[The potential role of lung dendritic cells and Th17/regulatory T cells in patients with chronic obstructive pulmonary disease].

Objective: To explore the role of lung dendritic cells (DCs) and Th17/regulatory T cells (Treg) pathway in the pathogenesis of chronic obstructive pulmonary disease(COPD). Methods: COPD patients who received lobectomy from Sep. 2015 to Mar. 2016 in our hospital were enrolled and classified into non-smoking non-COPD group, smoking without COPD group and COPD group. The expression of CD(80), chemokine recepter-6 (CCR6), interleukin-17A (IL-17A) and fork-head transcription factor P3 (FoxP3) were detected by immunohistochemistry (IHC) in lung tissue. Mature DCs (mDCs), immature DCs (imDCs), Th17 cells and Treg cells in lung tissue were detected by flow cytometry (FCM) and the correlation between Th17/Treg cells with lung function was analyzed. Results: (1) The expression of CD(80) and FoxP3 in COPD group was decreased, while the expression of CCR6 and IL-17A was increased (P<0.05). (2) The percentage of mDCs and Treg in lung tissue of COPD group was significantly decreased. In contrast, the proportion of imDCs and Th17 cells in COPD group was significantly increased (P<0.05). (3) The imbalance of Th17/Treg ratio in lung tissue was seen in patients with COPD, suggesting the potential mechanism of Th17 cell-mediated proinflammatory response. (4) The percentage of Th17 cells and Th17/Treg ratio in COPD patients was negatively correlated with forced expiratory volume in the first second (FEV(1)) as a percentage of predicted value (FEV(1)% pred), forced vital capacity(FVC) as a percentage of predicted value (FVC% pred), FEV(1)/FVC. On the other hand, the percentage of Treg cells was positively correlated with FEV(1)% pred, FVC% pred, FEV(1)/FVC. Conclusions: The data in this study demonstrate the maturation disorder of dendritic cells in lung tissue of COPD patients. The imbalance of Th17/Treg ratio suggests that Th17 cell-mediated proinflammatory response may be involved in the pathogenesis of COPD.

Selective replication of E1B55K-deleted adenoviruses depends on enhanced E1A expression in cancer cells.

E1B55K-deleted dl1520 could selectively replicate in cancer cells and has been used in clinical trials as an antitumor agent. The mechanism of virus selective replication in cancer cells, including a possible role of p53, is unclear. Studies with established cancer cell lines have demonstrated that some cancer cells are resistant to dl1520 replication, regardless of the p53 status. Hep3B cells supported the E1b-deleted adenoviruses to replicate, whereas Saos2 cells were resistant to viral replication. We applied p53-null Hep3B and Saos2 cells as models to clarify the replication ability of E1B55K-deleted adenoviruses with different expression levels of E1a. We show that lower E1A expression in Saos2 may be the reason for the poor replication in some cancer cells due to the fact that E1a promoter was less activated in Saos2 than in Hep3B. We also demonstrate that the E1B55K protein can increase E1A expression in Saos2 cells for efficient virus replication. In addition, the upstream regions of the E1a promoter have transcriptional activity in Hep3B cells but not in Saos2 cells. The viral E1B55K protein may activate cancer cellular factor(s) that targets the upstream regions of the E1a gene to increase its expression. This is the first study demonstrating that E1B55K protein affects the E1A production levels that is related to cancer selective replication. Our studies have suggested that increase of E1A expression from E1b-deleted adenoviruses may enhance killing cancer cells that otherwise are resistant to viral replication.

DNA vaccination against neu reduces breast cancer incidence and metastasis in mice.

