Computational Prediction of Usutu Virus E Protein B Cell and T Cell Epitopes for Potential Vaccine Development

Usutu virus (family Flaviviridae), once confined to Africa, has emerged in Europe a decade ago. The virus has been spreading throughout Europe at a greater pace mostly affecting avian species. While most bird species remain asymptomatic carriers of this virus, few bird species are highly susceptible. Lately, Usutu virus (USUV) infections in humans were reported sporadically with severe neuroinvasive symptoms like meningoencephalitis. As so much is unknown about this virus, which potentially may cause severe diseases in humans, there is a need for more studies of this virus. In this study, we have used computational tools to predict potential B cell and T cell epitopes of USUV envelope (E) protein. We found that amino acids between positions 68 and 84 could be a potential B cell epitope, while amino acids between positions 53 and 69 could be a potential major histocompatibility complex (MHC) class I‐ and class II‐restricted T cell epitope. By homology 3D modeling of USUV E protein, we found that the predicted B cell epitope was predominantly located in the coil region, while T cell epitope was located in the beta‐strand region of the E protein. Additionally, the potential MHC class I T cell epitope (LAEVRSYCYL) was predicted to bind to nearly 24 human leucocyte antigens (HLAs) (IC₅₀ ≤5000 nm ) covering nearly 86.44% of the Black population and 96.90% of the Caucasoid population. Further in vivo studies are needed to validate the predicted epitopes.     

Evolutionarily Conserved Function of CD28 in αβ T Cell Activation

The functional role of the chicken homologue of CD28 was studied. It is expressed on all thymocytes, and both Vβ1- and Vβ2-family expressing peripheral αβ T cells. Peripheral γβ T cells are CD28-negative. Monoclonal antibody against CD28 had a costimulatory effect on T cells stimulated by phorbol myristate acetate (PMA), concanavalin A or MoAb against TCR. Vβl and Vβ2 expressing cells responded equally well to stimulation with anti-CD28 in combination with PMA. These responses were resistant to cyclosporin A, but inhibited by herbimycin A, suggesting that CD28 employs a signalling pathway at least partly distinct from that triggered by TCR/CD3. These data indicate a striking conservation of the costimulatory function of CD28 and emphasize the importance of this costimulatory pathway.     

Heat‐Shock Protein gp96 Enhances T Cell Responses and Protective Potential to Bacillus Calmette‐Guérin Vaccine

The commonly used Bacillus Calmette‐Guérin (BCG) vaccine only induces moderate T cell responses and is less effective in protecting against pulmonary tuberculosis (TB) in adults and ageing populations. Thus, developing new TB vaccine candidates is an important strategy against the spread of Mycobacterium tuberculosis. Here, we demonstrated that immunization with heat‐shock protein gp96 as an adjuvant led to a significantly increased CD4⁺ and CD8⁺ T cell response to a BCG vaccine. Secretion of the Th1‐type cytokines was increased by splenocytes from gp96‐immunized mice. In addition, adding gp96 as an adjuvant effectively improved the protection against intravenous challenge with Mycobacterium bovis BCG in mice. Our study reveals the novel property of gp96 in boosting the vaccine‐specific T cell response and its potential use as an adjuvant for BCG vaccines against mycobacterial infection.     

Induction of Protective Immune Responses by Immunization with Linear Multiepitope Peptides Based on Conserved Sequences from Plasmodium falciparum Antigens

