In order to assess the number and function of macrophages in the placenta of pregnancy complicated with gestational diabetes mellitus (GDM) as well as those of normal pregnancies, placenta samples were collected from 15 GDM patients (GDM group) and 10 normal pregnant women (control group). The expression levels of macrophage markers (CD68/CD14) and inflammatory cytokines (IL-6/TNF-α) in placenta were detected using immunohistochemistry and PCR. The results showed that the number of CD68+ or CD14+ cells in the GMD group was remarkably higher than that in the control group (P<0.05), indicating that the number of macrophages in the GDM group was significantly greater than that in the control group. The mRNA expression levels of CD68+, IL-6 and TNF-α were higher in the GMD group than in the control group. In conclusion, more macrophages accumulate in placenta of pregnancy complicated with GDM, and the expression levels of pro-inflammation factors are also increased in GDM pregnancies, suggesting that macrophages and inflammatory mediators (IL-6 and TNF-α) may play an important role in GDM.
Adult ; Antigens, CD ; Antigens, Differentiation, Myelomonocytic ; Cell Count ; Cytokines ; genetics ; immunology ; metabolism ; Diabetes, Gestational ; genetics ; immunology ; metabolism ; Female ; Humans ; Immunohistochemistry ; Inflammation Mediators ; immunology ; metabolism ; Interleukin-6 ; genetics ; immunology ; metabolism ; Lipopolysaccharide Receptors ; genetics ; metabolism ; Macrophages ; immunology ; metabolism ; pathology ; Placenta ; immunology ; metabolism ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; genetics ; immunology ; metabolism

BACKGROUNDThe Fc receptor associated pathway might improve the immune responses against hepatitis B virus (HBV) as previously described by us. In addition, the Flt3 ligand (FL) has been reported to potentiate antigen presenting cells in vivo and may act as a potential adjuvant to boost antigen-specific immune responses. In this study, the immune efficacies of a set of fusion proteins of HBsAg and Fc and/or FL were evaluated in HBsAg transgenic mice.

METHODSThe fusion proteins composed of HBsAg and the Fc domain of murine IgG1 (HBsAg-Fc) and/or the Flt3 ligand, and yeast-derived recombinant HBsAg were used as immunogen to immunize HBsAg transgenic mice, respectively. Serum and liver HBsAg levels, serum anti-HBsAg and cytokine profile, and the activities of alanine aminotransferase (ALT)/AST were investigated after immunization.

RESULTSAfter six injections, the most pronounced decrease in serum and liver HBsAg levels was observed in the HBsAg-Fc immunized group. In addition, serum Th1 cytokines and ALT/AST activities were highest in this group, indicating an effective induction of a favorable cellular immune response. Interestingly, the fusion protein containing HBsAg-Fc and the Flt3 ligand stimulated an alternative Th1-type immune response featured with high level productions of tumor necrosis factor α (TNF-α) and monocyte chemoabstractant protein 1 (MCP-1), causing a more severe cytotoxicity in hepatocytes while showed less effective in reducing serum HBsAg level.

CONCLUSIONHBsAg-Fc is effective in eliciting both the humoral and cellular immune responses against HBsAg in HBsAg transgenic mice, which makes it a potential immunogen for the immunotherapy of chronic hepatitis B.

Animals ; Chemokine CCL2 ; metabolism ; Cytokines ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B Surface Antigens ; genetics ; immunology ; metabolism ; Immunity, Cellular ; immunology ; Immunity, Humoral ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Receptors, Fc ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo observe the effect of the antibody TSP-2 against a single epitope of mouse Toll-like receptor 2 extracellular domain (mTLR2ECD) on the expression of nuclear factor-kappa B (NF-κB) and cytokines in the intestinal tissue of septic mice.

METHODSMale BALB/c mice were randomly divided into 4 groups, namely the sham-operated group, model group, TSP-2 treatment group and rabbit IgG treatment group. Sepsis was induced by cecal ligation and puncture (CLP), and at 6, 12 or 24 h after the operation, the ileal tissues were harvested from the mice for HE staining. NF-κB expression was detected with immunohistochemistry. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA expressions were detected with qRT-PCR and their protein expressions by ELISA.

