BACKGROUND/AIMS: Combination treatment consisting of hepatic arterial infusion chemotherapy with epirubicin and cisplatin (HAIC-EC) and systemic infusion of low-dose 5-fluorouracil (5-FU) are sometimes effective against advanced hepatocellular carcinoma (HCC). However, there is no effective treatment for advanced HCCs with arterioportal shunts (APS) or arteriovenous shunts (AVS). METHODS: We investigated a response and adverse events of a new combination protocol of repeated HAIC-EC and percutaneous intratumoral injection chemotherapy with a mixture of recombinant interferon-gamma (IFN-gamma) and 5-FU (PIC-IF) in patients with far-advanced HCCs with large APSs or AVSs. RESULTS: There was a complete response (CR) for the large vascular shunts in all three patients and for all tumor burdens in two patients. Significant side effects were flu-like symptoms (grade 2) and bone marrow suppression (grade 2 or 3) after each cycle, but these were well-tolerated. CONCLUSIONS: These results suggest that the combination of HAIC-EC and PIC-IF is a new and promising approach for advanced HCC accompanied by a large APS or AVS.
Aged ; Angiography ; Antineoplastic Combined Chemotherapy Protocols/*therapeutic use ; Carcinoma, Hepatocellular/*drug therapy/pathology/radiography ; Cisplatin/administration & dosage ; Epirubicin/administration & dosage ; Fluorouracil/administration & dosage ; Hepatic Artery ; Humans ; Infusions, Intra-Arterial ; Injections, Intramuscular ; Interferon-gamma, Recombinant/administration & dosage ; Liver Neoplasms/*drug therapy/pathology/radiography ; Male ; Tomography, X-Ray Computed ; Tumor Burden

OBJECTIVETo synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice.

METHODSRecombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens.

RESULTSThe purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group.

CONCLUSIONSAg85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.

Acyltransferases ; administration & dosage ; isolation & purification ; therapeutic use ; Adjuvants, Immunologic ; therapeutic use ; Animals ; Antigens, Bacterial ; administration & dosage ; isolation & purification ; therapeutic use ; Bacterial Proteins ; administration & dosage ; isolation & purification ; therapeutic use ; Escherichia coli ; Immunity, Cellular ; Interferon-gamma ; Mice ; Mycobacterium tuberculosis ; immunology ; metabolism ; Recombinant Proteins ; administration & dosage ; isolation & purification ; therapeutic use

To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-&gamma;+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; Cross Protection ; Female ; Genetic Vectors ; biosynthesis ; genetics ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Interferon-gamma ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; administration & dosage ; genetics ; immunology ; Viral Core Proteins ; administration & dosage ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; genetics ; immunology ; Viral Nonstructural Proteins ; administration & dosage ; genetics ; immunology

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10(9)TCID50/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-gamma and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.
Adenoviridae/*genetics ; Animals ; Antibodies, Fungal/blood ; Antigens, Fungal/genetics/*immunology ; *Drug Carriers ; Fungal Proteins/genetics/*immunology ; Fungal Vaccines/administration & dosage/genetics/*immunology ; Immunoglobulin G/blood ; Interferon-gamma/blood ; Interleukin-4/blood ; Mice ; Mice, Inbred BALB C ; Neospora/genetics/*immunology ; Recombinant Fusion Proteins/genetics/immunology ; Vaccines, Synthetic/administration & dosage/genetics/immunology

Recent clinical evidence indicates that the non-eosinophilic subtype of severe asthma is characterized by fixed airway obstruction, which may be related to emphysema. Transgenic studies have demonstrated that high levels of IFN-gamma in the airways induce emphysema. Fibroblast growth factor 2 (FGF2), which is the downstream mediator of TGF-beta, is important in wound healing. We investigated the role of FGF2 in IFN-gamma-induced emphysema and the therapeutic effects of recombinant FGF2 in the prevention of emphysema in a severe non-eosinophilic asthma model. To evaluate the role of FGF2 in IFN-gamma-induced emphysema, lung targeted IFN-gamma transgenic mice were cross-bred with FGF2-deficient mice. A severe non-eosinophilic asthma model was generated by airway application of LPS-containing allergens twice a week for 4 weeks. To evaluate protective effects of FGF2, recombinant FGF2 (10 microg) was injected subcutaneously during allergen challenge in the severe asthma model. We found that non-eosinophilic inflammation and emphysema induced by transgenic overexpression of IFN-gamma in the airways were aggravated by the absence of FGF2. Airway challenge with LPS-containing allergens induced more inflammation in mice sensitized with LPS-containing allergens compared to challenge with allergens alone. In addition, LPS-induced lung inflammation and emphysema depended on IFN-gamma but not on IL-13. Interestingly, emphysema in the severe asthma model was significantly inhibited by treatment with recombinant FGF2 during allergen challenge, whereas lung inflammation was unaffected. Therefore, our present data suggest that FGF2 may help protect against IFN-gamma-induced emphysema, and that recombinant FGF2 may help lessen the severity of emphysema.
Animals ; Asthma/drug therapy/*prevention & control ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Emphysema/drug therapy/*prevention & control ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2/deficiency/*metabolism/*therapeutic use ; Flow Cytometry ; Inflammation/immunology ; Interferon-gamma/*biosynthesis/genetics ; Interleukin-13 ; Lipopolysaccharides/administration & dosage/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Pulmonary Eosinophilia ; Recombinant Proteins/administration & dosage/therapeutic use

OBJECTIVETo construct a recombinant plasmid containing Lgeionella pneumophila pilE gene, detect its expression in NIH3T3 cells and evaluate its immunogenicity.

METHODSPilE gene (LP) was amplified from Legionella pneumophila DNA by PCR and inserted into pcDNA3.1(+) vector to construct the recombinant plasmid pcDNA3.1-pilE, which as verified by restriction endonuclease digestion, PCR and DNA sequencing analysis. NIH3T3 cells were transfected with the recombinant plasmid with Lipofection strategy. Transient and stable pilE gene products were detected by immunofluorescence and Western blotting, respectively. To evaluate the immunogenicity of pcDNA3.1-pilE, the recombinant plasmid was used as a DNA vaccine to immunize female BALB/c mice intramuscularly and the specific antibodies, lymphocyte proliferation response, interferon (IFN)-gamma production and cytotoxic T-lymphocyte response of the immunized mice were detected and evaluated.

RESULTSThe pilE gene of 429 bp in length was amplified. After transfection of NIH3T3 cells with the recombinant plasmid, strong green fluorescence was observed on the cell membrane and inside the cell. A protein with relative molecular mass of 15.7 kD was detected in the transfected cells with Western blotting, suggesting successful protein expression of pilE gene. pcDNA3.1-pilE resulted in much stronger immune response in the immunized mice than pcDNA3.1(+) (P<0.01).

CONCLUSIONThe recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 cells, and can induce specific humoral and cellular immune responses in mice.

Animals ; Blotting, Western ; Cell Proliferation ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Fimbriae, Bacterial ; Fluorescent Antibody Technique ; Humans ; Immunization ; methods ; Injections, Intramuscular ; Interferon-gamma ; metabolism ; Legionella pneumophila ; genetics ; immunology ; metabolism ; Lymphocytes ; cytology ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; NIH 3T3 Cells ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection ; Vaccines, DNA ; administration & dosage ; genetics ; immunology