PURPOSE: The prognostic significance of tumor-infiltrating lymphocytes (TILs) has been determined in breast cancers. Interferons can affect T-cell activity through direct and indirect mechanisms. Myxovirus resistance A (MxA) is an excellent marker of interferon activity. Here,we evaluated TILs and MxA expression in human epidermal growth factor receptor 2 (HER2)–positive breast cancers. MATERIALS AND METHODS: Ninety cases of hormone receptor (HR)+/HER2+ tumors and 78 cases of HR–/HER2+ tumors were included. The TILs level was assessed using hematoxylin and eosin–stained full face sections, and MxA expressionwas evaluated by immunohistochemistrywith a tissue microarray. RESULTS: MxA protein expression was significantly higher in tumors with high histologic grade (p=0.023) and high levels of TILs (p=0.002). High levels of TILs were correlated with high histological grade (p=0.001), negative lymphovascular invasion (p=0.007), negative lymph node metastasis (p=0.007), absence of HR expression (p < 0.001), abundant tertiary lymphoid structures (TLSs) around ductal carcinoma in situ (p=0.018), and abundant TLSs around the invasive component (p < 0.001). High levels of TILs were also associated with improved disease-free survival, particularly in HR–/HER2+ breast cancers. However, MxA was not a prognostic factor. CONCLUSION: High expression of MxA in tumor cells was associated with high levels of TILs in HER2-positive breast cancers. Additionally, a high level of TILs was a prognostic factor for breast cancer, whereas the level of MxA expression had no prognostic value.
Breast Neoplasms ; Breast* ; Carcinoma, Intraductal, Noninfiltrating ; Disease-Free Survival ; Epidermal Growth Factor* ; Hematoxylin ; Humans* ; Interferons ; Lymph Nodes ; Lymphocytes, Tumor-Infiltrating* ; Myxovirus Resistance Proteins ; Neoplasm Metastasis ; Orthomyxoviridae* ; Receptor, Epidermal Growth Factor* ; T-Lymphocytes

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.
2',5'-Oligoadenylate Synthetase/genetics/metabolism ; Animals ; Cell Line ; *Cloning, Molecular ; Ducks ; Gene Expression Regulation/*physiology ; Humans ; Immunity, Innate ; Myeloid Differentiation Factor 88/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/metabolism ; Newcastle Disease/metabolism ; Newcastle disease virus/classification ; RNA, Messenger/genetics/metabolism ; Species Specificity ; Toll-Like Receptor 5/genetics/*metabolism

BACKGROUNDHuman myxovirus resistant protein A (MxA), encoded by the myxovirus resistance 1 (Mx1) gene, is an interferon (IFN)-triggered dynamin-like multi-domain GTPase involved in innate immune responses against viral infections. Recent studies suggest that MxA is associated with several human cancers and may be a tumor suppressor and a promising biomarker for IFN therapy. Mx1 gene mutations in the coding region for MxA have been discovered in many types of cancer, suggesting potential biological associations between mutations in MxA protein and corresponding cancers. In this study, we performed a systematic analysis based on the crystal structures of MxA and elucidated how these mutations specifically affect the structure and therefore the function of MxA protein.

METHODSCancer-associated Mx1 mutations were collected and screened from the COSMIC database. Twenty-two unique mutations that cause single amino acid alterations in the MxA protein were chosen for the analysis. Amino acid sequence alignment was performed using Clustal W to check the conservation level of mutation sites in Mx proteins and dynamins. Structural analysis of the mutants was carried out with Coot. Structural models of selected mutants were generated by the SWISS-MODEL server for comparison with the corresponding non-mutated structures. All structural figures were generated using PyMOL.

RESULTSWe analyzed the conservation level of the single-point mutation sites and mapped them on different domains of MxA. Through individual structural analysis, we found that some mutations severely affect the stability and function of MxA either by disrupting the intra-/inter-molecular interactions supported by the original residues or by incurring unfavorable configuration alterations, whereas other mutations lead to gentle or no interference to the protein stability and function because of positions or polarity features. The potential clinical value of the mutations that lead to drastic influence on MxA protein is also assessed.

CONCLUSIONSAmong all of the reported tumor-associated single-point mutations, seven of them notably affect the structure and function of MxA and therefore deserve more attention with respect to potential clinical applications. Our research provides an example for systematic analysis and consequence evaluation of single-point mutations on a given cancer-related protein.

