OBJECTIVE: To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines. METHODS: Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively. RESULTS: Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δβ value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R²=0.238, P<0.05; R²=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS. CONCLUSIONS: Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1β levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB. REGISTRATION NO ChiCTR-RCS-13004001.
Chemokine CCL3/genetics* ; Chemokine CCL4/genetics* ; Cytokines/genetics* ; DNA Methylation/genetics* ; HLA Antigens ; Hepatitis B, Chronic/genetics* ; Histocompatibility Antigens Class I ; Humans ; Interleukin-12/genetics* ; Medicine, Chinese Traditional ; RNA, Messenger ; Syndrome

To study the effect of anemoside B4 on rats with chronic obstructive pulmonary disease (COPD).Seventy-two SD male rats were randomly divided into blank group and model group.The method of exposure to cigarette smoke and combined with lipopolysaccharide (LPS) was used to replicate the rat model of COPD.After the model was maintained for 5 weeks,the rats were randomly divided into model group,dexamethasone group (0.81 mg·kg~(-1)) and anemoside B4 low,medium and high (2,4,8 mg·kg~(-1)) dose groups,a group of 12 animals were administered,and then the administration was started.The administration was maintained until the28th day,and the pulmonary function parameters of rats were measured by an animal pulmonary function instrument.After testing the rat lung function parameters,immediately draw rat alveolar lavage fluid (BALF),and use high-throughput protein chip technology to determined the expression levels of inflammatory cytokines in rat BALF.HE staining was used to observe the general pathological changes of rat lung and tracheal tissue.Masson staining was used to observe the collagen deposition in rat lung tissue.Real-time q PCR method was used to determine the mRNA expression level of related genes in rat lung tissue.Western blot method was used to determine the expression levels of related proteins in rat lung tissues.According to the findings,compared with the model group,the dexamethasone group and the anemoside B4 drug groups had different degrees of increase in the lung function parameters of rats (P<0.01,P<0.05),improved the expression level of inflammatory cytokines in the BALF of rats to varying degrees (P<0.01,P<0.05),and improved the pathological structure of rat lung tissue to varying degrees.Relative mRNA expressions of matrix metalloproteinase 2 (MMP-2),matrix metalloproteinase 12 (MMP-12),matrix metalloproteinase inhibitor 1 (TIMP-1),interleukin-6 (IL-6),and transforming growth factor-β1 (TGF-β1) were significantly reduced (P<0.01);whereas relative mRNA expressions of matrix metalloproteinase 9(MMP-9) and matrix metalloproteinase inhibitor 2 (TIMP-2) were increased significantly (P<0.01).The mRNA and protein expression levels of T-box transcription factor (T-bet),interleukin-12 (IL-12) and signal transducer and activator of transcription 4(STAT4) reduced to varying degrees (P<0.01,P<0.05).The mRNA of transcription factor GATA3 (binding protein-3),interleukin-4 (IL-4) and signal transducer and activator of transcription 6 (STAT6) in rat lung tissues and the protein expression levels of IL-4 and STAT6 were increased to varying degrees (P<0.01,P<0.05).In conclusion,anemoside B4 has a certain protective effect on COPD rats caused by cigarette smoke exposure and combined with LPS.The mechanism of action may be related to the regulation of IL-12/STAT4 and IL-4/STAT6 signaling pathways.
Animals ; Interleukin-12 ; Interleukin-4 ; Lung/metabolism* ; Male ; Matrix Metalloproteinase 2 ; Pulmonary Disease, Chronic Obstructive/genetics* ; Rats ; STAT4 Transcription Factor/metabolism* ; STAT6 Transcription Factor/metabolism* ; Saponins

OBJEVTIVETo study the effect of spvB/spvC gene on Salmonella virulence and the Host immune.

METHODSSTM.211, STM.211-Delta;spvB, STM.211-Delta;spvC, STM.211-Delta;spvB.spvC and PBS were infected with 0.2 mL 10(5) CFU corresponding strain respectively by intraperitoneal. We observed the mental status, movement, diarrhea, weight, pelage changed hair of the infected mouse. Then the level of IL-10, IL-12, IFN-γ were detected by ELISA. Finally, we observe the pathological changes of liver and spleen with the general view and the microscope.

