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

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

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

BACKGROUNDTo investigate expression of IL-6 in non?replicating vaccinia virus and its immune effects on recombinant virus.

METHODSThe recombinant non replicating vaccinia virus RVJ123 delta CK11 beta 75IL6 was constructed with non?replicating vaccinia virus vector pNEOCK11beta75IL6 and replicating vaccinia virus RVJ123. In animal model, immunization with the recombinant virus was carried out and its immune response was studied.

RESULTSThe recombinant virus could express IL-nd HBsAg simultaneously. Southern blot analysis demonstrated that the genes between vaccinia virus Hind? C and K fragments were deleted and IL-6 gene was integrated stably. Given intranasal inocula of the virus to immunize BALB/c mouse and New Zealand Rabbit, the elevated anti-HBsAg IgA and IgG antibody secreting cells in mouse lung lymphoid to vectors expressing IL-6 was at about two?fold higher level than those elicited by control virus at day 14 after immunization. Authors also could detect elevated anti-HBsAg IgA and IgG antibody conversion in mouse serum and lung fluid, rabbits serum, lung fluid, saliva, vagina and nasal washing samples.

CONCLUSIONSIL-6 expressed by non-replicating recombinant vaccinia virus could enhance the induced?immune effects, it could serve as the effective adjuvant for recombinant vector vaccine.

Adjuvants, Immunologic ; Animals ; Cells, Cultured ; Chick Embryo ; Female ; Genetic Vectors ; Humans ; Immunoglobulin A ; analysis ; Immunoglobulin G ; analysis ; Interleukin-6 ; biosynthesis ; genetics ; immunology ; Lung ; immunology ; Mice ; Mice, Inbred BALB C ; Rabbits ; Recombination, Genetic ; Vaccinia virus ; immunology ; metabolism ; physiology ; Virus Replication

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

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 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

OBJECTIVEGlucosyltransferase-B (GTF-B) of Streptococcus mutans has been implicated as a principal virulent factor in the development of dental caries. The objective was to use recombined plasmid pcDNA-gtfB expressing multiple antigen of glucosyltransferase-B as gene vaccine to immunize rats through different route, and to investigate the immunization effects of immunization routes.

METHODSA total of 18 Wistar rats were divided into 3 groups, including the quadriceps injection group, the intransal irrigation group and the submandibular gland-targeted injection group. The serum IgG and salivary IgA were assayed by using ELISA after pcDNA3-gtfB immunization. The serum IgG and salivary IgA in different groups were compared using statistical one-way ANOVA.

RESULTSCompared these 3 groups, the serum IgG in the quadriceps injection group was much higher than those of other two groups (P < 0.01), while the salivary IgA of the submandibular gland-targeted injection was much higher than those of other two groups (P < 0.01).

CONCLUSIONIt is indicated pcDNA3-gtfB is good candidate for anticarious gene vaccine, and submandibular gland-targeted injection is an effective immunization route for stimulating salivary IgA.

Animals ; Antibodies, Bacterial ; biosynthesis ; Bacterial Vaccines ; administration & dosage ; immunology ; Cloning, Molecular ; Dental Caries ; prevention & control ; Glucosyltransferases ; genetics ; immunology ; Immunization ; Immunoglobulin A, Secretory ; analysis ; Immunoglobulin G ; blood ; Male ; Plasmids ; genetics ; immunology ; Random Allocation ; Rats ; Rats, Wistar ; Recombination, Genetic ; Saliva ; immunology ; Streptococcus mutans ; genetics ; immunology ; Vaccines, DNA ; administration & dosage ; immunology

In order to understand the effect of hematopoietic stem cell mobilization agents, G-CSF and GM-CSF, on the expression of TNF-alpha mRAN and CD69 and secretion of IgG in SLE patients' peripheral blood mononuclear cells (PBMNC), expression of TNF-alpha mRNA and CD69 was measured by RT-PCR and flow cytometry, respectively, and IgG secretion by ELISA. The results showed that 0.1 - 2.0 microg/ml G-CSF did not affect the expression of TNF-alpha mRNA in active and static patients' PBMNC treated with 0.1 - 2.0 microg/ml cytokines, and 2.0 microg/ml GM-CSF increased the expression of TNF-alpha mRNA in active patients' PBMNC. G-CSF and GM-CSF did not interfere the expression of CD69 in active and static patients' PBMNC, however, the expression of CD69 was significantly increased in active patients' PBMNC treated with GM-CSF at more than 8 microg/ml. There was no obvious change of IgG secretion from PBMNC induced with 10 microg/ml G-CSF, while the IgG secretion was stimulated by 10 microg/ml GM-CSF. It was concluded that G-CSF as a mobilization agent could be safe to SLE patients, but GM-CSF may cause some harmful effects to patients because of the increase of the parameters relating to activity of lupus in active SLE patients.
Antigens, CD ; analysis ; Antigens, Differentiation, T-Lymphocyte ; analysis ; Dose-Response Relationship, Drug ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunoglobulin G ; biosynthesis ; Lectins, C-Type ; Leukocytes, Mononuclear ; immunology ; Lupus Erythematosus, Systemic ; immunology ; RNA, Messenger ; analysis ; Tumor Necrosis Factor-alpha ; genetics

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