RESULTSThe NF-κB expression in the intestinal tissue significantly increased in the model group as compared with that in the sham- operated group, and decreased after TSP-2 treatment. The model group also showed significantly increased expression levels of TNF-α and IL-6 mRNA and protein in the intestinal tissue (P<0.05), which were lowered by TSP-2 (P<0.05) but not by rabbit IgG treatment (P>0.05).

CONCLUSIONThe TSP-2 antibody can protect the intestine and delay the development of sepsis by inhibiting NF-κB activation and down-regulating TNF-α and IL-6 expressions in mice.

Animals ; Antibodies ; pharmacology ; Immunodominant Epitopes ; immunology ; Interleukin-6 ; genetics ; metabolism ; Intestinal Mucosa ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; genetics ; metabolism ; Random Allocation ; Receptors, Cell Surface ; immunology ; Sepsis ; metabolism ; Thrombospondins ; immunology ; Toll-Like Receptor 2 ; immunology ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Animals ; Food Hypersensitivity ; genetics ; immunology ; metabolism ; Forkhead Transcription Factors ; genetics ; metabolism ; Glucocorticoid-Induced TNFR-Related Protein ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; genetics ; Receptors, Nerve Growth Factor ; genetics ; metabolism ; Receptors, Tumor Necrosis Factor ; genetics ; metabolism ; T-Lymphocytes, Regulatory ; metabolism

OBJECTIVETo investigate the expression of TLR4/2 mRNA in neonatal cord blood mononuclear cells (MNC).

METHODSForty-six neonates without asphyxia and 40 neonates with asphyxia were divided into groups depending on the gestational age. In the neonates without asphyxia, there were 18 full term infants (the gestational age > or = 37 weeks), 16 preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 12 preterm infants whose gestational age was < 32 weeks. In the neonates with asphyxia, 11 were full term infants, 15 were preterm infants whose gestational age was > or = 32 weeks but < 37 weeks and 14 were preterm infants at gestational age < 32 weeks. MNCs were separated and cultured with LPS (1 microg/ml) for 3 h. Cells were collected for analysis of gene expression of TLR4/2 by RT-PCR technique. Cell supernatants were taken to measure TNF-alpha production following the ELISA protocol. Fifteen healthy adults were enrolled into the control group. In addition, the Pearson correlation analyses were carried out between the levels of TLR4, TLR2 mRNA and the levels of TNF-alpha.

RESULTSIn the neonates without asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.75 +/- 0.12, 0.63 +/- 0.08, 2502.6 +/- 273.1 ng/L, separately, in the full term infants, 0.37 +/- 0.04, 0.32 +/- 0.03, 1218.8 +/- 145.7 ng/L, separately, in the preterm infants whose gestational ages were > or = 32 weeks but < 37 weeks, and 0.26 +/- 0.03, 0.20 +/- 0.03, 811.8 +/- 105.2 ng/L separately, in the preterm infants whose gestational ages were < 32 weeks. In the neonates with asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.58 +/- 0.07, 0.50 +/- 0.06, 1946.4 +/- 244.2 ng/L, separately, in the full term infants, 0.29 +/- 0.03, 0.26 +/- 0.03, 970.0 +/- 94.3 ng/L, separately, in the preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 0.17 +/- 0.02, 0.14 +/- 0.02, 652.6 +/- 60.3 ng/L, separately, in the preterm infants whose gestational age was < 32 weeks. The levels of TLR4, TLR2 mRNA and TNF-alpha in the adults were 2.71 +/- 0.75, 2.61 +/- 0.33, 9270.1 +/- 1098.3 ng/L, separately. In the preterm infants and full term infants, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower in comparison to the adults. The lower the gestational age, the lower the levels of TLR4, TLR2 mRNA and TNF-alpha. There were significant differences between the levels of TLR4, TLR2 mRNA and TNF-alpha of the neonates without asphyxia and those of the neonates with asphyxia. In the neonates with asphyxia, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower than those in the neonates without asphyxia (P < 0.01). Whether the neonates were asphyxic or not, the levels of TLR4, TLR2 were paralleled with the levels of TNF-alpha.

