Heat shock protein gp96 is a highly conserved and monomorphic glycoprotein in the endoplasmic reticulum. It functions as molecular chaperone and can associate with a variety of antigenic peptides noncovalently in vivo and in vitro. Recent studies have indicated that gp96 molecules participate in major histocompatibility complex class I-restricted antigen presentation pathway. Immunization of mice with gp96 preparations isolated from cancer cells can elicit a cancer-specific protective T cell immune response that is recallable, which is a prerequisite for gp96 as a therapeutic vaccine against cancers. The immunogenicity of gp96 molecules has been attributed to the antigenic peptides associated with them. These phenomena provide a new pathway for cancer immunotherapy. The mechanism that the gp96-peptide complex induces specific immune response and the explorations for gp96-peptide complex as a therapeutic cancer vaccine are reviewed.
Animals ; Antigens, Neoplasm ; immunology ; therapeutic use ; Cancer Vaccines ; therapeutic use ; Humans ; Immunotherapy ; Membrane Glycoproteins ; immunology ; metabolism ; Molecular Chaperones ; immunology ; Neoplasms ; immunology ; therapy ; Peptides ; immunology ; metabolism

As a member of the HSP90 family, heat shock protein (HSP) Gp96 is one of the most abundant proteins in the endoplasmic reticulum (ER), which displayed important molecular chaperones function in cells. Gp96 can stimulate the production of cytokines by activating the antigen presentation cells (such as dendritic cell, et al) in innate immunity. It is capable of eliciting an antigen-specific cytotoxic T lymphocyte (CTL) immune response to eliminate pathogens and tumors by facilitating antigen cross-presentation in adaptive immunity. Gp96 is also an ideal adjuvant in many recent researches. Here, we review the progress that addresses the role of biological characteristics, immunogenic mechanism that may be involved in the induction of anti-infection immune response and antitumor immunity, which may guide the new vaccine strategies with the knowledge of Gp96-antigen complexes.
Adjuvants, Immunologic ; genetics ; metabolism ; Antigen-Presenting Cells ; physiology ; Communicable Diseases ; immunology ; Dendritic Cells ; immunology ; Endoplasmic Reticulum ; immunology ; Humans ; Membrane Glycoproteins ; immunology ; Neoplasms ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo explore the role and mechanism of B7 molecules in T cell anergy.

METHODSAnti-B7-1 (CD(80)) and anti-B7-2 (CD(86)) monoclonal antibodies were used to induce T cell anergy. T cell proliferation were assayed by mixed lymphocyte reaction (MLR) with (3)H-TdR incorporation, and cytokine mRNA transcripts were analyzed with reverse transcriptase-polymerase chain reaction (RT-PCR). B7-transfected-CHO cells were used as artificial antigen presentation cells (APCs) in MLR to exclude the effects of other costimulatory molecules.

RESULTSMLR results showed that the proliferation of T cells was inhibited to various extents by anti-CD(80) or anti-CD(86) monoclonal antibody, the effect of anti-CD(86) antibody was greater than that of anti-CD(80) antibody, and the proliferation was totally blocked when the two were used together. The results of RT-PCR demonstrated that IL-2 and IFN-gamma mRNA transcripts decreased whereas IL-4 mRNA transcripts increased in T cell after treatment with anti-B7 antibo-dies for 24 hours. In MLR with artificial APC, signal one (DR7) alone could stimulate T cell proliferation at a certain threshold intensity. Costimulator B7-1 molecule could help signal one in T cell proliferation. This effect was blocked by anti-CD(80).

CONCLUSIONB7 molecules play an important role in T cell immune response. Blockade of B7 family resulted in T cell anergy. The role of CD(86) may be more important than that of CD(80). The conversion of cytokine profile from Th1's to Th2's reflected that anergetic T cells were differentiated into Th2 cells by anti-B7 suggesting that anergetic blockade of costimulator molecules may be one of the mechanisms of T cell.

Animals ; Antigens, CD ; genetics ; B7 Antigens ; B7-1 Antigen ; metabolism ; Cricetulus ; Lymphocyte Activation ; immunology ; Membrane Glycoproteins ; T-Lymphocytes ; immunology

Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction. In this study, we obtained the soluble mZP3 fusion protein and identified its immunoreactivity. mZP3 cDNA was cloned into plasmid pMAL-p2x, and the recombinant plasmid was transformed into Escherichia coli BL21. To get the soluble mZP3 fusion protein, we tried to optimize the expression conditions, including additives, IPTG concentrations, temperatures and induction duration. Then, Western blotting and ELISA were used to identify the immunoreactivity of the purified protein. Based on the optimization experiments, we concluded that the best soluble expression conditions for the mZP3 fusion protein involved incubation to an A600 of 0.6, addition of glucose to a final concentration of 0.02 mol/L, addition of IPTG to a final concentration of 0.6 mmol/L and then further incubation for 4 h at 25 degrees C. Western blotting and ELISA showed that the mZP3 fusion protein retained immunoreactivity. The fusion protein can be used as solubility antigens for developing the immunocontraception vaccines of mZP3 and detecting the immune effects of the vaccine.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Vaccines, Contraceptive ; immunology ; Zona Pellucida Glycoproteins

OBJECTIVETo study the roles of granzyme B and perforin in diagnosing acute rejection after liver transplantation, and the relationship between their activity index (AI) and Banff's histological grading criteria.

