OBJECTIVETo evaluate the variety of non-myeloperoxidase-mediated system activity of neutrophils in newborns during bacterial infection and the effect of cord plasma on the activation of non-myeloperoxidase-mediated system.

METHODSAn infection model with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and a non-infection model with phorbol-12-myristate-13-acetate (PMA) were established to investigate the activation of non-myeloperoxidase-mediated system in neutrophils. According to the intensity of fluorescence, the activation of non-myeloperoxidase-mediated system of neutrophils was detected by flow cytometry (FCM). The blood cells and plasma were separated from cord blood and adult blood and cross-mixed in order to investigate the opsonic activity.

RESULTSIn the non-infection model, the activation of non-myeloperoxidase-mediated system with PMA stimulation in cord blood was lower compared with that in adult blood, the statistical difference was significant (t = 3.378, P < 0.01). In the infection model, the activations of non-myeloperoxidase-mediated system in cord blood were also lower compared with those in adult blood, while the statistical difference could only be found in the model with E. coli stimulation (t = 12.150, P < 0.001). Furthermore the experiments demonstrated that cord plasma could deeply depress the non-myeloperoxidase-mediated system activity with E. coli stimulation. On the contrary, adult plasma could successfully recruit the potential of non-myeloperoxidase-mediated system activity of neutrophils in newborns.

CONCLUSIONThe function of neonatal neutrophils might not developed very well. As a stimulant, E. coli failed to induce the non-myeloperoxidase-mediated system activity in neonates, which might be related to the lower level of immunoglobulins in cord blood. This result indicated that immunoglobulins played a more important modulating role in bacterial killing during gram-negative bacterial infections.

Escherichia coli ; immunology ; Fetal Blood ; immunology ; Flow Cytometry ; Humans ; Infant, Newborn ; Neutrophils ; enzymology ; immunology ; Peroxidase ; metabolism ; Staphylococcus aureus ; immunology

Fibronectin-binding protein (FnBPA) is a protein that expresses on cell surface of Staphylococcus aureus during early stage of infection. FnBPA was capable of promoting Staphylococcus aureus to invade cells and was viewed as a potential immune target. Based on the FnBPA-A gene two recombinant expression vectors with or without Kozak sequence were constructed. After identified and confirmed by restriction enzyme digestion and sequencing they were used to immunize C57BL/6 mice. Then induced antibody titer, T lymphocyte proliferative response and experiment mice challenge test were measured. Our result indicates that humoral immune responses and challenge experiment induced by recombinant DNA with Kozak sequence were better than those without Kozak sequence (P < 0.05). For T lymphocyte proliferative response the induced effect of recombinant DNA with Kozak sequence was higher than that without Kozak sequence, but there was no significant difference (P > 0.05). We conclude that Kozak sequence could play an important role in immune response induced by FnBPA-A recombinant DNA.
Adhesins, Bacterial ; genetics ; immunology ; Animals ; Immunization ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins ; immunology ; Staphylococcus aureus ; immunology ; T-Lymphocytes ; immunology ; Vaccines, DNA ; genetics ; immunology

Staphylococcus aureus (S. aureus) is an important human pathogen which can cause a chronic condition with a high relapse rate despite the aggressive antimicrobial treatment. Recent studies showed that intracellular pattern recognition receptors (including NOD) in response to bacteria or bacterial products play a proinflammatory role by activating nuclear transcription factor-κB (NF-κB). But how NOD2 mediates the proinflammatory response to S. aureus in mast cells (MCs) is unclear. So, in this study, we attempted to examine the role of NOD2 in inflammatory responses of MCs to S. aureus. P815 cells (a mouse mast cell line) were cultured. Real-time PCR was used to detect the NOD2 mRNA expression in P815 cells during S. aureus infection. The siRNA against NOD2 gene was synthesized and transfected into S. aureus-infected P815 cells. By using the methods of ELISA and flow cytometry, the effects of NOD2 gene silencing on cell phagocytosis, cytokine secretion, NF-κB activation and cell apoptosis of the S. aureus-infected P815 cells were examined. It was found that S. aureus infection could increase the expression of NOD2 mRNA in P815 cells. NOD2 gene interference in P815 cells reduced the number of S. aureus engulfed by P815 cells, the level of cytokines and the activation of NF-κB. In addition, S. aureus could induce the apoptosis of P815 cells, but NOD2 gene silencing did not affect the cell apoptosis rate. Our data suggested that NOD2 plays a key role in pathogen recognition, signal transduction, and NF-κB activation in the inflammatory responses of MCs infected by S. aureus.
Animals ; Cell Line ; Cytokines ; immunology ; Inflammation Mediators ; immunology ; Mast Cells ; immunology ; microbiology ; Mice ; NF-kappa B ; immunology ; Nod2 Signaling Adaptor Protein ; immunology ; Staphylococcus aureus ; physiology

