ABSTRACT
BACKGROUND: Melioidosis is a tropical disease caused by infection with Burkholderia pseudomallei. Here, we report an 11 year (2002-2013) retrospective study of melioidosis cases in China. METHODS: A total of 170 culture-confirmed melioidosis cases were included in our analysis, with culture-positive confirmation, biochemical identification and 16S DNA sequencing. A retrospective study design was employed and a correlational analysis of potential risk factors for mortality was carried out with logistic regression. RESULTS: We observed a year-over-year increasing trend in the incidence of melioidosis in Hainan, particularly after 2007 (annual peak of 64 cases in 2012). Farmers and fishers were the main group susceptible to melioidosis (75/170; 44.1%). Forty-six (27.1%) of the cases were fatal. Pneumonia (58/170; 34.1%) and septicaemia (44/170; 25.9%) were common presentations. Meanwhile, pre-existing diabetes (74/170; 43.5%) and being employed in a job that involves outdoor labour (148/170; 87.1%) emerged as common factors among affected patients. We did not observe a significant effect of seasonal variation on melioidosis mortality, but the greatest number of cases did occur in the rainiest season. CONCLUSIONS: This was the first clinical retrospective study of melioidosis in Hainan, China. The present data will be a useful resource to melioidosis researchers worldwide.
Subject(s)
Melioidosis/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , China/epidemiology , Female , Humans , Incidence , Infant , Male , Melioidosis/mortality , Middle Aged , Retrospective Studies , Risk FactorsABSTRACT
Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.
Subject(s)
Endocytosis/physiology , Lipoproteins, LDL/blood , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Receptors, LDL/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Substitution , Animals , CHO Cells , Clathrin/genetics , Clathrin/metabolism , Cricetinae , Cricetulus , Fibroblasts , Gene Knockdown Techniques , Humans , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Mutation, Missense , Receptors, LDL/geneticsABSTRACT
Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.
Subject(s)
Antigens, CD34 , Hematopoietic Stem Cells/cytology , Interleukin-3/pharmacology , Stem Cell Factor/pharmacology , Animals , Cell Line , Hematopoietic Stem Cells/metabolism , Humans , Interleukin-3/metabolism , Mice , Proto-Oncogene Proteins c-kit/metabolism , Receptors, Interleukin-3/metabolism , Stem Cell Factor/metabolismABSTRACT
AIM: To clarify the role of high in normal-1 (HIN-1) gene promoter methylation during gastric cancer development. METHODS: Gastric cancer cell lines and tissue specimens were analyzed for expression of HIN-1 mRNA and protein using the semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The methylation of the HIN-1 gene promoter was detected in gastric carcinoma cells and tissues using methylation-specific polymerase chain reaction. The 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay and flow cytometry were used to assess the changes in behaviors of gastric cancer cells with or without 5-aza-2'-deoxycytidine treatment. RESULTS: HIN-1 was not expressed in 4 of 5 gastric cancer cell lines. The demethylation reagent 5-aza-2'-deoxycytidine was able to induce or upregulate HIN-1 expression in gastric cancer cell lines, which is associated with reduction of tumor cell viability. Furthermore, methylation of the HIN-1 gene promoter was shown in 57.8% (26/45) of the primary gastric cancer and 42.1% (17/38) of adjacent tissue samples, but was not shown in normal gastric mucosa (0/10). From the clinicopathological data of the patients, methylation of the HIN-1 gene promoter was found to be associated with tumor differentiation (P = 0.000). CONCLUSION: High methylation of HIN-1 gene promoter results in silence of HIN-1 expression in gastric cancer. 5-aza-2'-deoxycytidine reverses HIN-1 methylation and reduces viability of gastric cancer cells.
Subject(s)
Cytokines/genetics , Cytokines/metabolism , DNA Methylation , Gene Silencing , Promoter Regions, Genetic , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Animals , Antimetabolites, Antineoplastic/pharmacology , Antimetabolites, Antineoplastic/therapeutic use , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Azacitidine/therapeutic use , Cell Line, Tumor , Decitabine , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Middle Aged , Stomach Neoplasms/drug therapyABSTRACT
Vascular smooth muscle cell (VSMC) proliferation is an important event in atherosclerosis and other vasculopathies. PDGF signaling is a key mediator of SMC proliferation, but the mechanisms that control its activity remain unclear. We previously identified a mutation in LDL receptor-related protein 6 (LRP6), LRP6(R611C), that causes early atherosclerosis. Examination of human atherosclerotic coronary arteries showed markedly increased expression of LRP6 and colocalization with PDGF receptor ß (PDGFR-ß). Further investigation showed that wild-type LRP6 inhibits but LRP6(R611C) promotes VSMC proliferation in response to PDGF. We found that wild-type LRP6 forms a complex with PDGFR-ß and enhances its lysosomal degradation, functions that are severely impaired in LRP6(R611C). Further, we observed that wild-type and mutant LRP6 regulate cell-cycle activity by triggering differential effects on PDGF-dependent pathways. These findings implicate LRP6 as a critical modulator of PDGF-dependent regulation of cell cycle in smooth muscle and indicate that loss of this function contributes to development of early atherosclerosis in humans.