METHODSLiver biopsies were processed as for routine surgical specimens and labeled with granzyme B and perforin monoclonal antibodies. The number of positive cells/mm(2) was determined as activity index (AI) by IPP image analysis software. Histologic findings were used as the "gold standard" in diagnosing acute rejection.

RESULTSOf 41 liver biopsy samples studied, acute rejection was noted in 21 cases, the remaining 20 cases showed no evidence of rejection. The AI of granzyme B and perforin in the acute rejection group was significantly higher than that in the non-acute rejection group (< 0.001). In the acute rejection group, the AI in moderate to severe acute rejection was higher than that in mild to indeterminate acute rejection (< 0.001). Compared with the "golden" histologic criteria, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of granzyme B in diagnosing acute rejection were 90.0%, 95.2%, 94.7%, 90.9% and 92.7% respectively. The values of these parameters for perforin were also above 80%.

CONCLUSIONSGranzyme B and perforin are key markers of activated immune cells in acute rejection and highly expressed during acute liver rejection episodes. As ancillary investigations, these parameters demonstrated high sensitivity and specificity in diagnosing acute rejection in allograft post-transplant liver biopsies.

Biomarkers ; Biopsy ; Graft Rejection ; diagnosis ; metabolism ; Granzymes ; metabolism ; Humans ; Liver ; metabolism ; pathology ; Liver Transplantation ; immunology ; Membrane Glycoproteins ; metabolism ; Perforin ; Pore Forming Cytotoxic Proteins ; metabolism ; Sensitivity and Specificity

OBJECTIVETo detect the immunoreactivity of targeted fusion anti-caries DNA vaccine pGJA-P in vitro, and the ability to enhance the immune responses compared with the non-targeted fusion anti-caries DNA vaccine pGLUA-P.

METHODSThe CHO cells were transfected with pGJA-P and the expression of recombinant protein in cultured supernatants were detected using Western blotting. 5 to 6-month-old female Japanese rabbits were immunized with either pGJA-P or pGLUA-P via either intramuscular injection (i.m.) or intranasal route (i.n.). The sera and saliva were collected and the antibody responses were checked by ELISA. The effect of immune sera on the synthesis of water-insoluble glucan by glucosyltransferase of S. mutans was examined.

RESULTSThe expressed protein could response to specific anti-GTF antibody. The antibody responses in serum generated by pGJA-P via i.m. were significantly higher than those generated by pGLUA-P (P < 0.01). The antibody responses in saliva generated by pGJA-P via i.n. were significantly higher than those generated by pGLUA-P (P < 0.01). The higher mucosal antibody response induced by pGJA-P via i.m. compared with pGLUA-P (P < 0.01) was detected. The immune sera of rabbits immunized by pGJA-P via i.m. most significantly inhibited the synthesis of water-insoluble glucan by glucosyltransferase.

CONCLUSIONSThe recombinant protein expressed by pGJA-P had the immunoreactivity to anti-GTF antibody. pGJA-P could induce faster and higher specific mucosal SIgA antibody responses via i.n. or serum IgG antibody responses via i.m. compared with non-targeted DNA vaccine, pGLUA-P. High titres of specific mucosal antibodies were found in rabbits immunized with pGJA-P via i.m. The immune sera of rabbits immunized by pGJA-P via i.m. displayed the ability of inhibiting the synthesis of water-insoluble glucan by glucosyltransferase.

Animals ; Bacterial Proteins ; immunology ; CHO Cells ; Cricetinae ; Dental Caries ; prevention & control ; Female ; Glucosyltransferases ; immunology ; metabolism ; Immunoglobulin G ; blood ; Membrane Glycoproteins ; immunology ; Rabbits ; Recombinant Fusion Proteins ; administration & dosage ; immunology ; Streptococcus mutans ; immunology ; Transfection ; Vaccines, DNA ; administration & dosage ; immunology

The aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen library for the epitope characterization of anti-S monoclonal antibodies (mAbs). First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage display vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7 x 10(6). After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possible epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a crucial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics.
Antibodies, Viral ; immunology ; Bacteriophages ; genetics ; metabolism ; Epitopes ; genetics ; immunology ; Humans ; Membrane Glycoproteins ; genetics ; immunology ; Peptide Library ; SARS Virus ; genetics ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; virology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; genetics ; immunology

OBJECTIVETo analyze the significance of Fas-FasL in NOD insulitis and to explore the mechanism of the autoimmune diabetes.