Amino Acid Sequence ; Cell Transformation, Neoplastic ; genetics ; Crystallography, X-Ray ; Databases, Genetic ; Humans ; Myxovirus Resistance Proteins ; genetics ; Neoplasm Proteins ; genetics ; Neoplasms ; genetics ; Point Mutation ; Protein Domains ; genetics ; Protein Folding ; Sequence Alignment ; Stereoisomerism ; Structure-Activity Relationship

Animals ; Binding Sites ; Capsid ; chemistry ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; HIV-1 ; chemistry ; immunology ; Humans ; Mice ; Myxovirus Resistance Proteins ; chemistry ; immunology ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Tertiary ; Recombinant Proteins ; chemistry ; immunology ; metabolism

Treatment with interferon beta (IFN-beta) induces the production of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in patients with multiple sclerosis (MS). NAbs against IFN-beta are associated with a loss of IFN-beta bioactivity and decreased clinical efficacy of the drug. The objective of this study was to evaluate the incidence and the prevalence of binding antibodies (BAbs) and neutralizing antibodies (NAbs) to IFN-beta in MS patients receiving CinnoVex, Rebif, or Betaferon. The presence of BAbs was studied in serum samples from 124 MS patients using one of these IFN-beta medications by ELISA. The NAbs against IFN-beta were measured in BAb-positive MS patients receiving IFN-beta using an MxA gene expression assay (real-time RT-PCR). Of the 124 patients, 36 (29.03%) had BAbs after at least 12 months of IFN-beta treatment. The proportion of BAb+ was 38.1% for Betaferon, 21.9% for Rebif, and 26.8% for CinnoVex. Five BAb-positive MS patients were lost to follow-up; thus 31 BAb-positive MS patients were studied for NAbs. NAbs were present in 25 (80.6%) of BAb-positive MS patients receiving IFN-beta. In conclusion, the three IFN-beta preparations have different degrees of immunogenicity.
Adolescent ; Adult ; Antibodies/*blood/immunology ; Antibodies, Neutralizing/*blood/immunology ; Cross Reactions ; DNA, Complementary/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interferon-beta/*immunology/therapeutic use ; Male ; Middle Aged ; Multiple Sclerosis/drug therapy/*immunology ; Myxovirus Resistance Proteins/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

OBJECTIVETo investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN alpha.

METHODSThe HepG2 cells were transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen, Pre-S antigen and core antigens. The infectious supernatant from HepG2.2.15 cells and the pured HBV proteins which contained the S, Pre-S antigens were used to treat the HepG2 cells. Northern blot and RT-PCR were applied to analyse the expressions of the antiviral proteins MxA, 2' -5' OAS, 9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN alpha treatment.

RESULTSThe HepG2 cells transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg, Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN alpha antiviral proteins MxA, 2' -5' OAS and 9-27. When transfected with pHBV-dimer, pHBS2-S, pHBc-EGFP plasmids, the IFN/A antiviral proteins MxA, 2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells, and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore, the expression of IFN alpha JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells.

CONCLUSIONSThe HBV and its antigens influence the expressions of IFN alpha JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is indicated that HBV might possess the activity to antagonise or counteract the IFN alpha antiviral action.

2',5'-Oligoadenylate Synthetase ; metabolism ; GTP-Binding Proteins ; metabolism ; Hep G2 Cells ; Hepatitis B Antigens ; immunology ; Hepatitis B virus ; immunology ; Humans ; Interferon-alpha ; metabolism ; Myxovirus Resistance Proteins ; RNA-Binding Proteins ; metabolism ; STAT1 Transcription Factor ; metabolism ; Signal Transduction ; Transfection

OBJECTIVETo study the human myxovirus resistant protein A (MxA), a specifically induced peptide by interferon I, and to use its level as a diagnostic criterion for viral infections.

METHODSAnti-MxA antisera from immunized mice were prepared with the expressed MxA protein of pET32a-MxA in E. coli BL-21(DE3). To confirm the antiserum activity and specificity, the expression product of BL21, wild type MxA pEGFP-C1-wMxA and site-directed mutant MxA pEGFP-C1-mMxA(N589S) stably transfected 3T3 cells and induced A549 cells were detected by Western blot with the antisera using non-MxA transfected or non-IFN-beta induced cells, intact A549, NIH 3T3 cells transfected with pEGFP-C1 and pET32a (+)-transformed BL-21 as controls.

RESULTSThe antisera had specific positive immunoreactivity to the NIH3T3 cells transformed with pEGFP-C1-wMxA and pEGFP-C1-mMxA, INF-beta induced A549 cells and BL21 proteins expressed with pET32a (+)-MxA. The hybridization signals from IFN-beta induced A549 cells depended on the IFN-beta inducing concentrations. Meanwhile, immunohistochemical assay showed that NIH 3T3 cells with pEGFP-C1-wMxA and pEGFP-C1-mMxA had > 98% of positive cells at 1:50 dilution of the serum and A549 cells induced by 20 ng/mL IFN-beta for 48 h showed 95% positive cells. pEGFP-C1-transfected NIH 3T3 cells were all negative.