RESULTSInfection symptoms of STM.211, STM.211-Delta;spvB and STM.211-Delta;spvC were significantly worse than PBS group, but there was no significant difference between STM.211-spvB.spvC group and PBS group. The secretion of IFN-γ and IL-12 of STM.211, STM.211-Delta;spvB, STM.211-Delta;spvC group were significantly lower than those in the STM.211-Delta;spvB.spvC group (P<0.05), but IL-10 secretion was significantly higher than STM.211-Delta;spvB.spvC group (P<0.05). There were no statistical significance among the STM.211, STM.211-Delta;SpvB, STM.211-Delta;spvC groups (P>0.05).

CONCLUSIONSSalmonella virulence can be affected obviously by spvB combined with spvC gene, but not by spvB or spvC. spvB/spvC gene can inhibit the TH1 cytokines (IFN-γ and IL-12) secretion but promote the TH2 cytokines (IL-10) expression, leading immune response trend to TH2 shift. It shows that spvB/spvC gene can help the bacteria evade the host immune defenses, leading to aggravation of infection.

Animals ; Cytokines ; immunology ; Interleukin-12 ; Mice ; Salmonella ; genetics ; pathogenicity ; Salmonella Infections ; immunology ; Virulence ; Virulence Factors ; genetics

OBJECTIVETo explore the molecular biology mechanism of acupuncture on improving immune function damage induced by chemotherapy in cancer-bearing mice.

METHODSSeventy-two mice (36 mice in 3-day treatment and 5-day treatment, respectively) which were successfully made into cancer-bearing model were divided into a blank group, a model group, an acupuncture group and a moxibustion group by stratified randomization method, 9 mice in each one. Except for the mice in the blank group, the remaining mice were treated with intra-peritoneal injection of cyclophosphamide (CTX, 150 mg/kg), to establish the cancer-bearing mice of CTX. The mice in the blank group were treated with intraperitoneal injection of 0.9% NaCl (identical dose as other groups). After 4 h, the mice in the acupuncture group and moxibustion group were treated with acupuncture and moxibustion at "Dazhui" (GV 14), "Geshu" (BL 17), "Shenshu" (BL 23), "Zusanli" (ST 36), once a day. The mice in the blank group and model group were treated with immobilization and fixation during the same time. On the next day of the end of 3-day and 5-day treatment, the sample was collected. The ELISA method was applied to measure the contents of IL-12 and TNF-α in spleen of all the mice.

RESULTSAfter 3-day and 5-day treatment, compared with the blank group, the contents of IL-12 and TNF-α in spleen in the model group were reduced (all P < 0.05); compared with the model group, the contents of IL-12 and TNF-α in spleen in the acupuncture group and moxibustion group were increased (all P < 0.05), but the content of IL-12 and TNF-α in the acupuncture group was not different from that in the moxibustion group (both P > 0.05).

CONCLUSIONAcupuncture and moxibustion could effectively increase the contents of IL-12 and TNF-α in spleen of CTX cancer-bearing mice, which could relieve chemotherapy-induced immune function damage to improve immune function.

Acupuncture Therapy ; Animals ; Cyclophosphamide ; adverse effects ; Humans ; Interleukin-12 ; genetics ; metabolism ; Male ; Mice ; Moxibustion ; Neoplasms ; chemically induced ; genetics ; metabolism ; therapy ; Spleen ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

OBJECTIVETo study the infiltration of macrophages and their phenotype in the healing process of full-thickness wound in rat.

METHODSThirty healthy SD rats were divided into control group (n = 6) and injury group (n = 24) according to the random number table. Two round full-thickness skin defects (11 mm diameter) were created on both sides of dorsal spine of rats in injury group with surgical scissors and homemade trephine. After injury, wound area was measured immediately. The wounds were disinfected with iodophor every day. Rats in control group received anesthesia and hair removal only. On post injury day (PID) 1, 3, 7, and 13, respectively, 6 rats of injury group were sacrificed after the measurement of wound area (wound healing rate was calculated). Wound samples were obtained by excision down to healthy fascia along wound edge. Histological study was done with HE staining. The expression of CD68 (the surface marker of macrophage) in the wound tissue was observed with immunohistochemical staining. The double positive expressions of induced nitric oxide synthase (iNOS) plus CD68 (type I macrophage) and arginase 1 (Arg-1) plus CD68 (type II macrophage) were observed with immunofluorescence staining. The levels of interferon-γ (IFN-γ), TNF-α, IL-4, IL-13, IL-10, and IL-12 in wound tissue were assayed by double-antibody sandwich ELISA, and the ratio of IL-10/IL-12 was calculated. Full-thickness skin tissues (11 mm diameter) in rats of control group were excised at the same site as rats in injury group, and the histological observation and cytokines assay were performed as well. Data were processed with one-way analysis of variance or LSD- t test.