A cysteine-containing peptide motif, EWSPCSVTCG, is found highly conserved in the circumsporozoite protein (CSP) and the thrombospondin-related anonymous protein (TRAP) of all the Plasmodium species analyzed so far and has been shown to be crucially involved in the sporozoite invasion of hepatocytes. We have recently shown that peptide sequences containing this motif, and also the antibodies raised against the motif, inhibit the merozoite invasion of erythrocytes. However, during natural infection, and upon immunization with recombinant CSP, this motif represents a cryptic epitope. Here we present the results of immunization studies with two linear multiepitopic constructs, a 60-residue (P60) and a 32-residue (P32) peptide, containing the conserved motif sequence. Both the peptides per se generated high levels of specific antibodies in BALB/c mice. P32 was found to be genetically restricted to H-2^d and H-2^b haplotypes of mice, whereas P60 was found to be immunogenic in five different strains of mice. The antibody response was predominantly targeted to the otherwise cryptic, conserved motif sequence in P60. Anti-P60 antibodies specifically stained the asexual blood stages of Plasmodium falciparum and Plasmodium yoelii in an immunofluorescence assay, recognized a 60- to 65-kDa parasite protein in an immunoblot assay, and blocked P. falciparum merozoite invasion of erythrocytes in a dose-dependent manner. Immunization with P60 also induced significant levels of the cytokines interleukin-2 (IL-2), IL-4, and gamma interferon in BALB/c mice. Moreover, >60% of mice immunized with P60 survived a heterologous challenge infection with a lethal strain of P. yoelii. These results indicate that appropriate medium-sized synthetic peptides might prove useful in generating specific immune responses to an otherwise cryptic but critical and putatively protective epitope in an antigen and could form part of a multicomponent malaria vaccine.     

HLA‐A2‐restricted Cytotoxic T Lymphocyte Epitopes from Human Hepsin as Novel Targets for Prostate Cancer Immunotherapy

Hepsin is a type II transmembrane serine protease that is overexpressed in prostate cancer, and it is associated with prostate cancer cellular migration and invasion. Therefore, HPN is a biomarker for prostate cancer. CD8⁺ T cells play an important role in tumour immunity. This study predicted and identified HLA‐A2‐restricted cytotoxic T lymphocyte (CTL) epitopes in human hepsin protein. HLA‐A2‐restricted CTL epitopes were identified using the following four‐step procedure: (1) a computer program generated predicted epitopes from the amino acid sequence of human hepsin; (2) an HLA‐A2‐binding assay detected the affinity of the predicted epitopes to the HLA‐A2 molecule; (3) the primary T cell response against the predicted epitopes was stimulated in vitro; and (4) the induced CTLs towards different types of hepsin‐ or HLA‐A2‐expressing prostate cancer cells were detected. Five candidate peptides were identified. The effectors that were induced by human hepsin epitopes containing residues 229 to 237 (Hpn229; GLQLGVQAV), 268 to 276 (Hpn268; PLTEYIQPV) and 191 to 199 (Hpn199; SLLSGDWVL) effectively lysed LNCaP prostate cancer cells that were hepsin‐positive and HLA‐A2 matched. These peptide‐specific CTLs did not lyse normal liver cells with low hepsin levels. Hpn229, Hpn268 and Hpn199 increased the frequency of IFN‐γ‐producing T cells compared with the negative peptide. These results suggest that the Hpn229, Hpn268 and Hpn199 epitopes are novel HLA‐A2‐restricted CTL epitopes that are capable of inducing hepsin‐specific CTLs in vitro. Hpn229, Hpn268 and Hpn199 peptide‐based vaccines may be useful for immunotherapy in patients with prostate cancer.     

乙型肝炎基因工程疫苗阻断乙型肝炎病毒母婴传播的研究

用转基因细胞（ＣＨＯ－Ｃ２８）分泌的乙型肝炎病毒表面抗原基因工程疫苗免疫母亲ＨＢｓＡｇ阳性的新生儿５０例，观察其阻断乙型肝炎病毒母婴传播的效果，随访１２个月，在３６例母亲为ＨＢｓＡｇ和ＨＢｅＡｇ均阳性的婴儿中仅１例为ＨＢｓＡｇ阳性，其余婴儿均有保护性抗体，预防保护率为９６．２％。１４例母亲单独ＨＢｓＡｇ阳性的所有婴儿抗ＨＢｓ均阳转，保护率达１００％。抗ＨＢｓ阳性的婴儿均具有较高抗体水平，抗ＨＢｓＧＭＴ为１１．１５６×１０５～１３．１３４×１０５ｍＩＵ／Ｌ。说明ＣＨＯ乙型肝炎基因工程疫苗具有较好的免疫原性和近期保护效果。     

