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.

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.

Objective To investigate the phenotypes and the HIV-1-specific T cell responses of KIR3DL1 positive CD8 cells in patients with early HIV-1 infection. Methods Fifty-six HIV-1 antibody negative individuals and thirty-two patients with early HIV-1 infection were enrolled in the study. Fluores-cence-activated cell sorting (FACS) was performed to detect the phenotypes of KIR3DL1 receptor expressed on the surface of CD8 cells. The levels of IFN-γwere measured by intracellular cytokine staining assay after the PBMCs were stimulated with an HIV-1 Gag peptide pool. Results The percentages of KIR3DL1+CD8 T cells in HIV-1 negative individuals and patients with early HIV-1 infection were 1. 45% (0. 12%-8. 4%) and 0. 82% (0. 14%-6. 14%), respectively, and there was no significant difference between them. The percentages of KIR3DL1+CD8 Temra cells in HIV-1 negative individuals and patients with early HIV-1 infec-tion were (4. 55±3. 84)% and (6. 71±8. 50)%, respectively, which were significantly higher than the per-centages of KIR3DL1+CD8 Tem cells, which were (0. 50±0. 59)% and (1. 18±1. 39)%, respectively (all P<0. 01). Moreover, the percentages of KIR3DL1+CD8 Tem cells in patients with early HIV-1 infection were higher than those in HIV-1 negative individuals (P=0. 001 2). The percentage of KIR3DL1+CD8 Temra cells was positively correlated with the HIV-1 viral load in patients with early HIV-1 infection ( rs=0. 576,P=0. 000 9). The percentages of KIR3DL1+CD8 Temra cells in HIV-1 patients, whose viral loads were larger than 4. 0log, were much higher than those in HIV-1 patients with viral loads less than 4. 0 log (P=0. 002). Additionally, the levels of IFN-γsecreted by KIR3DL1 positive CD8 cells were much lesser than those secreted by KIR3DL1 negative CD8 cells (P<0. 000 1). Conclusion The receptor of KIR3DL1 was mainly expressed on CD8 Temra cells in both HIV-1 negative subjects and patients with early HIV-1 infec-tion. High HIV-1 viremia was associated with the high percentage of KIR3DL1+CD8 Temra cells. The KIR3DL1 positive CD8 cells induced lower HIV-1-specific T cell responses.

Objective:To investigate the effects of rituximab on lymphocytes and immunoglobulin in the treatment of focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD).Methods:The subjects were FSGS and MCD patients admitted to Ruijin Hospital affiliated to Shanghai Jiaotong University on July 1, 2014 and July 1, 2019. All the enrolled patients were confirmed by clinical examination and renal biopsy, and received rituximab treatment (4 infusions of 375 mg/m 2 with the interval of 7-14 d). The levels of immunoglobulin IgA, IgG, IgM, and lymphocytes of CD19 +, CD20 +, CD3 +, CD3 +CD4 +, CD3 +CD8 + and natural killer cells (CD56 +CD16 +) were compared between baseline and the third month, the sixth month, the ninth month and the twelfth month after treatment. Results:Ninety-six patients with FSGS or MCD were enrolled in this study. The midian age was 28 years old (14-77 years old). The ratio of men to woman was 1.8∶1. There were 65 cases of MCD and 31 cases of FSGS. After rituximab treatment, the 24 h-proteinuria was significantly lower than that before treatment, and the serum albumin level was increased (both P<0.05). After rituximab treatment of 3 months, 6 months, 9 months and 12 months, CD19 + and CD20 + lymphocyte counts were significantly decreased (all P<0.01), and gradually recovered after 6 months. Compared with baseline, at 3, 6, 9, 12 months after rituximab treatment, the level of blood IgG was significantly increased ( P=0.004,<0.001,<0.001,<0.001, respectively), and the level of blood IgM was significantly decreased ( P<0.001, =0.008, =0.005,<0.001, respectively) but the median level still within the normal range (400-3 450 mg/L). The level of blood IgA was not significantly changed (all P<0.05). T lymphocytes (CD3 +, CD3 +CD4 + and CD3 +CD8 +) and natural killer cells (CD56 +CD16 +) showed no significant difference from baseline (all P>0.05). Conclusions:Rituximab can effectively eliminate CD19 + and CD20 + lymphocytes, and has little influence on peripheral blood lymphocyte count and immunoglobulin level except CD19 + and CD20 + lymphocytes. The standard administration of rituximab is safe for patients with FSGS and MCD.

