1) is known to promote cellular entry of enveloped viruses. Previous studies suggested that the polymorphisms of hTIM-1 affected its function. Here, we analyzed single nucleotide variants (SNVs) of hTIM-1 to determine their ability to promote cellular entry of viruses using pseudotyped vesicular stomatitis Indiana virus (VSIV). We obtained hTIM-1 sequences from a public database (Ensembl genome browser) and identified 35 missense SNVs in 3 loops of the hTIM-1 immunoglobulin variable (IgV) domain, which had been reported to interact with the Ebola virus glycoprotein (GP) and phosphatidylserine (PS) in the viral envelope. HEK293T cells transiently expressing wildtype hTIM-1 or its SNV mutants were infected with VSIVs pseudotyped with filovirus or arenavirus GPs, and their infectivities were compared. Eleven of the thirty-five SNV substitutions reduced the efficiency of hTIM-1-mediated entry of pseudotyped VSIVs. These SNV substitutions were found not only around the PS-binding pocket but also in other regions of the molecule. Taken together, our findings suggest that some SNVs of the hTIM-1 IgV domain have impaired ability to interact with PS and/or viral GPs in the viral envelope, which may affect the hTIM-1 function to promote viral entry into cells.     

Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or β-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R² = 0·15; slope = 9·09) and, less significantly, in HE (R² = 0·021; slope = 8·92) when compared with other haemolytic disorders (R² ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R² = 0·48; slope = −9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.     

A p-nitrophenylphosphatase activity associated with the human erythrocyte membrane.

A p-nitrophenylphosphatase activity has been identified as a component of the human erythrocyte membrane. This activity is distinct from that associated with the cell's Na(+)+K(+)-dependent ATPase, Ca(2+)-dependent ATPase, or spectrin phosphatase. The activity described here is stimulated by Mn2+ but not by Ca2+ with or without calmodulin. A potential erythrocyte membrane substrate for this activity is a 95 kDa phosphoprotein that can be shown to undergo Mn(2+)-stimulated but not Mg(2+)-stimulated dephosphorylation.     

抗细胞膜DNA抗体检测方法的建立及对系统性红斑狼疮诊断价值的临床应用研究

目的 应用间接免疫荧光法(IIF)检测抗细胞膜DNA(cmDNA)抗体,探讨抗cmDNA抗体对系统性红斑狼疮(SLE)的诊断价值.比较以人B淋巴细胞株Raji、人早幼粒白血病细胞株HL60为底物,抗cmDNA抗体在SLE中的检测效果.方法 选取306例SLE患者,192例非SLE疾病对照组,包括脊柱关节病(SPA) 52例,类风湿关节炎(RA) 70例,原发干燥综合征(pSS) 30例,其他结缔组织病(CTD)40例.50名健康志愿者作为对照组.采用IIF法观察培养的人B淋巴细胞株Raji、人早幼粒白血病细胞株HL60的细胞膜的荧光图形;用IIF法检测抗核抗体、抗双链DNA(dsDNA)抗体;联合应用免疫双扩散法和蛋白印迹法检测抗Sm抗体,酶联免疫吸附试验(ELISA)法测定抗核小体抗体(AnuA).配对资料比较用McNemarX2检验,相关性分析采用Spearman相关及Logistic回归分析.结果以Raji及HL60 2种细胞株为底物检测SLE患者抗cmDNA抗体,敏感性分别为72.5％、76.1％,特异性分别为91.7％、86.8％,差异均无统计学意义(P＞0.05).抗cmDNA抗体的敏感性明显高于抗Sm抗体及抗dsDNA抗体,差异有统计学意义(P＜0.01);特异性与抗dsDNA抗体相似(P＞0.05),日低于抗Sm抗体及AnuA(P＜0.01).抗cmDNA抗体分别与抗dsDNA抗体、抗Sm抗体及AnuA联合检测在SLE诊断中的敏感性均明显高于上述自身抗体单独检测,差异有统计学意义(P＜0.05).抗cmDNA抗体与SLE患者的黏膜溃疡之间有相关性(OR=2.343,P=0.029).抗cmDNA抗体与红细胞沉降率(ESR)有相关性(OR=1.031,P=0.012).抗cmDNA抗体与SLE疾病活动指数(SLEDAI)评分无相关性(r=0.070,P=0.600).本文研究还证实Raji细胞株较HL60细胞株更易复苏、荧光图形亮度更强,表达效果更好.结论 抗cmDNA抗体对SLE诊断的敏感性高,特异性强,可能成为SLE诊断的相对特异性抗体之一.抗cmDNA抗体与抗dsDNA抗体、抗Sm抗体及AnuA联合检测可提高对SLE诊断的敏感性.选择Raji细胞株为底物检测抗cmDNA抗体较HL60细胞株更有优势.     

