[Genetic characterization of haemagglutinin of novel H1N1 influenza viruses in Suzhou City].

OBJECTIVE: To investigate the genetic variation and molecular characteristics of HA gene of influenza A (H1N1) virus isolated in Suzhou during from2009 to 2011. METHODS: Viral RNA of 5 Suzhou isolates was isolated and their HA gene were amplified and sequenced by the primers and protocol recommended by WHO, and the sequences together with other sequences downloaded from GenBank were analyzed by several bioinformatics software. RESULTS: Compared with vaccine strain, the average homogeneity of nucleotide and amino acids of 5 Suzhou isolates were between 98.8-99.4% and 98.8-99.4% respectively. All of the 5 strains have 1 amino acids replacement in Sb region and 2 strains have 2 amino acids replacement in Ca region. Strains from and outside Suzhou both showed a trend of clustering by collection year. CONCLUSION: The Suzhou strains are still in stable condition although 1-2 amino acids replacement had happened in antigenic sites.

[The application of molecular differential diagnoses platform in identification of the respiratory pathogens].

AIM: To establish a MDD (molecular differential diagnoses) platform for diagnosing the pathogens that may cause respiratory infection by combination of the advanced Tem-PCR(Target enriched multiplex PCR)with xMAP(multiple analyses profiling), and to evaluate the reliability and further use the platform to test clinic samples. METHODS: 22 throat swab specimen from outpatient patients of respiratory department in First Affiliated Hospital of Suzhou University and 20 respiratory tract lavage fluid specimen from inpatients of respiratory department in Affiliated Children Hospital of Suzhou University were collected, and the nucleic acids of the samples were amplified by Tem-PCR and xMAP. RESULTS: Testing of the the known samples showed that the platform had excellent specificity and sensitivity. Testing of the clinic samples showed that the positive rate of the respiratory tract lavage fluid specimen was 63.6%, higher than that of the throat swab specimen, and that the positive rate of RNA pathogens was higher than that of DNA pathogens. CONCLUSION: A reliable MDD platform for detection of respiratory pathogens has been established with high-throughput detection capacity.

Generation of rat monoclonal antibodies against murine LAIR-1.

The leukocyte-associated Ig-like receptor 1 (LAIR-1), an inhibitory receptor bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIM), is expressed on the majority of peripheral leukocytes, including NK cells, T cells, B cells, monocytes, dendritic cells, granulocytes, and thymocytes and is involved in immunologic regulation and hematopoiesis. Murine LAIR-1 (mLAIR-1) is the homolog molecule of human LAIR-1. Using mLAIR-1-Fc as the immunogen and the technique of rat B lymphocyte hybridoma, we raised three hybridoma cell lines secreting monoclonal antibodies (MAb) to mLAIR-1, designated FMU-mLAIR-1.1, -1.2, and -1.3. Rat immunoglobulin class and subclass of the MAb FMU-mLAIR-1.1 approximately 3 were determined to be IgM, IgG1, and IgM, respectively. All these MAbs can bind the mLAIR-1 in immunocytochemistry and immunohistochemistry. FMU-m LAIR-1.2 worked well not only in Western blot assay but also in recognizing natural LAIR-1 molecules on the surface of P388D1, J774, and WEHI3 cells, and mLAIR-1 cDNA-transfected CHO cells detected by FCM. Thus, successful production of rat anti-murine LAIR-1 monoclonal antibodies provides a new powerful tool for investigation of murine LAIR-1 function in mouse model, both in vitro and in vivo.

[Comparison of the binding affinity of LAIR-1(CD305) and LAIR-2(CD306) with their ligands].

