ABSTRACT
BACKGROUND:Systemic erythematosus lupus (SLE) is closely associated with activity of dendritic cells (DCs) function.DCs derive from hematopoietic precursor cells (HPCs) in vivo and many factors might affect the differentiation and maturation of DCs.The cytokine network was in disturbance and the levels of various cytokines were anomalous in SLE serum.Previous studies mainly addressed effects of single factors.OBJECTIVE:To investigate the effects of joint action of serum interferon (IFN)-α and interieukin (IL)-6 in the serum of SLE patients on the differentiation and maturation of DCs derived from CD34~+ HPCs.DESIGN,TIME AND SETTING:The grouping,controlled and completely random designed study was performed at the Department of Microbiology and Immunology,Nanjing Medical University from May 2006 to October 2008.MATERIALS:Peripheral blood samples were collected from 50 SLE patients from the Department of Rheumatology,First Affiliated Hospital of Nanjing Medical University and 30 healthy volunteers from Nanjing Medical University.Cord blood samples were collected from 15 full-term normal delivery neonates from Nanjing Maternity and Children Health Care Hospital Affiliated to Nanjing Medical University.Informed consent was obtained from all patients and their family members.METHODS:Peripheral blood samples were collected from SLE patients and healthy volunteers.According to serum IFN-α and IL-6 concentrations of normal person,95% reference value range of serum IFN-α and IL-6 concentrations were calculated by the method of percentiles.Above this range represented abnormal increase.Cord blood mononuclear cells were harvested by Ficoll-Hypaque density gradient centrifugation.CD34~+HPCs were purified from cord blood by magnetic cell sorting system (MACS),and cultured to differentiate to DCs.There were 6 groups in this study.Normal serum,SLE serum with elevated levels of IFN-α,SLE serum with elevated levels of IL-6,SLE serum with elevated levels of IFN-α and IL-6,SLE serum with anti-IFN-α and anti-IL-6 neutralizing antibodies,or normal serum with exogenous IFN-α and IL-6 were respectively added to the culture medium in each group.10% volume fraction serum was used in each group,for totally 14 days incubation.MAIN OUTCOME MEASURES:The phenotype of DCs was analyzed by flow cytometry (FCM).Cytokine production was assessed by ELISA.The capacity of DCs to stimulate allogenic T lymphocyte proliferation and to regulate T cell differentiation was evaluated in a mixed lymphocyte reaction (MLR).RESULTS:Compared with normal serum,HLA-DR,CD80 and CD86 expression was significantly increased in the medium containing SLE serum with elevated levels of IFN-α,SLE serum with elevated levels of IL-6,SLE serum with elevated levels of IFN-α and IL-6 and normal serum with exogenous IFN-α and IL-6 (P＜0.05),whereas IL-12 levels were significantly decreased (P＜0.05),and capacity of DCs to stimulate allogenic T lymphocyte proliferation was significantly enhanced (P＜0.05).Above-mentioned indexes recovered to a normal level in the medium containing SLE serum with anti-IFN-α and anti-IL-6 neutralizing antibodies.Compared with normal serum,proportion of both CD3~+CD8~-IFN-γ~+ and CD3~+CD8~+IFN-γ~+ T cell subsets was significantly reduced in the medium containing SLE serum with elevated levels of IL-6 (P＜0.05).However,proportion of both CD3~+CD8~-IFN-γ~+ and CD3~+CD8~+IFN-γ~+ T cell subsets was significantly increased in the medium containing SLE serum with elevated levels Of IFN-α,SLE serum with elevated levels of IFN-α and IL-6,normal serum with exogenous IFN-α and IL-6 (P＜0.05).CONCLUSION:The joint action of IFN-α and IL-6 in SLE serum promotes the differentiation and maturation of DCs derived from CD34+HPCs and affects the DC-mediated T cell differentiation,which might contribute to the pathogenesis of SLE.
