Corticosterone rapidly promotes respiratory burst of mouse peritoneal macrophages via non-genomic mechanism

Background The immunomodulatory effects of glucocorticoids （GCs） have been described as bimodal. High concentration of GCs exerts immunosuppressive effects and low levels of GCs are immunopermissive. While the immunosuppressive mechanisms of GCs have been investigated intensely, the immunopermissive effects of GCs remain unclear. A lot of studies showed GCs could exert rapid non-genomic actions. We herein studied the rapid immunopromoting effects of GCs.Methods We observed the rapid （within 30 minutes） effects of corticosterone on respiratory burst of mouse peritoneal macrophages and studied their mechanisms. The superoxide anions were measured by cytochrome C reduction assay.Protein kinase C phosphorylation was measured by Western blotting and membrane fluidity was evaluated by fluorescence polarization measurement.Results The 10-8 mol/L and 10-7 mol/L corticosterone rapidly increased the superoxide anions production by macrophages, which were insensitive to GC-receptor antagonist, mifepristone, and protein-synthesis inhibitor,cycloheximide. Corticosterone coupled to bovine serum albumin was able to mimic the effects of corticosterone. The effects were independent of protein kinase C pathway and the change in membrane fluidity.Conclusions The results indicate that corticosterone rapidly promote the superoxide anions production by mouse peritoneal macrophages may through non-genomic mechanisms. This study may contribute to understanding the effects of GCs under stress condition and the physiological significance of nongenomic effects of GCs.

Modulation of postoperative immune and inflammatory response by immune-enhancing enteral diet in gastrointestinal cancer patients

AIM: To evaluate if the administration of an enteral diet supplemented with glutamine, arginine and omega-3-fatty acids modulates inflammatory and immune responses after surgery. METHODS: A prospective randomized double-blind, clinical trial was performed. Forty-eight patients with gastrointestinal cancer were randomized into two groups, one group was given an isocaloric and isonitrogenous standard diet and the other was fed with the supplemented diet with glutamine, arginine and omega-3-fatty acids. Feedings were started within 48 hours after operation, and continued until day 8. All variables were measured before operation and on postoperative day 1 and 8. Immune responses were determined by phagocytosis ability, respiratory burst of polymorphonuclear cells, total lymphocytes lymphocyte subsets, nitric oxide, cytokines concentration, and inflammatory responses by plasma levels of C-reactive protein, prostaglandin E2 level. RESULTS: Tolerance of both formula diets was excellent.There were significant differences in the immunological and inflammatory responses between the two groups. In supplemented group, phagocytosis and respiratory burst after surgery was higher and C-reactive protein level was lower (P【0.01) than in the standard group. The supplemented group had higher levels of nitric oxide, total lymphocytes, T lymphocytes, T-helper cells, and NK cells. Postoperative levels of IL-6 and TNF-alpha were lower in the supplemented group (P 【0.05). CONCLUSION: It was clearly established in this trial that early postoperative enteral feeding is safe in patients who have undergone major operations for gastrointestinal cancer. Supplementation of enteral nutrition with glutamine, arginine, and omega-3-fatty acids positively modulated postsurgical immunosuppressive and inflammatory responses.

Molecular and functional characterization of ferritin in abalone Haliotis diversicolor supertexta

Ferritin is an iron storage protein that plays a key role in the processes of physiology and pathology.In the present study,the authors reported the ferritin gene from abalone Haliotis diversicolor supertexta,which we named hds-ferritin.The full-length of hds-ferritin cDNA consisted of 879 bp with an ORF encoding a 171 amino acids.Amino acid sequence analysis revealed that hds-ferritin shared highly homology with other species.Real time PCR and western blot analysis showed that hds-ferritin was distributed ubiquitously in abalone tissues and had the highest expression level in digestive glands,but its transcripts are not modified remarkably by the stimulation with LPS.The recombinant protein was successfully expressed in Escherichia coli BL21 (DE3),and the titre of anti-ferritin antibody was about 1∶14000.The effects of ROS and RNS on ferritin were analyzed in the present study.The results showed that H2O2 played an important role in decreasing hds-ferritin,however NO cation appeared to have a protecting effect on H2O2-medied reduction of hds-ferritin.