METHODSThirty-two female NOD mice, 3-32 weeks of age, were selected. The blood glucose concentrations were recorded. The pathological data were obtained from the HE staining of the pancreatic sections and the immunohistochemical staining, in which insulin, Fas, FasL, CD8 were detected.

RESULTSDiabetes was found from the age of 14 weeks. In normal islets, insulin + cells accounted for (59.37 +/- 1.21)%, and some islet cells were observed expressing Fas. At the age of 6 weeks, insulitis lesions could be found. The average score of insulitis tended to rise with the increasing age (P < 0.0005). Meanwhile, insulin + cells decreased (P < 0.0005), and correlated negatively with scoring (P < 0.05). Fas+ islet cells increased (P < 0.0005), correlated positively with scoring (P < 0.01). In insulitis lesions, islet cells expressed FasL that increased gradually (P < 0.0005) and correlated positively with scoring (P < 0.01). The infiltrating cells were all Fas negative. But these mononucleated cells showed the expression of FasL and CD8, both increasing gradually (P < 0.0005). Furthermore, there was certain correlation between the expression of some antigens: in islet cells, between Fas and insulin (negative, P < 0.01), insulin and FasL (negative, P < 0.01), and Fas and FasL (positive, P < 0.01). In the infiltrating cells, the expression of CD8 was correlated with FasL (positively, P < 0.01); it was also found that there was a negative correlation between Fas+ islet cells and CD8+ mononucleated cells (P < 0.05).

CONCLUSIONTo sum up, there may be some important and complicated effects by Fas-FasL on the damage of beta cells and the regulation of autoreactive T cells in NOD insulitis, which will facilitate further studies in human type 1 diabetes.

Animals ; Autoimmune Diseases ; etiology ; immunology ; metabolism ; Diabetes Mellitus, Experimental ; etiology ; immunology ; Fas Ligand Protein ; Female ; Inflammation ; immunology ; metabolism ; Insulin ; blood ; Islets of Langerhans ; immunology ; metabolism ; Membrane Glycoproteins ; physiology ; Mice ; Mice, Inbred NOD ; fas Receptor ; physiology

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.
Animals ; Egg Proteins ; genetics ; metabolism ; Electroporation ; Fermentation ; Immunization ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Rabbits ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; secretion ; Swine ; Zona Pellucida Glycoproteins

OBJECTIVETo study the expression of specific anti- platelet glycoprotein autoantibodies GP II b/III a, GP I b/IX and GP I a/II a in primary immune thrombocytopenia (ITP), and to evaluate the relationship between the therapeutic effect and the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa.

METHODSAnti-GPIIb/IIIa, GPIb/ IX and GP I a/II a antibodies were assayed by ELISA for patients with ITP. Total 442 patients in our hospital, who were retrospectively investigated from December 2010 to November 2012, were divided into newly diagnosed ITP, persistent and chronic ITP. The expression of specific anti- platelet glycoprotein antibody in each group was measured separately. The newly diagnosed ITP patients were treated with intravenous IgG (IVIG) and corticosteroids. The relationship between the expression of specific anti- platelet glycoprotein antibodies GPIIb/IIIa, GPIb/IX and GPIa/IIa and the complete response (CR) was studied.

RESULTSPositive rates of anti- platelet glycoprotein antibodies were 59.09%, 26.97% and 37.35% respectively in newly diagnosed ITP, persistent and chronic ITP, the difference was statistical significant (P<0.05). In newly diagnosed ITP, positive rate of antibody against GPIIb/IIIa was 38.64%, double positive rate of antibodies against both GP II b/III a and GP I a/II a was 15.91%, there was statistical significance (P<0.05) compared with that of persistent and chronic ITP. The complete response (CR) rate in newly diagnosed ITP patients with positive antibody against GP II b/III a was 80.39% after treatment with IVIG and corticosteroids. There was statistical significance compared with that in patients having no antibodies (P<0.05).

CONCLUSIONThe expression of antibodies against GP II b/III a and double positive for both GP II b/III a and GP I a/II a autoantibodies increased in newly diagnosed ITP patients. Patients with anti-GP II b/III a autoantibody had good response to medication with IVIG and corticosteroids.

Adolescent ; Adult ; Aged ; Autoantibodies ; metabolism ; Child ; Child, Preschool ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Platelet Membrane Glycoproteins ; immunology ; Retrospective Studies ; Thrombocytopenia ; drug therapy ; immunology ; metabolism ; Treatment Outcome ; Young Adult