optimized method for ex vivo generation of dendritic cells

Dendritic cells are the most potent antigen presenting cells. They capture, process and present antigens to T cells and secrete various cytokines and soluble factors to initiate adaptive immune responses. Dendritic cells are also important in induction of immunological tolerance to self- antigens. Due to their crucial role in immune responses during infections, cancers, transplantations, allergies, and autoimmune diseases, they have become important targets for many biological and clinical studies. Usually large numbers of dendritic cells is essential for these studies; however, small numbers of these cells in blood and tissues makes the isolation very difficult. Therefore, In vitro generation of these cells is useful for research and clinical applications. The aim of this study was in vitro generation of dendritic cells from bone marrow-hematopoietic progenitor cells using a simple and efficient method. Murine bone marrow cells were cultured in medium supplemented with Granulocyte-macrophage colony-stimulating factor and Interleukin 4 without depletion of any cell population for 9 days. Fresh medium containing cytokines was added every 3 days. Analysis of the morphology and immunophenotype of cultured cells showed the generation of dendritic cells from day 2 of the culture period. But, the number of cells that possess morphological characteristics and typical cell surface markers [CD11c, MHC-II and CD86] of dendritic cells was elevated by increasing the culture period. The purity of dendritic cells was 86% by the end of 9 days culture. This method can be used as an efficient method for ex vivo generation of dendritic cells

Expressions of fractalkine and CD11c on common carotid artery atherosclerotic plaques from apoE(-/-) mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the association of fractalkine (FKN) and CD11c expressions oncommon carotid artery atherosclerotic plaques from apoE(-/-) mice with the severity of atherosclerotic lesions.</p><p><b>METHODS</b>Totally 24 apoE(-/-) mice were divided into two groups and fed on a high-fat diet or a normal diet for 12 weeks. Then the blood lipids as well as the plaque area and vascular stenosis rate of the common carotid artery were measured to evaluate the severity of atherosclerotic lesions of the animals. Moreover, immunohistochemical staining was performed to examine the levels of FKN and CD11c expression.</p><p><b>RESULTS</b>The plaque areas and vascular stenosis rates of the common carotid artery in the experimental group were remarkably larger than those in control group (about 4-fold and 2-fold, respectively). The level of FKN expression in the experimental group was 2 times of that in the control group (P<0.05), and the number of CD11c (+) cells in the plaques in the experimental group was about 4 times of than in the control group (P<0.05).</p><p><b>CONCLUSION</b>The expressions of chemokine and FKN remarkably increase in apoE (-/-) atherosclerotic plaques, suggesting that chemokine and FKN may paly important roles in the development of atherosclerosis.</p>

Anti-thrombosis effect and its mechanism of Qingkailing injection / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the anti-thrombosis effect and its mechanism of Qingkailing injection (QKL).</p><p><b>METHOD</b>SD rats were randomly divided into control group, model group and QKL 2.5, 5.0, 10 groups. QKL were given (i.p.) to rats once a day for successively 4 days. The rats in all groups but control were pretreated with carrageenin (Ca) i.p. at 16 h before the last dose of QKL and followed by intravenous injection of endotoxin ( LPS fom E. coli O111:B4) 50 microg x kg(-1) 30 min after the last dosing of QKL. Thrombosis in rat tails were observed at 24 h after injection of LPS. The number of white blood cells and platelets, serum TNF-alpha, IL-6 level, CD11b/CD18 expression of white blood cells and platelet aggregation were analysed.</p><p><b>RESULT</b>QKL obviously inhibited the LPS/Ca-induced thrombosis as showed a reduced infarction range due to thrombosis in tails. The sera concentration of TNF-alpha and IL-6, expression of CD11b/CD18 in WBC and platelet coagulation rate were reduced after QKL treatment.</p><p><b>CONCLUSION</b>The anti-thrombosis action of QKL is associated with inhibition of WBC activation and adherence, reduction of inflammatory factor release and abating of platelet coagulation rate. The anti-thrombosis mechanism of QKL is consistent with its function of clearing away heat-evil and toxic materials.</p>

[Leukocyte adhesion deficiency]

Leukocyte adhesion deficiency [LAD] is a rare functional leukocyte disorder, which is divided into two separate types: LAD-1 and LAD-2. LAD-1 results from lack of beta2 integrin molecules [CD 11 and CD 18] on the leukocyte cell surface. These molecules are essential for leukocyte adhesion to endothelial cells and chemotaxis. The present case report is about a 42-month-old girl with recurrent otitis, pneumonia and gingivitis. On physical examination, patient was pale and malnourished. Multiple desquamated erythematous plagues were found on her body and extremities. Blood investigations revealed persistent leukocytosis with normal serum Immunoglobulin profile and complement. The diagnosis of LAD1 was made based on Flow cytometry finding; showing decreased in GD11 and CD 18 markers of PMN-a. When a patient has persistent leukocytosis and recurrent infections, investigation for the primary immune deficiency, specially leukocyte adhesion deficiency must be considered

[Leukocyte adhesion deficiency syndrome and report of three cases]

Leukocyte adhesion deficiency syndrome [LADS] is a rare inherited immunodeficiency in which the frunction of leukocytes, particularly the phagocytes are disturbed. In the present study, we report three cases with LADS [2 male infants and 1 female child] who had a history of delay in separation of umbilical cord. One infant admitted for skin infection and two patients admitted in hospital following bacterial and fungal infections in nose and finger. All cases had a remarkable leukocytosis, neutrophilia and decreased of CD 11 and CD 18. LADS is a congenital syndrome causes recurrent bacterial and fungal infections. Usually all patients have a history of delayed separation of umbilical cord

