In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
Animals ; Antibodies, Viral ; genetics ; immunology ; Cricetinae ; Cytomegalovirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin M ; immunology ; Mice ; Protein Sorting Signals ; Recombinant Fusion Proteins ; immunology

Biomarkers ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; Cytomegalovirus Retinitis ; immunology ; Female ; Humans ; Male ; Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; metabolism ; virology

OBJECTIVETo investigate the percentages of peripheral blood γδ T cells and regulatory T cells (Treg) and the expression of associated cytokines, interleukin 17 (IL-17) and transforming growth factor-β1 (TGF-β1), in infants with human cytomegalovirus (HCMV) infection.

METHODSTwenty-two infants with HCMV infection (HCMV group) and 22 healthy infants who underwent physical examination (control group) were enrolled in this study. The percentages of peripheral blood γδ T cells and Treg cells were determined by flow cytometry. The levels of IL-17 and TGF-β1 in plasma were measured using ELISA.

RESULTSCompared with the control group, the HCMV group had significantly higher percentage of γδ T cells and IL-17 level (P<0.01) and significantly lower percentage of Treg cells and TGF-β1 level (P<0.01). In the HCMV group, the percentage of γδ T cells was negatively correlated with the percentage of Treg cells and TGF-β1 level (P<0.05), but positively correlated with IL-17 level (P<0.05); the percentage of Treg cells was positively correlated with TGF-β1 level (P<0.05), but negatively correlated with IL-17 level (P<0.05); there was no correlation between IL-17 level and TGF-β1 level (P>0.05).

CONCLUSIONSThere is an imbalance between γδ T cells and Treg cells in the peripheral blood of infants with HCMV infection, and γδ T cells may be involved in the secretion of IL-17.

Cytokines ; blood ; Cytomegalovirus Infections ; immunology ; Female ; Humans ; Infant ; Interleukin-17 ; blood ; Male ; Receptors, Antigen, T-Cell, gamma-delta ; analysis ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta1 ; blood

OBJECTIVETo study the relationship between the expression of peripheral blood HLA-DR, CD4CD25 regulatory T cells, IL-17 and IL-27 with liver damage in children with human cytomegalovirus (HCMV) infection.

METHODSTwenty-one HCMV children with liver damage and twenty-one HCMV children without liver damage were enrolled in this study. The expression of peripheral blood HLA-DR and CD4CD25 regulatory T cells was detected by flow cytometry. Plasma levels of IL-17 and IL-27 were measured using ELISA.

RESULTSThe plasma levels of IL-17 and IL-27 in children with liver damage were significantly higher than in those without liver damage, while the expression of peripheral blood CD4CD25 regulatory T cells was lower than in those without liver damage (P<0.05). Plasma IL-17 and IL-27 levels were negatively correlated with the expression of peripheral blood CD4CD25 regulatory T cells (P<0.01).

CONCLUSIONSImmune imbalance mediated by CD4CD25 regulatory T cells and over-expression of IL-17 and IL-27 may be involved in the pathogenesis of liver damage in children with HCMV infection.

CD4 Antigens ; immunology ; Cytomegalovirus ; physiology ; Cytomegalovirus Infections ; blood ; complications ; genetics ; Female ; Flow Cytometry ; HLA-DR Antigens ; genetics ; immunology ; Humans ; Infant ; Interleukin-17 ; blood ; genetics ; Interleukin-2 Receptor alpha Subunit ; immunology ; Interleukins ; blood ; genetics ; Liver ; injuries ; metabolism ; Liver Diseases ; blood ; etiology ; immunology ; Male ; T-Lymphocytes, Regulatory ; immunology