In order to characterize the immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein Clumping factor A (ClfA), we amplified clfa genes from S. aureus Newman strain, Wood46 strain and HLJ23-1. The clfa gene from Newman strain was subsequently inserted into pQE-30 vector and the recombinant plasmid was transformed into Escherichia coli strain M15 (pREP4). The recombinant ClfA protein was expressed and purified. Then, we immunized mice with the purified recombinant protein. The antibody level and the concentration of cytokines were measured by enzyme-linked immunosorbent assay. Finally, immunized mice were challenged with S. aureus Newman, Wood46 and HLJ23-1. These results suggested that clfa gene sequences were highly conserved, and the recombinant ClfA was expressed correctly with good antigenicity. The antibody titer and the concentration of cytokines in the immunized groups increased significantly (P < 0.05) compared with control, and the mice in the immunized groups were protected against the challenge strains to some extent. These results showed that the ClfA had high immunogenicity and immunoprotective potential.
Animals ; Coagulase ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Immunization ; Mice ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Staphylococcus aureus ; metabolism ; pathogenicity

OBJECTIVETo explore the immuno-effect of plasmacytoid dendritic cells (pDC) on bacteria infection induced spontaneous remission (SR) of leukemia.

METHODSBoth pDC and myeloid dendritic cells (mDC) were isolated and purified from leukemic patient with SR and healthy donor by combination of immunomagnetic beads and flow cytometry. pDC were cultured in RPMI1640 medium and stimulated with different bacteria. The T cells proliferation was detected by MTT, and cytokine production by ELISA kits.

RESULTSThe human bacterial pathogen Staphylococcus aureus and Pseudomonas aeruginosa stimulation for 48 h resulted in the maturation of pDC with production of high quantity of IFN-α at (15.34 ± 2.91) ng/ml and (10.38 ± 1.41) ng/ml, respectively, comparing with that of negative group at (1.36 ± 0.13) ng/ml (P<0.01). Activated pDC could promote the differentiation of naive CD4⁺ T cells to Th1 cells with secretion of IFN-γ at (2.16 ± 0.37) ng/ml and (2.73 ± 1.11) ng/ml, respectively, comparing with that of positive control at (2.55 ± 0.23) ng/ml (P > 0.05). Activated pDC showed higher T cell stimulatory capacities [proliferation index (PI) was 4.36 and 4.05, respectively] than that of non-activated pDC (PI was 1.23 and 0.13, respectively) (P < 0.01).

CONCLUSIONStaphylococcus aureus and Pseudomonas aeruginosa activated pDC may play a key role in SR of leukemia following severe infections.

CD4-Positive T-Lymphocytes ; Dendritic Cells ; immunology ; Flow Cytometry ; Humans ; Interferon-alpha ; Leukemia ; diagnosis ; immunology ; Lymphocyte Activation ; Pseudomonas aeruginosa ; immunology ; Remission, Spontaneous ; Staphylococcus aureus ; immunology

OBJECTIVETo prepare and identify monoclonal antibodies against staphylococcal enterotoxin I (SEI).

METHODSSpleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells (SP2/0). Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and the stable monoclonal hybridomas were isolated by limiting dilution at least three times. The characters of purified monoclonal antibodies were identified by indirect ELISA and Western blotting.