Objective To evaluate the relationship between plasma cystatin C concentration (PcyC) and coronary artery diseases (CAD). Method A total of 126 subjects with CAD evidenced by coronary angiography admitted from April 2007 to March 2009 were divided into three groups: stable angina pectoris (SAPs, n = 34),unstable angina pectoris (UAPs, n = 56) and acute myocardial infarction (AMIs, n = 36), according to the diag-nostic criteria of CAD set by WHO. Another 34 subjects without CAD were taken as controls. There were no statis-tical differences in demographics among four groups. Serum lipids profile, uric acid (UA), PcyC and high-sensi-tive C-reactive protein (hs-CRP) were determined. And in the meantime, all patients were followed up for six months and adverse cardiovascular events were recorded. Comparisons were made between groups with a number of independent-sample t -tests. Data were processed with analysis of variance to test the differences in means among four groups, and the means were compared with chi-square test. Statistical significance was established at a P val-ue of less than 0.05. Results Cystatin C levels were significantly higher in UAPs than that in SAPs and in controls (P < 0.05), but were much lower than that in AMIs (P < 0.05). And much higher concentration of hs-CRP was found in UAPs (P < 0.05) and in AMIs (P < 0.01). Cystatin C was positively and significantly corre-lated with age, hs-CRP, WBC, creatinine and UA (r > 0, P < 0.05), whereas a significantly negative correla-tion with high-density lipoprotein cholesterol was found (r = - 0.227, P < 0.05). These coefficients were obvi-ously high for creatinine (r = + 0. 612), and WBC (r = + 0.459). During the period of six-month follow-up, 26 patients with adverse cardiovascular events were found, and had significantly higher cystatin C levels than 22 con-trols at admission (P < 0.01). Conclusions Cystatin C plays a pivotal role in the course of CAD, and the PcyC is a strong predictor for the risk of cardiovascular events.

OBJECTIVETo evaluate the association between chemical exposure, DNA methylation status and gene-environment interactions in the development of childhood acute leukemia (AL).

METHODSFrom January 1st 2009 to December 31st 2010, an exploratory case-control study was conducted on childhood AL among children who were less than 15 years of age in Shanghai, China. A total of 131 patients with newly diagnosed AL were recruited from 3 Shanghai children hospitals. The controls selected from the same hospital were healthy children who attended the physical check-up held by the department of Children's Healthcare, or who visited the clinic of developmental pediatrics or orthopedics (excluding blood diseases and malignant tumors). 140 controls matched with cases in gender and age were included in this study. Chemical exposure were investigated by questionnaires, methylation specific polymerase chain reaction (MSP) was adopted to analyze the methylation or deletion status of 8 genes, and gene-environment interactions were analyzed by relative excess risk of interaction (RERI), attributable proportion of interaction (API) and synergy index (S).