Resistance of Neisseria meningitidis to Human Serum Depends on T and B Cell Stimulating Protein B

The ability of the human bacterial pathogen Neisseria meningitidis to cause invasive disease depends on survival in the bloodstream via mechanisms to suppress complement activation. In this study, we show that prophage genes coding for T and B cell stimulating protein B (TspB), which is an immunoglobulin-binding protein, are essential for survival of N. meningitidis group B strain H44/76 in normal human serum (NHS). H44/76 carries three genes coding for TspB. Mutants having all tspB genes inactivated did not survive in >5% NHS or IgG-depleted NHS. TspB appeared to inhibit IgM-mediated activation of the classical complement pathway, since survival of the tspB triple knockout was the same as that of the parent strain or a complemented mutant when the classical pathway was inactivated by depleting NHS of C1q and was increased in IgM-depleted NHS. A mutant solely carrying tspB gene nmbh4476_0681 was as resistant as the parent strain, while mutants carrying only nmbh4476_0598 or nmbh4476_1698 were killed in ≥5% NHS. The phenotype associated with TspB is formation of a matrix containing TspB, IgG, and DNA that envelopes aggregates of bacteria. Recombinant proteins corresponding to particular subdomains of TspB were found to have human IgG Fcγ- and/or DNA-binding activity, but only TspB derivatives containing both domains formed large, biofilm-like aggregates when combined with purified IgG and DNA. Recognizing the role of TspB in serum resistance may lead to a better understanding of why strains that carry tspB genes are associated with invasive meningococcal disease.     

Detachment‐Based Equilibrium of Anoikic Cell Death and Autophagic Cell Survival Through Adaptor Protein p66Shc

Anoikis (detachment‐induced cell death) confers a tumor‐suppressive function in metastatic cancer cells. Autophagy, a conserved self‐degradative process, enhances the anoikis resistance of detached cancer cells by maintaining cellular homeostasis. However, the mechanism of regulating cell fate‐decision by balancing anoikis and autophagy has been poorly understood. Our previous studies have shown that the adaptor protein p66^Shc mediates anoikis through RhoA activation and inhibits tumor metastasis in vivo. We also found that p66^Shc depletion mitigates nutrient‐deprivation‐induced autophagy. These findings suggest p66^Shc may coordinately regulate these two processes. To verify this hypothesis, we investigated the effect of p66^Shc on the cell death of detached lung cancer cells, and measured autophagy markers and autophagic flux. Results showed that p66^Shc depletion significantly inhibited anoikis, and reduced the formation of LC3B‐II and the degradation of Sequestosome 1 (SQSTM1, p62) in detachment‐induced cells. Using monodansylcadaverine (MDC)‐LysoTracker double staining and monomeric Cherry (mCherry)‐GFP‐LC3 assay, we found that the autophagic flux was also mitigated by p66^Shc depletion. In addition, p66^Shc knockdown increased the formation of full‐length X‐linked inhibitor of apoptosis (XIAP)‐associated factor 1 (XAF1), which enhances anoikis sensitivity. In conclusion, p66^Shc plays an essential role in detachment‐based equilibrium of anoikic cell death and autophagic cell survival. Anat Rec, 299:325–333, 2016. © 2015 Wiley Periodicals, Inc.     

Nucleotide Sequences and Functions of the Epstein-Barr Virus Latent Membrane Protein 1 Genes Isolated from Salivary Gland Lymphoepithelial Carcinomas

