OBJECTIVETo investigate the effect of heat shock factor 1 (HSF1) on airway hyperresponsiveness and airway inflammation in mice with asthma and possible mechanisms.

METHODSA total of 36 mice were randomly divided into four groups: control, asthma, HSF1 small interfering RNA negative control (siHSF1-NC), and siHSF1 intervention (n=9 each). Ovalbumin (OVA) sensitization and challenge were performed to induce asthma in the latter three groups. The mice in the siHSF1-NC and siHSF1 groups were treated with siHSF1-NC and siHSF1, respectively. A spirometer was used to measure airway responsiveness at 24 hours after the last challenge. The direct count method was used to calculate the number of eosinophils. ELISA was used to measure the serum level of OVA-specific IgE and levels of interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), and interferon-γ (IFN-γ) in lung tissues and bronchoalveolar lavage fluid (BALF). Quantitative real-time PCR was used to measure the mRNA expression of HSF1 in asthmatic mice. Western blot was used to measure the protein expression of HSF1, high-mobility group box 1 (HMGB1), and phosphorylated c-Jun N-terminal kinase (p-JNK).

RESULTSThe asthma group had significant increases in the mRNA and protein expression of HSF1 compared with the control group (P<0.05). The siHSF1 group had significantly reduced mRNA and protein expression of HSF1 compared with the siHSF1-NC group (P<0.05). The knockdown of HSF1 increased airway wall thickness, airway hyperresponsiveness, OVA-specific IgE content, and the number of eosinophils (P<0.05). Compared with the siHSF1-NC group, the siHSF1 group had significantly increased levels of IL-4, IL-5, and IL-13 and significantly reduced expression of IFN-γ in lung tissues and BALF (P<0.05), as well as significantly increased expression of HMGB1 and p-JNK (P<0.05).

CONCLUSIONSKnockdown of HSF1 aggravates airway hyperresponsiveness and airway inflammation in asthmatic mice, and its possible mechanism may involve the negative regulation of HMGB1 and JNK.

Animals ; Asthma ; etiology ; Bronchial Hyperreactivity ; etiology ; immunology ; Cytokines ; biosynthesis ; DNA-Binding Proteins ; analysis ; physiology ; Eosinophils ; physiology ; Female ; HMGB1 Protein ; analysis ; Heat Shock Transcription Factors ; Immunoglobulin E ; blood ; Mice ; Mice, Inbred BALB C ; Transcription Factors ; analysis ; physiology

OBJECTIVESTo construct and to express a human-mouse chimeric antibody against Aβ peptide involved in Alzheimer disease by genetic antibody engineering with reducing of its human anti-mouse antibody response.

METHODSTotal RNA was extracted from a murine hybridoma cell line that secreted anti-Aβ monoclonal antibody. The entire gene coding heavy and light chains were amplified using RT-PCR and analyzed by Genebank Blast. The chimeric antibody gene was acquired by variable region gene of the monoclonal antibody with constant region gene of human IgG, in which point mutations were incluced by recombinant PCR technology, respectively. The eukaryotic expression vectors established by cloning chimeric antibody genes of the heavy and light chains into 3.1 were co-transfected into COS-7 cells. The expressed products were analyzed using ELISA and immunohistochemistry subsequently.

RESULTSGenebank Blast analysis showed that the entire cloned antibody genes were in accordance with the murine antibody genes. DNA sequencing confirmed that the expression vectors of chimeric antibody were constructed successfully after splicing the variable region and constant region sequences. By co-transfecting COS-7 cells, a chimeric antibody was produced and collected in the culture medium. The antibody was humanized and bound Aβ specifically by ELISA and immunohistochemistry evaluations.

CONCLUSIONSExpression vector of chimeric antibody against Aβ was constructed successfully and expressed in the eukaryotic cells. It provides a solid base for developing diagnostic and therapeutic methods for Alzheimer's disease in clinic and paves a way for a further humanization in the future.

