This study was aimed to explore the method for induction and expansion of EB virus specific cytotoxic T lymphocytes (EBV-CTL) in vitro, and to detect their killing effect. Peripheral blood mononuclear cells (PBMNC) were collected from 6 EBV seropositive healthy donors, and EBV-transformed B lymphoblastoid cells (BLCL)were used as the antigen-presenting cells and antigen stimulant which was irradiated by 40 Gy (60)Co irradiator. The autologous PBMNC and irradiated BLCL were cultured to induce and expand the EBV-CTL, and the immunophenotype was identified by the flow cytometry. The killing effect of the EBV-CTL against the autologous BLCL (autoBLCL), the autologous PHA cultured B lymphoblastoid cells( PHA-BLCL), the allogeneic BLCL (alloBLCL) and the K562 cells were measured with LDH release assay under different effector-to-target ratio. The results showed that the 6 cell lines of EBV-CTL were induced and expanded from the EBV seropositive healthy donors, the overall increase in cell numbers varied from 18.6 to 55.0 times. After 10 stimulations, the specific killing efficiency of the EBV-CTL for the autoBLCL were 59.4%, 43.2% and 29.0% under the effector-to-target ratio of 20: 1, 10: 1 and 5: 1. The nonspecific killing efficiency for the PHA-blast, alloBLCL and K562 cells were 7.1%, 9.4% and 10.3% (P < 0.05) under the 20: 1 ratio; 6.6%, 8.3% and 8.1% (P < 0.05) under 10: 1; 5.4%, 7.3% and 6.3% (P < 0.05) under 5: 1, respectively. It is concluded that the EBV-CTL can be successfully induced and expanded ex vivo for specific killing of HLA matched BLCL and may become a potential treatment for EBV related post-transplant lymphoproliferative disorders.
B-Lymphocytes ; immunology ; Cell Line, Transformed ; Herpesvirus 4, Human ; immunology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; immunology ; virology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; virology

OBJECTIVETo investigate the effect of HBV antigens and pathological mechanism of chronic HBV infection by analyzing the cellular immune function of peripheral blood mononuclear cells (PBMCs) from HBsAg carriers.

METHODSPBMCs were prepared from individuals with chronic asymptomatic HBV infection and cultured in the presence of different antigens and/ or cytokines. The levels of cytokines in culture supernatants were detected by ELISA method. The phenotype of the cells was detected by FACS.

RESULTSThe levels of IFN y secreted by PBMCs from HBsAg carriers were (48.3+/-19.8) pg/ml, significantly lower than that from healthy controls (t = 3.023, P less than 0.05); The IFN y produced by PBMCs from HBeAg positive patients due to HBsAg and HBcAg stimulation were (50.4+/-51.6) pg/ml and (63.2+/-36.9) pg/ml, significantly lower than that of HBeAg negative patients (t = 2.468 and 3.184, P less than 0.05, respectively). The IL-12p70 secreted by PBMCs from HBeAg positive patients was also significantly lower than that of HBeAg negative patients (P less than 0.05); Exogenous IL-12 promoted significantly PBMCs to secrete IFN y (P less than 0.01) and IL-12 combined with HBV antigens activated CD8+CD45RA+CCR7+ and CD8+CD45RA-CD62L+ cells. IL-12 secreted by PBMCs decreased in HBeAg positive patients, which may be the crucial reason of viral persistence in chronic HBV carriers. Exogenous IL-12 combined with specific HBV antigen could promote the central memory CD8+ T cells to produce IFN y.

Adolescent ; Adult ; Carrier State ; blood ; immunology ; virology ; Case-Control Studies ; Hepatitis B ; blood ; immunology ; Hepatitis B Antigens ; blood ; Hepatitis B virus ; immunology ; Humans ; Interferon-gamma ; blood ; Interleukin-12 ; blood ; immunology ; Leukocytes, Mononuclear ; immunology ; T-Lymphocytes ; immunology ; Young Adult