RESULTSThere were 131 and 140 subjects in case and control group, who were aged (6.9 ± 3.8) and (6.9 ± 3.9) years old (t = 0.01, P = 0.911), respectively. After adjusting age and other potential confounding factors, chemical substances' exposure of children/mother/father were all significantly higher in cases than that in controls (Children: OR = 3.90, 95% CI: 1.69-9.02; Mother: OR = 2.71, 95% CI: 1.12-6.52; Father: OR = 1.91, 95% CI: 1.05-3.47). For the 8 genes analyzed, the methylation status of DAPK and PTEN and P73 in case group were significantly higher than that in control group (cases: 3.1% (4 cases), 16.0% (21 cases), 7.6% (10 cases); controls: 0.7% (1 case), 2.9% (4 cases), 0.7% (1 case); χ²: 7.11, 16.90, 11.38; P value: 0.029, 0.000, 0.003). The methylation status of P16 in case group was significantly lower than that in control group (cases: 3.8% (5 cases); controls: 8.6% (12 cases), χ² = 10.33, P = 0.007). The interactions of children chemical substances' exposure and 3 genes' (PTEN, P16 and P73) methylation status were probably existed after adjusted for confounding factors (PTEN: RERI = -7.01, API = -2.14, S = 0.24; P16: RERI = 4.08, API = 0.53, S = 2.59; P73: RERI = 4.32, API = 0.48, S = 2.19), we also found the potential interaction between maternal chemical substances' exposure and PTEN, P16 gene methylation status (PTEN: RERI = -1.30, API = -0.38, S = 0.65; P16: RERI = 1.70, API = 0.38, S = 1.97).

CONCLUSIONThe study suggested the strong combined effects of chemical substances exposure of children and abnormal methylation status were risk factors of childhood AL, and there existed different interaction between them, which may indicate the important role in the pathogenesis process of childhood AL.

Acute Disease ; Case-Control Studies ; Child ; Child, Preschool ; China ; DNA Methylation ; Environmental Exposure ; Female ; Gene-Environment Interaction ; Humans ; Leukemia ; epidemiology ; Maternal Exposure ; Polymerase Chain Reaction ; Risk Factors

OBJECTIVETo investigate the association between children's and their parents' lifestyles, household environmental exposures and risk of childhood acute leukemia (AL).

METHODSA 1:2 matched case-control study of childhood AL was conducted in Shanghai between April 2011 and January 2014. The study enrolled 66 cases aged < 15, diagnosed with AL and 132 controls matched by age, gender and residence. All of the controls had no hematological diseases or previous history of malignancy. Children who had been adopted and had congenital genetic syndromes such as Down's syndrome or a positive HIV test result were not eligible as either cases or controls. Information was obtained from standardized face-to-face interviews of their parents/guardians with detailed questions on demographic characteristics, lifestyle, and household environment. Conditional logistic regression models were used to analyze effecting factors of childhood AL, odds ratios (OR) and their 95% confidence intervals (CI) were calculated.

RESULTSAmong 198 cases, 66 cases were aged (5.0 ± 3.7) years old, and 132 controls were aged (6.0 ± 3.8) years old (t = 0.48, P = 0.523). The paternal drink frequencies of cases and controls were 57.6% (38/66), and 31.1% (41/132), respectively (χ² = 4.91, P = 0.027). And the frequencies of household insecticides usage in the last year in the two groups were 78.8% (52/66), and 65.2% (86/132) (χ² = 3.87, P = 0.049). Chemical exposure during childhood (OR = 4.76, 95% CI: 1.34-16.89), maternal exposure to chemicals (OR = 4.51, 95% CI: 1.65-12.33), household insecticides use during 0-3 years of child (OR = 2.90, 95% CI: 1.31-6.39), and renovating after their children's birth (OR = 3.12, 95% CI: 1.26-7.74) were associated with an increased risk of childhood AL and these differences between the cases and the controls have statistical significance. Besides, we found that frequent contaction with other children during 0-3 years old (OR = 0.32, 95% CI: 0.15-0.69) and ventilation during sleeping in summer (OR = 0.43, 95% CI: 0.18-0.98) were associated with a decreased risk of childhood AL.

CONCLUSIONOur results support the association between children's and their parents' lifestyles, household environmental exposures and childhood AL.