SMMC-7721肝癌细胞膜上β_2糖蛋白I可能的受体

研究β2糖蛋白I(β2GPI)与肝细胞相互作用的过程,以进一步探讨β2GPI在乙型肝炎病毒感染肝细胞过程中所发挥的作用。采用L igand b lot技术,从SMMC-7721、HL-60及SGC-7901三个细胞株中,筛选出具有与人β2GPI特异结合蛋白成分的细胞。SMMC-7721细胞在40kD处出现一特异染色带,而HL-60及SGC-7901二种细胞则无此反应。在SMMC-7721细胞中存在有与人β2GPI特异结合的蛋白,这种蛋白可能参与了HBV感染肝细胞的过程。     

Circulating Th17 cells frequency is associated with the disease progression in HBV infected patients

Background and Aims: Th17 cells have been shown to mediate host defensive mechanisms in various infections, but their role in HBV infection in humans has not been well characterized. In this study, we analyzed the frequency and cytokines secretion of circulating Th17 cells in HBV infected patients with different statuses, and also evaluated the potential association of Th17 frequency with the levels of liver injury. Methods: The study population consisted of 133 subjects, including 40 mild chronic hepatitis B (CHB) patients, 37 severe CHB patients, 20 acute hepatitis B (AHB) patients and 36 healthy controls. The frequency of circulating Th17 cells were carried out by intracellular cytokine staining analysis and serum IL‐10 levels were measured by ELISA. Results: Our data shown that AHB and severe CHB patients had a significant increase of Th17 cells frequency in peripheral blood compared with mild CHB patients and healthy control (both P < 0.05). The elevated prevalence of Th17 cells is positively associated with the increased serum ALT levels in severe CHB patients (r= 0.457, P= 0.004) but had no correlation with serum HBV DNA load. In addition, the serum IL‐10 were negatively correlated with the frequency of Th17 cells in PBMC from patients with chronic HBV infection (r=?0.452, P < 0.01). Conclusion: Th17 cells may contribute to the disease progression and pathogenesis of liver injury in HBV infected patients, and the induction of IL‐10 may be one mechanism of constraining pro‐inflammatory Th17 responses.     

A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer

BACKGROUND & AIMS:Helicobacter pylori is the main cause of peptic ulceration and gastric adenocarcinoma. The vacuolating cytotoxin gene, vacA, is a major determinant of virulence. Two naturally polymorphic sites in vacA, the signal region and midregion, are well-characterized determinants of toxicity and markers of pathogenesis. The aim of this study was to characterize a new vacA polymorphic site, the intermediate (i) region. METHODs: The vacA i-region was identified and characterized by constructing isogenic vacA exchange mutants and determining their vacuolating activity on HeLa, AGS, and RK13 cell lines. The vacA i-region types of H pylori isolates from patients undergoing routine endoscopy were determined by nucleotide sequencing and allele-specific polymerase chain reaction. RESULTS: Two i-region types were identified, i1 and i2, and both were common among 42 Western clinical isolates. Interestingly, only naturally occurring s1/m2 strains varied in i-type; s1/m1 and s2/m2 strains were exclusively i1 and i2, respectively. Vacuolation assays showed that i-type determined vacuolating activity among these s1/m2 strains, and exchange mutagenesis confirmed that the i-region itself was directly responsible. Using a simple i-region polymerase chain reaction-based typing system, it was shown for 73 Iranian patients that i1-type strains were strongly associated with gastric adenocarcinoma (P < 10(-3)). Finally, logistic regression analysis showed this association to be independent of, and larger than, associations of vacA s- or m-type or cag status with gastric adenocarcinoma. CONCLUSIONS: Together these data show that the vacA i-region is an important determinant of H pylori toxicity and the best independent marker of VacA-associated pathogenicity.     