Classical swine fever (CSF) is a contagious swine disease charactered by hemorrhagic fever and leukopenia,usually leading to substantial economic losses. To obtain a insight of leucopenia caused by CSFV infection, DNA microarray analyses of peripheral blood leucocytes (PBL) of the infected pigs was performed. Three health pigs were inoculated with a lethal dose of CSFV Shimen strain and their PBLs were isolated when the onset of typical clinical signs and then subjected to total RNA extraction followed by microarray analysis with Affymetrix Porcine Genome Array GeneChips. The results showed that the significant differences were observed in cellular apoptotic genes expression at 7 days post-infection (p. i.). The changes of the genes expression were confirmed by real time RT-PCR of some selected apoptosis-related genes. This study provided a valuable information for further investigating the molecular mechanism of apoptosis caused by CSFV infection.
Animals ; Apoptosis ; Cells, Cultured ; Classical Swine Fever ; genetics ; immunology ; virology ; Classical swine fever virus ; immunology ; physiology ; Gene Expression Profiling ; Leukocytes, Mononuclear ; cytology ; immunology ; virology ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Sus scrofa

It is known that CD28, a positive costimulatory receptor, plays a very important role in inducing the optimal stimulation of T lymphocytes. CTLA-4 (CD152), however, acts as a negative regulator in T lymphocyte activation. The effect of an feline immunodeficiency virus (FIV) infection on the expression of feline CD28 and CTLA-4 was studied with FIV-infected and uninfected peripheral blood mononuclear cells (PBMC) using a competitive PCR assay. The nature of CD28 and CTLA-4 expression was also examined with fresh and antigen-stimulated PBMC. FIV infection induced a lower expression of CD28, but a higher expression of CTLA-4 in the infected PBMC than in the uninfected PBMC. Relatively high levels of CD28 expression were demonstrated in both the fresh and the antigen-stimulated PBMC. The expression level of CTLA-4 in the freshly isolated PBMC was rather low, however, FIV antigen stimulation induced a relatively high expression of CTLA-4 in feline PBMC.
Animals ; Antigens, CD ; Antigens, CD28/*biosynthesis ; Antigens, Differentiation/*biosynthesis ; Antigens, Viral/*immunology ; Cats ; Cell Survival ; Cells, Cultured ; Gene Expression ; Immunodeficiency Virus, Feline/immunology/*physiology ; Leukocytes, Mononuclear/immunology/*virology ; Polymerase Chain Reaction/veterinary

OBJECTIVETo study the effect and mechanism of the peripheral blood mononuclear cell (PBMC) invasion by HBV on artificial immunization in newborns.

METHODSFifty-two newborns of HBsAg positive mothers were immunized with HBIG (hepatitis B immunoglobulin) and HBVac (hepatitis B vaccine) and were followed up for 7 months. The newborns' HBV-DNA in serum and in the PBMCs was detected with nested-PCR; anti-HBs was tested with solid phase radioimmunoassay (SP-RIA). PBMCs isolated from newborn peripheral blood were incubated in the presence of PHA or purified HBsAg. Interleukin-2 (IL-2) level in culture supernatants of activated cells was detected by ELISA.

RESULTSThe failure rate of immunization was higher in infants with positive HBV-DNA in PBMCs than those with negative HBV-DNA (P < 0.05); IL-2 level in PBMC culture supernatants was lower in former than in the latter and in normal controls (P < 0.05). The level of IL-2 in the immunization failure newborns was lower than that in the successfully immunized newborns and in normal controls (P < 0.05).

CONCLUSIONSIntrauterine invasion of PBMCs by HBV is one of the important reasons for immunization failure in newborns. IL-2 production is closely related to the invasion of PBMCs by HBV, which may contribute to the failure of artificial immunization in newborns.

DNA, Viral ; blood ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; adverse effects ; Hepatitis B virus ; isolation & purification ; physiology ; Humans ; Immunization ; adverse effects ; Immunoglobulins ; immunology ; Infant, Newborn ; Interleukin-2 ; biosynthesis ; blood ; Leukocytes, Mononuclear ; virology

OBJECTIVETo study whether dendritic cells (DCs) derived from the peripheral blood in chronic hepatitis B patients can induce specific T cell immune response.