The gene for HER2/neu is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. The HER2/neu gene product, a transmembrane protein kinase member of the EGF receptor family, has significant potential as a tumor antigen for vaccination. We inserted the sequence for neu into a novel plasmid called ELVIS containing a Sindbis virus replicon that reproduces multiple copies of mRNA. Mice vaccinated one time intramuscularly demonstrated a strong antibody response against A2L2, a murine breast cancer cell line transfected to express neu. Vaccinated mice challenged in the mammary fatpad with A2L2 had reduced tumor incidence and reduced tumor mass compared to mice challenged with tumor cells lacking the neu insert. Intradermal vaccination was also protective and required 80% less plasmid for a similar level of protection. Vaccination reduced the incidence of lung metastasis from mammary fatpad tumors and reduced the number of lung metastases resulting from intravenous injection of A2L2 cells. Cytotoxic T lymphocytes cultures of immune spleen cells with P815-neu cells produced high levels of interferon-gamma indicating an antigen-specific Th1-type immune response resulting from the vaccination. In a spontaneous breast tumor model using neu transgenic mice, vaccination with ELVIS-neu protected against development of spontaneous breast tumors. Our preclinical data indicate that therapeutic vaccination of patients with ELVIS-neu may reduce metastasis from HER2/neu-expressing breast and ovarian tumors.

A Cre-expressing cell line and an E1/E2a double-deleted virus for preparation of helper-dependent adenovirus vector.

Adenoviral vectors are attractive for the delivery of transgenes into mammalian cells because of their efficient transduction, high titer, and stability. The major concerns with using E1-deleted adenoviral vectors in gene therapy are the pathogenic potential of the virus backbone and the leaky viral protein synthesis that leads to host immune responses and a short duration of transgene expression. Helper-dependent (HD) adenoviral vectors that are devoid of all viral protein-coding sequences have significantly increased the safety and reduced the immunogenicity of these vectors. Currently available HD vectors depend on an E1-deleted adenovirus as a helper to provide viral proteins in trans. As a consequence, contamination with helper virus cannot be avoided in the HD vector preparation though it can be decreased to 0.01% using a Cre/loxP mechanism. Since the presence of E1-deleted helper virus may have substantial unwanted effects, we have developed a new Cre-expressing cell line based on an E1- and E2a-complementing cell. This new cell line can efficiently cleave the packaging region in the helper virus genome. We have also developed an E1 and E2a double-deleted helper virus. By using the CreE cell with the helper virus deleted in both the E1 and the E2a genes it may be possible to further improve the safety of the vectors.

Quantitative and bioluminescent assay to measure efficacy of conventional and DNA vaccinations against Helicobacter pylori.

Vaccination against Helicobacter pylori using DNA sequences encoding Urease A and B subunits was compared to immunization with urease antigen and MTP-PE in a liposome formulation. To determine the effectiveness of a vaccine against H. pylori in a mouse model it is essential to quantify the number of H. pylori remaining in the stomachs following challenge with an inoculum of live bacteria. Culture assays and enzymatic assays produce inconsistent results often unsuitable to conclude if vaccine candidates are protective. To overcome this problem, we developed two assays: 1) a competitive quantitative PCR using a colorimetric readout and 2) a non-competitive direct quantitative PCR using a highly sensitive bioluminescent readout. The competitive PCR requires coamplification of a segment of the urease C sequence and an internal control standard in a competitive manner using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, bioluminescent assay measures the amplified DNA directly using a flash-type luminescent tag and a specific probe. The Sydney strain of H. pylori was used for the mouse infection model. Quantification of H. pylori by either the bioluminescent assay or the competitive PCR was reliable, specific and sensitive compared to quantitative growth assays which often gave false results. The bioluminescent assay was much more sensitive and less labor/time intensive than the competitive PCR. The bioluminescent assay was able to quantitate as few as 100 bacteria, while the competitive assay could not detect less than 10(3) bacteria per mouse stomach. Quantification of H. pylori by bioluminescent assay was superior to the competitive assay and may be used for research applications, such as the development of vaccines, pathogenesis of gastric disease and monitoring of antibiotic treatment.

Quantitation of Helicobacter pylori in the stomach using quantitative polymerase chain reaction assays.