Epstein-Barr virus (EBV) infection is associated with salivary gland lymphoepithelial carcinoma (SLEC) and nasopharyngeal carcinoma (NPC). EBV is a ubiquitous herpes virus world wide, but EBV-associated SLEC and NPC are prevalent in restricted regions such as south areas of China, Southeastern Asia and Greenland (Eskimos). To examine whether particular EBV variants play roles in the development of SLEC and NPC, we isolated the complete EBV LMP1 genes from 12 paraffin-embedded biopsy samples of SLECs isolated from China, Taiwan and Russia, and compared these LMP1 genes with those of NPC (CAO) and the prototype B95-8 EBV. Nucleotide sequence analysis showed that SLECs LMP1 is more similar to that of CAO than that of prototype B95-8. The analysis also identified several conserved (67–100%) variations in SLEC-LMP1 and CAO-LMP1 distinct from B95-8-LMP1. These included 10-amino acid deletion, 5-amino acid deletion and 12-single amino acid variations. A SLEC-LMP1 gene with the aforementioned conserved variations inhibited the growth of an embryonic kidney cell line (293T), highly activated the NF-B pathway, and these activities were equivalent to those of B95-8 and CAO. These findings suggest that the biological functions of SLEC-LMP 1 are similar to those of B95-8-LMP1 and CAO-LMP1, and that these amino acid variations including the well-known 10-aa deletion did not affect these two prominent activities. While the present results could not uncover functional differences between SLEC-LMP1 and B95-8-LMP1, the nucleotide sequences and the molecular clone of LMP1 directly isolated from SLEC patients will be a useful tool to identify the high-pathogenic EBV strain(s), associated with SLEC and NPC.     

Identification of Aurora Kinase B and Wee1-Like Protein Kinase as Downstream Targets of V600EB-RAF in Melanoma