Alzheimer Disease ; immunology ; metabolism ; Amyloid beta-Peptides ; immunology ; metabolism ; Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; COS Cells ; Cell Line ; Cercopithecus aethiops ; Genetic Vectors ; Humans ; Hybridomas ; cytology ; immunology ; Immunoglobulin Heavy Chains ; genetics ; metabolism ; Immunoglobulin Light Chains ; genetics ; metabolism ; Mice ; Point Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, DNA ; Transfection

TNFR-Fc is an important fusion protein that has great potential in therapeutic and diagnostic applications. We developed an efficient fed-batch process for GS-CHO cells to produce TNFR-Fc. The rationale of this fed-batch process relies on the supply of sufficient nutrients to meet the requirements of cell metabolism. The optimal feed medium was designed through ration design. A metabolically responsive feeding strategy was designed and dynamically adjusted based on the residual glucose concentration determined off-line. In this process, the maximal viable cell density and antibody concentration reached above 9.4x10(6) cells/mL and 207 mg/L, respectively. Compared with the batch process, the newly developed fed-batch process increased the cell yield by 3.4 fold and the final antibody concentration by 3 fold. This fed-batch process would therefore facilitate the production of therapeutic antibody by GS-CHO cells.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media ; Etanercept ; Glucose ; analysis ; Immunoglobulin G ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

In order to determine the role of Peyer's patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.
Animals ; Antibodies, Protozoan/analysis/metabolism ; Cattle ; Cryptosporidiosis/*immunology/parasitology ; Cryptosporidium parvum/*immunology ; Feces/parasitology ; Female ; Humans ; Immunoglobulin A/analysis/biosynthesis ; Immunoglobulin G/analysis/biosynthesis ; Interferon-gamma/analysis/biosynthesis ; Interleukin-2/analysis/biosynthesis ; Lymphocytes/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Peyer's Patches/cytology/*immunology ; Specific Pathogen-Free Organisms

The mRNA expression of several cytokines was evaluated in splenocytes and mesenteric lymph node (MLN) cells of rats infected with Capillaria hepatica by reverse-transcription (RT)-PCR until week 12 after infection. IgG1 and IgG2a, which are associated with Th1 and Th2 response, respectively, were also assessed by ELISA. The results indicated that the majority of cytokines, including the Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4, IL-5 and IL-10) were expressed at maximal levels during the early stage of infection (after week 1-2), and the ELISA data also evidenced a similar pattern of changes in IgG1 and IgG2a. Th1 and Th2 cytokines responded in a similar fashion in this rat model. The expression of cytokines in splenocytes was significantly higher than that in MLN cells, thereby indicating that cytokine production is controlled more by spleen than by MLN. In addition, the observation that IFN-gamma expression increased unexpectedly at the time of maximal egg production (6 weeks after infection) indicated that IFN-gamma is a cytokine reacting against egg production. However, increased IL-5 expression occurring in tandem with worm activity indicated that the activity of C. hepatica might be controlled by IL-5 expression.
Animals ; Antibodies, Helminth/*blood ; Capillaria/*immunology ; Cytokines/*biosynthesis/genetics ; Disease Models, Animal ; Enoplida Infections/*immunology ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Immunoglobulin G/*blood ; Lymph Nodes/immunology ; Lymphocytes/immunology ; RNA, Messenger/analysis/genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen/cytology/immunology ; Th1 Cells/immunology ; Th2 Cells/immunology

OBJECTIVE: To evaluate the expression of hCTLA4-Ig and their biological function in newborn porcine islets (NPIs) transfected with AAV-hCTLA4-Ig. METHODS: Cultured NPIs were transfected with AAV-hCTLA4-Ig. The expression of CTLA4-Ig in these NPIs was assayed by RT-PCR and immunocytochemistry. The levels of IL-2, IFN-gamma, and TNF-alpha in the culture medium were assayed by ELISA after these cells the co-cultured with human. The response of glucose-stimulated insulin secretion was observed in the transgene group and the control group. RESULTS: The expressions of CTLA4-Ig mRNA and protein were detected in the transgene group. The levels of cytokines were obviously lower in the transgene group than those in the control group (P<0.01). There was no significant difference in the response of glucose-stimulated insulin release between the transgene group and the control group (P>0.05). CONCLUSION AAV mediated hCTLA4-Ig expression in NPIs could inhibit T lymphocyte to produce cytokines, while the endocrine functions of the NPIs were not significantly affected.
Animals ; Animals, Newborn ; Antigens, CD ; biosynthesis ; genetics ; Antigens, Differentiation ; biosynthesis ; genetics ; CTLA-4 Antigen ; Cells, Cultured ; Dependovirus ; genetics ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunohistochemistry ; Interferon-gamma ; analysis ; Interleukin-2 ; analysis ; Islets of Langerhans ; cytology ; immunology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Transfection ; Tumor Necrosis Factor-alpha ; analysis