AIM: To compare the binding affinity of LAIR-1 and LAIR-2 with their ligands. METHODS: The ligand-expressing cells were incubated with the LAIR-1 and/or LAIR-2 fusion proteins at different concentration at the same time or in succession. Then the interaction changes and competition were measured by influorescent staining and flow cytometric analysis with the specific mAbs against LAIR-1 or LAIR-2. RESULTS: The membrane ligand for LAIR-1 and LAIR-2 was expressed extensively and ligand recognition was of cross-species. LAIR-2 blocked the interaction of LAIR-1 with its ligand but LAIR-1 didn't block the interaction of LAIR-2 with its ligand. CONCLUSION: LAIR-1 and LAIR-2 probably bind to the same ligand with different affinity, which provides some significant evidence for investigating the molecular mechanisms of LAIR-1 family in modulating immune response.

[Expression and function of CD226 on NK cells activated by Superantigens].

AIM: To study the regulation of superantigen in expression and function of CD226 on NK cells. METHODS: Double fluorescent staining and flow cytometry analysis were employed to detect the expression of CD226 on NK cells from human peripheral blood mononuclear cells (PBMC) stimulated by staphylococcus enterotoxin A (SEA) or SEB. (51)Cr release assay was performed to compare the cytotoxicities of NK cells in different activation models. Laser scanning confocal microscope was used to observe the distribution of CD226 on NK cells activated by superantigens at killing stage. RESULTS: In resting NK cells, the percentage of CD56(+) NK cells and CD56(+) CD226(+) cells were 12.3% and 1.4% respectively, and the cytotoxicity of NK cells against K562 cells was (3.2+/-0.2)% at the ratio of 5:1. After PBMC were stimulated by 0.1 microg/mL SEA and SEB for 1 day, the percentage of CD56(+) NK cells was 13.5% and 14.1% respectively, and the percentage of CD56(+) CD226(+) cells were increased. Interestingly, CD226 was mainly expressed on CD56(dim) NK cells. On day 2 after SEA/SEB stimulation, the proportions of CD56(+) CD226(+) cells among CD56(+) cells were 69.1% and 64.3% in SEA and SEB group respectively. On day 3 after SEA/SEB stimulation, the expression of CD226 on NK cells decreased. Furthermore, the cytotoxicity of NK cells stimulated by SEA or SEB for 3 days against K562 cells were much higher than that of the resting NK cells as well as NK cells cultured without SAg for the same culture time (P<0.05), and reached to the peak at day 2 (82.3%+/-6.9% and 80.6%+/-7.5%, respectively). Additionally, we observed that CD226 molecules were colocalized with LFA-1 at the interface of NK cells which contacted K562 cells. CONCLUSION: The cytotoxicity of NK cells enhanced by SEA or SEB may be correlation with the increased expression of CD226 molecule, and CD226 may be involved in synapse formation of NK cells at killing stage.

[The influence of TLSFJM on the proportion of Th1/Th2-like cell subsets].

AIM: To explore the influence of TLSF(JM) on the proportion of alloantigen activated Th1 and Th2-like cell subsets. METHODS: TLSF(JM) or IL-4 was added to mixed lymocyte reaction(MLR) system. The influence of TLSF(JM) on the proportion of Th1 and Th2-like cell subsets was analyzed by intracellular immunofluorescence staining and FACS. RESULTS: In the TLSF(JM) group, the proportion of IFN-gamma(+) cells differentiated from activated lymphoblast descended from 49.8% to 43.1%, IL-4(+) cells from 75.4% to 43.7% and IL-6(+) cells from 67.8% to 52.6%. The similar tendency was also observed in the unactivated small lymphocytes. CONCLUSION: TLSF(JM) can inhibit both the Th1 and Th2-like cell subsets, but mainly inhibit the Th2-like cell subset, thereby reducing the proportion of Th2-like cell subsets.

[Using flow cytometry to detect peripheral blood eosinophils and their related molecules].