BRAF is the most mutated gene in melanoma, with approximately 50% of patients containing V600E mutant protein. ^V600EB-RAF can be targeted using pharmacological agents, but resistance develops in patients by activating other proteins in the signaling pathway. Identifying downstream members in this signaling cascade is important to design strategies to avoid the development of resistance. Unfortunately, downstream proteins remain to be identified and therapeutic potential requires validation. A kinase screen was undertaken to identify downstream targets in the ^V600EB-RAF signaling cascade. Involvement of aurora kinase B (AURKB) and Wee1-like protein kinase (WEE1) as downstream proteins in the ^V600EB-RAF pathway was validated in xenografted tumors, and mechanisms of action were characterized in size- and time-matched tumors. Levels of only AURKB and WEE1 decreased in melanoma cells, when ^V600EB-RAF, mitogen-activated protein kinase 1/2, or extracellular signal–regulated kinase 1/2 protein levels were reduced using siRNA compared with other identified kinases. AURKB and WEE1 were expressed in tumors of patients with melanoma at higher levels than observed in normal human melanocytes. Targeting these proteins reduced tumor development by approximately 70%, similar to that observed when inhibiting ^V600EB-RAF. Furthermore, protein or activity levels of AURKB and WEE1 decreased in melanoma cells when pharmacological agents targeting upstream ^V600EB-RAF or mitogen-activated protein kinase were used to inhibit the ^V600EB-RAF pathway. Thus, AURKB and WEE1 are targets and biomarkers of therapeutic efficacy, lying downstream of ^V600EB-RAF in melanomas.Melanoma remains the most common cause of skin cancer–related deaths worldwide.¹ The incidence of melanoma increases with age, with a 28% probability of disease for individuals <40 years and a ≥70% probability for those >60 years.² Approaches to manage advanced melanoma include surgery, radiation, immunotherapy, chemotherapy, or combinations of these approaches. Patients in the advanced stages of this disease have few treatment options for long-term management of the disease, with average 5-year survival being 10%.³ Therefore, a better understanding of the genes and processes regulating melanoma that could be used for selection of therapeutic targets as biomarkers for particular drug efficacy or prognostic indicators to assist in therapeutic agent selection and for overcoming resistance to targeted agents is needed.Kinases play a key role regulating cellular proliferation and drug resistance development.⁴ In the mitogen-activated protein (MAP) kinase pathway, 50% and 25% of sporadic melanomas harbor BRAF or NRAS mutations, respectively, which activate the MAP kinase pathway measured through the activation of extracellular signal–regulated kinase (ERK).⁵ These mutations rarely occur in the same cell, but both mutations activate pathways to regulate diverse cellular processes aiding cancer development, with the most prominent being regulation of cellular proliferation.⁶ The most frequent BRAF mutation is a valine to glutamic acid substitution at residue 600 (V600E), which increases basal kinase activity.⁷ The most common NRAS mutation is a glutamine to leucine substitution (Q61L), which impairs GTP hydrolysis and maintains a constitutively active protein.⁸Pharmacological agents have been developed to inhibit the activity of various proteins in the deregulated MAP kinase signaling pathway.^9–12 Recent FDA approval of Zelboraf (vemurafenib; formerly known as PLX4032), is a major breakthrough for individuals with mutant ^V600EB-RAF.^13–16 Vemurafenib leads to a high response rate in patients, but in most cases, more invasive resistant disease eventually recurs by circumventing ^V600EB-RAF, leading to mortality.^13,16,17 Therefore, a better understanding of downstream members of the ^V600EB-RAF pathways is needed so that these proteins could be targeted together with vemurafenib or inhibited after the development of resistance to more effectively manage this disease.To identify novel kinases regulating the proliferative potential of melanoma cells and then pinpoint those lying downstream of ^V600EB-RAF in this signaling cascade, an siRNA-based screen of a library of 636 kinases was undertaken. AURKB, Wee1-like protein kinase (WEE1), glycogen synthase kinase-3α (GSK3A), thiamin pyrophosphokinase 1 (TPK1), and B-RAF were identified as potential modulators of melanoma cell survival. The aurora kinase family consists of aurora kinase A (AURKA), aurora kinase B (AURKB), and aurora kinase C (AURKC).¹⁸ Involvement of AURKA in melanoma development has been reported, but it is not known whether AURKB and AURKC play roles in melanoma pathogenesis or development of drug resistance.¹⁹ WEE1 is a dual-specificity protein kinase involved in regulating cell cycle progression by phosphorylating and deactivating cyclin-associated CDKs.^20,21 WEE1 currently has no known role in melanoma development. Two isoforms of GSK-3, called GSK3A and GSK-3β (GSK3B), have been identified. Although GSK3B has been shown to play a role in melanoma development and drug resistance,²² GSK3A has not been identified as a melanoma therapeutic target. The TPK catalyzes phosphorylation of thiamin to thiamin pyrophosphate and also has no known role in melanoma development.²³This study shows that AURKB, WEE1, GSK3A, and TPK1 were all expressed in tumors of patients with melanoma at higher levels than observed in normal human melanocytes. However, only AURKB and WEE1 levels decreased when ^V600EB-Raf, mitogen-activated protein kinase (MEK) 1/2, or ERK1/2 were targeted using siRNA, demonstrating that these proteins were downstream of ^V600EB-RAF in the deregulated MAP kinase signaling pathway. Subsequent studies confirmed that targeting AURKB or WEE1 reduced melanoma tumor development and led to a phenotype similar to that observed when inhibiting ^V600EB-RAF in this deregulated signaling cascade. Furthermore, AURKB or WEE1 levels decreased when pharmacological agents inhibiting ^V600EB-Raf or MEK were used to target melanoma cells. Thus, AURKB and WEE1 can be used as downstream therapeutic targets and as biomarkers of efficacy of agents targeting the ^V600EB-RAF signaling cascade in melanomas.     

Differential Effect of TPA on Cell Growth and Epstein-Barr Virus Reactivation in Epithelial Cell Lines Derived from Gastric Tissues and B Cell Line Raji

We characterized the cell growth and Epstein-Barr virus (EBV) reactivation for EBV infected epithelial cell lines, GT38, GT39, and GTC-4 using 12-O-tetradecanoylphorbol-13-acetate (TPA). These cell lines grew similarly in liquid medium, and formed colonies in soft agar. The cell growth was inhibited with TPA, dose-dependently in liquid medium. The colony formation was enhanced with low concentrations of TPA, but was inhibited with high concentrations. The latent EBV was reactivated with high concentrations of TPA as shown by the expression of EBV BZLF1 gene product ZEBRA. The effects of TPA on GTC-4 were compared with a Burkitt's lymphoma cell line Raji. The mode of actions of TPA in GTC-4 was different from Raji in terms of cell growth and EBV reactivation. The effective concentrations of TPA for cell growth inhibition and EBV reactivation were higher in Raji than GTC-4. Cell cycle analysis showed that TPA (20 ng/ml) induced cell cycle arrest to Raji but not to GTC-4; however, the rate of trypan blue stained cells increased in the TPA treated GTC-4 but not Raji. These results demonstrated that TPA affects differentially for the stimulation and inhibition of cell growth, and also EBV reactivation depends on TPA concentrations and cell types.     