OBJECTIVETo investigate possible protective effect of maternal immunoglobulin G (IgG) against N-methyl-D-aspartate-mediated neurotoxicity on primary-cultured rat hippocampal neurons and the mechanism of the effect.

METHODSAn in vitro system had been developed for the study of hippocampal neurons. Intracellular lactic dehydrogenase (LDH) release was used as a marker to measure the rates of neuronal damage. The cells were stained with Trypan blue to measure the rate of neuronal death.

RESULTSN-methyl-D-aspartate (NMDA) at a concentration of 50 micromol/L resulted in increased release of LDH and the cell mortality (P < 0.01, respectively). Maternal IgG of different concentration (10 mg/L, 100 mg/L) inhibited NMDA-induced intracellular LDH release (P < 0.01, respectively) and cell mortality (P < 0.05, 0.01, respectively), and larger dose had stronger effect (P < 0.05).

CONCLUSIONSMaternal IgG had protective effect on primary-cultured rat hippocampal neurons injured by NMDA and the effect was dose-dependent.

Animals ; Animals, Newborn ; Cell Death ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Excitatory Amino Acid Agonists ; Female ; Hippocampus ; cytology ; drug effects ; metabolism ; pathology ; Immunity, Maternally-Acquired ; immunology ; Immunoglobulin G ; biosynthesis ; isolation & purification ; pharmacology ; Immunologic Factors ; biosynthesis ; isolation & purification ; pharmacology ; L-Lactate Dehydrogenase ; analysis ; biosynthesis ; Male ; N-Methylaspartate ; Neurons ; drug effects ; metabolism ; pathology ; Organ Culture Techniques ; Pregnancy ; Rats ; Rats, Wistar

BACKGROUNDDNA immunization is a promising novel type of immunotherapy against allergy. An estimated 79.2% patients with asthma, wheezing and/or rhinitis suffer from Dermatophagoides pteronyssinus group 2 (Der p 2) allegen. The aim of the present study was to determine whether DNA vaccine encoding Der p 2 could generate immunologic protection in recombinant Der p 2 (rDer p 2) allergen-induced allergic airway inflammation mice model and to understand the role of DNA vaccination in specific-allergen immunotherapy for asthma.

METHODSAfter DNA vaccination, BALB/c mice were sensitized by intraperitoneal injection (i.p) and challenged by intranasal instillation of rDer p 2. The lung tissues were assessed using hematoxylin and eosin. Mucus-producing goblet cells were identifed using periodic acid-Schiff (PAS)/alcian blue. The total cell number and composition of bronchoalveolar lavage samples were determined. The levels of the cytokines IL-4 and IFN-gamma, as well as IgE and IgG2a in the serum were determined by enzyme-linked immunosorbent assay. Allergen-specific IL-4 and IFN-gamma production by spleen cells were also measured by enzyme-linked immunosorbent assay. Expression of signal transducer and activator of transcription 6 (STAT6) in splenocytes were determined by Western blot.

RESULTSDNA vaccine encoding Der p 2 allergen inhibited extensive infiltration of inflammatory cells and production of mucin induced by allergen. The influx of eosinophils into the lung interstitium was significantly reduced after administration of DNA vaccine. Significant reductions of IL-4 and increase in levels of IFN-gamma in bronchoalveolar lavage fluid were observed. The allergen-specific IgE was markedly decreased in mice receiving DNA vaccination. Allergen could induce higher IFN-gamma, weaker IL-4 in cultured spleen cells from mice receiving DNA vaccine. DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen.