RESULTThe monoclonal antibodies secreted by two hybridomas 8F7 and D8 belonged to IgG(2b) and IgG(1) subtypes. Both had high titer and specificity with no cross reaction to SEG, SEE and SEC.

CONCLUSIONThe monoclonal antibodies against SEI has been successfully prepared and identified in this study.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Enterotoxins ; immunology ; Hybridomas ; secretion ; Immunoglobulin G ; biosynthesis ; immunology ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; Staphylococcus aureus ; immunology

A 13-year-old girl presented with multiple skin abscesses. She was diagnosed as having juvenile dermatomyositis (DM) at the age of 7 years. She had suffered from recurrent skin infections, atypical pruritic dermatitis and pneumonia since the age of 8 years. Bacteriologic and fungal cultures for skin abscesses and oral mucosa were positive S. aureus and C. albicans, respectively. Chemotactic defect in peripheral blood neutrophils was observed. The level of serum IgE was markedly elevated, and anti-S.aureus specific IgE was found. A diagnosis of hyperimmunoglobulin E-recurrent infection syndrome (HIE) was made and she was successfully treated with surgical drainage and antibiotics. To our knowledge, this is the first case report of HIE in a patient with juvenile dermatomyositis.
Adolescence ; Case Report ; Dermatomyositis/complications* ; Female ; Human ; IgE/blood ; Job's Syndrome/immunology ; Job's Syndrome/diagnosis ; Job's Syndrome/complications* ; Staphylococcal Infections/immunology ; Staphylococcal Infections/complications ; Staphylococcus aureus/immunology

Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.
Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Escherichia coli ; Flow Cytometry ; Immune Sera ; Immunoglobulin G ; blood ; Opsonin Proteins ; immunology ; Phagocytosis ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

Lysostaphin, a specific endopeptidase enzyme derived from Staphylococcus aureus, is a bactericidal agent against Staphylococcus and difficult to be drug-resistant. This study established the monoclonal antibody affinity chromatography to obtain lysostaphin of high purity for drug-use standard. The purified Lysostaphin was of > 95% purity and its recovery rate more than 90%. Moreover, the affinity column kept its efficiency of purification invariable after more than 30 times repeat. Also, the dye release assay validated that the purified lysostaphin had significant bactericidal activity. This method was simple and of high efficacy for the lysostaphin purification and showed its potency in commercial production.
Antibodies, Monoclonal ; immunology ; Chromatography, Affinity ; methods ; Lysostaphin ; biosynthesis ; isolation & purification ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Staphylococcus aureus ; enzymology

Clinical application of staphylococcal enterotoxin C2 (SEC2) was restricted during the cure of malignant tumor due to its side-effects. The aim of this study was to obtain SEC2 mutant, preserving the important functional sites responsible for the T-cell stimulatory activities but removing the sites responsible for emetic activity, through truncation of SEC2. It would efficiently solve the question of SEC2 side-effect. According to the results of methyl thiazol tetrazolium (MTT) assay in vitro, novel truncated SEC2 mutant (NSM) efficiently stimulated T-cell proliferation and inhibited the growth of such tumor cells as human colorectal cancer cells (Cx-1) and human breast cancer cells (MCF-7) in vitro. Activities of T cell stimulating and anti-tumor of NSM were similar to those of SEC2. According to results of animal experiments, the mutant no longer induced emetic response even if the dose was a 10-fold excess of the amount of SEC2 required. And also, NSM obviously inhibited the tumor growth in tumor-bearing mice. Therefore, we obtained novel truncated staphylococcal enterotoxin C2 mutant, which could efficiently inhibit the growth of tumor cells. It will become novel anti-tumor agents with the lowest side-effects and best treatment effects in clinic.
Animals ; Antineoplastic Agents ; adverse effects ; pharmacology ; Breast Neoplasms ; immunology ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; immunology ; pathology ; Enterotoxins ; genetics ; immunology ; Humans ; Mice ; Mutant Proteins ; immunology ; Staphylococcus aureus ; immunology ; Superantigens ; immunology ; T-Lymphocytes ; immunology ; Vomiting ; prevention & control