ABSTRACT
BACKGROUND: Blood stem cells (BSCs) are the most primitive cells in the immune system and can be differentiated into many kinds of cells. As the regulatory cells of immune response, dendritic cells (DCs) have been attracted more and more attention in the field of autoimmune diseases. Due to different resources of precursor cells, DCs have different cytokines, ideal cytokine matching,applying orders, and experimental cultured conditions. Furthermore, development, phenotypic expression, and mature degree are still different.OBJECTIVE: To investigate the influence of tumor necrosis factor- α (TNF- a ) and interleukin-4 (IL-4) on the culture system and provide improved method for inducing functional DCs in vitro, derived from cord blood CD34+ hematopoietic precursor cells (HPC).DESIGN, TIME AND SETTING: Observational study, which was performed in the Department of Microbiology and Immunology, Nanjing Medical University from March 2005 to November 2005.MATERIALS: The cord blood was collected from neonatal umbilical cord in the 81 Hospital of Nanjing City. CD34 monoclonal antibody coated magnetic bead system (MACS) was provided by Miltenyi Biotec Company, Germany; human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF), human recombinant interleukin-4 (rhlL-4), and human recombinant tumor necrosis factor-α (rhTNF-α) by Pepro Tech Company, USA.METHODS: CD34+HPC were isolated and purified from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS). Then the cells were cultured with different culture medium which contained different combinations of cytokines:GM-CSF and TNF-α (GT) or GM-CSF, TNF-α and IL-α (GTI) in order to be induced to differentiate into dendritic cells (DCs).The DCs derived from CD34+HPC were identified for their morphology and phcnotype by FACS and laser scanning con_focal fluorescence microscopy, and also for their abilities of inducing proliferation of allogenic T cells by 3H-TdR incorporation assay.RESULTS: The purity of selected CD34+ cells with MACS was more than 90%. DCs could be obtained from CD34+HPC by the culture in presence of GM-CSF and TNF-α or GM-CSF, TNF- o and IL-4. With the time of culture lasting, the cells expressed lower level surface antigen of CD34 and HLA-DR (P ＜ 0.05), and possessed the phenotypes of DCs characterized by higher expression of CD80, CD86, CD83 and CDIa. At 13-15 days, the cells possessed higher level of phenotypes of DCs compared to 7-9 days and 10-12 days. DCs induced with GTI culture system expressed higher levels of surface antigen CD80, CD86, CD83,CD 1 a and a lower level of CD 14 than those induced with GT culture system. DCs displayed typical morphology and property and expressed higher level surface antigen of CD86 and CD80, especially expressed CD86 when they were induced with proper cytokines of GM-CSF and TNF- α added at 0 hour, and of IL-4 added at 48 hours.CONCLUSION: DCs can be generated from CD34+HPC by proper culture system, the design of GM-CSF + TNF- α + IL-4(GM-CSF and TNF- α were added at 0 hour, IL-4 was added at 48 hours) is preferred.
ABSTRACT
Objective To review the preliminary experience of primary laparoscopic pull-through for Hirschsprung's Disease in children.Methods Since May 2000,25 patients with Hischsprung's disease underwent Laparoscopic Pull-Through. The oldest age was 6 years old and the youngest was 20 days. All patients underwent anesthesia and tracheal intuition, carbon dioxide was insufflated into abdominal cavity via a Veers needle, reaching a insulation pressure of 1.33～1.86 kPa (10～14 mmHg). One umbilical camera port one-instrument port at left abdomen and one instrument port at right abdomen were made.Results The mean operative time was 165-min. Oral feeding was started 3 days postoperatively. The average hospitalization was 9 days (7～10 days).No complication was encountered.Conclusions Primary laparoscopic Pull-Through for Hirschsprung's Disease in children is safe and feasible.
ABSTRACT
Objective To present our experience with laparoscopic pyloromyotomy and laparoscopically assisted endorectal pull through for Hirschsprung's disease (HD) in newborns and infants.Methods Twenty newborns or infants with hypertrophic pyloric stenosis and HD,age ranged from 16～120 days and weight from 2.7～8 kg.Under epidural anesthesia and tracheal intubation,carbon dioxide was insufflated into abdominal cavity via a Veress needle,reaching an insufflative pressure of 12～14 mm Hg.One umbilical and two or three subcostal canulas for instrumentation were demanded.The pyloromyotomy was performed in patients with hypertrophic pyrolic stenosis and in patients with HD the affected colon and rectum were mobilized by dissecting the supplying vessels and cutting the peritoneal reflection,and pull-through procedure was carried out.Results The operation time was 25～150 minutes with rare complications.Oral feeding was resumed on the following day postoperation.The patients recovered and discharged from hospital 3～7 days after operation.Conclusions Laparoscopic pyloromyotomy and laparoscopic endorectal pull-through for HD in newborns and infants are safe and feasible.The advantages of this procedure include minimal trauma to abdomen,rapid restoration of stomach and bowel function and quick rehabilitation.