Immune strategies of phagoctytic cells stimulated in vitro with live and heat-inactivated Streptococcus pyogenes

Streptococcus pyogenes(group A Streptococcus)is frequently involved in a wide range of human diseases.Here we evaluated polymorphonuclear neutrophils and mononuclear cells from healthy subjects for their bactericidal function after stimulation with live and inactivated Streptococcus pyogenes(Streptococcus Group A).Mononuclear cells and Neutrophils were isolated from heparinized blood samples(n=18)using a Ficoll-Hypaque gradient and cultured in RPMI 1640 for 18 hours with a suspension of either live or inactivated Streptococcus pyogenes.Both the respiratory burst(flow cytometry)and nitrite,TNF and IL17 production(ELISA)were measured in the cell culture supernatants.An increased respiratory burst(expressed as R index)was induced by both live and inactivated bacteria.Also,increased nitrite,TNF and IL17 concentrations were found in cell culture supernatants in both cases.These findings may provide some explanation as to the roles played by neutrophils and mononuclear cells in Streptococcus pyogenes immunopathogenicity。

Alternative Splicing and Differential Expression of Two Transcripts of Nicotine Adenine Dinucleotide Phosphate Oxidase B Gene from Zea mays

With the exception of rice, little is known about the existence of respiratory burst oxidase homolog （rboh） gene in cereals. The present study reports the cloning and analysis of a novel rboh gene, termed ZmrbohB, from maize （Zea mays L.）. The full-length cDNA of ZmrbohB encodes a 942 amino acid protein containing all of the respiratory burst oxidase homolog catalytically critical motifs. Alternative splicing of ZmrbohB has generated two transcript isoforms, ZmrbohB-α and -β. Spliced transcript ZmrbohB-β retains an unspliced intron 11 that carries a premature termination codon and probably leads to nonsense-mediated mRNA decay. Expression analysis showed that two splice isoforms were differentially expressed in various tissues and at different developmental stages, and the major product was ZmrbohB-e. The transcripts of ZmrbohB-α accumulated markedly when the maize seedlings were subjected to various abiotic stimuli, such as wounding, cold （4℃）, heat （40℃）, UV and salinity stress. In addition, several abiotic stimuli also affected the alternative splicing pattern of ZmrbohB except wounding. These results provide new insight into roles in the expression regulation of plant rboh genes and suggest that ZrnrbohB gene may play a role in response to environmental stresses.

Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis

Production of reactive oxygen species(ROS)is a conserved immune response primarily mediated by NADPH oxidases(NOXs),also known in plants as respiratory burst oxidase homologs(RBOHs).Most microbe-associated molecular patterns(MAMPs)trigger a very fast and transient ROS burst in plants.However,recently,we found that lipopolysaccharides(LPS),a typical bacterial MAMP,triggered a biphasic ROS burst.In this study,we isolated mutants defective in LPS-triggered biphasic ROS burst(delt)in Arabidopsis,and cloned the DELT1 gene that was shown to encode RBOHD.In the delt1-2 allele,the antepenultimate residue,glutamic acid(E919),at the C-terminus of RBOHD was mutated to lysine(K).E919 is a highly conserved residue in NADPH oxidases,and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease.Consistently,we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure.It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein’s stability and complex assembly.However,we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association,suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs.Taken together,our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.

PMN apoptosis and its relationship with the lung injury after chest impact trauma

Background Polymorphonuclear neutrophil (PMN),one of the most important inflammatory cells,functions throughout the initiation,progression and resolution of inflammation. This study aimed at investigating the relationship between PMN apoptosis and the lung injury after chest impact trauma. Methods PMNs were purified from rabbits subjected to the chest impact trauma and their apoptosis,necrosis,survival and respiratory burst were detected by flow cytometry. Meanwhile,lactate dehydrogenase and (LDH) [Ca 2+ ]i were measured. Results The delayed apoptosis of PMNs in bronchoalveolar lavage fluid was observed from 2 hours to 12 hours after trauma,and viable cells increased. Respiratory burst of PMNs in bronchoalveolar lavage fluid was increased significantly from 2 hours with the peak at 8 hours. Meanwhile,lactate dehydrogenase in bronchoalveolar lavage fluid was higher than that in control ( P <0.05) from 4 hours to 24 hours,and intracellular free Ca 2+ in PMN was increased temporarilly. Conclusions Retention of PMN in tissues and the abnormality in apoptotic pathway inevitably generate persistent activation of PMN and excessive release of toxic substances,resulting in tissue injury. The temporary increase of intracellular free Ca 2+ may be responsible for the delayed apoptosis of PMN.