Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics

OBJECTIVETo construct a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein, ScFv/tP, carrying small interfering (si)RNA directed against the heat shock protein Hsp47, a collagen-binding glycoprotein, in order to evaluate the role Hsp47 in apoptosis of hepatic stellate cells.

METHODSA single chain of the human variable fragment was obtained by phage display and fused with the tP gene and with or without (negative control) the Hsp47 siRNA sequences. Following expression and purification of the scFv/tP fusion protein and the scFv/tPHsp47 siRNA fusion protein, internalization capabilities were tested in isolated human hepatic stellate cells and the QSG-7701 human hepatocyte cells with visualization by immunofluorescent staining. The DNA binding ability of the fusion proteins were verified by gel shift assay.Following ScFv/tP-Hsp47 siRNA fusion protein transfection into the human hepatic stellate cells, the levels of Hsp47 mRNA and protein expression were tested by RT-PCR and Western blotting; in addition, effects of siRNA-mediated silencing of Hsp47 on cell proliferation and apoptosis were analyzed by the cell counting kit (CCK)-8, flow cytometry and Western blot detection of the apoptosis marker poly (ADP-ribose) polymerase (PARP).

RESULTSIndirect immunofluorescence revealed that the ScFv/tP fusion proteins were internalized into human hepatic stellate cells but not into the QSG-7701 cells.The ScFv/tP-Hsp47 siRNA fusion protein caused reduced expression of Hsp47 mRNA and protein expression in the human hepatic stellate cells, as well as increased the cells' apoptosis remarkably.

CONCLUSIONThe ScFv/tP fusion protein can be used as a transfection reagent to deliver Hsp47 siRNA into hepatic stellate cells and to mediate apoptosis via blockade of Hsp47 expression.

Apoptosis ; Cell Proliferation ; HSP47 Heat-Shock Proteins ; genetics ; Hepatic Stellate Cells ; cytology ; Humans ; Protamines ; metabolism ; RNA, Messenger ; RNA, Small Interfering ; Receptor, Epidermal Growth Factor ; immunology ; Single-Chain Antibodies ; Transfection

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by abnormal proliferation of synoviocytes, leukocyte infiltration, and angiogenesis. The endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded proteins in the ER leads to a condition known as ER stress. Failure of the ER's adaptive capacity results in abnormal activation of the unfolded protein response. Recently, we have demonstrated that ER stress-associated gene signatures are highly expressed in RA synovium and synovial cells. Mice with Grp78 haploinsufficiency exhibit the suppression of experimentally induced arthritis, suggesting that the ER chaperone GRP78 is crucial for RA pathogenesis. Moreover, increasing evidence has suggested that GRP78 participates in antibody generation, T cell proliferation, and pro-inflammatory cytokine production, and is therefore one of the potential therapeutic targets for RA. In this review, we discuss the putative, pathophysiological roles of ER stress and GRP78 in RA pathogenesis.
Animals ; Arthritis, Rheumatoid/genetics/*pathology ; Autoantibodies/immunology ; Cell Proliferation ; Cytokines/biosynthesis/immunology ; Endoplasmic Reticulum/immunology/pathology ; Endoplasmic Reticulum Stress/*immunology ; Haploinsufficiency/genetics ; Heat-Shock Proteins/*genetics/*immunology ; Humans ; Lymphocyte Activation ; Mice ; Neovascularization, Pathologic/genetics ; Protein Folding ; Synovial Membrane/cytology ; T-Lymphocytes/immunology ; Unfolded Protein Response/*immunology

Heat shock protein 27 (HSP27) is a member of the small heat shock proteins (sHSP) and has multiple functions, it also plays an important role in the life cycle of some viruses. To investigate the regulatory effect of HSP27 during influenza virus infection, we cloned and expressed human HSP27 in both prokaryotic and eukaryotic cells, and demonstrated that HSP27 interacted with influenza A virus NS1 protein both in vivo and in vitro. Luciferase assay showed that HSP27 inhibited the expression of interferon-beta (IFN-beta) in infected cells, and independent of its phosphorylation. Moreover, HSP27 enhanced the inhibitory effect of NS1 on the expression of IFN-beta. Further analysis indicated that HSP27 exerted the inhibitory effect probably through influencing MDA5 of the RIG-I like helicase (RLH) pathway. The results suggested that HSP27 play a role in the innate immunity of infected cells, contributed to our understanding of the regulatory effect of host factors during influenza virus infection.
Cell Line ; Gene Expression Regulation, Viral ; HEK293 Cells ; HSP27 Heat-Shock Proteins ; genetics ; pharmacology ; Humans ; Influenza, Human ; genetics ; immunology ; Interferon-beta ; antagonists & inhibitors ; metabolism ; Viral Nonstructural Proteins ; genetics ; pharmacology

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2 in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.
Adjuvants, Immunologic ; Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; immunology ; Carcinoma, Ehrlich Tumor ; immunology ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Diphtheria Toxin ; genetics ; immunology ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Immunoglobulin G ; immunology ; Immunotherapy ; Mice ; Neovascularization, Pathologic ; Peptide Fragments ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tandem Repeat Sequences

OBJECTIVETo study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes.

METHODSPlasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group.

RESULTSIn vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly.