CONCLUSIONAnti-sera are highly specific to diversified MxAs. The antibody is detectable by Western blot, immunocytochemistry and immunofluorescence assay.

Animals ; Antibody Specificity ; Cell Line, Tumor ; GTP-Binding Proteins ; genetics ; immunology ; metabolism ; Gene Expression Regulation ; Humans ; Mice ; Myxovirus Resistance Proteins ; NIH 3T3 Cells ; Species Specificity

OBJECTIVESTo identify the host single nucleotide polymorphisms (SNP) of myxovirus resistance A (MxA) protein and eukaryote initiation factor 2alfa regulatory region 2(eIF-2a-reg2) and to predict interferon (IFN) treatment responses in patients with chronic hepatitis B.

METHODSTwo hundred sixty-two patients with chronic hepatitis B (CHB) were treated with interferon alfa (IFN ) for 12 months. Six months later the therapeutic effectiveness was evaluated. All the patients had signed a formal consent form. The patients were grouped into a sustained response (SR) group and a non-sustained response (NSR) group according to their responses to the IFNa treatment. Single nucleotide polymorphisms of the antiviral protein MxA promoter -88,-123 and protein kinase(PKR) activated eIF-2a-reg2 sites were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and were compared with the responsiveness to IFN treatment of these CHB patients.

RESULTSAmong the 262 patients, 212 (80.9%) were non-sustained responders to IFNa and 50 (19.1%) were sustained responders. The rate of sustained responders with GT heterozygote at MxA promoter -88 was higher than that of the GG genotype (OR: 5.3, 95% CI: 2.46-11.43, P less than 0.01) and also higher than that of the TT genotype (OR: 4.1, 95% CI: 1.86-9.09, P less than 0.01). There were no statistically significant differences in IFN therapeutic effectiveness among the patients with different genotypes at MxA promoter -123, eIF-2a-reg2 and haplotypes made by MxA promoter -88 G/T, and -123 C/A alleles (P more than 0.05).

CONCLUSIONPatients with GT genotype at MxA promoter -88 responded well to IFN treatment. SNP as a potential marker could be used to predict IFN treatment responses of patients with chronic hepatitis B.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; Eukaryotic Initiation Factor-2 ; genetics ; Female ; GTP-Binding Proteins ; genetics ; Genotype ; Hepatitis B virus ; Hepatitis B, Chronic ; drug therapy ; genetics ; Humans ; Interferon-alpha ; therapeutic use ; Male ; Middle Aged ; Myxovirus Resistance Proteins ; Polymorphism, Single Nucleotide ; Regulatory Sequences, Nucleic Acid ; genetics ; Young Adult

OBJECTIVETo study the relationship between a G/T substitution at position -88 of myxovirus resistance-1 gene (MxA) and the self-limiting or chronic infection of HBV.

METHODSBlood samples from 100 patients with self-limiting HBV infection (positive anti-HBs and anti-HBc) and from 340 patients with chronic HBV infection were collected. MxA-88 G/T polymorphism was typed using a protocol based on competitively differentiated-polymerase chain reaction. For statistical analysis, odds ratio and chi-square test were used.

RESULTSThe detective rate of G/G genotype (low expression genotype) of MxA-88 G/T was 50.2% (221/440), those of T/T genotype (high expression genotype) and G/T heterozygous genotype were 5.5% (24/440) and 44.3% (195/440). Compared to patients with chronic infection, patients with self-limiting infection had lower frequency of G/G genotype (41.0% vs 52.9%, P < 0.05) or G allele (62.5% vs 75.9%, P < 0.01) and had higher frequency of T/T genotype (16.0% vs 2.4%, P < 0.01) or T allele (37.5% vs 24.1%, P < 0.01), but there was no significant difference in the G/T heterozygous genotype.

CONCLUSIONSMxA gene -88 G/T polymorphism influences the natural outcomes of HBV infection to some extent. This SNP of MxA gene may be used as a clinical prognostic marker of HBV infection.

Adult ; Biomarkers ; Female ; GTP-Binding Proteins ; biosynthesis ; genetics ; Genotype ; Hepatitis B, Chronic ; genetics ; Humans ; Male ; Myxovirus Resistance Proteins ; Polymorphism, Single Nucleotide ; genetics ; Prognosis

Animals ; Antiviral Agents ; therapeutic use ; GTP-Binding Proteins ; therapeutic use ; Hepatitis, Viral, Human ; drug therapy ; Humans ; Interferon-alpha ; therapeutic use ; Interferon-gamma ; therapeutic use ; Interferons ; therapeutic use ; Myxovirus Resistance Proteins ; Polyethylene Glycols ; Recombinant Proteins