Lipopolysaccharide preconditioning induces protection against lipopolysaccharide-induced neurotoxicity in organotypic midbrain slice culture / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To identify the protective effect of lipopolysaccharide (LPS) preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms.</p><p><b>METHODS</b>After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations (0, 1, 3, 6 or 10 ng/mL) of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was determined by measurement of the activity of lactic acid dehydrogenase (LDH). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons and CD11b/c equivalent-immunoreactive (OX-42-IR) microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-alpha (TNF-alpha) levels in the culture media were detected by enzyme-linked immunosorbent assays (ELISA).</p><p><b>RESULTS</b>In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191+/-12 in the control slices to 46+/-4, and the LDH activity elevated obviously (P < 0.01), along with remarkably increased number of OX-42-IR cells and production of TNF-alpha (P < 0.01). Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss (the number of TH-IR neurons increased to 126+/-12 and 180+/-13, respectively) and markedly reduced LDH levels (P < 0.05), accompanied by significant decreases of OX-42-IR microglia activation and TNF-alpha production (P < 0.05).</p><p><b>CONCLUSION</b>Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-alpha production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson's disease.</p>

Antitumor immunity by a dendritic cell vaccine encoding secondary lymphoid chemokine and tumor lysate on murine prostate cancer / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To investigate the antitumor immunity by a dendritic cell (DC) vaccine encoding secondary lymphoid chemokine gene and tumor lysate on murine prostate cancer.</p><p><b>METHODS</b>DC from bone marrow of C57BL/6 were transfected with a plasmid vector expressing secondary lymphoid chemokine (SLC) cDNA by Lipofectamine 2,000 liposome and tumor lysate. Total RNA extracted from SLC+lysate-DC was used to verify the expression of SLC by reverse transcriptase-polymerase chain reaction (RT-PCR). The immunotherapeutic effect of DC vaccine on murine prostate cancer was assessed.</p><p><b>RESULTS</b>We found that in the prostate tumor model of C57BL/6 mice, the administration of SLC+lysate-DC inhibited tumor growth most significantly when compared with SLC-DC, lysate-DC, DC or phosphate buffer solution (PBS) counterparts (P < 0.01). Immunohistochemical fluorescent staining analysis showed the infiltration of more CD4(+), CD8(+) T cell and CD11c(+) DC within established tumor treated by SLC+lysate-DC vaccine than other DC vaccines (P < 0.01).</p><p><b>CONCLUSION</b>DC vaccine encoding secondary lymphoid chemokine and tumor lysate can elicit significant antitumor immunity by infiltration of CD4(+), CD8(+) T cell and DC, which might provide a potential immunotherapy method for prostate cancer.</p>

Evaluation of CD11b expression on peripheral blood neutrophils for early detection of neonatal sepsis

Neonatal sepsis is a disease of infants who are less than 1 month of age. These infants are clinically ill, and their blood culture are positive for bacteria. The reported incidence of neonatal sepsis for allinfants is 1 to 10 per 1000 live births. The mortality rate is 4.2-26%. The clinical signs are not specific and diagnosis of neonatal sepsis is one of the most difficult tasks in clinical medicine. The aim of this work was determination of CD11b sensitivity and specificity for early detection of neonatal sepsis. We studied 65 neonates with gestational age of 27 to 38 weeks who were suspected for sepsis within the 28 days of life. Whole blood was obtained from neonates to determine CD11b expression on peripheral blood neutrophils by flow cytometry. C-Reactive protein [CRP] was measured qualitatively. Neonates were divided into two groups. Classification was based on the result of the blood culture. In the sepsis group all of the neonates [n = 8] showed positive blood culture and clinical symptoms. In the suspected group [n = 57] the neonates showed clinical signs but blood cultures were negative. Sensitivity and specificity of CD11b were 75%, 100% respectively. Also positive and negative predictive values of CD11b were 100% and 86% respectively. Results of present study and previous studies showed that measurement of neutrophil surface markers can be useful for diagnosis of infection in the early phases. Also, the quantitative measurement of CRP in addition to CD11b further enhances the ability to diagnose infections and improves sensitivity and negative predictive value by 100%

CD137 induces adhesion and cytokine production in human monocytic THP-1 cells

CD137, which is expressed on activated T cells, plays a critical role in inflammatory responses. However, the exact role that CD137 plays in monocytes is not fully known. Here we studied the expression and function of CD137 in human monocytic THP-1 cells, which we found constitutively expresses CD137 at the mRNA and protein level. Cross-linking of CD137 increased the secretion of IL-8 and TNF-alpha, promoted the expression of CD54 and CD11b, and increased adhesion to extracellular matrix (ECM) proteins. In particular CD137-induced adhesion of THP-1 cells was inhibited by an inhibitor of mitogen-activated protein kinase kinase (MEK), but not by a p38 kinase inhibitor. Taken together, these results show that the adhesion and cytokine production of THP-1 cells induced by CD137 occur via activation of MEK, which results in the activation of ERK-1/2 signaling pathways. Therefore, this study suggests that CD137 induces an activating and migrating signal during inflammatory processes.