CONCLUSIONThese results indicated that DNA vaccine encoding Der p 2 allergen generates immunologic protection in recombinant Der p 2 allergen-induced allergic airway inflammation mice model with regulating the immune response towards a Th1-type reaction.

Animals ; Antigens, Dermatophagoides ; genetics ; immunology ; Arthropod Proteins ; Asthma ; immunology ; therapy ; Eosinophilia ; prevention & control ; Humans ; Immunoglobulin E ; blood ; Immunoglobulin G ; blood ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Mice ; Mice, Inbred BALB C ; STAT6 Transcription Factor ; Th1 Cells ; immunology ; Trans-Activators ; analysis ; Vaccination ; Vaccines, DNA ; immunology

OBJECTIVETo prepare the polyclonal antibody to amelogenin.

METHODSThe fetal porcine dental enamel was collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7.4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund's adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test.

RESULTSThe Gel filtration showed that amelogenin components is at molecular weights of 15 kD and 13 kD apparently, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1:1000.

CONCLUSIONThe antibody prepared in this study can be used for the detection of amelogenin.

Amelogenin ; Animals ; Animals, Newborn ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Dental Enamel ; chemistry ; Dental Enamel Proteins ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; immunology ; Immunoglobulin G ; analysis ; biosynthesis ; Rabbits ; Swine ; Tooth Germ ; chemistry

OBJECTIVETo investigate the role of IL-17 in the overproduction of autoantibodies and IL-6 overexpression by peripheral blood mononuclear cells (PBMC) of lupus nephritis (LN) patients.

METHODSFifteen consecutively hospitalized LN patients were selected as subjects and 15 healthy adults as normal controls. PBMC were obtained by Ficoll density gradient centrifugation. IgG, anti-dsDNA antibody and IL-6 protein levels were assessed using enzyme-linked immunosorbent assays (ELISA) on the supernatant of cultured PBMC of LN patients or normal controls. IL-6 mRNA levels in PBMC were measured using reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSIn medium culture, IgG, anti-dsDNA and IL-6 protein levels of the supernatant of PBMC from LN patients were significantly higher than those from normal controls (1492.1 +/- 73.2 ng/ml vs 636.7 +/- 51.9 ng/ml for IgG, 306.6 +/- 53.7 IU/ml vs 95.8 +/- 11.6 IU/ml for anti-dsDNA and 50.92 +/- 15.92 ng/ml vs 1.77 +/- 0.73 ng/ml for IL-6, all P < 0.001). In LN patients, IgG, anti-dsDNA and IL-6 protein levels were higher in the supernatants of PBMC in the IL-17-stimulated culture than the medium culture, but in normal controls, only the IL-6 protein levels were significantly higher. The increase in IgG, anti-dsDNA and IL-6 protein levels induced by IL-17 was dose-dependent and could be completely blocked by IL-17 monoclonal antibody mIgG(28) and partially blocked by dexamethasone. Similarly, IL-6 mRNA overexpression of PBMC in LN patients or normal controls induced by IL-17 was both dose- and time-dependent. During medium culture, IL-6 mRNA levels in LN patients were significantly higher than those in normal controls (1.80 +/- 0.11 vs 0.36 +/- 0.07). During stimulation with IL-17, IL-6 mRNA levels in LN patients were higher than those in normal controls (3.21 +/- 0.24 vs 1.30 +/- 0.14, P < 0.05) and also significantly higher when comparing the stimulated culture with the medium culture either in LN patients or normal control.

CONCLUSIONSIL-17 may play an important role in the pathogenesis of LN through the induction of IgG, anti-dsDNA overproduction and IL-6 overexpression of PBMC in LN patients.

Adolescent ; Adult ; Antibodies, Antinuclear ; biosynthesis ; Autoantibodies ; biosynthesis ; Female ; Humans ; Immunoglobulin G ; biosynthesis ; Interleukin-17 ; pharmacology ; Interleukin-6 ; biosynthesis ; genetics ; Leukocytes, Mononuclear ; metabolism ; Lupus Nephritis ; immunology ; Male ; RNA, Messenger ; analysis