Identification of a Novel B Cell Epitope on the Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus by Phage Display

A phage display peptide library targeting the nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-1a was generated and used for epitope mapping. After 3 rounds of biopanning with the monoclonal antibody (MAb) N3H2 directed against the N protein, 3 positive phages were screened and sequenced. These phages share a consensus sequence, IQTAFNQGA, which corresponds to the amino acid (AA) 79–87 segment of the CH-1a N protein. A small DNA fragment coding for IQTAFNQGA was expressed as a fusion product, and reacted to N3H2 in Western blots and indirect ELISA. Four truncated peptides (IQTAFNQG, IQTAFNQ, QTAFNQGA, and TAFNQGA) expressed as GST fusion products failed to react with N3H2. The sequences around the N3H2-binding site among the N proteins of 57 PRRSV strains were compared. Our results indicate that the IQTAFNQGA motif is highly conserved among North American and European isolates. We concluded that the precisely defined nona-peptide epitope is a novel conserved Linear B cell epitope on the N protein of PRRSV.     

T and B cell clonal expansion in Ras‐associated lymphoproliferative disease (RALD) as revealed by next‐generation sequencing

Ras‐associated lymphoproliferative disease (RALD) is an autoimmune lymphoproliferative syndrome (ALPS)‐like disease caused by mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) or neuroblastoma RAS viral (V‐Ras) oncogene homologue (NRAS). The immunological phenotype and pathogenesis of RALD have yet to be studied extensively. Here we report a thorough immunological investigation of a RALD patient with a somatic KRAS mutation. Patient lymphocytes were analysed for phenotype, immunoglobulin levels and T cell proliferation capacity. T and B cell receptor excision circles (TREC and KREC, respectively), markers of naive T and B cell production, were measured serially for 3 years. T and B cell receptor repertoires were studied using both traditional assays as well as next‐generation sequencing (NGS). TREC and KREC declined dramatically with time, as did T cell receptor diversity. NGS analysis demonstrated T and B clonal expansions and marked restriction of T and B cell receptor repertoires compared to healthy controls. Our results demonstrate, at least for our reported RALD patient, how peripheral T and B clonal expansions reciprocally limit lymphocyte production and restrict the lymphocyte receptor repertoire in this disease. Decreased naive lymphocyte production correlated with a clinical deterioration in our patient's immune status, suggesting that TREC and KREC may be used as an aid in monitoring disease progression. Both the methodologies used here and the conclusions regarding immune homeostasis may be applicable to the research of ALPS and other immune dysregulation syndromes.     

Epstein–Barr virus‐driven B Cell Proliferation with CD4+ T Cell Expansion: A Lymphomatoid Granulomatosis‐like Disease Related to Hyperinterleukin‐10 Secretion of Remarkably Favourable Outcome with Rituximab

Granulomatous lymphomatosis is an Epstein–Barr virus (EBV)‐driven B cell proliferation associated with an exuberant CD4⁺ T cell reaction with usually histopathological pictures of angiocentrism. So far, the characteristics of CD4⁺ T cells in granulomatous lymphomatosis and the mechanism leading to their expansion remain poorly explored. We report a 56‐year‐old female with a past history of cold agglutinin disease, which was successfully treated with 4 weekly infusions of rituximab. She presented one year later with features of granulomatous lymphomatosis that resulted in severe lung and bone marrow infiltration. We provide evidence that CD4⁺ T cell expansion was oligoclonal, involved anergic cells and did not result from an EBV‐driven stimulation. Rather, it resulted possibly from a high production of interleukin‐10 by immunoblastic EBV‐positive B cells. The outcome was remarkably favourable with rituximab and steroids. Our results suggest that an EBV‐driven B cell proliferation should be investigated in patients presenting with a CD4⁺ T cells alveolitis or other systemic manifestations resulting from a CD4⁺ T cell expansion. These features should prompt to introduce an immunosuppressive therapy including steroids and rituximab. Our results deserve further investigations to confirm our pathophysiological hypotheses in CD4⁺ T cell expansions associated with EBV‐driven B cell proliferations and to assess whether granulomatous lymphomatosis could result from comparable mechanisms.     