RESULTSWound area of rats in injury group was gradually reduced after injury, and the overall difference of the wound healing rate on each PID was statistically significant (F = 358.55, P < 0.01). No abnormal appearance of skin tissue was observed in rats of control group. In injury group, inflammatory cell infiltration was obvious in wound tissue on PID 1 and 3; vascular structure and fresh collagen were observed in wound tissue on PID 7 and 13. Numbers of CD68 positive cells in skin tissue of rats in control group and wound tissue of rats in injury group on PID 1, 3, 7, and 13 were respectively (2.7 ± 1.5), (31.8 ± 3.5), (40.8 ± 4.7), (20.8 ± 2.8), (3.2 ± 2.4) per 200 times visual field (F = 180.55, P < 0.01). Compared with that in control group, the number of CD68 positive cells of rats in injury group was increased on PID 1, 3, and 7 (with t values respectively 18.81, 18.79, 14.05, P values below 0.01). No double positive expression of iNOS plus CD68 or Arg-1 plus CD68 was observed in normal tissue of rats in control group. In injury group, proportions of iNOS plus CD68 double positive cells on PID 1, 3, 7, and 13 were respectively (12.2 ± 2.8)%, (16.5 ± 2.9)%, (4.2 ± 2.3)%, (0.7 ± 0.8)% (F = 72.50, P < 0.01); proportions of Arg-1 plus CD68 double positive cells on PID 1, 3, 7, and 13 were respectively 0, (8.2 ± 1.9)%, (21.5 ± 3.4)%, (4.7 ± 2.0)% (F = 120.93, P < 0.01). In injury group, proportion of iNOS plus CD68 double positive cells on PID 3 was significantly higher than that on other PID (with t values respectively 2.65, 8.17, 12.95, P values below 0.05); proportion of Arg-1 plus CD68 double positive cells on PID 7 was higher than that on other PID (with t values respectively 15.27, 8.25, 10.38, P values below 0.01). Compared with that of Arg-1 plus CD68 double positive cells, proportion of iNOS plus CD68 double positive cells was higher on PID 1 and 3 (with t values respectively 10.71 and 5.88, P values below 0.01) and lower on PID 7 and 13 (with t values respectively 10.24 and 4.60, P values below 0.01). The overall differences of IFN-γ, TNF-α, IL-4, IL-13, and IL-10/IL-12 ratio in skin tissue of rats in control group and wound tissue of rats in injury group on every PID were statistically significant (with F values from 14.08 to 631.03, P values below 0.01). Compared with those in control group, levels of IFN-γ, TNF-α, IL-4, and IL-13 in wound tissue of rats in injury group were significantly higher on every PID (with t values from 4.58 to 9.17, P values below 0.05), while IL-10/IL-12 ratio was significantly higher on PID 1, 3, and 7 (with t values respectively 27.70, 30.51, 9.49, P values below 0.05) . In injury group, IFN-γ level on PID 1 [(61 ± 5) pg/mL] and IL-10/IL-12 ratio on PID 3 (1.647 ± 0.098) were significantly higher than those of control group and those on other PID in injury group [with IFN-γ level respectively (32 ± 4), (54 ± 6), (46 ± 7), (47 ± 4) pg/mL and IL-10/IL-12 ratio respectively 0.328 ± 0.045, 0.960 ± 0.034, 0.530 ± 0.028, 0.289 ± 0.040, with t values respectively from 3.19 to 8.20 and from 16.59 to 31.84, P values below 0.05].

CONCLUSIONSMacrophage infiltration increases in the healing process of full-thickness wound in rat with different phenotypes, among which type I macrophage appears in the inflammatory stage, and type II macrophage predominates in the proliferative stage.

Animals ; Antigens, CD ; genetics ; metabolism ; Antigens, Differentiation, Myelomonocytic ; genetics ; metabolism ; Collagen ; Enzyme-Linked Immunosorbent Assay ; Interferon-gamma ; Interleukin-10 ; Interleukin-12 ; Interleukin-13 ; Interleukin-4 ; Macrophages ; Male ; Phenotype ; Rats ; Skin ; injuries ; Tumor Necrosis Factor-alpha ; blood ; Wound Healing ; genetics

OBJECTIVETo explore different microRNA expression profiles between chronic hepatitis B (CHB) patients of Pi-Wei dampness-heat syndrome (PWDHS) and Gan depression Pi deficiency syndrome (GDPDS).