No abstract available.
Asian Continental Ancestry Group/*genetics ; Base Sequence ; Bone Marrow Cells/cytology/pathology ; Cytomegalovirus Infections/diagnosis ; Epstein-Barr Virus Infections/diagnosis ; Female ; Flow Cytometry ; Heterozygote ; Humans ; Infant ; Killer Cells, Natural/cytology/immunology ; Lymphohistiocytosis, Hemophagocytic/*diagnosis/genetics ; Perforin/*genetics ; Phagocytosis ; Polymorphism, Single Nucleotide ; Republic of Korea ; Sequence Analysis, DNA

BACKGROUNDRheumatic diseases involve multiple organs that are affected by immunological mechanisms. Treatment with corticosteroids and immunosuppressive agents may also increase the frequency of infection. Cytomegalovirus (CMV) is a widespread herpes virus and a well-recognized pathogen, which causes an opportunistic and potentially fatal infection in immunocompromised patients. This retrospective study aimed to investigate the clinical and laboratory characteristics of CMV pneumonia in patients with rheumatic diseases after immunosuppressive therapy in a single center in Shanghai, China.

METHODSEight hundred and thirty-four patients with rheumatic diseases who had undergone CMV-DNA viral load tests were included, and the medical records of 142 patients who were positive for CMV-DNA in plasma samples were evaluated. GraphPad Prism version 5.013 (San Diego, CA, USA) was used to conduct statistical analysis. The correlation between CMV-DNA viral loads and lymphocyte counts was assessed using the Spearman rank correlation coefficient test. Significance between qualitative data was analyzed using Pearson's Chi-squared test. The cut-off thresholds for CMV-DNA viral load and lymphocyte count were determined by receiver operating characteristic (ROC) curve analysis.

RESULTSOne hundred and forty-two patients had positive CMV viral load tests. Of these 142 patients, 73 patients with CMV pneumonia were regarded as symptomatic, and the other 69 were asymptomatic. The symptomatic group received higher doses of prednisolone (PSL) and more frequently immunosuppressants than the asymptomatic group (P < 0.01). The symptomatic group had lower lymphocyte counts, especially CD4+ T-cells, than the asymptomatic group (P < 0.01). By ROC curve analysis, when CD4+ T-cell count was <0.39 × 109/L, patients with rheumatic diseases were at high risk for symptomatic CMV infection. The CMV-DNA load was significantly higher in the symptomatic patients than that in asymptomatic patients (P < 0.01; threshold viral loads: 1.75 × 104 copies/ml). Seven patients had a fatal outcome, and they had lower peripheral lymphocyte counts (P < 0.01), including CD4+ and CD8+ T-cells (P < 0.01).

CONCLUSIONSWhen CD4+ T-cell count is <0.39 × 109/L, patients are at high risk for pulmonary CMV infection. Patients are prone to be symptomatic with CMV-DNA load >1.75 × 104 copies/ml. Lymphopenia (especially CD4+ T-cells), presence of symptoms, and other infections, especially fungal infection, are significant risk factors for poor outcome, and a higher PSL dosage combined with immunosuppressants may predict CMV pneumonia.

CD4-Positive T-Lymphocytes ; metabolism ; China ; Cytomegalovirus ; pathogenicity ; Cytomegalovirus Infections ; genetics ; immunology ; therapy ; virology ; Humans ; Immunosuppression ; methods ; Pneumonia ; genetics ; immunology ; therapy ; virology ; Polymerase Chain Reaction ; Retrospective Studies ; Rheumatic Diseases ; genetics ; immunology ; therapy ; virology ; Viral Load

OBJECTIVETo study the possible immunological mechanism of wheezing attack in children with cytomegalovirus (CMV) infection.

METHODSA total of 25 under-5-year-old children with wheezing following CMV infection were enrolled. The expression of serum regulatory T cells (Treg)/T helper 17 (Th17) cytokines interleukin (IL)-10, IL-6, and IL-17, and peripheral blood lymphocyte subsets were determined. Twenty age-matched healthy children were selected as the control group.