Association of Inducible T Cell Costimulator Polymorphisms with Susceptibility and Outcome of Hepatitis B Virus Infection in a Chinese Han Population

Inducible T cell costimulator (ICOS) functions to regulate cell–cell signalling, immune responses and cell proliferation. ICOS single nucleotide polymorphism (SNP) may affect protein expression and functions. This study investigated the association of ICOS SNPs with hepatitis B virus (HBV) infection and outcome in a Chinese population. A total of 1290 Chinese Han individuals were enrolled, including 63 asymptomatic HBV carriers, 220 chronic hepatitis B patients (CHB), 249 HBV‐related liver cirrhosis patients (LC), 108 patients with HBV‐related hepatocellular carcinoma (HCC), 338 patients with natural HBV clearance and 312 healthy subjects (as controls). DNA samples from these subjects were genotyped for four ICOS SNPs (rs11883722, rs10932029, rs1559931 and rs4675379) using TaqMan SNP Genotyping Assay and analysed. The data showed that genotype and allele frequencies of ICOS SNPs in cases and controls followed the Hardy–Weinberg distribution. The CC genotype of rs4675379 was higher in patients with HBV infection (including AC, CHB, LC and HCC) than in patients with HBV clearance (P = 0.006). Furthermore, the genotype ‘GA’ and the minor allele ‘A’ of rs1559931 were associated with a decreased HCC susceptibility (P < 0.001). Haplotype analysis data showed that ‘GC’ haplotype in block 2 (rs1559931 and rs4675379) had a lower frequency in patients than in HBV‐cleared subjects (P = 0.034), although its overall frequency was only 1.6%. Our study found that ICOS rs1559931 SNP was associated with decreased HBV‐related HCC risk in the studied Chinese Han population, except for patients with natural clearance of HBV.     

Envelope and Long Terminal Repeat Sequences of an Infectious Murine Leukemia Virus from a Human SCLC Cell Line: Implications for Gene Transfer

Development of methods for gene transfer into specific cell types or tissues is important for experimental research as well as clinical therapeutical approaches. We report here the cloning and characterization of the envelope (env) gene and the U3 region of a retrovirus from an infected human Small Cell Lung Cancer (SCLC) cell line. The replication of this murine retrovirus is also fully supported by other lung cancer cell lines of different histological origin. We present evidence that a long terminal repeat (LTR)-β-galactosidase (β-Gal) reporter construct performed as well as an analogous cytomegalovirus (CMV) promoter β-Gal construct in the human lung epithelial cell line A549 and in the human larynx carcinoma cell line HEp2. This revised version was published online in July 2006 with corrections to the Cover Date.     

T细胞免疫球蛋白和黏蛋白结构域分子1(Tim-1)促进马尔堡病毒感染的作用研究

目的 体外评价Tim-1对马尔堡假病毒感染细胞的促进作用。方法 通过转染MARV-GP质粒制备马尔堡假病毒,并通过瞬转Tim-1表达质粒制备高表达Tim-1的HEK-293T细胞作为靶细胞,采用生物发光法评价Tim-1及其多态性对MARV感染的促进作用,最后检测Tim-1-ECD重组蛋白对Tim-1促进作用的阻断效果。结果 成功制备了马尔堡假病毒且对HEK-293T细胞有较高的感染性,Tim-1能丰度依赖性地增强马尔堡假病毒的感染。Tim-1-359aa和-364aa均有促进作用,Tim-1-359aa的增强效应强于Tim-1-364aa。Tim-1-ECD重组蛋白可显著抑制Tim-1对马尔堡假病毒感染的促进作用。结论 Tim-1能够显著增强马尔堡假病毒的感染性。