A new polymorphism in the GRP78 is not associated with HBV invasion

AIM: To examine the association between-86 bp (T > A) in the glucose-regulated protein 78 gene ( GRP78) and hepatitis B virus (HBV) invasion.METHODS: DNA was genotyped for the single-nucleotide polymorphism by polymerase chain reaction followed by sequencing in a sample of 382 unrelated HBV carriers and a total of 350 sex-and age-matched healthy controls. Serological markers for HBV infection were determined by enzyme-linked immunosorbent assay kits or clinical chemistry testing.RESULTS: The distributions of allelotype and genotype in cases were not signi.cantly different from those in controls. In addition, our .ndings suggested that neither alanine aminotransferase/hepatitis B e antigen nor HBV-DNA were associated with the allele/genotype variation in HBV infected individuals.CONCLUSION:-86 bp T > A polymorphism in GRP78 gene is not related to the clinical risk and acute exacerbation of HBV invasion.     

Hereditary xerocytosis: a report of six unrelated Spanish families with leaky red cell syndrome and increased heat stability of the erythrocyte membrane

Summary. Hereditary xerocytosis (HX) is a rare haemolytic disease due to dehydrated red blood cells (RBCs). A unique feature of this syndrome is that affected members often show normal or near normal haemoglobin levels despite clinical and laboratory evidence of mild to moderate haemolysis. The diagnostic clue is the association of markedly increased RBC Na⁺+K⁺ fluxes with low total cation (Na⁺ +K⁺) content. 11 patients of six unrelated families of Spanish origin with HX have been studied from clinical, genetical and biological points of view. In addition, we have investigated the sensitivity of RBC membrane to heat at three different incubation times (15, 30 and 60min) and two different temperature values (46°C and 49°C). Under these conditions control RBCs (50 normal subjects) exhibited at 49°C and 30min a maximum of 30% fragmented RBCs. This value increased to 80% after 60min of incubation. In contrast, patients with HX showed significantly lower percentages of fragmented RBCs at both 30 and 60min of incubation (maximum 10% and 30%, respectively). In an attempt to determine if increased heat stability was unique to HX RBCs, several other congenital membranopathies with haemolytic anaemia were also studied. The degree of fragmentation, except in one case of HPP (which was strongly increased), did not differ from the control group. Electrophoretic studies of membrane proteins performed in RBCs of all the patients with HX did not explain any qualitative nor quantitative abnormality.
In addition to its physiopathological interest, study of RBC heat stability, together with other haematological parameters (increased MCHC and decreased RBC osmotic fragility), may be useful for HX diagnosis, especially in laboratories which are not equipped to evaluate RBC membrane permeability.     

Baseline cholesterol is associated with the response to antiviral therapy in chronic hepatitis C

Background: Hepatitis C virus (HCV) partially interacts with low‐density lipoprotein (LDL) receptors, suggesting a role for lipids in regulating HCV clearance. Our aim was to study if baseline lipids can discriminate responders from non‐responders among patients with HCV infection. Methods: A total of 109 HCV patients were studied. Laboratory measurements included serum lipids, aminotransferases and viral load, as well as HCV genotype determinations. Results: Responders (n = 53) had significantly higher serum baseline levels of total cholesterol, LDL cholesterol and apolipoprotein B compared to non‐responders (n = 56). Multivariate logistic regression analysis showed that a 10 mg/dL increase in total cholesterol was associated with 3.02 higher odds of responding to treatment (95% CI 1.74–5.32, P < 0.001), while a 10 mg/dL increase in apolipoprotein B levels was associated with 1.81 higher odds of responding to treatment (95% CI 1.37–2.54, P < 0.001), after adjustment for age, sex, body mass index (BMI), smoking habits, baseline viral load, liver histology and administration of pegylated interferon. An inverse association between BMI and response to treatment was also evident (adjusted odds ratio 0.73, 95% CI 0.55–0.96; P = 0.03). Conclusion: Baseline serum total cholesterol levels and BMI could be helpful in discriminating responders to antiviral therapy among patients with HCV infection.     

Genetic susceptibility to Hodgkin's lymphoma associated with the human leukocyte antigen region

Abstract:  Based on the presence of an abundant inflammatory infiltrate, expression of a broad spectrum of cytokines and the professional antigen presenting phenotype of Hodgkin Reed-Sternberg cells it can be anticipated that immunological mechanisms play a major role in the pathogenesis of Hodgkin's lymphoma (HL). Genetic susceptibility to HL probably relates to functionality of the immune system and the large number of associations with the human leukocyte antigen (HLA) region in family and population-based studies supports this relation. In Epstein–Barr virus (EBV) positive HL cases, which usually demonstrate HLA class I expression, HRS cells should be able to present EBV derived antigenic peptides and trigger the immune system. This process depends on the affinity of the HLA binding groove for binding immunogenic peptides and thus on the HLA alleles. It can be anticipated that certain combinations of alleles predispose to or protect from the development of EBV positive HL. In EBV negative HL cases other antigenic peptides, related to malignant transformation, in combination with other HLA alleles may be involved. In addition, differential attraction and activation of inflammatory cells may influence HL subtype. In this article, possible roles of HLA in HL pathogenesis are explored and genetic associations of HLA with HL are reviewed and commented on.     