BACKGROUND: The density of Helicobacter pylori is thought to correlate with the degree of inflammation and thus indirectly with the outcome of the infection. Rapid quantitative assays of H. pylori in gastric or duodenal mucosa are lacking. The aim was to develop quantitative assays using the polymerase chain reaction to assess the quantity of H. pylori in the gastric mucosa. METHODS: Competitive PCR was based on coamplification of a segment of the ureC sequence and an internal control using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, noncompetitive PCR assay does not use coamplification and measures the amplified DNA sequence using a flash-type luminescent tag and a specific probe. The mouse infected model using H. pylori strain SS-1 was used to develop the assays. RESULTS: Quantification of H. pylori using either the competitive or noncompetitive PCR was reliable, highly sensitive and specific. CONCLUSIONS: The ability to rapidly quantitate H. pylori from gastric mucosa should be useful to investigate the role of H. pylori density and infection outcome, as well as to monitor the effectiveness of antibiotic treatment or vaccines against H. pylori.

Immunoliposomes containing antibodies to costimulatory molecules as adjuvants for HIV subunit vaccines.

Immunoliposomes containing monoclonal antibodies (MAbs) to the costimulatory molecules CD28 and CTLA4 and their counterreceptors B7-1 (CD80) and B7-2 (CD86) were evaluated for the ability to increase the immune response to recombinant envelope protein rgp120 of the MN strain of human immunodeficiency virus type 1 (HIV-1) during vaccination. MAbs were attached to rgp120-containing liposomes via a biotin-avidin-biotin bridge. Mice vaccinated with immunoliposomes were found to have a strong delayed-type hypersensitivity (DTH) response to the weakly immunogenic gp120 that was dependent on the presence of the MAbs. However, this vaccination protocol did not induce humoral immunity. The DTH response was not accompanied by increased production of interferon gamma (IFN-gamma) or interleukin 4 (IL-4), implying that the primary cellular interaction was between the immunoliposomes and cells of the reticuloendothelial system and not helper T (Th) cells. This strategy of incorporating antibodies to costimulatory molecules on the surface of antigen-containing particulates, such as liposomes or microspheres, can be used to increase DTH immune responses to protein or peptide vaccines.

Development of a murine model of Helicobacter pylori infection.

BACKGROUND: A murine model for Helicobacter pylori infection could facilitate vaccine development. This study was designed to determine the effect of various conditions of dose, frequency of administration, and fasting on H. pylori infection of mice. MATERIALS AND METHODS: Balb/c and C3H/HeN mice were inoculated orogastrically with clinical isolates of H. pylori grown in liquid culture. At 2-week intervals, the stomachs were removed for secondary culture on horse blood agar and for histological analysis. H. pylori from secondary cultures or homogenized stomach tissue from infected mice was inoculated a second time in naïve animals. RESULTS: H. pylori was cultured with high frequency only from the stomachs of C3H/HeN mice. Fasting the mice and increasing the number of organisms inoculated did not increase the rate of infection. Histological analysis detected no inflammation, but mucus depletion and erosion were present in the stomachs of C3H/HeN mice. H. pylori organisms were not observed. Secondary cultures of H. pylori or homogenized infected stomach tissue did not cause infection when inoculated in naïve mice. CONCLUSIONS: Clinical isolates of H. pylori transiently infect C3H/HeN mice. This murine model is suitable for testing oral vaccines. Effective vaccination against H. pylori could prevent transient infection and reduce subsequent gastritis.

Cytokine-containing liposomes as vaccine adjuvants.

The ability of cytokines to regulate and augment an immune response makes them likely agents to be included in vaccines. The systemic use of cytokines as vaccine adjuvants has been hampered by toxicity at effective doses. More precise delivery of cytokines and immunogen can be achieved through the use of microparticle carriers such as liposomes. Several cytokines, including IL-2, IL-7, IL-6 and IFN-gamma have been shown to increase the adjuvant activity of liposomes. It may be possible to use certain cytokines to induce immunoglobulin production, others to induce cytotoxic T lymphocytes (CTL) and still others to promote IgA production at mucosal surfaces. An alternative to liposomes containing cytokines may be liposomes containing cytokine-inducing adjuvants such as MTPPE, MPL and QS21. This approach may produce undesirable immunomodulators as well as beneficial cytokines.