METHODS(1)The subjects were divided into 3 groups: chronic hepatitis B group (CHB), acute hepatitis B group (AHB), and normal donor group (ND). The peripheral blood mononuclear cells (PBMCs) isolated from those subjects were stimulated with HBcAg 18 to 27 CTL epitope peptide, and intracellular cytokine staining (ICCS) was used for detecting IFN-gamma, IL-2 and TNF-alpha produced by CD8+ T cell. (2) DCs generated from PBMCs were pulsed with HBcAg 18 to 27 CTL epitope peptide, then were cocultured with autologous lymphocytes for 10 days to induce antigen-specific T cell, which was assessed by ICCS and cytotoxic assay.

RESULTS(1) The memory effect of the PBMCs from AHB group to HBcAg 18 to 27 CTL epitope peptide was stronger than that from CHB or ND group (t=2.508-3.305, P<0.05). (2)After lymphocytes were cocultured with DC treated with HBcAg 18 to 27 CTL epitope peptide, antigen-specific T cell effect was induced. And the killing rates were (57.0+/-23.0)%, (49.5+/-20.2)%, (21.8+/-12.9)% at the effector/target of 30:1, 10:1, 3:1, which were higher than that in control group.

CONCLUSIONSThe memory T cells against HBV antigen lacks in CHB patients. DCs from CHB patients pulsed with HBcAg 18 to 27 epitope peptide can induce HBV antigen-specific T cell, which can kill specific target cells and produce cytokines involved in virus clearance.

Adult ; CD8-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; virology ; Epitopes, T-Lymphocyte ; immunology ; Female ; Hepatitis B Core Antigens ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Leukocytes, Mononuclear ; immunology ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo study the mechanism and significance of peripheral blood mononuclear cell (PBMC) of neonates infected with hepatitis B virus (HBV).

METHODSEighty-four HBsAg-positive and HBeAg-negative mothers and their newborns were recruited in this study. Sixteen hepatitis B virus markers (HBVM)-negative mothers and their neonates were served as control. All these cases had no symptoms of hepatitis, serious pregnancy complications and preexisting disease. Age, gestational age and the method of delivery were matched in two groups (P > 0.05). Five ml blood samples were taken from the peripheral vein of the pregnant women before delivery and from neonates within 24 hours after birth, before inoculation of HBV vaccine (HBVac). Serum and PBMC were isolated from 2 ml and 3 ml samples respectively. The sera, PBMC and the last supernatant of PBMC washing were stored at -80 degrees C. HBVM of neonates were detected by using enzyme linked immunosorbent assay (ELISA). HBV DNA in serum, PBMC and the last supernatant of PBMC washing of mothers and neonates were detected by using a nested-polymerase chain reaction (n-PCR). Two pairs of oligonucleotide primers, the outer primer pair for first PCR and inner primer pair for second PCR, designed according to region S of HBV genome were synthesized at Shanghai Cell Biology Institute of Chinese Academy of Sciences. The neonates who were HBV DNA positive in PBMC but HBsAg and HBV DNA negative in serum were followed up for one year, HBsAb in serum and HBV DNA in PBMC were observed in the neonates.

RESULTS(1) The positive rate of HBV DNA in 84 serum and PBMC of mothers were 53.57% and 40.48%, respectively (chi(2) = 2.891, P > 0.05). All the results were weakly positive. (2) Twenty-four (28.57%) newborns in the study group were infected, including 7 who were only HBV DNA positive in serum, 11 only HBV DNA positive in PBMC and 6 in both, all the results were weakly positive. HBsAg was negative in all the newborns. None of the neonates in control group was infected with HBV. There was significant difference between the two groups (chi(2) = 4.55, P < 0.05). (3) Of all the study cases, 11 (13.10%) neonates were HBV DNA weakly positive in PBMC but HBsAg and HBV DNA negative in serum. Of their mothers, 5 were only HBV DNA positive in serum, 2 only positive in PBMC and 4 positive in both serum and PBMC. Seven of the 11 neonates were followed up for one year and at the end of follow-up, 4 were HBsAb positive and HBV DNA negative in PBMC; 3 were HBsAb negative, and among the 3 cases HBV DNA in 2 was still positive in PBMC, HBsAg and HBV DNA in serum were negative in all the 7 neonates.

CONCLUSION(1) HBV DNA positivity either in serum or in PBMC in mothers can result in infection of PBMC with HBV in their neonates. (2) PBMC infection with HBV can exist for a long time in neonates while HBsAg and HBV DNA are negative in serum, and may result in vaccination failure in neonates.