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.Commitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1–4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2–associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7–10). These novel “symmetric” homo-dimers can multimerize via association of α6 helices (8, 11, 12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x_L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two hydrophobic core helices as a hairpin across the membrane (14), suggesting that the central helices of Bak and Bax (α5 and α6) might penetrate the MOM similarly (reviewed in refs 3, 15, and 16). Consistent with this hairpin insertion model, α5 and α6 peptides can permeabilize membranes (17–19). More pertinently, Bax α5 and α6 were reported to insert into and span the MOM as a hairpin before oligomerization (20).This longstanding model, however, does not fit well with recent evidence on the structure of Bak and Bax oligomers, as recently reviewed (2, 4). Analysis of Bak oligomers in liposomes by electron paramagnetic resonance (EPR) suggests that α6 inserts only shallowly in the lipid bilayer (21). Additionaly, the first 3D structures of activated forms of Bax (10) suggest that, early in its activation, α5 and α6 separate. Moreover, a Bax core domain containing only helices α2 to α5 generated a BH3:groove symmetric dimer in which two α4 and two α5 helices form an aromatic face that might sit on the bilayer (10). These findings fit better with an “in-plane model” in which only α9 is a TM domain and other helices (including α5 and α6) insert only shallowly into the bilayer.The nature of the apoptotic pores remains uncertain. Some findings favor a proteinaceous pore (22), but studies with model membranes suggest that Bax oligomers can perturb the bilayer and produce lipidic pores (i.e., pores not bounded entirely by protein) (23–26).These important unresolved questions about the pivotal event in apoptosis prompted us to explore the membrane topology of Bak, before, during, and after an apoptotic signal, and to reinvestigate that of Bax. In accord with recent Bax structures (10) and recent EPR studies on Bak (21, 27), the results show that neither oligomeric Bak nor Bax inserts an α5–α6 hairpin across the MOM. We propose instead that the α5 and α6 helices lie in the bilayer plane and disrupt membrane integrity by imposing tension and curvature to the membrane that provoke its permeabilization.     

Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase

BACKGROUND & AIMS: Adefovir dipivoxil effectively inhibits both hepatitis B virus (HBV) replication and disease activity in patients with chronic hepatitis B. Resistance to treatment was not observed in 2 recent large placebo-controlled 48-week studies with this drug. The aim of this study was to characterize adefovir resistance in a patient who developed clinical and virologic evidence of breakthrough during a 96-week course of treatment. METHODS: HBV DNA was PCR amplified and sequenced. Phenotypic studies used patient-derived HBV as well as specific mutations created by site-directed mutagenesis of a HBV/baculovirus recombinant. RESULTS: Following the commencement of treatment with adefovir dipivoxil, the patient initially responded with a 2.4 log(10) decrease in serum HBV DNA and normalization of alanine aminotransaminase levels by week 16. During the second year of treatment, however, serum HBV DNA rose progressively, eventually returning to near-pretreatment levels. This increase in viral replication was associated with a marked increase in alanine aminotransferase and mild changes in bilirubin, albumin, and prothrombin time. Comparison of pretreatment and posttreatment HBV DNA by polymerase chain reaction sequencing identified a novel asparagine to threonine mutation at residue rt236 in domain D of the HBV polymerase. In vitro testing of a laboratory strain encoding the rtN236T mutation and testing of patient-derived virus confirmed that the rtN236T substitution caused a marked reduction in susceptibility to adefovir. CONCLUSIONS: The development of this novel mutation in the HBV polymerase confers resistance to adefovir dipivoxil. The patient responded to subsequent lamivudine therapy, achieving normalization of alanine aminotransferase and a significant decrease in serum HBV DNA.     

A case of late-onset polymyositis with autoantibodies to the signal recognition particle

We describe a case of polymyositis (PM) that developed in an 87-year-old woman with autoantibodies to the signal recognition particle. She complained of neither muscular weakness nor myalgia, although muscular weakness was present. Muscle biopsy was not performed because her consent was not obtained. The presence of myositis-specific autoantibodies is helpful in the diagnosis of PM/dermatomyositis lacking the characteristic clinical features or histopathologic confirmation.