Acute Disease ; Case-Control Studies ; Child ; Child, Preschool ; China ; Environmental Exposure ; Female ; Humans ; Insecticides ; Leukemia ; epidemiology ; Life Style ; Logistic Models ; Maternal Exposure ; Neoplasms ; Odds Ratio ; Parents ; Risk Factors

Objective To investigate clinicopathological features of pancreatic carcinoma with or without lymph node metastasis,and to explore the relationship between the lymph node metastasis and the prognosis of pancreatic carcinoma.Methods The clinical and follow up data of 216 patients with pancreatic carcinoma from 2001 to 2015 were retrospectively analyzed.Kaplan-Meier method was used to estimate survival rates and plot survival curves.Results The postoperative survival time was 4-86 months,the median survival time was 19 months,and the postoperative 1,3 and 5 year survival rates were 65.1％,33.8％,20.5％,respectively.Patients with positive lymph node metastasis were with 1,3,5 year survival rates of 36.5％,12.2％,0％,those with no lymph node metastasis were with 1,3,5 year survival rates of 70.3％,38.0％,21.4％ (x2 =15.803,P ＜ 0.001).Conclusions Lymph node metastasis in patients with pancreatic cancer is worse than that without lymph node metastasis.Lymph node metastasis is one of the main prognostic factors in patients after radical resection of the pancreatic cancer.

Objective To analyze the correlation between the status of the donation after brain and cardiac death (DBCD)donors and postoperative recovery of the organ function in the liver and renal transplant recipients.Methods The assessment data and organ protection measures of 12 DBCD donors admitted to the Organ Transplantation Center in Sichuan Provincial People’s Hospital from August 2011 to November 2013 were retrospectively analyzed.The parameters of postoperative recovery of 12 liver and 22 renal transplant recipients were also assessed.The correlation between the parameters of the donors and postoperative recovery of the liver and renal transplant recipients was statistically analyzed.Results Among 12 liver transplant recipients,1 patient had primary non-function (PNF)(1 /12,8%)and 11 cases developed delayed graft function (DGF) after renal transplantation (11 /22,50%).Intensive care unit (ICU)period,liver function,maintaining systolic blood pressure (SBP),blood coagulation function,blood glucose level and electrolyte (Na +/K +) were significantly correlated with postoperative recovery of the liver and kidney function in the recipients (all in P <0.05 ).Age,cause of brain death,maintaining diastolic blood pressure (DBP),activated partial thromboplastin time (APTT)and pH of arterial blood gas (ABG)were associated with postoperative recovery of the liver function.Total bilirubin and white blood cell count (WBC)were correlated with postoperative recovery of kidney function.Conclusions DBCD donors cater to the specific conditions in China.The incidence of postoperative PNF in liver recipients is relatively low whereas and the incidence of DGF after renal transplantation is relatively high.Assessment of the DBCD donors and organ protection measures should be specifically taken to enhance the clinical efficacy of liver and renal transplantation from DBCD donors.

The clinical data, laboratory testing, genetic testing results, diagnosis and treatment process of a child with PERCHING syndrome diagnosed and treated in the Department of Neonatology, the Third Affiliated Hospital of Zhengzhou University in June 2022 were retrospectively analyzed, and the relevant literatures were reviewed.The proband mainly presented with dyspnea and feeding difficulties after delivery, facial nevus flammeus, protrusion of eyes, small fissure of eyes, wide nasal root, limited opening of mouth, slightly high palatal arch, special posture, cryptorchid, hypospadias, and high muscle tone of limbs.Magnetic resonance imaging of the brain suggested possible agenesis of corpus callosum.Genetic testing showed complex heterozygous variations in the KLHL7 gene, and the two mutation sites have not been previously reported.A case of PERCHING syndrome caused by the KLHL7 gene mutation in China was reported for the first time, which provided new ideas for the diagnosis and treatment of children with PERCHING syndrome and reliable genetic evidence for family reproduction.