METHODSBy applying gene chip technology, blood samples from CHB patients of PWDHS (3 cases), GDPDS (3 cases), and healthy volunteers (3 cases) were withdrawn and microRNA detected. The microRNA was screened and functional analyses performed by using SAS system.

RESULTSTotally 77 microRNAs with differential expression were screened from CHB patients of PWDHS and healthy volunteers, including 60 up-regulated microRNAs and 17 down-regulated microRNAs. Functions of target genes were mainly associated with transcription factors, gas exchange, adverse stimulating, regulation of enzyme activities, developing of the immune system, and the process of actin filaments. Totally 41 microRNAs with differential expression were screened from CHB patients of GDPDS and healthy volunteers, including 32 up-regulated microRNAs and 9 down-regulated microRNAs. Functions of target genes were mainly associated with binding to nucleotide or chromatin, inhibition and activation of transcription, biosynthesis, regulation of metabolic process, regulation of enzyme activities, developing of the immune system, the process of actin filaments, and IL-12. Totally 6 microRNAs with differential expression were screened from CHB patients of PWDHS and CHB patients of GDPDS, including 1 up-regulated microRNA and 5 down-regulated microRNAs. Functions of target genes were mainly associated with transmembrane transport, regulation of transcription factors, metabolism of hormones, developing of the immune system, the process of actin filaments, regulation of metabolic process, response to exterior stimulation, and so on.

CONCLUSIONThere existed differentially expressed microRNAs (spectrum) between CHB patients of PWDHS and CHB patients of GDPDS.

Depression ; Hepatitis B, Chronic ; genetics ; metabolism ; Hot Temperature ; Humans ; Interleukin-12 ; metabolism ; Medicine, Chinese Traditional ; MicroRNAs ; metabolism ; Oligonucleotide Array Sequence Analysis ; Research ; Syndrome

BACKGROUNDHow the transcriptional factors regulated the innate and adaptive immune system in pregnancy and pre-eclampsia are less understood. Nevertheless, what the plasma work in the development of this disease was not sure. The present study was design to evaluate what the transcriptional factors change in innate and adaptive immune system and what the plasma do in this filed.

METHODSPeripheral blood mononuclear cells (PBMC) from non-pregnant women (n = 18), women with clinically normal pregnancies (n = 23) and women with pre-eclampsia (n = 20) were separated from peripheral blood to isolate monocytes and T cells. The purity of monocytes and T cells were analysed by flow cytometry. Monocytes and T cells were stimulated in either lipopolysaccharides (LPS) or phorbol-myristate-acetate (PMA), respectively. Transcription Factor Arrays were used to screen the transcription factors of interest in comparing of different groups. PBMC were isolated from another 8 non-pregnant samples were co-incubated with different groups of plasma. Polymerase chain reaction (PCR) was performed using whole cell extractions of the samples.

RESULTSNuclear factor of activated T-cells-1 (NFAT-1), signal transducers and activators of transcription-1 (STAT-1) and activator protein-1 (AP-1) are up-regulated in monocytes in pregnancy and more so in pre-eclampsia. On the the contrary, NFAT-1, STAT-1 and AP-1 are down-regulated in T cells in pregnancy and more so in pre-eclampsia. A reduction was observed in interferon (IFN)-γ, interleukin (IL)-12 and IL-4 expression in T cells incubated with pre-eclamptic plasma. An elevation was observed in tumor necrosis factor (TNF)-α, IL-1 and IL-12 expression in monocytes incubated with pre-eclamptic plasma.

CONCLUSIONSInnate immunity is over activated and adaptive immunity is over suppressed in the development of pre-eclampsia. NFAT-1, STAT-1 and AP-1 might be the central transcription factors in the pathogenesis of pre-eclampsia. They induced some changes in plasma and "educate" the monocytes and T cells for relevant cytokine production. Successful completion of this study will enhance our understanding of pre-eclampsia and will discover new knowledge beyond pregnancy. The work will inform future therapies for the treatment of a wide range of condition such as transplantation immunology and a wide range of immune and inflammatory conditions.

Adult ; Female ; Humans ; Immunity, Innate ; physiology ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Male ; NFATC Transcription Factors ; genetics ; metabolism ; Pre-Eclampsia ; immunology ; metabolism ; Pregnancy ; STAT1 Transcription Factor ; genetics ; metabolism ; Transcription Factor AP-1 ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Young Adult

OBJECTIVETo observe the effect of Huanglian Jiedu IJecoction (HJU) on systemic and vascular immune responses of high fat diet fed apoE deficient (apoE(-/-)) mice.