Case-Control Studies ; DNA, Viral ; blood ; Female ; Hepatitis B ; diagnosis ; immunology ; Hepatitis B Vaccines ; administration & dosage ; Hepatitis B virus ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; Leukocytes, Mononuclear ; virology ; Pregnancy

OBJECTIVETo study the relationship between HBeAg expression and HBV-DNA in serum and peripheral blood mononuclear cells (PBMCs).

METHODS208 patients with chronic hepatitis B were included in this present study. HBV-DNA in the PBMCs were performed by polymerase chain reaction (PCR), with the serum HBV-DNA level being determined by the way of fluoresces quantities PCR (FQ-PCR). Meanwhile, HBV-GM was also detected via enzyme-linked immunosorbent assay (ELISA).

RESULTSThere were 106 patients for positivity in the HBV-DNA level of PBMCs with 102 for negativity, in which the HBV-DNA high levels (HBV DNA load > or = 1.0E5) in serum were 91.5%, 45.1% (chi2=52.12, P>0.01) respectively, with 76.4% and 50.9% (chi2=21.55, P>0.01) for the positive percentage of HBeAg expression.

CONCLUSIONA significantly positive correlation was found between HBV-DNA in PBMCs and serum HBV-DNA along with the positive percentage of HBeAg, indicating that obvious PBMCs' increase infected by HBV in patients with positivity of HBeAg and high level of serum HBV-DNA.

Adolescent ; Adult ; Aged ; Antibodies, Viral ; blood ; DNA, Viral ; blood ; genetics ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B ; genetics ; immunology ; Hepatitis B e Antigens ; immunology ; Hepatitis B, Chronic ; blood ; virology ; Humans ; Leukocytes, Mononuclear ; virology ; Male ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Young Adult

OBJECTIVERotavirus is the single most common cause of severe dehydrating diarrhea in young children worldwide, but the pathogenesis and immunity against this disease are not completely understood. A prospective study was conducted to assess gene expression of toll-like receptors (TLR) in children with acute rotavirus diarrhea.

METHODSSeventy-five children with acute rotavirus diarrhea and 38 control children were enrolled in this study from Sep. 2004 to Jan. 2005. All the 75 patients had detailed records of clinical characteristics. Rotavirus antigen was detected by ELISA from stools. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll reagent and RNA was extracted by Trizol. The levels of mRNA for five TLRs in PBMC were examined by fluorescent quantitative RT-PCR.

RESULTSPatients with acute rotavirus infection had elevated mean levels of TLR 2, 3, 4, 7, 8 mRNA expressions in PBMC within 3 days since onset of the disease, P less than 0.05. But only TLR 2, 3, 8 mRNA levels remained increased in patients within 7 or 14 days since onset (P less than 0.05). Mean levels of mRNA for TLR 4 in PBMC was higher in patients with more severe diarrhea including longer duration of diarrhea, more episodes of diarrhea per day and higher severity scores (P less than 0.05).

CONCLUSIONManifold TLR may play roles in the start-up and regulation of immune responses in children with acute rotavirus diarrhea. These findings will be helpful to further recognize immune response in Chinese children with rotavirus diarrhea and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.

Acute Disease ; Antigens, Viral ; analysis ; Child, Preschool ; Diarrhea ; genetics ; virology ; Enzyme-Linked Immunosorbent Assay ; Feces ; virology ; Gene Expression ; Host-Pathogen Interactions ; Humans ; Infant ; Leukocytes, Mononuclear ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus ; immunology ; physiology ; Rotavirus Infections ; genetics ; virology ; Toll-Like Receptors ; genetics

OBJECTIVETo observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1.

METHODSLeukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR).

RESULTSThe optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05).

CONCLUSIONSThe RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Coculture Techniques ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; virology ; Diterpenes ; pharmacology ; Fetal Blood ; cytology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; immunology ; Influenza, Human ; drug therapy ; immunology ; virology ; Interferon-beta ; genetics ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; drug effects ; immunology ; virology ; Macrophages ; drug effects ; virology ; NF-kappa B ; genetics ; metabolism ; Promoter Regions, Genetic ; drug effects ; immunology ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; genetics ; immunology