OBJECTIVE: To investigate an improved culturing method for karyotyping analysis, and increase the detection rate of cytogenetic abnormalities in patients with multiple myeloma (MM), so as to provide more powerful information for the clinical diagnosis, prognosis stratification, and individualized treatment of MM patients. METHODS: Eighty newly-diagnosed MM patients were enrolled and divided into two groups. In observation group, IL-6 (10 ng/ml) and GM-CSF (30 ng/ml) were supplemented in the culture medium, while no stimulating factor was added in control group. The samples from both groups were cultured for 72 hours under the same conditions, and their karyotypes were analyzed by G-banding. The detection rate of the cytogenetic abnormalities, as well as the corresponding characteristics were compared between the two groups. RESULTS: The detection rate of the chromosome aberrations was greatly increased in the observation group compared with the control group, the overall detection rate was 72.5% and 22.5%, respectively, as well as 80.0% and 19.2% in the subgroup of ≤60 years old, 68.0% and 28.6% in the subgroup of > 60 years old, which showed significant statistical differences (P<0.05). CONCLUSION The modification of the culturing method with the addition of IL-6 (10 ng/ml) and GM-CSF (30 ng/ml) dual stimulating factors followed by incubation for 72 hours can effectively increase the detection rate of abnormal karyotypes in MM patients.
Chromosome Aberrations ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Interleukin-6 ; Karyotype ; Karyotyping ; Middle Aged ; Multiple Myeloma/genetics*

OBJECTIVE: To identify differentially expressed genes in peripheral blood mononuclear cells between patients with continuous mild-to-moderate asthma and healthy controls using mRNA microarray in order to explore the underlying signaling pathways and clarify the roles of CD4+ T cells in the pathogenesis of asthma. METHODS: Global transcriptomic profiles of the CD4+ T cells were defined by using Agilent Sure Print G3 Human GE 8×60K microarray. Enrichment pathways were analyzed with Ingenuity Pathway Analysis (IPA) software. RESULTS: Compared with controls, 805 genes were up-regulated, 192 were down-regulated in asthma patients. Among these, the expression of 38 annotated genes have varied by 4 times or more. Expression of CD300A was inversely proportional to the absolute value of eosinophils (r=-0.89, P=0.02) as well as the proportion of eosinophils (r=-0.94, P=0.004), while CSF1R was inversely proportional to PD20 (r=-0.83, P=0.04) and AQLQ (r=-0.88, P=0.02) by correlation analysis. CONCLUSION Numerous pathophysiological pathways may be involved in the pathogenesis of asthma. Above findings have provided a basis for the delineation the pathogenesis of asthma.
Antigens, CD ; genetics ; Asthma ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Case-Control Studies ; Eosinophils ; Gene Expression Profiling ; Humans ; Leukocytes, Mononuclear ; Oligonucleotide Array Sequence Analysis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Immunologic ; genetics ; Transcriptome

Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in traditional Chinese medicines (TCM). Two isomers, paeoniflorin (PF) and albiflorin (AF), are isolated from P. lactiflora. The present study aimed to investigate the protective effects of PF and AF on myelosuppression induced by chemotherapy in mice and to explore the underlying mechanisms. The mouse myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CP, 200 mg·kg(-1)). The blood cell counts were performed. The thymus index and spleen index were also determined and bone morrow histological examination was performed. The levels of tumor necrosis factor-α (TNF-α) in serum and colony-stimulating factor (G-CSF) in plasma were measured by Enzyme-Linked Immunosorbent Assays (ELISA) and the serum levels of interleukin-3 (IL-3), granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), and interleukin-6 (IL-6) were measured by radioimmunoassay (RIA). The levels of mRNA expression protein of IL-3, GM-CSF and G-CSF in spleen and bone marrow cells were determined respectively. PF and AF significantly increased the white blood cell (WBC) counts and reversed the atrophy of thymus. They also increased the serum levels of GM-CSF and IL-3 and the plasma level of G-CSF and reduced the level of TNF-α in serum. PF enhanced the mRNA level of IL-3 and AF enhanced the mRNA levels of GM-CSF and G-CSF in the spleen. PF and AF both increased the protein levels of GM-CSF and G-CSF in bone marrow cells. In conclusion, our results demonstrated that PF and AF promoted the recovery of bone marrow hemopoietic function in the mouse myelosuppression model.
Animals ; Antineoplastic Agents ; adverse effects ; Bridged-Ring Compounds ; administration & dosage ; Cyclophosphamide ; adverse effects ; Drugs, Chinese Herbal ; administration & dosage ; Glucosides ; administration & dosage ; Granulocyte Colony-Stimulating Factor ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Hematologic Diseases ; etiology ; genetics ; metabolism ; prevention & control ; Humans ; Interleukin-3 ; genetics ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Monoterpenes ; administration & dosage ; Paeonia ; chemistry ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