CONCLUSIONResults of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; pharmacology ; Genetic Therapy ; methods ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Melanoma, Experimental ; microbiology ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Mycobacterium tuberculosis ; genetics ; Salmonella typhimurium ; genetics ; immunology ; Simplexvirus ; enzymology ; genetics ; Skin Neoplasms ; therapy ; Thymidine Kinase ; genetics ; immunology ; Vaccines, Attenuated ; genetics ; immunology ; pharmacology ; Vaccines, DNA ; genetics ; immunology ; pharmacology

OBJECTIVE: To determine the immune-protective effect of Japan Schistosoma (Chinese mainland strain) 23 kD membrane protein-heat shock protein (SjC23-Hsp70) DNA vaccine plus adjuvantinduced interleukin-12 (IL-12) plasmid DNA on Schistosoma japonicum infection in water buffalos. METHODS: Forty-five health water buffalos (8-10 months old) in non-endemic area of schistosomiasis were randomly assigned into group A (SjC23-Hsp70+IL-12, 300 μg), group B (SjC23+IL-12, 300 μg) and group C (pVAX+IL-12, 300 μg), 15 in each group. Each buffalo was immuned by shoulder intramuscular injection for 3 times, at an interval of 28 days. Twenty-eight days after the last immunization, each buffalo was infected with 1000 Japan cercariae of Schistosoma. Fecal examinations were conducted 2 days and 1 day before the perfusion, and on the day of perfusion. The number of hatching miracidia and eggs per gram feces was recorded. Fifty-six days after the infection, the buffalos were sacrificed and perfused via the descending aorta. The recovered adult worms and eggs in the liver tissue were counted. RESULTS: We compared group A and B with group C: the estrogen reduction rate was 45.7% and 26.61%; bug reduction rate was 44.51% and 25.84%; the fecal egg reduction rate was 41.1% and 31.63%; the miracidium reduction rate was 48.11% and 38.07%; and the liver egg reduction rate was 43.39% and 31.95%. The above rates in group A were higher than those in group B (P<0.05). CONCLUSION SjC23-Hsp70 DNA vaccine combined with IL-12 may have a significant immunoprotective effect on buffalos.
Animals ; Antigens, Helminth ; immunology ; Buffaloes ; Cattle ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Helminth Proteins ; immunology ; Immunization ; methods ; Interleukin-12 ; genetics ; immunology ; Membrane Proteins ; immunology ; Schistosomiasis japonica ; immunology ; prevention & control ; veterinary ; Vaccines, DNA ; administration & dosage ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2.

METHODSTo construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2.

RESULTSThe pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05).

CONCLUSIONSGPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.

Cancer Vaccines ; immunology ; Carcinoma, Transitional Cell ; immunology ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-2 ; metabolism ; Melanoma-Specific Antigens ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Urinary Bladder Neoplasms ; immunology ; metabolism ; pathology

OBJECTIVETo investigate the expression of heat shock protein 90 (HSP90) on the cell surface of highly invasive human prostate cancer cells PC3 and its possible molecular mechanisms of its effect on cell invasion through analyzing FAK/Src signaling pathway.

METHODSThe expression of cell surface HSP90 on PC3 cells was studied by immunofluorescence staining and surface biotinylation assay respectively. A specific HSP90 antibody was used to inhibit the cell surface HSP90. In vitro cell invasion was assessed by modified Boyden chambers. Phosphorylated FAK on tyr 397, 576, 577 and 925, and phosphorylated c-Src on tyr 416 were examined by Western blot assay. The association between FAK and c-Src was analyzed by immunoprecipitation. The effects of FAK knockdown by siRNA or Src kinases inhibitor PP2, with or without anti-HSP90 antibody, on PC3 cell invasion were also evaluated.

RESULTSA pool of HSP90 was detected on the cell surface of PC3 cells. A specific HSP90 antibody significantly retarded tumor cell invasion. Concomitant with this finding, targeting cell surface HSP90 significantly inhibited the phosphorylations of FAK and c-Src, and also the interactions between FAK and c-Src. FAK knockdown or PP2 dramatically suppressed cell invasion, however, anti-HSP90 antibody didn't further inhibit cell invasion.

CONCLUSIONSCell surface HSP90 promotes human prostate cancer cell invasion through a FAK/c-Src signaling, with may be a novel therapeutic target against metastatic tumors.

Antibodies ; pharmacology ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Gene Knockdown Techniques ; HSP90 Heat-Shock Proteins ; immunology ; metabolism ; Humans ; Male ; Neoplasm Invasiveness ; Phosphorylation ; Prostatic Neoplasms ; metabolism ; pathology ; Pyrimidines ; pharmacology ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; src-Family Kinases ; antagonists & inhibitors ; metabolism

Heat shock protein gp96 isolated from tumor tissues holds great promise for tumor immunotherapy. However, at present only very limited amount of gp96 protein can be isolated from tumor tissues. Here, we reconstituted the yeast-expressed gp96 (recombinant gp96, rgp96) with B16.F10 melanoma antigens in vitro to prepare new gp96 tumor vaccine on large-scale, and analyzed its induction of specific anti-tumor immunoresponses by ELISPOT, IFN-gamma intracellular staining and cytotoxicity assays. Immunization with rgp96-tumor antigen complexes significantly inhibited B16 tumor growth compared with either rgp96 or tumor antigens alone and led to enhancement of tumor-specific T-cell activities, which was found similar to that of tumor tissue derived gp96. Our results therefore may provide bases for large-scale preparation of the new generation of gp96 tumor vaccines.
Animals ; Cancer Vaccines ; biosynthesis ; genetics ; immunology ; therapeutic use ; Female ; Heat-Shock Proteins ; biosynthesis ; genetics ; immunology ; therapeutic use ; Melanoma, Experimental ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; therapeutic use ; Skin Neoplasms ; therapy ; Yeasts ; genetics ; metabolism