Cytokine-containing liposomes as adjuvants for HIV subunit vaccines.

Dehydration-rehydration liposome vesicles (DRVs) containing various cytokines were evaluated for their ability to induce delayed-type hypersensitivity (DTH) and humoral immunity to the recombinant envelope protein rgp120 of the MN strain of human immunodeficiency virus type 1 (HIV-1). The DRVs trapped approximately 25% of the radiolabeled cytokines and approximately 17% of the radiolabeled rgp120 that were added. The level of trapping was greater than the aqueous volume of the DRVs, indicating association of the proteins with the lipid bilayer. Flow cytometric analysis using antibody to rgp120 or the V3 loop of rgp120 showed the diameter of the DRVs to be 2-7.5 microns. Transmission electron microscopy confirmed the heterogeneity in size of the DRVs and revealed morphological heterogeneity. Transmission electron microscopy with immunogold labeling also revealed the presence of rgp120 on the surface of the DRVs. In vitro bioassays demonstrated slow leakage of biologically active cytokines from DRVs soaked in tissue culture medium containing serum. Mice injected subcutaneously three times at 14-day intervals with DRVs containing 15 micrograms of rgp120 plus interleukin 6 (IL-6) or interferon gamma (IFN-gamma) produced significantly greater DTH responses than mice injected with DRVs containing rgp120 alone. Soluble rgp120 plus soluble IFN-gamma produced DTH in some experiments, but of lower magnitude than the comparable DRVs. Interleukin 6, but not IFN-gamma, increased the antibody titer to rgp120 when included in the DRVs. The mice did not develop antibodies to IFN-gamma or IL-6. Induction of DTH by vaccines may increase protection from viral pathogens such as HIV. Cytokine-containing liposomes may be an effective adjuvant for the induction of a DTH response to envelope-antigen subunit vaccines.

Conjugal mobilization of plasmid DNA: termination frequency at the origin of transfer of plasmid R1162.

Conjugal transfer of plasmid R1162 is initiated and terminated at a 38-bp origin of transfer (oriT). Plasmids containing two directly repeated copies of oriT were used to determine the actual frequency of termination at this site. This frequency was calculated both for oriTnic, a mutated origin that cannot act as the initiation site of transfer, and for an unmutated oriT where transfer had been initiated. In both cases, the termination frequency decreased as the distance between the initiation and termination sites became greater and was significantly less than one for plasmids the size of R1162. A substantial proportion of recipient cells received more than one plasmid copy during transfer. Our results indicate that termination is inefficient but that this is partly compensated for by the transmission of multiple plasmid copies.

Role of the origin of transfer in termination of strand transfer during bacterial conjugation.

Conjugal transfer of the broad-host-range plasmid R1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (oriT). Termination involves ligation of the transferred single strand by the plasmid-encoded MobA protein. Several different assays were used to identify the oriT DNA required for termination. For plasmids containing two oriTs, with transfer initiated at one and terminated at the other, the inverted repeat within oriT is important for termination. Deletion of the outer arm reduces the termination frequency; those terminations that do occur probably depend upon nicking at this oriT prior to transfer. The locations of second-site suppressor mutations indicate that base pairing between the arms of the inverted repeat is important for termination. In vitro, the inverted repeat is not required for specific cleavage of single-stranded DNA at nic, but competition experiments indicate that oriTs with the inverted repeat are preferentially cleaved. We propose that the function of the oriT inverted repeat is to trap the plasmid-encoded MobA protein at the end of a round of strand transfer, thus ensuring that the protein is available for the ligation step.