RING finger protein 13 (RNF13) is a novel E3 ubiquitin ligase whose expression is associated with cancer development. However, its specific role in cancer progression and metastasis remains unclear. Here, a B16F10/LLC experimental pulmonary metastatic model was developed to examine the formation of metastatic foci in the lung. A greater number of tumor colonies were observed in the lungs of RNF13-knockout (KO) mice than in their wild-type (WT) littermates, whereas no significant differences in tumor size were observed between the two groups. In short-term experiments, the number of fluorescently-labeled B16F10 cells increased remarkably in RNF13-KO lungs at early time points, whereas clearance of tumor cells from the blood was not affected. These results indicated that RNF13 may inhibit the colonization of B16F10 cells in the lung. Assessment of the concentration of various cytokines in tumor bearing lungs and blood did not detect significant differences between the blood of RNF13-KO and WT mice; however the levels of GM-CSF were significantly reduced in RNF13-KO tumor bearing lungs, which may have guided more B16F10 cells to migrate to the lungs. This was confirmed by lower GM-CSF concentrations in conditioned media from the culture of RNF13-KO lung slices. Collectively, our results suggest that host RNF13 affects the concentration of GM-CSF in tumor-bearing lungs, leading to a reduction in the colonization of metastatic tumor cells in the lung.
Animals ; Cell Line, Tumor ; Gene Knockout Techniques ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Lung ; metabolism ; pathology ; Lung Neoplasms ; pathology ; Mice ; Mice, Knockout ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Ubiquitin-Protein Ligases ; deficiency ; genetics

Granulocyte colony stimulating factor (G-CSF) restores neutrophil count in patients with chemotherapy-induced neutropenia. G-CSF can also induce production of epithelial neutrophil activating protein-78 (ENA-78) and interleukin-8 (IL-8), chemotactic factors from neutrophils in vitro. This study was purposed to investigate whether this effect is also observed in vivo. 10 lymphoma patients were selected who received chemotherapy and G-CSF (nartograstim) administration. Blood was obtained before chemotherapy [Time Point 1 (TP1)], at neutropenic phase before G-CSF administration (TP2), and at neutrophil recovery phase after G-CSF (TP3). ENA-78 and IL-8 mRNA in neutrophils were quantified by real-time PCR. Phagocytosis and reactive oxygen species (ROS) generation were examined by flow cytometry. The results showed that ENA-78 and IL-8 mRNA expression at TP2 increased in 5 and 8 patients, respectively. The ENA-78 mRNA expression at TP3 was increased in 3 and decreased in 6 patients, and IL-8 mRNA expression at TP3 decreased in 7 patients. G-CSF did not affect phagocytosis and normalized ROS generation in all of the patient. It is concluded that increase of ENA-78 and IL-8 expression in neutrophils is common in chemotherapy-induced neutropenic patients. G-CSF administration does not significantly increase ENA-78 and IL-8 expression.
Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; Chemokine CXCL5 ; metabolism ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-8 ; metabolism ; Lymphoma ; metabolism ; Male ; Middle Aged ; Neutropenia ; chemically induced ; metabolism ; Neutrophils ; drug effects ; metabolism ; RNA, Messenger ; genetics

This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF. In this study, plasmid DNA (pJME/GM-CSF) was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process. Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection. The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly. The killing activity of CTLs was assessed using the lactate dehydrogenase (LDH) release assay. The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results. Our results also found that, after pJME/GM-CSF injection, the incoming cells were a mixture of macrophages, neutrophils, and immature DCs. Meanwhile, pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs, and enhanced their Ag capture and presentation functions. Cell-mediated immunity was induced by the vaccine. Furthermore, chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment, antigen processing and presentation, and vaccine enhancement. These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations, and enhance pJME/GM-CSF-induced cellular immune responses.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Chitosan ; administration & dosage ; immunology ; Dendritic Cells ; immunology ; virology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; genetics ; immunology ; Humans ; Immunity, Cellular ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; administration & dosage ; Spleen ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

OBJECTIVETo investigate the effects of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) on wound healing and microRNA expression in diabetic rats.

METHODSEighteen male SD rats of clean grade were used to reproduce diabetes model. Four weeks later, a total of 64 full-thickness skin wounds were created on the back of 16 rats with established diabetes, with 4 wounds on each rat. Two symmetrical wounds on either side of the spine were created as a pair according to paired design. Then the wounds were divided into groups A and B according to the random number table and blind method (red and blue tags on the rhGM-CSF or the gel vehicle), with 32 wounds in each group. The ointment with red tag was applied on the wounds of group A and the blue one on group B. The application was conducted once a day, with a thickness of 3 mm, up to post injury day (PID) 14. Gross observation of wound healing was conducted on PID 3, 7, 14. The wound healing rate was determined on PID 3 and 7. On PID 3, 7, 14, tissues from 2, 4, and 8 wounds were harvested from each group respectively for the observation of the histopathological changes with HE staining, and also for analyzing the expression of proliferating cell nuclear antigen (PCNA) and CD31 with immunohistochemical staining (denoted as absorbance value). On PID 7, tissues from 6 wounds in each group were harvested for microarray gene chip to screen the differentially expressed microRNAs. Enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway on the differentially expressed microRNAs were performed after the microRNA screening results were validated by real-time fluorescent quantitative RT-PCR. Data were processed with paired t test or two-sample t test.