CONCLUSIONSThe expression of TLRs in the neonates, especially in the preterm infants was lower than that in the adults, which probably contributes to the susceptibility of neonates to infections.

Blood Cells ; metabolism ; Gene Expression ; Humans ; Infant ; Infant, Newborn ; RNA, Messenger ; genetics ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptors ; metabolism ; Tumor Necrosis Factor-alpha ; immunology

Oxidative stress plays critical roles in airway inflammation that is usually accompanied by increased vascular permeability and plasma exudation. VEGF increases vascular permeability and leads to airway inflammation. In addition, VEGF has been shown to enhance receptor activator of NF-kappaB (RANK) expression in endothelial cells. An aim of the study was to determine the potential role of antioxidant in the regulation of RANK expression in murine model of asthma. We have used a C57BL/6 mouse model of allergic asthma to evaluate the effect of L-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, which acts as an antioxidant, and VEGF receptor inhibitor on RANK mRNA expression. The mice develop the following pathophysiological features of asthma in the lungs: increased expression of RANK mRNA, increased number of inflammatory cells of the airways, increased vascular permeability, and increased levels of VEGF. Administration of OTC and VEGF receptor inhibitor markedly reduced plasma extravasation and VEGF levels in allergen-induced asthmatic lungs. We also showed that the increased RANK mRNA expression at 72 h after ovalbumin inhalation were reduced by the administration of OTC or VEGF receptor inhibitor. The results indicate that OTC and VEGF receptor inhibitor which inhibit up-regulation of VEGF expression modulate RANK expression that may be in association with the regulation of vascular permeability, and suggest that VEGF may regulate the RANK expression. These findings provide a crucial molecular mechanism for the potential use of antioxidants to prevent and/or treat asthma and other airway inflammatory disorders.
Vascular Endothelial Growth Factor A/analysis/antagonists & inhibitors/metabolism ; Thiazolidines ; Thiazoles/*pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors ; Receptors, Tumor Necrosis Factor/genetics/*metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Reactive Oxygen Species/metabolism ; RNA, Messenger/genetics/metabolism ; Pyrrolidonecarboxylic Acid ; Proto-Oncogene Proteins c-akt/metabolism ; Protein Kinase Inhibitors/pharmacology ; Prodrugs/pharmacology ; Phosphorylation/drug effects ; Ovalbumin/immunology ; Osteoprotegerin ; Mice, Inbred C57BL ; Mice ; Immunohistochemistry ; Glycoproteins/genetics/*metabolism ; Gene Expression/drug effects ; Female ; Capillary Permeability/drug effects ; Bronchoalveolar Lavage Fluid/chemistry/cytology ; Blotting, Western ; Asthma/*drug therapy/immunology/metabolism ; Antioxidants/*pharmacology ; Animals

BACKGROUNDRev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE).

METHODSMolecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope.

RESULTSThe new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect expression of TNF-R1 from pT128 was slower than the direct expression of that from Hu p60 TNFR1 in pDC302 (pT60), but all those pT60 or pT128 transfected cells showed apoptosis at last while TNF-R1 was sufficiently expressed. Other kinds of Rev expression construct such as pAD8 and a chimeric HIV vaccine also can switched on the selective expression of pT128. Not only Rev-dependent expression in Helas, pT128 also normally expressed its TNF-R1 in keratinocytes. Co-transfected with pRev or pAD8 that expressed Rev, pT128 expressed TNF-R1 and induced apoptosis of green fluorescent keratinocytes in skin explant. The number of green fluorescent keratinocytes co-transfected by pT128 plus pRev or pAD8 was gradually outnumbered by that co-transfected by pT128 only. The difference was more significant after culturing for 72 hours.