Subject(s)
Atherosclerosis/physiopathology , Cell Proliferation , LDL-Receptor Related Proteins/physiology , Muscle, Smooth, Vascular/cytology , Platelet-Derived Growth Factor/physiology , Atherosclerosis/pathology , Cyclin D1/metabolism , Humans , Immunohistochemistry , Low Density Lipoprotein Receptor-Related Protein-6 , RNA, Messenger/genetics , Receptor, Platelet-Derived Growth Factor beta/genetics , Signal TransductionABSTRACT
Transfection of human cells with DNA in biomedical applications carries the risk of insertional mutagenesis. Transfection with mRNA avoids this problem; however, in vitro production of mRNA, based on preliminary DNA template cloning in special vectors, is a laborious and time-consuming procedure. We report an efficient vectorfree method of mRNA production from polymerase chain reaction-generated DNA templates. For all cell types tested mRNA was transfected more readily than DNA, and its expression was highly uniform in cell populations. Even cell types relatively resistant to transfection with DNA could express transfected mRNA well. The level of mRNA expression could be controlled over a wide range by changing the amount of input RNA. Cells could be efficiently and simultaneously loaded with several different transcripts. To test a potential clinical application of this method, we transfected human T lymphocytes with mRNA encoding a chimeric immune receptor directed against CD19, a surface antigen widely expressed in leukemia and lymphoma. The transfected mRNA conferred powerful cytotoxicity to T cells against CD19+ targets from the same donor. These results demonstrate that this method can be applied to generate autologous T lymphocytes directed toward malignant cells.
Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors/standards , Polymerase Chain Reaction/methods , RNA, Messenger/therapeutic use , Antigens, CD19/genetics , Cells, Cultured , DNA/genetics , HeLa Cells , Humans , Jurkat Cells , RNA, Messenger/biosynthesis , RNA, Messenger/standards , T-Lymphocytes/metabolism , Transcription, Genetic , TransfectionABSTRACT
Mature B cells, upon activation, progressively differentiate through centroblasts into centrocytes and finally to plasmacytes that express large amounts of selected immunoglobulins. A significant part of this maturation is thought to involve induction of the unfolded protein response (UPR). We have compared gene expression in normal germinal center centroblasts, centrocytes, lymphoblastoid cells undergoing induced UPR, and the CCL155 plasmacytoma cell line. In the centroblast to centrocyte transition there is a change in the expression of a relatively small number of genes. These include a limited subset of the genes upregulated by a fully activated UPR as well as a small number of other transcription factors, some disulphide isomerases, and other genes. This is consistent with a model in which this transition is mediated by changes in the levels of expression of transcription factor B-lymphocyte-induced maturation protein 1 (Blimp1) (PRDM1), BACH2, X-box binding protein 1 (XBP1), interferon regulatory factor 4 (IRF4), and possibly vitamin D receptor (VDR) expression, together with post-transcriptional changes and a limited induction of aspects of the UPR.
Subject(s)
B-Lymphocytes/physiology , Cell Differentiation/genetics , Germinal Center/cytology , Animals , B-Lymphocytes/cytology , Cell Line , Child , Child, Preschool , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dithiothreitol/metabolism , Gene Expression Profiling , Humans , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Palatine Tonsil/cytology , Palatine Tonsil/immunology , Protein Folding , Regulatory Factor X Transcription Factors , Transcription Factors , Tunicamycin/metabolism , X-Box Binding Protein 1ABSTRACT
The biochemistry of early stages of hematopoietic differentiation is difficult to study because only relatively small numbers of precursor cells are available. The murine EML cell line is a multipotential cell line that can be used to model some of these steps. We found that the lineage- EML precursor cells can be separated into two populations based on cell surface markers including CD34. Both populations contain similar levels of stem cell factor (SCF) receptor (c-Kit) but only the CD34+ population shows a growth response when treated with SCF. Conversely, the CD34- population will grow in the presence of the cytokine IL-3. The human beta-globin locus control region hypersensitive site 2 plays different roles on beta-globin transcription in the CD34+ and CD34- populations. The two populations are present in about equal amounts in culture, and the CD34+ population rapidly regenerates the mixed population when grown in the presence of SCF. We suggest that this system may mimic a normal developmental transition in hematopoiesis.