RESULTS(1) On PID 3, the wound area was significantly decreased, and the wound granulation was significantly proliferated in both groups. On PID 7, the wound area was further decreased, and the wound area was almost filled by granulation in both groups; the conditions in group A were better. On PID 14, all the wounds in group A were almost healed, while a small area of raw wound with incrustation still remained in some wounds of group B. On PID 3 and 7, the wound healing rates of group A were (41 ± 5)% and (75 ± 4)%, significantly higher than those of group B [(31 ± 9)% and (71 ± 4)%, with t values respectively 10.13 and 8.06, P values below 0.001]. (2) On PID 3, the epidermal cells, endothelial cells, and Fbs in the wounds of 2 groups were sparse, with heavy infiltration of inflammatory cells. The above condition in the wounds was better in group A than in group B. On PID 7, the epidermal cells, endothelial cells, and Fbs were gradually well arranged in group A; infiltration of inflammatory cells decreased, and the condition was better than that of group B. On PID 14, the wounds of group A were completely covered by epidermis, while infiltration of inflammatory cells still remained in some wounds of group B. (3) On PID 3, 7, 14, the positive expressions of CD31 and PCNA in group A were respectively 0.275 ± 0.018, 0.345 ± 0.034, 0.305 ± 0.023; 0.406 ± 0.063, 0.223 ± 0.011, 0.045 ± 0.022. They were significantly higher than those of group B (0.222 ± 0.020, 0.229 ± 0.018, 0.197 ± 0.015; 0.324 ± 0.039, 0.162 ± 0.012, 0.018 ± 0.020, with t values from 2.281 to 9.652, P < 0.05 or P < 0.01). (4) According to the microRNAs detection and screening, as compared with group B, 18 microRNAs were up-regulated while 13 were down-regulated in the wounds of group A. (5) The results of real-time fluorescent quantitative RT-PCR had good consistency with the results of microRNAs detection. (6) Enrichment analysis of KEGG signaling pathway showed that among the 31 differentially expressed microRNAs, 4 took part in the MAPK signaling pathway, 3 took part in the Wnt signaling pathway, 1 took part in the TGF-β signaling pathway, 3 took part in the epidermal growth factor receptor signaling pathway, 2 took part in the cell cycle pathway, 5 took part in the axon guidance signaling pathway, 6 took part in the focal adhesion pathway, 3 took part in the regulation of actin cytoskeleton pathway, 1 took part in the extracellular cell matrix receptor pathway, 3 took part in the adherens junction pathway, and 1 took part in the cell adhesion molecules pathway. After disclosing the blind, it showed that the ointment with red tag was the rhGM-CSF gel and the blue one was gel vehicle.

CONCLUSIONSThe rhGM-CSF gel can promote wound healing in diabetic rats, producing significant differential microRNA expression in wounds, and they may be the target at gene post-transcriptional level of rhGM-CSF gel in promoting wound healing.

Animals ; Bacteria ; isolation & purification ; Burns ; drug therapy ; microbiology ; pathology ; Diabetes Mellitus, Experimental ; complications ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Male ; MicroRNAs ; genetics ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Recombinant Proteins ; Signal Transduction ; Wound Healing ; drug effects

OBJECTIVETo investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX- tG250FcGB.

METHODSThe DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared.

RESULTSThe vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection.

CONCLUSIONElectroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.

Animals ; Antibody Formation ; Antibody Specificity ; Antigens, Neoplasm ; genetics ; immunology ; Electroporation ; Gene Fusion ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; HEK293 Cells ; Humans ; Injections, Intramuscular ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Transfection ; Vaccines, DNA ; genetics ; immunology

To investigate the adjuvant effect of granulocyte macrophage colony stimulating factor (GM-CSF) in Flaviviridae virus DNA vaccines. After DNA immunization, the antibody levels of serum from mice were detected by ELISA and indirect immunofluorescence assay. Co-immunization of GM-CSF suppressed the immune responses induced by DV1 and DV2 candidate vaccines whereas enhanced the immune response induced by HCV C and E1 DNA vaccines. As genetic adjuvant for DNA vaccines, GM-CSF might display complex diversity on the immune responses: an augmentation or suppression due to different immunogens. Therefore, GM-CSF should be used with some cautions in clinic.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Antibodies, Viral ; immunology ; DNA, Viral ; administration & dosage ; genetics ; immunology ; Dengue ; immunology ; prevention & control ; virology ; Dengue Vaccines ; administration & dosage ; genetics ; immunology ; Dengue Virus ; genetics ; immunology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; immunology ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

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