RESULTSThe wheezing group had a significantly reduced serum IL-10 level, significantly increased IL-6 and IL-17 levels, significantly reduced levels of natural killer cells, and significantly increased levels of CD8+ T cells and CD19+ B cells, as compared with the control group.

CONCLUSIONSWheezing children with CMV infection have Treg/Th17 imbalance and cellular immune dysfunction, which may be an important immunological mechanism of the development of wheezing in children after CMV infection.

Child, Preschool ; Cytokines ; blood ; Cytomegalovirus Infections ; immunology ; Female ; Humans ; Infant ; Male ; Respiratory Sounds ; etiology ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology

The human cytomegalovirus (HCMV) is a widespread herpesvirus. Virus reactivation from latency can lead to stillbirth, miscarriage, fetal anomalies, and intrauterine growth retardation. During latent infection with the HCMV, the virus can be cleared by the immune response or apoptosis of host cells. However, the HCMV has developed several strategies to manipulate expression of its genes and the microenvironment of host cells. Recent studies have shown that latent infection with the HCMV is associated with viral: regulation of early expression of genes; evasion of cell death; evasion of the immune response; regulatin of non-coding RNAs. This review summarizes recent research progress on the mechanisms underpinning latent infection with the HCMV.
Animals ; Cytomegalovirus ; genetics ; physiology ; Cytomegalovirus Infections ; immunology ; virology ; Humans ; Viral Proteins ; genetics ; metabolism ; Virus Latency

To explore the association between T-cell receptor beta variable (TCR BV) complementarity determining region 3 (CDR3) spectratyping and CMV activation in the recipients of allogeneic hematopoietic stem cell transplantation (HSCT).Fluorescence quantitative PCR melting curve analysis was used to sequence 24 TCR BV families in 7 HSCT recipients and 3 healthy controls. CMV-pp65 antigenemia was measured by immunohistochemical staining. Plasma IgM specific for CMV was identified using ELISA. Relationship between TCR BV families and CMV activation was statistically analyzed.Twenty-four TCR BV families were expressed in 3 healthy controls, while TCR BV CDR3 sequencing results in 7 recipients turned out to be BV9, BV11, BV17, BV20 and so on. Amino acid sequence features were as follows:TCR BV9 contained "QVRGGTDTQ", TCR BV11 contained "VATDEQ" and "LGDEQ", TCR BV17 contained "IGQGNTEA", and TCR BV20 contained "VGLAANEQ". Five recipients suffered from pp65 antigenemia in 3 month after transplantation, and pp65-positive cells ranged from 2 to 15 per 5×10white blood cells. Three recipients were CMV-IgM positive. No significant differences were found in TCR BV families between pp65-positive recipients and pp65-negative recipients (all>0.05). But there was statistically significant difference in frequency of TCR BV11 between CMV-IgM negative recipients and CMV-IgM positive recipients (<0.05).T cell immune response was characterized by special TCR BV CDR3 spectratyping in HSCT recipients, and TCR BV11 expression may be associated with CMV activation.
Amino Acid Sequence ; Complementarity Determining Regions ; genetics ; immunology ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; genetics ; Genotype ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Lymphocyte Activation ; genetics ; Phosphoproteins ; Polymerase Chain Reaction ; Polymorphism, Genetic ; immunology ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; immunology ; Spleen ; T-Lymphocytes ; immunology ; virology ; Viral Matrix Proteins

BACKGROUND: Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. METHOD: 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. RESULTS: The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. CONCLUSIONS: Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients.
Antiviral Agents/therapeutic use ; Cytomegalovirus/*genetics ; Cytomegalovirus Infections/drug therapy/pathology/virology ; DNA, Viral/*blood/metabolism ; Ganciclovir/therapeutic use ; Humans ; *Immunoassay ; Organ Transplantation ; Phosphoproteins/genetics/immunology/*metabolism ; *Real-Time Polymerase Chain Reaction ; Viral Matrix Proteins/genetics/immunology/*metabolism ; Virology/*methods