AIM: To set up an accurate and rapid method to detect the phenotype of peripheral blood eosinophils using flow cytometry. METHODS: 10(-4) mol/L of theophylline, 10(-4) of dexamethasone and 10(-8) mol/L of rhIL-5 were added to peripheral blood sampled from normal human subjects. Anti-CD16-PE mAb and FITC-labelled mAbs to eosinophil's molecules were used to perform double labeling staining. Simultaneously, CD16-FL2 was used to install gating so as to accurately locate eosinophils. And then their molecules were examined and analyzed. RESULTS: The eosinophils were accurately located. Theophylline and dexamethasone effectively inhibited activation of eosinophils and shedding of CD62L on eosinophils caused by IL-5. CONCLUSION: Our method can accurately detect eosinophils in peripheral blood samples and their molecules. In addition, only a tiny amount of blood sample is needed and few artificial factors are involved in the detection. Therefore, this method may be an ideal both in basic immunological research and clinical laboratory examination.

Preparation and characterization of a set of monoclonal antibodies to TRAIL and TRAIL receptors DR4, DR5, DcR1, and DcR2.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo 2L) is a novel cytotoxic ligand belonging to TNF superfamily. Among TRAIL receptors, death receptor 4 (DR4) and DR5 containing death domain (DD) in their cytoplasmic region mediate apoptosis-signaling upon TRAIL binding, while decoy receptor 1 (DcR1) and DcR2 with a truncated or non-functional DD play "decoy" role. The interaction of TRAIL and TRAIL receptors plays important roles both in immunoregulation and immune pathogenesis of some diseases. In this study, we raised hybridomas secreting monoclonal antibodies against TRAIL (FMU1.1, 1.2, 1.3), DR4 (FMU1.4), DR5 (FMU1.5, 1.6), DcR1 (FMU1.7) and DcR2 (FMU1.8, 1.9). These MAbs could be used for fluorescent staining and flow cytometry (FCM) analysis as well as immunohistochemistry (IHC). Moreover, FMU1.1, 1.3, 1.4 and 1.5 could be used as coating antibodies paring corresponding polyclonal antibodies to develop sandwich ELISAs to quantitate the soluble TRAIL (sTRAIL), sDR4 or sDR5 in serum samples respectively. In addition, cross-linking of DR4/DR5 by FMU1.4 or FMU1.5 MAbs could induce apoptosis of some DR4/DR5-expressing tumor cells. Thus, this set of monoclonal antibodies against TRAIL or TRAIL receptors may be useful in expression phenotypic and functional study of TRAIL and TRAIL receptors.

Localization of TRAIL/TRAILR in fetal pancreas.

AIM: To observe the localization of TRAIL/TRAIR (DR4, DR5, DcR1, DcR2) in the fetal pancreas. METHODS: Fetal pancreas of 32 weeks of pregnancy were obtained from induced abortions, embedded in paraffin, and 4-microm sections were prepared. The localization of TRAIL/TRAILR in fetal pancreas was investigated by fluorescence immunohistochemical method combined with laser scanning confocal microscopy. RESULTS: TRAIL immunoreactive cells were mainly located on the periphery of the pancreas islets. There were a few DcR1 and DcR2 positive cells whereas there were no immunoreactive cells of DR4 and DR5 in the pancreas islets. In the acini and the ducts of the exocrine pancreas there were no TRAIL/TRAILR immunoreactive cells. CONCLUSION: This study not only describes the distribution of TRAIL/TRAILR in the fetal pancreas, but also provides a morphological basis for deducing the function of TRAIL/TRAILR in pancreas, suggesting that in normal pancreatic islets, the pancreatic cells are resistant towards apoptosis too.

[Establishment and characterization of human alloantigen induced T cell clones].

AIM: To estabilish and analyze stable human T cell clones cultured in-vitro. METHODS: The activated T cells from mixed lymphocyte culture (MLC) were cloned by limiting dilution using irradiated PBMCs as feeder cells. T cell clones were then characterized by immunofluorescence staining and flow cytometry analysis. RESULTS: 16 clones were obtained, of which all but one were alphabetaT cells. Among the 15 alphabetaT cell clones, 9 clones belonged to Th subset, including 3 Th0 and 6 Th2, and 6 clones belonged to Tc subset, including 5 Tc0 and 1 Tc2. CONCLUSION: Fifteen human T cell clones were successfully established, which lays the foundation for study on markers and function of Tc1 and Tc2 subsets.