METHODSEight wild type C57BL6 mice were recruited as the wild type common food group. Totally 24 apoE(-/-) mice were randomly divided into the ApoE'common food group, the ApoE(-/-) hyperlipidemia group, and the ApoE(-/-) hyperlipidemia plus HJD group, 8 in each group. In the present study, the common food mice and high fat fed mice were fed with a chow diet or a high cholesterol diet for 4 weeks. HJD was given to mice in the ApoE(-/-) hyperlipidemia plus HJD group at the daily dose of 5 g/kg by gastrogavage, while equal volume of pure water was given to mice in the rest groups by gastrogavage. Four weeks later, the plasma levels of blood lipids, the ratio of peripheral blood mononuclear cells, and expressions of Toll-like receptor 4 (TLR-4) and CD36 on the monocytes were detected. The pathological changes and expressions of cytokines in local aorta were detected. The plasma cytokine levels in response to lipopolysaccharide (LPS) were analyzed. Results (1) Compared with the wild type common food group, TO, TG, and LDL-O significantly increased in the ApoE(-/-) common food group (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, TC and LDL-C significantly increased in the hyperlipidemia group (P < 0. 05). There was no statistical difference in each index between the ApoE(-/-) hyperlipidemia group and the ApoE(-/-) hyperlipidemia plus HJD group (P > 0.05). (2) Compared with the wild type common food group, no obvious change of the ratio of peripheral blood mononuclear cells happened, the TLR4 expression level significantly increased in the ApoE'common food group (P < 0. 05). Compared with the ApoE common food group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly increased in the ApoE' hyperlipidemia group (P < 0.05). Compared with the ApoE(-/-) hyperlipidemia group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly decreased. Besides, the CD36 expression level also significantly decreased (P<0.05). (3) After stimulated by LPS for 3 h, compared with the wild type common food group, plasma TNF-ct and IL-b expressions significantly increased in the ApoE(-/-) common food group (P < 0.05). Compared with the ApoE(-/-) common food group, plasma expressions of IL-12, TNF-alpha, MCP-1, and IL-10 increased, but with no statistical difference in the ApoE(-/-) hyperlipidemia group (P > 0.05). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, the MCP-1 expression was significantly down-regulated, while the IL-10 expression significantly increased, showing statistical difference (P < 0.05). Compared with the wild type common food group, mRNA expression levels of IFN-gamma, MCP-1 , TNF-alpha, IL-10, and IL-1beta significantly increased (P < 0. 05, P < 0.01). Compared with the ApoE(-/-) common food group, not only mRNA expression levels of IFN-gamma, MCP-1, TNF-alpha, and IL-1beta, further significantly increased, but also IL-12, IL-10, and TGF-beta significantly increased (P < 0. 05, P < 0. 01). After 4-week intervention of HJD, compared with the ApoE(-/-) hyperlipidemia group, mRNA expression levels of MCP-1, TNF-alpha, IL-1beta, and IL-12 significantly decreased in the ApoE(-/-) hyperlipidemia plus HJD group (P < 0.05, P < 0.01).

CONCLUSIONSHigh fat diet induced systemic reaction and inflammatory reactions of local vessels. The local inflammatory response of vessels exceeded systemic inflammatory response. Intervention of HJD could attenuate inflammatory response, especially in local arteries. Meanwhile, it enhanced systemic anti-inflammatory reactions.

Animals ; Aorta ; pathology ; Apolipoproteins E ; genetics ; CD36 Antigens ; metabolism ; Chemokine CCL2 ; metabolism ; Dietary Fats ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hyperlipidemias ; blood ; etiology ; immunology ; Inflammation ; Interleukin-10 ; blood ; Interleukin-12 ; blood ; Interleukin-1beta ; blood ; Leukocytes, Mononuclear ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Systemic Inflammatory Response Syndrome ; blood ; etiology ; immunology ; Toll-Like Receptor 4 ; metabolism ; Transforming Growth Factor beta ; blood ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.

METHODSHuman IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1, and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000. The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA, and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay. The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation, and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.

RESULTSEnzyme digestion, PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB. IL-12, GM-CSF and B7.1 expressions were all detected in transfected 293T cells, and the expression of IL-12 was also detected in the transfected mouse muscular tissues.

CONCLUSIONA novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro, which facilitates further studies of tumor immunogene therapy.

Animals ; B7-1 Antigen ; genetics ; immunology ; Cancer Vaccines ; genetics ; immunology ; Electroporation ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Humans ; Interleukin-12 ; genetics ; immunology ; Mice ; Plasmids ; Transfection