CONCLUSIONSRev dependent pT128 is able to selectively induce apoptosis of HIV-infected or Rev-expressed target cells by expression of TNF-R1. The new strategy based on manipulation of the regulatory protein of HIV may be valuable in design of new HIV vaccine.

AIDS Vaccines ; immunology ; Apoptosis ; Biolistics ; Cell Line, Tumor ; Gene Products, rev ; physiology ; Genes, env ; physiology ; Genetic Vectors ; Humans ; Keratinocytes ; metabolism ; Plasmids ; Receptors, Tumor Necrosis Factor, Type I ; genetics

Recent studies revealed that apoptotic cells are actively involved in immunosuppression and anti-inflammation. After being phagocytosed by macrophages, apoptotic cells can actively regulate cytokines secretion from lipopolysaccharide (LPS)-stimulated macrophages, in which the secretion of immunosuppressive cytokines such as interleukin-10 (IL-10) is increased while the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFa), interleukin-1beta (IL-1b) and leukin-8 (IL-8) are suppressed. In this paper, we first present evidence that phagocytosed apoptotic cells regulate cytokine secretion of LPS-stimulated macrophages, but also inhibit the activation of T lymphocytes stimulated by ConA. These data suggest that apoptotic cells can alter the biological behavior of macrophages which gain immunosuppressive property.
Animals ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Apoptosis ; immunology ; Chemokine CXCL2 ; Chemokines ; biosynthesis ; genetics ; Concanavalin A ; pharmacology ; Cytokines ; biosynthesis ; Female ; Humans ; Immune Tolerance ; In Vitro Techniques ; Jurkat Cells ; Lectins, C-Type ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; drug effects ; Macrophages ; drug effects ; immunology ; Mice ; Mice, Inbred ICR ; Phagocytosis ; Receptors, Interleukin-2 ; metabolism ; Signal Transduction ; immunology ; T-Lymphocytes ; drug effects ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4+CD25+ regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4+ and CD8+ T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4+ or CD8+ T cells with a CD4+ or CD8+ T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.
Animals ; Antibodies, Monoclonal/pharmacology ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cell Proliferation ; Female ; Glucocorticoids/*pharmacology ; Herpes Simplex/*immunology ; Herpesvirus 1, Human/pathogenicity ; *Immunity, Cellular ; Interferon Type II/secretion ; *Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Peptide Fragments/metabolism ; Receptors, Interleukin-2/metabolism ; Receptors, Nerve Growth Factor/genetics/immunology/*metabolism ; Receptors, Tumor Necrosis Factor/genetics/immunology/*metabolism ; Research Support, Non-U.S. Gov't ; T-Lymphocytes/*immunology/metabolism/virology

OBJECTIVEThe purpose of this study was to clone the soluble form of the mouse BlyS (msBlyS) and insert it into a eukaryotic expression vector pSecTag2B in order to further elucidat the antitumor activity induced by msBlyS expressed by the recombined plasmid pSecTag2B-msBlyS.

METHODSFull length cDNA of mouse soluble BlyS (msBlyS) was amplified by reverse transcription-PCR from total RNA of mouse spleen. The PCR product was ligated directly with linearized vector pCR2.1 supplied in the TA cloning kit. The recombined plasmid pCR2.1-msBlyS which was selected and identified using blue-white screening method and restriction map analysis and the purified original plasmid pSecTag2B were both cut by HindIII and EcoR I. The digested fragments were extracted and purified from low-melting temperature agarose and ligated by T4DNA ligase. The recombined plasmid pSecTag2B-msBlyS were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing.

RESULTSThe sequencing data indicated that inserted msBlyS gene had correct DNA sequence and orientation.

CONCLUSIONEukaryotic expression vector pSecTag2B. Expressing mouse BlyS have successfully been cloned. This will provide us an opportunity to do further research work on BlyS.

Animals ; B-Cell Activating Factor ; Cloning, Molecular ; Epitopes, B-Lymphocyte ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis, DNA ; Spleen ; cytology ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics