Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion.

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Zuopeng Wu

2016-05-01

Full Text Available Most humans harbor both CD177neg and CD177pos neutrophils but 1-10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1, which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation.

Histone methylation mediates plasticity of human FOXP3(+) regulatory T cells by modulating signature gene expressions.

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He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-03-01

CD4(+) FOXP3(+) regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4(+) CD25(high) CD127(low/-) Treg cells convert to two subpopulations with distinctive FOXP3(+) and FOXP3(-) phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. © 2013 John Wiley & Sons Ltd.

Histone methylation mediates plasticity of human FOXP3+ regulatory T cells by modulating signature gene expressions

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He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-01-01

CD4+ FOXP3+ regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4+ CD25high CD127low/− Treg cells convert to two subpopulations with distinctive FOXP3+ and FOXP3− phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. PMID:24152290

Aminopeptidase N/CD13 as a Potential Therapeutic Target in Malignant Pleural Mesothelioma.

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Otsuki, Takahiko; Nakashima, Taku; Hamada, Hironobu; Takayama, Yusuke; Akita, Shin; Masuda, Takeshi; Horimasu, Yasushi; Miyamoto, Shintaro; Iwamoto, Hiroshi; Fujitaka, Kazunori; Miyata, Yoshihiro; Miyake, Masayuki; Kohno, Nobuoki; Okada, Morihito; Hattori, Noboru

2018-03-08

Angiogenesis is a crucial factor in the progression of malignant pleural mesothelioma (MPM), and antiangiogenic strategies might be effective against MPM. Aminopeptidase N/CD13 (APN/CD13) promotes tumour angiogenesis and is associated with poor prognosis; however, its clinical significance in MPM remains unclear.In 37 consecutive patients with surgically resected MPM, we evaluated the association between immunohistochemical APN/CD13 expression in resected tumours and survival. Additionally, the antitumour and antiangiogenic effects of MT95-4, a fully humanized anti-APN/CD13 monoclonal antibody, were evaluated in mice orthotopically implanted with EHMES-10 (abundantly expressing APN/CD13) and MSTO-211H (scarcely expressing APN/CD13) MPM cells.High tumour APN/CD13 expression was associated with poor prognosis in MPM patients ( P =0.04), and MT95-4 treatment reduced tumour growth and angiogenesis in mice harbouring EHMES-10, but not MSTO-211H, cells. Furthermore, in mice harbouring EHMES-10 cells, MT95-4 combined with cisplatin more effectively suppressed tumour progression than cisplatin alone.Taken together these results suggested that APN/CD13 is implicated in the aggressiveness of MPM. Here, MT95-4 treatment reduced tumour progression likely by inhibiting angiogenesis, suggesting APN/CD13 as a potential molecular target for MPM treatment. Additionally, combination treatment with MT95-4 and cisplatin could represent a promising approach to treating MPM exhibiting high APN/CD13 expression. Copyright ©ERS 2018.

High Aminopeptidase N/CD13 Levels Characterize Human Amniotic Mesenchymal Stem Cells and Drive Their Increased Adipogenic Potential in Obese Women

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Iaffaldano, Laura; Nardelli, Carmela; Raia, Maddalena; Mariotti, Elisabetta; Ferrigno, Maddalena; Quaglia, Filomena; Labruna, Giuseppe; Capobianco, Valentina; Capone, Angela; Maruotti, Giuseppe Maria; Pastore, Lucio; Di Noto, Rosa; Martinelli, Pasquale; Del Vecchio, Luigi

2013-01-01

Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P=0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2=0.84, P<0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P=0.05 and P=0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P<0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers. PMID:23488598

Selection of suitable housekeeping genes for real-time quantitative PCR in CD4(+ lymphocytes from asthmatics with or without depression.

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Ting Wang

Full Text Available OBJECTIVE: No optimal housekeeping genes (HKGs have been identified for CD4(+ T cells from non-depressive asthmatic and depressive asthmatic adults for normalizing quantitative real-time PCR (qPCR assays. The aim of present study was to select appropriate HKGs for gene expression analysis in purified CD4(+ T cells from these asthmatics. METHODS: Three groups of subjects (Non-depressive asthmatic, NDA, n = 10, Depressive asthmatic, DA, n = 11, and Healthy control, HC, n = 10 respectively were studied. qPCR for 9 potential HKGs, namely RNA, 28S ribosomal 1 (RN28S1, ribosomal protein, large, P0 (RPLP0, actin, beta (ACTB, cyclophilin A (PPIA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, phosphoglycerate kinase 1 (PGK1, beta-2-microglobulin (B2M, glucuronidase, beta (GUSB and ribosomal protein L13a (RPL13A, was performed. Then the data were analyzed with three different applications namely BestKeeper, geNorm, and NormFinder. RESULTS: The analysis of gene expression data identified B2M and RPLP0 as the most stable reference genes and showed that the level of PPIA was significantly different among subjects of three groups when the two best HKGs identified were applied. Post-hoc analysis by Student-Newman-Keuls correction shows that depressive asthmatics and non-depressive asthmatics exhibited lower expression level of PPIA than healthy controls (p<0.05. CONCLUSIONS: B2M and RPLP0 were identified as the most optimal HKGs in gene expression studies involving human blood CD4(+ T cells derived from normal, depressive asthmatics and non-depressive asthmatics. The suitability of using the PPIA gene as the HKG for such studies was questioned due to its low expression in asthmatics.

Susceptibility to experimental biliary atresia linked to different hepatic gene expression profiles in two mouse strains.

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Leonhardt, Johannes; Kuebler, Joachim F; Turowski, Carmen; Tschernig, Thomas; Geffers, Robert; Petersen, Claus

2010-02-01

To compare hepatic gene expression during the development of experimental biliary atresia (BA) in two different mouse strains. Balb/c mice and C57Black/6 (Black/6) mice were infected with rhesus rotavirus (RRV) postpartum, clinical signs of BA and survival were noted. Liver sections were assessed for cluster of differentiation antigen (CD) 3, CD4 and CD8 expression, and the hepatic virus load was determined. Second, mice of both strains were sacrificed three days after infection. Isolated hepatic RNA was subjected to gene expression analysis using Affymetrix Gene Chip MOE 430 2.0. The incidence of BA was significantly lower in Black/6 mice compared to Balb/c mice (13.5% vs. 67%, P < 0.05). The mean virus titers were higher in mice with BA compared to mice without BA. Different gene profiles three days after virus infection were noted, with differential expression of 201 genes, including those regulating apoptosis, nucleic acid binding, transport function and particularly the immune response (chemokine C-C motif ligand 2, toll-like receptor 3, CD antigen 14, chemokine (C-X-C motif) ligands 10 and 11). This correlated with a significant increase of CD4 positive cells only in Balb/c mice with BA compared to healthy mice (13.5 vs. 5.0; P < 0.05). Black/6 mice did not exhibit any significant increase of CD3 or CD4 leukocytes despite cholestasis. The different susceptibility to experimental BA was associated with an increase of CD4 T-cells in the liver of Balb/c mice, which is linked to different gene profiles at the onset of bile duct obstruction.

Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/− Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice

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Séverine Bézie

2018-01-01

Full Text Available Both CD4+ and CD8+ Tregs play a critical role in the control of immune responses and immune tolerance; however, our understanding of CD8+ Tregs is limited while they are particularly promising for therapeutic application. We report here existence of highly suppressive human CD8+CD45RClow/− Tregs expressing Foxp3 and producing IFNγ, IL-10, IL-34, and TGFβ to mediate their suppressive activity. We demonstrate that total CD8+CD45RClow/− Tregs can be efficiently expanded in the presence of anti-CD3/28 mAbs, high-dose IL-2 and IL-15 and that such expanded Tregs efficiently delay GVHD and human skin transplantation rejection in immune humanized mice. Robustly expanded CD8+ Tregs displayed a specific gene signature, upregulated cytokines and expansion in the presence of rapamycin greatly improved proliferation and suppression. We show that CD8+CD45RClow/− Tregs are equivalent to canonical CD4+CD25highCD127low/− Tregs for suppression of allogeneic immune responses in vitro. Altogether, our results open new perspectives to tolerogenic strategies in human solid organ transplantation and GVHD.

CD150 is a member of a family of genes that encode glycoproteins on the surface of hematopoietic cells.

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Wang, N; Morra, M; Wu, C; Gullo, C; Howie, D; Coyle, T; Engel, P; Terhorst, C

2001-07-01

Human CD150 (SLAM) is a glycoprotein expressed on the surface of T, B, natural killer, and dendritic cells. The extracellular domain of CD150 is the receptor for measles virus and CD150 acts as a co-activator on T and B cells. We characterized the mouse and human CD150 genes, each of which comprises seven exons spanning approximately 32 kb. Mouse CD150 mRNA was detected in T cells and in most thymocyte subsets, except CD4-8- cells. Surprisingly, the CD4-8- thymocytes of CD3gammadeltanull mice, but not of Ragnull or severe combined immunodeficiency mice, expressed CD150. Whereas high levels of CD150 were found in Th1 cells, only small amounts were detectable in Th2 cells. CD150 expression was up-regulated upon in vitro activation of mouse T cells by anti-CD3. The complete mouse CD150 gene is highly homologous to its human orthologue in terms of nucleotide sequences and intron/exon organization. The human genomic sequences indicate that all isoforms detected so far have arisen from alternative splicing events. As judged by fluorescence in situ hybridization, mouse CD150 mapped to Chromosome (Chr) 1, band 1H2.2-2.3, and human CD150 was found on Chr 1q22. Human and mouse CD150 share sequence homologies with six other genes, five of which - CD84, CD229 (Ly-9), CD244 (2B4), CD48, and 19A - are localized in a 250-kb segment in close proximity to the human gene. Their location and their sequence similarities strongly suggest that the CD150 family of cell surface receptors arose via successive duplications of a common ancestral gene.

Molecular pathways of early CD105-positive erythroid cells as compared with CD34-positive common precursor cells by flow cytometric cell-sorting and gene expression profiling

International Nuclear Information System (INIS)

Machherndl-Spandl, S; Suessner, S; Danzer, M; Proell, J; Gabriel, C; Lauf, J; Sylie, R; Klein, H-U; Béné, M C; Weltermann, A; Bettelheim, P

2013-01-01

Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34 + ) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34 + progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository

Use of a human chromosome 11 radiation hybrid panel to map markers at 11q13

International Nuclear Information System (INIS)

Withers, D.; Richard, C. III; Meeker, T.C.; Maurer, S.; Evans, G.; Myers, R.M.; Cox, D.R.

1990-01-01

A human/hamster hybrid cell line containing human chromosome 11 was X-irradiated and 102-independent derivative lines were recovered. These 'radiation hybrids' contain random fragments of human chromosome 11. This radiation hybrid panel was used to score the retention of markers at band 11q13. Statistical analysis of marker co-retention patterns in the radiation hybrid panel permits a preliminary ordering and mapping of the markers used. The best order for six scored markers is: proximal - CD5 - CD20 - PGA - HST - BCL1 - SEA - distal. Additional markers are currently being scored. The six 11q13 markers above are spread over approximately 10-12 mB of DNA. The mapping data has implications for the identification of the bcl-1 gene. bcl-1 is the site of chromosome breakage in translocations associated with B lymphocytic malignancy. bcl-1 markers map at least 4 Mb away from any of four genes previously hypothesized to be activated by such translocations, thereby making them unlikely candidates for activation

[The Role of 5-Aza-CdR on Methylation of Promoter in RASSF1A Gene in Endometrial Carcinoma].

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Huang, Li-ping; Chen, Chen; Wang, Xue-ping; Liu, Hui

2015-05-01

To explore the effect of demethylating drug 5-Aza-2'-deoxycytidine (5-Aza-CdR) on methtylation status of the Ras-association domain familylA gene (RASSF1A) in human endometrial carcinoma. Randomly'assign the human endometrial carcinoma cell line HEC-1-B into groups and use demethylating drug 5-Aza-CdR of different concentration to treat them. Then Methylation-specific polymerase chain reaction (MSP), real-time PCR, Western blot, TUNEL technology were used to analyze methylation status of RASSF1A promoter CpG islands, RASSF1A mRNA expression, RASSF1A protein expression and apoptosis of HEC-1-B cell. High DNA methylation in RASSF1A gene promoter region, low RASSF1A mRNA level and protein expression and out of control of human endometrial carcinoma cell HEC-1-B apoptosis were observed. 5-Aza-CdR of different concentration could reverse RASSF1A gene's methylation status, recover the expression of mRNA and protein, and control the growth of HEC-1-B by inducing apoptosis. Aberrant methylation of RASSF1A in endometrial cancer as a therapeutic target, demethylating agent 5-Aza-CdR could be an effective way of gene therapy.

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13

OpenAIRE

Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2015-01-01

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but ...

Isolation and characterization of a primary proximal tubular epithelial cell model from human kidney by CD10/CD13 double labeling.

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Cynthia Van der Hauwaert

Full Text Available Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

Bone marrow-derived CD13+ cells sustain tumor progression

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Dondossola, Eleonora; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2014-01-01

Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b+CD13+ myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b+CD13+ myeloid cells could become a non-malignant target for the development of novel anticancer regimens. PMID:25339996

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

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Wheeler, Lee Adam; Trifonova, Radiana; Vrbanac, Vladimir; Basar, Emre; McKernan, Shannon; Xu, Zhan; Seung, Edward; Deruaz, Maud; Dudek, Tim; Einarsson, Jon Ivar; Yang, Linda; Allen, Todd M.; Luster, Andrew D.; Tager, Andrew M.; Dykxhoorn, Derek M.; Lieberman, Judy

2011-01-01

The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission. PMID:21576818

Distinct Transcriptional and Alternative Splicing Signatures of Decidual CD4+ T Cells in Early Human Pregnancy

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Weihong Zeng

2017-06-01

Full Text Available Decidual CD4+ T (dCD4 T cells are crucial for the maternal-fetal immune tolerance required for a healthy pregnancy outcome. However, their molecular and functional characteristics are not well elucidated. In this study, we performed the first analysis of transcriptional and alternative splicing (AS landscapes for paired decidual and peripheral blood CD4+ T (pCD4 T cells in human early pregnancy using high throughput mRNA sequencing. Our data showed that dCD4 T cells are endowed with a unique transcriptional signature when compared to pCD4 T cells: dCD4 T cells upregulate 1,695 genes enriched in immune system process whereas downregulate 1,011 genes mainly related to mRNA catabolic process and the ribosome. Moreover, dCD4 T cells were observed to be at M phase, and show increased activation, proliferation, and cytokine production, as well as display an effector-memory phenotype and a heterogenous nature containing Th1, Th17, and Treg cell subsets. However, dCD4 T cells undergo a comparable number of upregulated and downregulated AS events, both of which are enriched in the genes related to cellular metabolic process. And the changes at the AS event level do not reflect measurable differences at the gene expression level in dCD4 T cells. Collectively, our findings provide a comprehensive portrait of the unique transcriptional signature and AS profile of CD4+ T cells in human decidua and help us gain more understanding of the functional characteristic of these cells during early pregnancy.

Selective in vivo radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells transduced with the E. coli cytosine deaminase (CD) gene

International Nuclear Information System (INIS)

Gabel, M.; Kim, J.H.; Kolozsvary, A.; Khil, M.; Freytag, S.

1998-01-01

Purpose: The E. coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer. Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy. Methods and Materials: WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model. After administration of 5-FC, tumors received 10-30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone. Results: Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT. Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation. There was no appreciable toxicity using this new approach. In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization. Conclusion: The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity. This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers

Radiosensitivity of CD4 and CD8 positive human T lymphocytes by an in vitro colony formation assay

International Nuclear Information System (INIS)

Nakamura, Nori; Kusunoki, Yoichiro; Akiyama, Mitoshi.

1989-12-01

The recent development of an in vitro lymphocyte colony assay provides a new opportunity to examine possible variations in human radiosensitivity using peripheral blood lymphocytes (PBL) in place of the hitherto used skin fibroblast assay. Our recent study showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4 + and CD8 + cells in PBL differs among individuals, it was suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4 + and CD8 + cells differed. In the present study, CD4 + lymphocytes (helper/inducer T cells) and CD8 + lymphocytes (suppressor/cytotoxic T cells) were isolated from PBL and their dose-survival curves were determined. The results showed that the D 10 (the dose required to reduce the surviving fraction to 10 %) was quite similar for these two types of cells (3.13 ± 0.10 Gy [mean ±SD] for CD4 + , 3.34 ± 0.50 Gy for CD8 + and 3.07 ± 0.05 Gy for the unsorted cells), supporting the use of a whole PBL population for screening of individuals with altered radiosensitivity. (author)

CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

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Harkness, Linda; Zaher, Walid; Ditzel, Nicholas

2016-01-01

BACKGROUND: Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous hMSC...... population. METHODS: Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high...... and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146(+) and hMSC-CD146(-) cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146(-) cells (12.6 % versus 8.1 %) and bone...

The human cumulus--oocyte complex gene-expression profile

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Assou, Said; Anahory, Tal; Pantesco, Véronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Hervé; De Vos, John; Hamamah, Samir

2006-01-01

BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors. PMID:16571642

Loss of CD34 expression in aging human choriocapillaris endothelial cells.

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Elliott H Sohn

Full Text Available Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD. In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40, age-matched controls (> age 60 without AMD, or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization. Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10(-6. Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse or a trend toward decreased CD34 (human in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.

Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees.

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Chabot, Adrien; Shrit, Ralla A; Blekhman, Ran; Gilad, Yoav

2007-08-01

Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human-chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.

Cholesteryl ester hydroperoxides increase macrophage CD36 gene expression via PPARα

International Nuclear Information System (INIS)

Jedidi, Iness; Couturier, Martine; Therond, Patrice; Gardes-Albert, Monique; Legrand, Alain; Barouki, Robert; Bonnefont-Rousselot, Dominique; Aggerbeck, Martine

2006-01-01

The uptake of oxidized LDL by macrophages is a key event in the development of atherosclerosis. The scavenger receptor CD36 is one major receptor that internalizes oxidized LDL. In differentiated human macrophages, we compared the regulation of CD36 expression by copper-oxidized LDL or their products. Only oxidized derivatives of cholesteryl ester (CEOOH) increased the amount of CD36 mRNA (2.5-fold). Both oxidized LDL and CEOOH treatment increased two to fourfold the transcription of promoters containing peroxisome-proliferator-activated-receptor responsive elements (PPRE) in the presence of PPARα or γ. Electrophoretic-mobility-shift-assays with nuclear extracts prepared from macrophages treated by either oxidized LDL or CEOOH showed increased binding of PPARα to the CD36 gene promoter PPRE. In conclusion, CEOOH present in oxidized LDL increase CD36 gene expression in a pathway involving PPARα

Cre-/IoxP-Mediated Recombination between the SIL and SCL Genes Leads to a Block in T-Cell Development at the CD4-CD8- to CD4+CD8+ Transition

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Yue Cheng

2007-04-01

Full Text Available In the most common form of stem cell leukemia (SCL gene rearrangement, an interstitial deletion of 82 kb brings SCL under the control of regulatory elements that normally govern expression of the ubiquitously expressed SCL interrupting locus (SIL gene, which is located directly upstream of SCL. To investigate the effect of this fusion in a mouse model, a bacterial artificial chromosome (BAC clone containing both human SIL and SCL genes was isolated, and IoxP sites were inserted into intron 1 of both the SIL and SCL genes, corresponding to the sites at which recombination occurs in human T-cell acute lymphocytic leukemia patients. This BAC clone was used to generate transgenic SILIoxloxSCL mice. These transgenic mice were subsequently bred to Lck-Cre mice that express the Cre recombinase specifically in the thymus. The BAC transgene was recombined between the two IoxP sites in over 50% of the thymocytes from SILIoxloxSCL/Cre double-transgenic mice, bringing the SCL gene under the direct control of SIL regulatory elements. Aberrant SCL gene expression in the thymus was verified by reverse transcription- polymerase chain reaction. Using FACS analysis, we found that mice carrying both SILIoxloxSCL and Cre transgenes have increased CD4-/CD8- thymocytes compared with transgenenegative mice. In the spleen, these transgenic mice show a marked reduction in the number of mature CD4+ or CD8+ cells. These results demonstrate that conditional activation of SCL under control of SIL regulatory elements can impair normal T-cell development.

Characterization of death of human fetal bone marrow CD34+ cells after different dose of γ-irradiation

International Nuclear Information System (INIS)

Xiang Yingsong; Yang Rujun; Tang Gusheng

2001-01-01

Objective: To investigate the characterization of death of the human hematopoietic stem cells after irradiation. Methods: Human fetal bone marrow mononuclear cells were irradiated with different doses of 60 Co γ-rays at different high dose rates. Apoptosis and necrosis of CD34 + cells were analyzed by flow cytometry, following three-color labelling with PE-CD34/FITC-Annexin V/7AAD at different times after irradiation. Results: The death of CD34 + cells after 5 Gy and 8 Gy irradiation showed a continuous process of reproductive death during the first week,and the main death type was apoptosis. A majority of CD34 + cells died of necrosis during the first day after 10 Gy and 12 Gy irradiation, and all of them died within a week. Conclusion: Niches are continuously vacated every day within a week following irradiation and reproductive death of hematopoietic stem cells occurred

CD133-targeted Gene Transfer Into Long-term Repopulating Hematopoietic Stem Cells

NARCIS (Netherlands)

Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwaeble, Joachim; Kaufmann, Kerstin B.; Mueller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J.; Grez, Manuel

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem

Cloning analysis of HBV-specific CD8 T cell receptor gene in patients with acute hepatitis B

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Ning DING

2011-05-01

Full Text Available Objective To investigate the molecular mechanism of T cell receptor(TCR in CD8 T cell-mediated immune response to HBV in patients with acute hepatitis B(AHB.Methods Peripheral blood mononuclear cells(PBMCs were collected from HLA-A2-positive AHB patients.To determine HBsAg183-191 and HBsAg335-343-specific CD8 T cell frequencies,the PBMCs were stained by fluorescence-labeled anti-CD3,anti-CD8 and pentamers,and analyzed by flow cytometry.PBMCs from 6 patients were stimulated with epitopic peptide HBsAg335-343 in vitro for 3 to 4 weeks.HBV-specific CD8 T cells were isolated by magnetic activated cell sorting followed by flow florescence activated cell sorting.The mRNA of sorted cells was extracted after expanding by IL-2,anti-CD3 and anti-CD8.The full-length gene fragments of variable region of TCR α and β chains were gained by 5’-RACE,and then cloned and sequenced(≥50 clones for single chain of each sample.The gene families of TCR α and β chains were identified and the sequence characters of CDR3 were compared.Results Analysis of more than 600 cloned gene sequences of TCR α and β chains showed that the proliferated HBV-specific CD8 T cells from 6 AHB patients presented a predominant expression in TCR α and chains,with 2-4 α chain families and 1-4 chain families in each case.The α2,α14,α15,β3,β13 and 23 families were detected in more than one case.The chain genes were all 13 for all tested clones in one case.For the same α chain or-chain family,CDR3 sequences tended to be identical in one case but different among cases.Conclusions HBV-specific CD8 T cells with antigenic peptide-induced proliferation present predominance in the usage of TCR α and β chains.This property might be one of the important molecular factors influencing anti-HBV immunity.

Prognostic values of soluble CD30 and CD30 gene polymorphisms in heart transplantation.

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Frisaldi, Elisa; Conca, Raffaele; Magistroni, Paola; Fasano, Maria Edvige; Mazzola, Gina; Patanè, Francesco; Zingarelli, Edoardo; Dall'omo, Anna M; Brusco, Alfredo; Amoroso, Antonio

2006-04-27

Pretransplant soluble CD30 (sCD30) is a predictor of kidney graft outcome. Its status as a predictor of heart transplant (HT) outcome has not been established. We have studied this question by assessing sCD30 levels and the number of (CCAT)n repeats of the microsatellite in the CD30 promoter region, which is able alone to repress gene transcription, in the sera of 83 HT patients and 77 of their donors. sCD30 was non-significantly increased in the patients, whereas there were no differences in the CD30 microsatellite allele frequencies. A negative correlation between the number of (CCAT)n and sCD30 levels was evident in the donors. Patients with pretransplant sCD30sCD30 levels are predictive of HT outcome.

Molecular characterization and expression profiling of cd-responsive genes in triticum durum

OpenAIRE

Cebeci, Özge; Cebeci, Ozge

2006-01-01

Cadmium (Cd) is a toxic heavy metal which has detrimental effects both in plants and human. There is a lack of knowledge on the molecular mechanisms of Cd toxicity in crop plants. The objective of this study was to identify and clone expressed Cd-responsive genes from two Triticum durum cvs. Balcah-85 and Balcah-2000 using mRNA differential display technique. We identified 10 cDNA clones whose level of expression significantly changed upon Cd exposure and thus isolated for further characteriz...

Glucose metabolism in pigs expressing human genes under an insulin promoter.

Science.gov (United States)

Wijkstrom, Martin; Bottino, Rita; Iwase, Hayoto; Hara, Hidetaka; Ekser, Burcin; van der Windt, Dirk; Long, Cassandra; Toledo, Frederico G S; Phelps, Carol J; Trucco, Massimo; Cooper, David K C; Ayares, David

2015-01-01

Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. This preliminary study did not show definite evidence of β-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

CD13 is a novel mediator of monocytic/endothelial cell adhesion

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Mina-Osorio, Paola; Winnicka, Beata; O'Conor, Catherine

2008-01-01

During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown...... to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal...... rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept...

Discretization of Gene Expression Data Unmasks Molecular Subgroups Recurring in Different Human Cancer Types.

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Manfred Beleut

Full Text Available Despite the individually different molecular alterations in tumors, the malignancy associated biological traits are strikingly similar. Results of a previous study using renal cell carcinoma (RCC as a model pointed towards cancer-related features, which could be visualized as three groups by microarray based gene expression analysis. In this study, we used a mathematic model to verify the presence of these groups in RCC as well as in other cancer types. We developed an algorithm for gene-expression deviation profiling for analyzing gene expression data of a total of 8397 patients with 13 different cancer types and normal tissues. We revealed three common Cancer Transcriptomic Profiles (CTPs which recurred in all investigated tumors. Additionally, CTPs remained robust regardless of the functions or numbers of genes analyzed. CTPs may represent common genetic fingerprints, which potentially reflect the closely related biological traits of human cancers.

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

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Fisher-Adams, G; Wong, K K; Podsakoff, G; Forman, S J; Chatterjee, S

1996-07-15

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

OpenAIRE

Paquette, Y; Doyon, L; Laperrière, A; Hanna, Z; Ball, J; Sekaly, R P; Jolicoeur, P

1992-01-01

The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to...

Responses of different water spinach cultivars and their hybrid to Cd, Pb and Cd-Pb exposures.

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Xin, Junliang; Huang, Baifei; Yang, Zhongyi; Yuan, Jiangang; Dai, Hongwen; Qiu, Qiu

2010-03-15

A pot experiment was conducted to investigate the stability of Cd and/or Pb accumulation in shoot of Cd and Pb pollution-safe cultivars (PSCs), the hereditary pattern of shoot Cd accumulation, and the transfer potentials of Cd and Pb in water spinach (Ipomoea aquatica Forsk.). A typical Cd-PSC, a typical non-Cd-PSC (Cd accumulative cultivar), a hybrid from the former two cultivars, and two typical Cd+Pb-PSCs were grown in seven soils with different concentrations of Cd and Pb. The results showed that concentrations of Cd and Pb in shoot of the PSCs were always lower than the non-PSC and the highest Cd and Pb transfer factors were also always observed in the non-PSC, indicating the stability of the PSCs in Cd and Pb accumulation. Shoot Cd concentration seemed to be controlled by high Cd dominant gene(s) and thus crossbreeding might not minimize Cd accumulation in water spinach. Interaction between Cd and Pb in soils affected the accumulations of the metals in shoot of water spinach. Under middle Cd and Pb treatments, the presence of higher Pb promoted the accumulation of Cd. However, under high Pb treatment, accumulations of Cd and Pb were both restricted. (c) 2009 Elsevier B.V. All rights reserved.

SLC25A13 gene analysis in citrin deficiency: sixteen novel mutations in East Asian patients, and the mutation distribution in a large pediatric cohort in China.

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Yuan-Zong Song

Full Text Available BACKGROUND: The human SLC25A13 gene encodes citrin, the liver-type mitochondrial aspartate/glutamate carrier isoform 2 (AGC2, and SLC25A13 mutations cause citrin deficiency (CD, a disease entity that encompasses different age-dependant clinical phenotypes such as Adult-onset Citrullinemia Type II (CTLN2 and Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD. The analyses of SLC25A13 gene and its protein/mRNA products remain reliable tools for the definitive diagnoses of CD patients, and so far, the SLC25A13 mutation spectrum in Chinese CD patients has not been well-characterized yet. METHODS AND RESULTS: By means of direct DNA sequencing, cDNA cloning and SNP analyses, 16 novel pathogenic mutations, including 9 missense, 4 nonsense, 1 splice-site, 1 deletion and 1 large transposal insertion IVS4ins6kb (GenBank accession number KF425758, were identified in CTLN2 or NICCD patients from China, Japan and Malaysia, respectively, making the SLC25A13 variations worldwide reach the total number of 81. A large NICCD cohort of 116 Chinese cases was also established, and the 4 high-frequency mutations contributed a much larger proportion of the mutated alleles in the patients from south China than in those from the north (χ(2 = 14.93, P<0.01, with the latitude of 30°N as the geographic dividing line in mainland China. CONCLUSIONS: This paper further enriched the SLC25A13 variation spectrum worldwide, and formed a substantial contribution to the in-depth understanding of the genotypic feature of Chinese CD patients.

Spatially conserved regulatory elements identified within human and mouse Cd247 gene using high-throughput sequencing data from the ENCODE project

DEFF Research Database (Denmark)

Pundhir, Sachin; Hannibal, Tine Dahlbæk; Bang-Berthelsen, Claus Heiner

2014-01-01

. In this study, we have utilized the wealth of high-throughput sequencing data produced during the Encyclopedia of DNA Elements (ENCODE) project to identify spatially conserved regulatory elements within the Cd247 gene from human and mouse. We show the presence of two transcription factor binding sites...

DNA microarray revealed and RNAi plants confirmed key genes conferring low Cd accumulation in barley grains

DEFF Research Database (Denmark)

Sun, Hongyan; Chen, Zhong-Hua; Chen, Fei

2015-01-01

Background Understanding the mechanism of low Cd accumulation in crops is crucial for sustainable safe food production in Cd-contaminated soils. Results Confocal microscopy, atomic absorption spectrometry, gas exchange and chlorophyll fluorescence analyses revealed a distinct difference in Cd...... with a substantial difference between the two genotypes. Cd stress led to higher expression of genes involved in transport, carbohydrate metabolism and signal transduction in the low-grain-Cd-accumulating genotype. Novel transporter genes such as zinc transporter genes were identified as being associated with low Cd...... accumulation. Quantitative RT-PCR confirmed our microarray data. Furthermore, suppression of the zinc transporter genes HvZIP3 and HvZIP8 by RNAi silencing showed increased Cd accumulation and reduced Zn and Mn concentrations in barley grains. Thus, HvZIP3 and HvZIP8 could be candidate genes related to low...

Haplotype frequencies in a sub-region of chromosome 19q13.3, related to risk and prognosis of cancer, differ dramatically between ethnic groups

DEFF Research Database (Denmark)

Schierup, M.H.; Mailund, T.; Li, H.

2009-01-01

Background: A small region of about 70 kb on human chromosome 19q13.3 encompasses 4 genes of which 3, ERCC1, ERCC2, and PPP1R13L (aka RAI) are related to DNA repair and cell survival, and one, CD3EAP, aka ASE1, may be related to cell proliferation. The whole region seems related to the cellular...

Expression of two isoforms of CD44 in human endometrium.

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Behzad, F; Seif, M W; Campbell, S; Aplin, J D

1994-10-01

The distribution of the cell-surface adhesion glycoprotein CD44 in human endometrium was examined by immunofluorescence using six monoclonal antibodies to epitopes common to all forms of the molecule, and by reverse transcription-polymerase chain reaction (RT-PCR). Immunoreactivity was observed throughout the menstrual cycle in stroma, vessels, glandular, and luminal epithelium. Variations in staining intensity were observed, especially in the epithelial compartment. CD44 was also expressed strongly by decidualized stromal cells of first-trimester pregnancy. No systematic variation of immunoreactivity was observed with stages of the normal cycle, but a fraction (25%) of the specimens lacked reactivity in the epithelium. To determine the molecular size of the epithelial isoform, an immunoprecipitation technique was developed using surface-radioiodinated, detergent-extracted glands. This indicated the presence at the cell surface of a single dominant CD44E species with an approximate molecular mass of 130 kDa. RT-PCR was used to investigate the isoforms present in whole endometrial tissue, isolated gland fragments, and Ishikawa endometrial carcinoma cells. Complementary DNA produced from total endometrial mRNA was PCR-amplified across the splice junction between exons 5 and 15. Transcripts corresponding to the hyaluronate receptor CD44H as well as a larger isoform were identified. CD44H was absent, or very scarce, in cDNA from purified gland epithelium. In contrast, Ishikawa cells expressed this form abundantly. The glands and Ishikawa cells also expressed CD44E containing sequences encoded by exons 12, 13, and 14. These data demonstrate the presence of CD44 in human endometrium and decidua, and show that different isoforms of CD44 are associated with tissue compartments in which different functional roles can be anticipated.

Microarray evaluation of gene expression profiles in inflamed and healthy human dental pulp: the role of IL1beta and CD40 in pulp inflammation.

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Gatta, V; Zizzari, V L; Dd ' Amico, V; Salini, L; D' Aurora, M; Franchi, S; Antonucci, I; Sberna, M T; Gherlone, E; Stuppia, L; Tetè, S

2012-01-01

Dental pulp undergoes a number of changes passing from healthy status to inflammation due to deep decay. These changes are regulated by several genes resulting differently expressed in inflamed and healthy dental pulp, and the knowledge of the processes underlying this differential expression is of great relevance in the identification of the pathogenesis of the disease. In this study, the gene expression profile of inflamed and healthy dental pulps were compared by microarray analysis, and data obtained were analyzed by Ingenuity Pathway Analysis (IPA) software. This analysis allows to focus on a variety of genes, typically expressed in inflamed tissues. The comparison analysis showed an increased expression of several genes in inflamed pulp, among which IL1β and CD40 resulted of particular interest. These results indicate that gene expression profile of human dental pulp in different physiological and pathological conditions may become an useful tool for improving our knowledge about processes regulating pulp inflammation.

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes.

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Tsai, Wei-Jern; Chang, Chu-Ting; Wang, Guei-Jane; Lee, Tzong-Huei; Chang, Shwu-Fen; Lu, Shao-Chun; Kuo, Yuh-Chi

2011-03-25

Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

Study of association of CD40-CD154 gene polymorphisms with disease susceptibility and cardiovascular risk in Spanish rheumatoid arthritis patients.

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Mercedes García-Bermúdez

Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disease associated with increased cardiovascular (CV mortality. Since CD40-CD154 binding has direct consequences on inflammation process initiation, we aimed to replicate previous findings related to disease susceptibility in Spanish RA population. Furthermore, as the major complication in RA disease patients is the development of CV events due to accelerated atherosclerosis, and elevated levels of CD40L/CD154 are present in patients with acute myocardial infarction, we assessed the potential association of CD40 and CD154/CD40L gene variants with CV risk in Spanish RA patients.One thousand five hundred and seventy-five patients fulfilling the 1987 ACR classification criteria for RA and 1600 matched controls were genotyped for the CD40 rs1883832, rs4810485 and rs1535045 and CD154 rs3092952 and rs3092920 gene polymorphisms, using predesigned TaqMan single nucleotide polymorphism genotyping assays. Afterwards, we investigated the influence of CD40-CD154 gene variants in the development of CV events. Also, in a subgroup of 273 patients without history of CV events, we assessed the influence of these polymorphisms in the risk of subclinical atherosclerosis determined by carotid ultrasonography.Nominally significant differences in the allele frequencies for the rs1883832 CD40 gene polymorphism between RA patients and controls were found (p=0.038. Although we did not observe a significant association of CD40-CD154 gene variants with the development of CV events, an ANCOVA model adjusted for sex, age at the time of the ultrasonography assessment, follow-up time, traditional CV risk factors and anti-cyclic citrullinated peptide antibodies disclosed a significant association (p=0.0047 between CD40 rs1535045 polymorphism and carotid intima media thickness, a surrogate marker of atherosclerosis.Data from our pilot study indicate a potential association of rs1883832 CD40 gene polymorphism with susceptibility

Effect of granulocyte colony-stimulating factor (G-CSF) in human immunodeficiency virus-infected patients: increase in numbers of naive CD4 cells and CD34 cells makes G-CSF a candidate for use in gene therapy or to support antiretroviral therapy

DEFF Research Database (Denmark)

Nielsen, S D; Afzelius, P; Dam-Larsen, S

1998-01-01

The potential of granulocyte colony-stimulating factor (G-CSF) to mobilize CD4 cells and/or CD34 cells for use in gene therapy or to support antiretroviral therapy was examined. Ten human immunodeficiency virus-infected patients were treated with G-CSF (300 microg/day) for 5 days. Numbers of CD4.......01/microL (P CSF induced increases in numbers of CD34 cells and CD4 cells in HIV-infected patients...

Structure and function of the human metallothionein gene family: Final technical report

International Nuclear Information System (INIS)

Karin, M.

1986-01-01

The full nucleotide sequence of two additional human metallothionein (hMT) genes has been determined. These genes, hMT-I/sub B/ and hMT-I/sub F/, are located within the MT-I gene cluster we have described originally. The hMT-I/sub F/ gene is the first hMT-I gene whose amino acid sequence is in complete agreement with the published sequence of the human MT-I proteins. Therefore it is likely to be an active gene encoding a functional protein. However, since we have just completed the sequence analysis, we have not characterized this gene further yet. The hMT-I/sub B/ gene is closely linked to the hMT-I/sub A/ gene, and two pseudogenes, hMT-I/sub C/ and hMT-I/sub D/ separate the two. From its nucleotide sequence hMT-I/sub B/ seems to be an active gene, encoding a functional protein even though it differs in four positions from the published sequence of human MT-I proteins. This gene is expressed in a human hepatoma cell line, HepG2, and its expression is stimulated by Cd ++ . Using gene fusions to the viral thymidine-kinase gene we find that hMT-I/sub B/, like the hMT-I/sub A/ and hMT-II/sub A/ genes, contains a heavy metal responsive promoterregulatory element within its 5' flanking region. We analyzed the level of hMT-I/sub B/ mRNA in a variety of human cell lines by the S1 nuclease technique, and compared it to the expression of the hMT-II/sub A/ gene. While the hMT-II/sub A/ gene was expressed in all of the cell lines analyzed, the hMT-I/sub B/ gene was expressed in liver and kidney derived cell lines cells. This suggest that the expression of the hMT-I/sub B/ gene is controlled in a tissue specific manner. 13 refs

Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

Science.gov (United States)

Nomura, M; Tsujimura, A; Begum, N A; Matsumoto, M; Wabiko, H; Toyoshima, K; Seya, T

2000-01-01

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46. PMID:11023821

Haplotype frequencies in a sub-region of chromosome 19q13.3, related to risk and prognosis of cancer, differ dramatically between ethnic groups

DEFF Research Database (Denmark)

Schierup, M.H.; Mailund, T.; Li, H.

2009-01-01

Background: A small region of about 70 kb on human chromosome 19q13.3 encompasses 4 genes of which 3, ERCC1, ERCC2, and PPP1R13L (aka RAI) are related to DNA repair and cell survival, and one, CD3EAP, aka ASE1, may be related to cell proliferation. The whole region seems related to the cellular r...

Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

Science.gov (United States)

Kreuzer, J; Denger, S; Reifers, F; Beisel, C; Haack, K; Gebert, J; Kübler, W

1996-07-01

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and

CD177: A member of the Ly-6 gene superfamily involved with neutrophil proliferation and polycythemia vera

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Bettinotti Maria

2004-03-01

Full Text Available Abstract Genes in the Leukocyte Antigen 6 (Ly-6 superfamily encode glycosyl-phosphatidylinositol (GPI anchored glycoproteins (gp with conserved domains of 70 to 100 amino acids and 8 to 10 cysteine residues. Murine Ly-6 genes encode important lymphocyte and hematopoietic stem cell antigens. Recently, a new member of the human Ly-6 gene superfamily has been described, CD177. CD177 is polymorphic and has at least two alleles, PRV-1 and NB1. CD177 was first described as PRV-1, a gene that is overexpressed in neutrophils from approximately 95% of patients with polycythemia vera and from about half of patients with essential thrombocythemia. CD177 encodes NB1 gp, a 58–64 kD GPI gp that is expressed by neutrophils and neutrophil precursors. NB1 gp carries Human Neutrophil Antigen (HNA-2a. Investigators working to identify the gene encoding NB1 gp called the CD177 allele they described NB1. NB1 gp is unusual in that neutrophils from some healthy people lack the NB1 gp completely and in most people NB1 gp is expressed by a subpopulation of neutrophils. The function of NB1 gp and the role of CD177 in the pathogenesis and clinical course of polycythemia vera and essential thrombocythemia are not yet known. However, measuring neutrophil CD177 mRNA levels has become an important marker for diagnosing the myeloproliferative disorders polycythemia vera and essential thrombocythemia.

Horizontal transmission of malignancy: in-vivo fusion of human lymphomas with hamster stroma produces tumors retaining human genes and lymphoid pathology.

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David M Goldenberg

Full Text Available We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5-6 years GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH, lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b suggests that this uniform population may be the clonal initiating (malignant cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.

Synthesis of 1-[13CD3]-9-cis-retinoic acid

International Nuclear Information System (INIS)

Bennani, Y.L.

1995-01-01

1-[ 13 CD 3 ]-9-cis-Retinoic acid was prepared in 8 steps from 2,6-dimethylcyclohexanone. Alkylation of 2,6-dimethylcyclohexanone under LiHMDS/MnBr 2 / 13 CD 3 I gave the corresponding labeled 2-[ 13 CD 3 ]-2,2,6-trimethylcyclohexanone 4 in good yield. Further functionalization of 4 to 6-[ 13 CD 3 ]-β-cyclocitral 6 proceeded through a Shapiro reaction. Aldehyde 6 was condensed with ethyl 3,3-dimethylacrylate to afford the corresponding bicyclic pyranone 7. Reduction of 7 to lactol 8, followed by acid-catalyzed ring opening gave the 9-cis-aldehyde 9. Wittig-Horner olefination and saponification afforded the title compound in good overall yield and in excellent isotopic purity. (Author)

Gene expression profiles of cryopreserved CD34{sup +} human umbilical cord blood cells are related to their bone marrow reconstitution abilities in mouse xenografts

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Sudo, Kazuhiro [Cell Engineering Division, RIKEN BioResource Center, Tsukuba (Japan); Yasuda, Jun, E-mail: yasuda-jun@umin.ac.jp [Omics Science Center, RIKEN, Yokohama (Japan); Department of Cell Biology, The JFCR-Cancer Institute (Japan); Nakamura, Yukio, E-mail: yukionak@brc.riken.jp [Cell Engineering Division, RIKEN BioResource Center, Tsukuba (Japan)

2010-07-09

Human umbilical cord blood (UCB) cells are an alternative source of hematopoietic stem cells for treatment of leukemia and other diseases. It is very difficult to assess the quality of UCB cells in the clinical situation. Here, we sought to assess the quality of UCB cells by transplantation to immunodeficient mice. Cryopreserved CD34{sup +} UCB cells from twelve different human donors were transplanted into sublethally irradiated NOD/shi-scid Jic mice. In parallel, the gene expression profiles of the UCB cells were determined from oligonucleotide microarrays. UCB cells from three donors failed to establish an engraftment in the host mice, while the other nine succeeded to various extents. Gene expression profiling indicated that 71 genes, including HOXB4, C/EBP-{beta}, and ETS2, were specifically overexpressed and 23 genes were suppressed more than 2-fold in the successful UCB cells compared to those that failed. Functional annotation revealed that cell growth and cell cycle regulators were more abundant in the successful UCB cells. Our results suggest that hematopoietic ability may vary among cryopreserved UCB cells and that this ability can be distinguished by profiling expression of certain sets of genes.

Chromosomal localization of the gonadotropin-releasing hormone receptor gene to human chromosome 4q13. 1-q21. 1 and mouse chromosome 5

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Kaiser, U.B.; Dushkin, H.; Beier, D.R.; Chin, W.W. (Harvard Medical School, Boston, MA (United States)); Altherr, M.R. (Los Alamos National Lab., NM (United States))

1994-04-01

The gonadotropin-releasing hormone receptor (GRHR) is a G-protein-coupled receptor on the cell surface of pituitary gonadotropes, where it serves to transduce signals from the extracellular ligand, the hypothalamic factor gonadotropin-releasing hormone, and to modulate the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. The authors have localized the GRHR gene to the q13.1-q21.1 region of the human chromosome 4 using mapping panels of human/rodent somatic cell hybrids containing different human chromosomes or different regions of human chromosome 4. Furthermore, using linkage analysis of single-strand conformational polymorphisms, the murine GRHR gene was localized to mouse chromosome 5, linked to the endogenous retroviral marker Pmv-11. This is consistent with the evolutionary conservation of homology between these two regions, as has been previously suggested from comparative mapping of several other loci. The localization of the GRHR gene may be useful in the study of disorders of reproduction. 22 refs., 2 figs.

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

Science.gov (United States)

2011-01-01

Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT. PMID:21435270

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

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Chang Shwu-Fen

2011-03-01

Full Text Available Abstract Background Arctium lappa (Niubang, a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC, isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2 and interferon-γ (IFN-γ production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

Lipopolysaccharide-Elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14+ CD1c+ Monocytes.

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Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Vastolo, Viviana; Petrosino, Giuseppe; Visconte, Feliciano; Raia, Maddalena; Scalia, Giulia; Loffredo, Stefania; Varricchi, Gilda; Galdiero, Maria Rosaria; Granata, Francescopaolo; Del Vecchio, Luigi; Portella, Giuseppe; Marone, Gianni

2017-05-01

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14 + monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14 + CD16 - monocytes, TSLPR + monocytes (TSLPR + mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR + mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., GAS6 , ALOX15B , FCGR2B , LAIR1 ). Strikingly, TSLPR + mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14 + CD1c + cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14 + CD16 - monocytes and prompt further ontogenetic and functional analysis of CD14 + CD1c + and LPS-activated CD14 + CD1c + TSLPR + mono. Copyright © 2017 by The American Association of Immunologists, Inc.

Gene expression patterns in CD4+ peripheral blood cells in healthy subjects and stage IV melanoma patients.

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Felts, Sara J; Van Keulen, Virginia P; Scheid, Adam D; Allen, Kathleen S; Bradshaw, Renee K; Jen, Jin; Peikert, Tobias; Middha, Sumit; Zhang, Yuji; Block, Matthew S; Markovic, Svetomir N; Pease, Larry R

2015-11-01

Melanoma patients exhibit changes in immune responsiveness in the local tumor environment, draining lymph nodes, and peripheral blood. Immune-targeting therapies are revolutionizing melanoma patient care increasingly, and studies show that patients derive clinical benefit from these newer agents. Nonetheless, predicting which patients will benefit from these costly therapies remains a challenge. In an effort to capture individual differences in immune responsiveness, we are analyzing patterns of gene expression in human peripheral blood cells using RNAseq. Focusing on CD4+ peripheral blood cells, we describe multiple categories of immune regulating genes, which are expressed in highly ordered patterns shared by cohorts of healthy subjects and stage IV melanoma patients. Despite displaying conservation in overall transcriptome structure, CD4+ peripheral blood cells from melanoma patients differ quantitatively from healthy subjects in the expression of more than 2000 genes. Moreover, 1300 differentially expressed genes are found in transcript response patterns following activation of CD4+ cells ex vivo, suggesting that widespread functional discrepancies differentiate the immune systems of healthy subjects and melanoma patients. While our analysis reveals that the transcriptome architecture characteristic of healthy subjects is maintained in cancer patients, the genes expressed differentially among individuals and across cohorts provide opportunities for understanding variable immune states as well as response potentials, thus establishing a foundation for predicting individual responses to stimuli such as immunotherapeutic agents.

Aberrant Splicing of Estrogen Receptor, HER2, and CD44 Genes in Breast Cancer

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Kazushi Inoue

2015-01-01

Full Text Available Breast cancer (BC is the most common cause of cancer-related death among women under the age of 50 years. Established biomarkers, such as hormone receptors (estrogen receptor [ER]/progesterone receptor and human epidermal growth factor receptor 2 (HER2, play significant roles in the selection of patients for endocrine and trastuzumab therapies. However, the initial treatment response is often followed by tumor relapse with intrinsic resistance to the first-line therapy, so it has been expected to identify novel molecular markers to improve the survival and quality of life of patients. Alternative splicing of pre-messenger RNAs is a ubiquitous and flexible mechanism for the control of gene expression in mammalian cells. It provides cells with the opportunity to create protein isoforms with different, even opposing, functions from a single genomic locus. Aberrant alternative splicing is very common in cancer where emerging tumor cells take advantage of this flexibility to produce proteins that promote cell growth and survival. While a number of splicing alterations have been reported in human cancers, we focus on aberrant splicing of ER , HER2 , and CD44 genes from the viewpoint of BC development. ERα36 , a splice variant from the ER1 locus, governs nongenomic membrane signaling pathways triggered by estrogen and confers 4-hydroxytamoxifen resistance in BC therapy. The alternative spliced isoform of HER2 lacking exon 20 (Δ16HER2 has been reported in human BC; this isoform is associated with transforming ability than the wild-type HER2 and recapitulates the phenotypes of endocrine therapy-resistant BC. Although both CD44 splice isoforms ( CD44s , CD44v play essential roles in BC development, CD44v is more associated with those with favorable prognosis, such as luminal A subtype, while CD44s is linked to those with poor prognosis, such as HER2 or basal cell subtypes that are often metastatic. Hence, the detection of splice variants from these loci

Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

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Aoki, Yuki; Watanabe, Takashi; Saito, Yoriko; Kuroki, Yoko; Hijikata, Atsushi; Takagi, Masatoshi; Tomizawa, Daisuke; Eguchi, Mariko; Eguchi-Ishimae, Minenori; Kaneko, Akiko; Ono, Rintaro; Sato, Kaori; Suzuki, Nahoko; Fujiki, Saera; Koh, Katsuyoshi; Ishii, Eiichi; Shultz, Leonard D.; Ohara, Osamu; Mizutani, Shuki

2015-01-01

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34+CD38+CD19+ and CD34−CD19+ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19+ cells were LICs. In MLL-ENL patients, either CD34+ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34+CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34+CD38+ and CD34− LICs but not in CD34+CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia. PMID:25538041

Profiling calcium signals of in vitro polarized human effector CD4+ T cells.

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Kircher, Sarah; Merino-Wong, Maylin; Niemeyer, Barbara A; Alansary, Dalia

2018-06-01

Differentiation of naïve CD4 + T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca 2+ signals, mainly mediated by the store operated Ca 2+ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4 + T cell subsets show differential Ca 2+ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4 + effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca 2+ release activated Ca 2+ currents (I CRAC ) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca 2+ profiles of helper CD4 + Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4 + T cells. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone

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Walseth Timothy F

2008-03-01

Full Text Available Abstract Background CD38 is expressed in human airway smooth muscle (HASM cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α. CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene. Methods We cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-κB and several AP-1 and glucocorticoid response element (GRE motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies. Results TNF-α induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to

Frequent amplification of CENPF, GMNN and CDK13 genes in hepatocellular carcinomas.

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Hye-Eun Kim

Full Text Available Genomic changes frequently occur in cancer cells during tumorigenesis from normal cells. Using the Illumina Human NS-12 single-nucleotide polymorphism (SNP chip to screen for gene copy number changes in primary hepatocellular carcinomas (HCCs, we initially detected amplification of 35 genes from four genomic regions (1q21-41, 6p21.2-24.1, 7p13 and 8q13-23. By integrated screening of these genes for both DNA copy number and gene expression in HCC and colorectal cancer, we selected CENPF (centromere protein F/mitosin, GMNN (geminin, DNA replication inhibitor, CDK13 (cyclin-dependent kinase 13, and FAM82B (family with sequence similarity 82, member B as common cancer genes. Each gene exhibited an amplification frequency of ~30% (range, 20-50% in primary HCC (n = 57 and colorectal cancer (n = 12, as well as in a panel of human cancer cell lines (n = 70. Clonogenic and invasion assays of NIH3T3 cells transfected with each of the four amplified genes showed that CENPF, GMNN, and CDK13 were highly oncogenic whereas FAM82B was not. Interestingly, the oncogenic activity of these genes (excluding FAM82B was highly correlated with gene-copy numbers in tumor samples (correlation coefficient, r>0.423, indicating that amplifications of CENPF, GMNN, and CDK13 genes are tightly linked and coincident in tumors. Furthermore, we confirmed that CDK13 gene copy number was significantly associated with clinical onset age in patients with HCC (P = 0.0037. Taken together, our results suggest that coincidently amplified CDK13, GMNN, and CENPF genes can play a role as common cancer-driver genes in human cancers.

CD1 and mycobacterial lipids activate human T cells.

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Van Rhijn, Ildiko; Moody, D Branch

2015-03-01

For decades, proteins were thought to be the sole or at least the dominant source of antigens for T cells. Studies in the 1990s demonstrated that CD1 proteins and mycobacterial lipids form specific targets of human αβ T cells. The molecular basis by which T-cell receptors (TCRs) recognize CD1-lipid complexes is now well understood. Many types of mycobacterial lipids function as antigens in the CD1 system, and new studies done with CD1 tetramers identify T-cell populations in the blood of tuberculosis patients. In human populations, a fundamental difference between the CD1 and major histocompatibility complex systems is that all humans express nearly identical CD1 proteins. Correspondingly, human CD1 responsive T cells show evidence of conserved TCRs. In addition to natural killer T cells and mucosal-associated invariant T (MAIT cells), conserved TCRs define other subsets of human T cells, including germline-encoded mycolyl-reactive (GEM) T cells. The simple immunogenetics of the CD1 system and new investigative tools to measure T-cell responses in humans now creates a situation in which known lipid antigens can be developed as immunodiagnostic and immunotherapeutic reagents for tuberculosis disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells.

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Solstad, Therese; Bains, Simer Jit; Landskron, Johannes; Aandahl, Einar Martin; Thiede, Bernd; Taskén, Kjetil; Torgersen, Knut Martin

2011-11-10

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.

Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

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Aspord, Caroline; Pedroza-Gonzalez, Alexander; Gallegos, Mike; Tindle, Sasha; Burton, Elizabeth C.; Su, Dan; Marches, Florentina; Banchereau, Jacques; Palucka, A. Karolina

2007-01-01

We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development. PMID:17438063

Life stage differences in mammary gland gene expression profile in non-human primates.

Science.gov (United States)

Stute, Petra; Sielker, Sonja; Wood, Charles E; Register, Thomas C; Lees, Cynthia J; Dewi, Fitriya N; Williams, J Koudy; Wagner, Janice D; Stefenelli, Ulrich; Cline, J Mark

2012-06-01

Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer

Differential expression of granulopoiesis related genes in neutrophil subsets distinguished by membrane expression of CD177

DEFF Research Database (Denmark)

Hu, Nan; Mora-Jensen, Helena; Theilgaard-Mønch, Kim

2014-01-01

OBJECTIVE: Differential gene expression in CD177+ and CD177- neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV). METHODS: Neutrophils were isolated from healthy controls (HC......) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177- subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177- subsets in microarray analysis were re-assessed using...... quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry. RESULTS: The proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177...

Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media.

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Kleijkers, Sander H M; Eijssen, Lars M T; Coonen, Edith; Derhaag, Josien G; Mantikou, Eleni; Jonker, Martijs J; Mastenbroek, Sebastiaan; Repping, Sjoerd; Evers, Johannes L H; Dumoulin, John C M; van Montfoort, Aafke P A

2015-10-01

Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein degradation, metabolism and cell-cycle regulation. Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal models of human development, it has been demonstrated that culture of preimplantation embryos in vitro affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days. In a multicenter trial, women were randomly assigned to two culture medium groups [G5 and human tubal fluid (HTF)]. Data on embryonic development were collected for all embryos. In one center, embryos originating from two pronuclei (2PN) zygotes that were not selected for transfer or cryopreservation on Day 2 or 3 because of lower morphological quality, were cultured until Day 6 and used in this study, if couples consented. Ten blastocysts each from the G5 and HTF study groups, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were examined individually for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity. Expression of 951 genes differed significantly (P differences observed between the study groups are caused by factors that we did not investigate. Extrapolation of these results to embryos used for transfer demands caution as in the present study embryos that were not selected for either embryo transfer or cryopreservation have been used for the

Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

Science.gov (United States)

Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

2015-05-01

Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy. © 2015 AlphaMed Press.

T helper-independent activation of human CD8+ cells: the role of CD28 costimulation.

Science.gov (United States)

Van Gool, S W; Zhang, Y; Kasran, A; de Boer, M; Ceuppens, J L

1996-07-01

The concept that activation of MHC class I-restricted CD8+ cells entirely depends on help from MHC class II-restricted CD4+ T cells has recently been supplemented with an alternative model in which CD8+ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8+ T cells in two different in vitro models. Freshly isolated CD8+ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting Fc gamma R+ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8+ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8+ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8+ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8+ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.

Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma.

Science.gov (United States)

Rissetto, K C; Rindt, H; Selting, K A; Villamil, J A; Henry, C J; Reinero, C R

2010-05-15

T regulatory cells (Tregs) are a unique subset of T helper cells that serve to modify/inhibit effector cells of the immune system and thus are essential to prevent autoimmunity. Overzealous Treg activity may contribute to impaired immune responses to cancer. Tregs can be phenotypically identified by proteins expressed on the cell surface (CD4 and CD25) and inside the cell (forkhead box3 (FoxP3)), although in dogs, no anti-canine CD25 antibody exists. We hypothesized that a mouse anti-human CD25 antibody definitively recognizes the canine protein and can be used to identify Tregs in dogs. We describe cloning and transfection of the canine CD25 gene into human HeLa cells with subsequent expression of the canine protein on the cell surface detected using an anti-human CD25 antibody in a flow cytometric assay. Validation of this antibody was used to identify CD4+CD25+FoxP3+ Tregs in 39 healthy dogs and 16 dogs with osteosarcoma (OSA). Results were expressed in five different ways and showed significantly fewer %CD4+CD25+ T lymphocytes expressing FoxP3 in blood of older dogs (>/=7 years) compared with the other two age groups (<2 and 2-6 years) (p<0.001) and fewer %CD4+CD25+FoxP3+ Tregs in the tumor draining lymph nodes of OSA patients compared to the unrelated lymph node (p=0.049). However, there was no significant difference in % Tregs in the peripheral blood or lymph nodes between the control dogs and those with OSA. While the CD25 antibody can be successfully used in a flow cytometric assay to identify Tregs, this study does not support clinical utility of phenotypic recognition of Tregs in dogs with OSA. Copyright 2010 Elsevier B.V. All rights reserved.

Characterization of cancer stem cell properties of CD24 and CD26-positive human malignant mesothelioma cells

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Yamazaki, Hiroto; Naito, Motohiko; Ghani, Farhana Ishrat [Division of Clinical Immunology, Institute of Medical Science, University of Tokyo, Tokyo (Japan); Dang, Nam H. [Division of Hematology/Oncology, University of Florida Shands Cancer Center, Gainesville, FL 32610 (United States); Iwata, Satoshi [Division of Clinical Immunology, Institute of Medical Science, University of Tokyo, Tokyo (Japan); Morimoto, Chikao, E-mail: morimoto@ims.u-tokyo.ac.jp [Division of Clinical Immunology, Institute of Medical Science, University of Tokyo, Tokyo (Japan)

2012-03-16

Highlights: Black-Right-Pointing-Pointer We focused on CD24 and CD26 for further analysis of CSC properties in MM. Black-Right-Pointing-Pointer Their expressions were correlated with chemoresistance, cell growth, and invasion. Black-Right-Pointing-Pointer Their expressions were also correlated with several cancer related genes. Black-Right-Pointing-Pointer The expression of each marker was correlated with different CSC property in Meso1. Black-Right-Pointing-Pointer Phosphorylation of ERK by EGF was regulated by expression of CD26, but not CD24. -- Abstract: Malignant mesothelioma (MM) is an asbestos-related malignancy characterized by rapid growth and poor prognosis. In our previous study, we have demonstrated that several cancer stem cell (CSC) markers correlated with CSC properties in MM cells. Among these markers, we focused on two: CD24, the common CSC marker, and CD26, the additional CSC marker. We further analyzed the CSC properties of CD24 and CD26-positve MM cells. We established RNAi-knockdown cells and found that these markers were significantly correlated with chemoresistance, proliferation, and invasion potentials in vitro. Interestingly, while Meso-1 cells expressed both CD24 and CD26, the presence of each of these two markers was correlated with different CSC property. In addition, downstream signaling of these markers was explored by microarray analysis, which revealed that their expressions were correlated with several cancer-related genes. Furthermore, phosphorylation of ERK by EGF stimulation was significantly affected by the expression of CD26, but not CD24. These results suggest that CD24 and CD26 differentially regulate the CSC potentials of MM and could be promising targets for CSC-oriented therapy.

Various domains of the B-cell regulatory molecule CD72 has diverged at different rates in mammals

DEFF Research Database (Denmark)

Petersen, Cathrine Bie; Hillig, Ann-Britt Nygaard; Fredholm, Merete

2007-01-01

72 has been shown to be a negatively regulating BCR co-receptor. We isolated and sequenced three porcine CD72 transcript variants. Using a pig radiation hybrid panel we found the porcien CD72 gene to be located on chromosome 1q21-28 in a region syntenic to human chromosome 9. The porcine CD72 gene......We report the cloning of the porcine B-cell co-receptor CD72, as well as genomic mapping and examination of transcription. The B-cell receptor (BCR) complex mediates signalling upon antigen recognition by the membrane bound BCR. Several co-receptors modulate this signal positively or negatively. CD...

Expression of CD44 splice variants in human primary brain tumors

NARCIS (Netherlands)

Kaaijk, P.; Troost, D.; Morsink, F.; Keehnen, R. M.; Leenstra, S.; Bosch, D. A.; Pals, S. T.

1995-01-01

Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastatic potential in a number of different animal and human cancers. Although differential expression of CD44 standard epitopes (CD44s) in human brain tumors has been reported, the expression of

Immobilization remediation of Cd-polluted soil with different water condition.

Science.gov (United States)

Li, Jianrui; Xu, Yingming

2017-05-15

To demonstrate effects of water management on soil Cd immobilization using palygorskite, the investigation evaluated impacts of palygorskite on uptake of Cd present in soils with different water condition by rice plant. Pot experiment results showed that, pH, available Fe and P in untreated soils were higher in continuous flooding than in traditional irrigation and wetting irrigation, which were reasons for lower soil exchangeable Cd and plant Cd in continuous flooding. In control group (untreated soils), compared to traditional irrigation, continuous flooding reduced brown rice Cd by 37.9%, that in wetting irrigation increased by 31.0%. At palygorskite concentrations of 5 g kg -1 , 10 g kg -1 and 15 g kg -1 , brown rice Cd reduced by 16.7%, 44.4% and 55.6% under continuous flooding, 13.8%, 34.5% and 44.8% under traditional irrigation, 13.1%, 36.8% and 47.3% under wetting irrigation (p soils. Copyright © 2017 Elsevier Ltd. All rights reserved.

Induction of CD3 delta epsilon omega by phorbol 12-myristate 13-acetate

DEFF Research Database (Denmark)

Vangsted, A; Neisig, A; Wallin, H

1993-01-01

The effect of phorbol 12-myristate 13-acetate (PMA) on the synthesis, assembly and processing of the components of the T cell receptor (TcR) was studied with special focus on the CD3 omega chain. Treatment of the human leukemic T cell line Jurkat with PMA increased the synthesis of the Ti alpha, CD......3 gamma and CG3 zeta chains two- to threefold and the synthesis of Ti beta and CD3 delta epsilon omega complexes five- to sevenfold as assessed by metabolic labeling, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by scanning densitometry. The amount...... of TcR complexes expressed on the cell surface was decreased after 16 h of PMA treatment. Based on these results we propose a role of CD3 omega in retention of TcR complexes. From PMA-treated CEM cells more than 50-fold the amount of CD3 delta epsilon omega complexes was immunoprecipitated as compared...

Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells.

Science.gov (United States)

Matsuoka, Yoshikazu; Nakatsuka, Ryusuke; Sumide, Keisuke; Kawamura, Hiroshi; Takahashi, Masaya; Fujioka, Tatsuya; Uemura, Yasushi; Asano, Hiroaki; Sasaki, Yutaka; Inoue, Masami; Ogawa, Hiroyasu; Takahashi, Takayuki; Hino, Masayuki; Sonoda, Yoshiaki

2015-05-01

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. © 2014 AlphaMed Press.

EDAG promotes the expansion and survival of human CD34+ cells.

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Ke Zhao

Full Text Available EDAG is multifunctional transcriptional regulator primarily expressed in the linloc-kit+Sca-1+ hematopoietic stem cells (HSC and CD34+ progenitor cells. Previous studies indicate that EDAG is required for maintaining hematopoietic lineage commitment balance. Here using ex vivo culture and HSC transplantation models, we report that EDAG enhances the proliferative potential of human cord blood CD34+ cells, increases survival, prevents cell apoptosis and promotes their repopulating capacity. Moreover, EDAG overexpression induces rapid entry of CD34+ cells into the cell cycle. Gene expression profile analysis indicate that EDAG knockdown leads to down-regulation of various positive cell cycle regulators including cyclin A, B, D, and E. Together these data provides novel insights into EDAG in regulation of expansion and survival of human hematopoietic stem/progenitor cells.

Tissue-specific expression of the human laminin alpha5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation

DEFF Research Database (Denmark)

Durkin, M E; Loechel, F; Mattei, M G

1997-01-01

, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra...

TLR5 signaling enhances the proliferation of human allogeneic CD40-activated B cell induced CD4hiCD25+ regulatory T cells.

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Ping-Lung Chan

Full Text Available Although diverse functions of different toll-like receptors (TLR on human natural regulatory T cells have been demonstrated recently, the role of TLR-related signals on human induced regulatory T cells remain elusive. Previously our group developed an ex vivo high-efficient system in generating human alloantigen-specific CD4(hiCD25(+ regulatory T cells from naïve CD4(+CD25(- T cells using allogeneic CD40-activated B cells as stimulators. In this study, we investigated the role of TLR5-related signals on the generation and function of these novel CD4(hiCD25(+ regulatory T cells. It was found that induced CD4(hiCD25(+ regulatory T cells expressed an up-regulated level of TLR5 compared to their precursors. The blockade of TLR5 using anti-TLR5 antibodies during the co-culture decreased CD4(hiCD25(+ regulatory T cells proliferation by induction of S phase arrest. The S phase arrest was associated with reduced ERK1/2 phosphorylation. However, TLR5 blockade did not decrease the CTLA-4, GITR and FOXP3 expressions, and the suppressive function of CD4(hiCD25(+ regulatory T cells. In conclusion, we discovered a novel function of TLR5-related signaling in enhancing the proliferation of CD4(hiCD25(+ regulatory T cells by promoting S phase progress but not involved in the suppressive function of human CD40-activated B cell-induced CD4(hiCD25(+ regulatory T cells, suggesting a novel role of TLR5-related signals in the generation of induced regulatory T cells.

Genetic subspecies diversity of the chimpanzee CD4 virus-receptor gene

DEFF Research Database (Denmark)

Hvilsom, Christina; Carlsen, Frands; Siegismund, Hans R

2008-01-01

six among the subspecies of chimpanzees. We found the CD4 receptor to be conserved in individuals belonging to the P. t. verus subspecies and divergent from the other three subspecies, which harbored highly variable CD4 receptors. The CD4 receptor of chimpanzees differed from that of humans. We...... question whether the observed diversity can explain the species-specific differences in susceptibility to and pathogenicity of SIV/HIV....

Introduction of the human proα1(I) collagen gene into proα1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

International Nuclear Information System (INIS)

Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

1987-01-01

The Mov-13 mouse strain carries a retroviral insertion in the proα1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of proα2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse proα1(I) collagen gene into homozygous cell lines to assess whether the human or mouse proα1(I) chains can associate with the endogenous mouse proα2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human α1 chains and one mouse α2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both α1(I) and α2(I) chains in the human-mouse hybrid molecules were retarded, compared to the α(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse α1 and α2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human α chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected proα1(I) genes have on the synthesis, assembly, and function of collagen I

A computational profiling of changes in gene expression and transcription factors induced by vFLIP K13 in primary effusion lymphoma.

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Vasu Punj

Full Text Available Infection with Kaposi's sarcoma associated herpesvirus (KSHV has been linked to the development of primary effusion lymphoma (PEL, a rare lymphoproliferative disorder that is characterized by loss of expression of most B cell markers and effusions in the body cavities. This unique clinical presentation of PEL has been attributed to their distinctive plasmablastic gene expression profile that shows overexpression of genes involved in inflammation, adhesion and invasion. KSHV-encoded latent protein vFLIP K13 has been previously shown to promote the survival and proliferation of PEL cells. In this study, we employed gene array analysis to characterize the effect of K13 on global gene expression in PEL-derived BCBL1 cells, which express negligible K13 endogenously. We demonstrate that K13 upregulates the expression of a number of NF-κB responsive genes involved in cytokine signaling, cell death, adhesion, inflammation and immune response, including two NF-κB subunits involved in the alternate NF-κB pathway, RELB and NFKB2. In contrast, CD19, a B cell marker, was one of the genes downregulated by K13. A comparison with K13-induced genes in human vascular endothelial cells revealed that although there was a considerable overlap among the genes induced by K13 in the two cell types, chemokines genes were preferentially induced in HUVEC with few exceptions, such as RANTES/CCL5, which was induced in both cell types. Functional studies confirmed that K13 activated the RANTES/CCL5 promoter through the NF-κB pathway. Taken collectively, our results suggest that K13 may contribute to the unique gene expression profile, immunophenotype and clinical presentation that are characteristics of KSHV-associated PEL.

Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK Expression with [124I]FIAU and PET

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Trevor Hackman

2002-01-01

Full Text Available Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CDherpes simplex virus type 1 thymidine kinase (HSV1-tk fusion gene (CD/TK with 5-fluorocytosine (5FC, ganciclovir (GCV, and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil and positron emission tomography (PET for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells. The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

Science.gov (United States)

Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Gene Transfer Properties and Structural Modeling of Human Stem Cell-derived AAV

OpenAIRE

Smith, Laura J; Ul-Hasan, Taihra; Carvaines, Sarah K; Van Vliet, Kim; Yang, Ethel; Wong, Kamehameha K; Agbandje-McKenna, Mavis; Chatterjee, Saswati

2014-01-01

Adeno-associated virus (AAV) vectors are proving to be remarkably successful for in vivo gene delivery. Based upon reports of abundant AAV in the human marrow, we tested CD34+ hematopoietic stem cells for the presence of natural AAV. Here, we report for the first time, the presence of novel AAV variants in healthy CD34+ human peripheral blood stem cells. The majority of healthy peripheral blood stem cell donors were found to harbor AAV in their CD34+ cells. Every AAV isolated from CD34+ cells...

Suppression of human breast tumors in NOD/SCID mice by CD44 shRNA gene therapy combined with doxorubicin treatment

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Pham PV

2012-05-01

Full Text Available Phuc Van Pham1, Ngoc Bich Vu1, Thuy Thanh Duong1, Tam Thanh Nguyen1, Nhung Hai Truong1, Nhan Lu Chinh Phan1, Tue Gia Vuong1, Viet Quoc Pham1, Hoang Minh Nguyen1, Kha The Nguyen1, Nhung Thi Nguyen1, Khue Gia Nguyen1, Lam Tan Khat1, Dong Van Le2, Kiet Dinh Truong1, Ngoc Kim Phan11Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, HCM City, 2Military Medical University, Ha Noi, VietnamBackground: Breast cancer stem cells with a CD44+CD24- phenotype are the origin of breast tumors. Strong CD44 expression in this population indicates its important role in maintaining the stem cell phenotype. Previous studies show that CD44 down-regulation causes CD44+CD24- breast cancer stem cells to differentiate into non-stem cells that are sensitive to antitumor drugs and lose many characteristics of the original cells. In this study, we determined tumor suppression in non-obese severe combined immunodeficiency mice using CD44 shRNA therapy combined with doxorubicin treatment.Methods: Tumor-bearing non-obese severe combined immunodeficiency mice were established by injection of CD44+CD24- cells. To track CD44+CD24- cells, green fluorescence protein was stably transduced using a lentiviral vector prior to injection into mice. The amount of CD44 shRNA lentiviral vector used for transduction was based on CD44 down-regulation by in vitro CD44 shRNA transduction. Mice were treated with direct injection of CD44 shRNA lentiviral vector into tumors followed by doxorubicin administration after 48 hours. The effect was evaluated by changes in the size and weight of tumors compared with that of the control.Results: The combination of CD44 down-regulation and doxorubicin strongly suppressed tumor growth with significant differences in tumor sizes and weights compared with that of CD44 down-regulation or doxorubicin treatment alone. In the combination of CD44 down-regulation and doxorubicin group, the tumor weight was

CD13-positive bone marrow-derived myeloid cells promote angiogenesis, tumor growth, and metastasis.

Science.gov (United States)

Dondossola, Eleonora; Rangel, Roberto; Guzman-Rojas, Liliana; Barbu, Elena M; Hosoya, Hitomi; St John, Lisa S; Molldrem, Jeffrey J; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2013-12-17

Angiogenesis is fundamental to tumorigenesis and an attractive target for therapeutic intervention against cancer. We have recently demonstrated that CD13 (aminopeptidase N) expressed by nonmalignant host cells of unspecified types regulate tumor blood vessel development. Here, we compare CD13 wild-type and null bone marrow-transplanted tumor-bearing mice to show that host CD13(+) bone marrow-derived cells promote cancer progression via their effect on angiogenesis. Furthermore, we have identified CD11b(+)CD13(+) myeloid cells as the immune subpopulation directly regulating tumor blood vessel development. Finally, we show that these cells are specifically localized within the tumor microenvironment and produce proangiogenic soluble factors. Thus, CD11b(+)CD13(+) myeloid cells constitute a population of bone marrow-derived cells that promote tumor progression and metastasis and are potential candidates for the development of targeted antiangiogenic drugs.

Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.)

International Nuclear Information System (INIS)

Wang Xiaojuan; Song, Yu; Ma Yanhua; Zhuo Renying; Jin Liang

2011-01-01

In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible. - Highlights: → Evaluate Cd tolerance in wide sources of alfalfa accessions. → Identify Cd-hyperaccumulators potentially useful for restoring Cd-contaminated environments. → Cloned differentially expressed metallothionein (MT) genes. → Characteristics and deduced protein sequence of MsMT2a and MsMT2b were analyzed. → MsMT2a might be a universally gene of alfalfa but MsMT2b might be an inductive gene. - Two Cd tolerant alfalfa genotypes were screened and their metallothionein genes were cloned which showed that MsMT2a was universally expressed but MsMT2b was Cd inducible expression.

Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.)

Energy Technology Data Exchange (ETDEWEB)

Wang Xiaojuan, E-mail: xiaojuanwang@lzu.edu.cn [School of Pastoral Agriculture Science and Technology, Lanzhou University, P.O. Box 61, Lanzhou 730020 (China); Song, Yu [School of Pastoral Agriculture Science and Technology, Lanzhou University, P.O. Box 61, Lanzhou 730020 (China); Environment Management College of China, Qinhuangdao 066004 (China); Ma Yanhua [Hebei Normal University of Science and Technology, Qinhuangdao 066004 (China); Zhuo Renying [Key Lab of Tree Genomics, Research Institute of Subtropical of Forest, Chinese Academy of Forest, Fuyang 311400 (China); Jin Liang [School of Pastoral Agriculture Science and Technology, Lanzhou University, P.O. Box 61, Lanzhou 730020 (China)

2011-12-15

In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible. - Highlights: > Evaluate Cd tolerance in wide sources of alfalfa accessions. > Identify Cd-hyperaccumulators potentially useful for restoring Cd-contaminated environments. > Cloned differentially expressed metallothionein (MT) genes. > Characteristics and deduced protein sequence of MsMT2a and MsMT2b were analyzed. > MsMT2a might be a universally gene of alfalfa but MsMT2b might be an inductive gene. - Two Cd tolerant alfalfa genotypes were screened and their metallothionein genes were cloned which showed that MsMT2a was universally expressed but MsMT2b was Cd inducible expression.

Synthesis of 1-[{sup 13}CD{sub 3}]-9-cis-retinoic acid

Energy Technology Data Exchange (ETDEWEB)

Bennani, Y.L. [Ligand Pharmaceuticals Inc., San Diego, CA (United States). Dept. of Medicinal Chemistry

1995-12-31

1-[{sup 13}CD{sub 3}]-9-cis-Retinoic acid was prepared in 8 steps from 2,6-dimethylcyclohexanone. Alkylation of 2,6-dimethylcyclohexanone under LiHMDS/MnBr{sub 2}/{sup 13}CD{sub 3}I gave the corresponding labeled 2-[{sup 13}CD{sub 3}]-2,2,6-trimethylcyclohexanone 4 in good yield. Further functionalization of 4 to 6-[{sup 13}CD{sub 3}]-{beta}-cyclocitral 6 proceeded through a Shapiro reaction. Aldehyde 6 was condensed with ethyl 3,3-dimethylacrylate to afford the corresponding bicyclic pyranone 7. Reduction of 7 to lactol 8, followed by acid-catalyzed ring opening gave the 9-cis-aldehyde 9. Wittig-Horner olefination and saponification afforded the title compound in good overall yield and in excellent isotopic purity. (Author).

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

International Nuclear Information System (INIS)

Ahn, Kwang Sung; Won, Ji Young; Park, Jin-Ki; Sorrell, Alice M.; Heo, Soon Young; Kang, Jee Hyun; Woo, Jae-Seok; Choi, Bong-Hwan; Chang, Won-Kyong; Shim, Hosup

2010-01-01

Research highlights: → Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. → hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. → hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

Energy Technology Data Exchange (ETDEWEB)

Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

2010-10-01

Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.

Science.gov (United States)

Masiuk, Katelyn E; Brown, Devin; Laborada, Jennifer; Hollis, Roger P; Urbinati, Fabrizia; Kohn, Donald B

2017-09-06

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 - cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 - cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 - cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 - cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Transcriptional profiling of human monocytes identifies the inhibitory receptor CD300a as regulator of transendothelial migration.

Directory of Open Access Journals (Sweden)

Sharang Ghavampour

Full Text Available Local inflammatory responses are characterized by the recruitment of circulating leukocytes from the blood to sites of inflammation, a process requiring the directed migration of leukocytes across the vessel wall and hence a penetration of the endothelial lining. To identify underlying signalling events and novel factors involved in these processes we screened for genes differentially expressed in human monocytes following their adhesion to and passage through an endothelial monolayer. Functional annotation clustering of the genes identified revealed an overrepresentation of those associated with inflammation/immune response, in particular early monocyte to macrophage differentiation. Among the gene products so far not implicated in monocyte transendothelial migration was the inhibitory immune receptor CD300a. CD300a mRNA and protein levels were upregulated following transmigration and engagement of the receptor by anti-CD300a antibodies markedly reduced monocyte transendothelial migration. In contrast, siRNA mediated downregulation of CD300a in human monocytes increased their rate of migration. CD300a colocalized and cosedimented with actin filaments and, when activated, caused F-actin cytoskeleton alterations. Thus, monocyte transendothelial migration is accompanied by an elevation of CD300a which serves an inhibitory function possibly required for termination of the actual transmigration.

Chromosomal localization of the human and mouse hyaluronan synthase genes

Energy Technology Data Exchange (ETDEWEB)

Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

1997-05-01

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

CD63 tetraspanin is a negative driver of epithelial-to-mesenchymal transition in human melanoma cells.

Science.gov (United States)

Lupia, Antonella; Peppicelli, Silvia; Witort, Ewa; Bianchini, Francesca; Carloni, Vinicio; Pimpinelli, Nicola; Urso, Carmelo; Borgognoni, Lorenzo; Capaccioli, Sergio; Calorini, Lido; Lulli, Matteo

2014-12-01

The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFβ. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

Science.gov (United States)

Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F; Greber, Urs F; Hemmi, Silvio

2004-05-01

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

Dioxin exposure of human CD34+ hemopoietic cells induces gene expression modulation that recapitulates its in vivo clinical and biological effects

International Nuclear Information System (INIS)

Fracchiolla, Nicola Stefano; Todoerti, Katia; Bertazzi, Pier Alberto; Servida, Federica; Corradini, Paolo; Carniti, Cristiana; Colombi, Antonio; Cecilia Pesatori, Angela; Neri, Antonino; Deliliers, Giorgio Lambertenghi

2011-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has a large number of biological effects, including skin, cardiovascular, neurologic diseases, diabetes, infertility, cancers and immunotoxicity. We analysed the in vitro TCDD effects on human CD34 + cells and tested the gene expression modulation by means of microarray analyses before and after TCDD exposure. We identified 257 differentially modulated probe sets, identifying 221 well characterized genes. A large part of these resulted associated to cell adhesion and/or angiogenesis and to transcription regulation. Synaptic transmission and visual perception functions, with the particular involvement of the GABAergic pathway were also significantly modulated. Numerous transcripts involved in cell cycle or cell proliferation, immune response, signal transduction, ion channel activity or calcium ion binding, tissue development and differentiation, female or male fertility or in several metabolic pathways were also affected after dioxin exposure. The transcriptional profile induced by TCDD treatment on human CD34 + cells strikingly reproduces the clinical and biological effects observed in individuals exposed to dioxin and in biological experimental systems. Our data support a role of dioxin in the neoplastic transformation of hemopoietic stem cells and in immune modulation processes after in vivo exposure, as indicated by the epidemiologic data in dioxin accidentally exposed populations, providing a molecular basis for it. In addition, TCDD alters genes associated to glucidic and lipidic metabolisms, to GABAergic transmission or involved in male and female fertility, thus providing a possible explanation of the diabetogenic, dyslipidemic, neurologic and fertility effects induced by TCDD in vivo exposure.

5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4+ T Cells

Directory of Open Access Journals (Sweden)

Colm E. Nestor

2016-07-01

Full Text Available 5-methylcytosine (5mC is converted to 5-hydroxymethylcytosine (5hmC by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4+ T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4+ memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4+ T cell biology in humans, with important implications for gene regulation and lineage commitment.

Ectoenzymes and innate immunity: the role of human CD157 in leukocyte trafficking.

Science.gov (United States)

Funaro, Ada; Ortolan, Erika; Bovino, Paola; Lo Buono, Nicola; Nacci, Giulia; Parrotta, Rossella; Ferrero, Enza; Malavasi, Fabio

2009-01-01

CD157 is a glycosylphosphatidylinositol-anchored molecule encoded by a member of the CD38/ADP-ribosyl cyclase gene family, involved in the metabolism of NAD. Expressed mainly by cells of the myeloid lineage and by vascular endothelial cells, CD157 has a dual nature behaving both as an ectoenzyme and as a receptor. Although it lacks a cytoplasmic domain, and cannot transduce signals on its own, the molecule compensates for this structural limit by interacting with conventional receptors. Recent experimental evidence suggests that CD157 orchestrates critical functions of human neutrophils. Indeed, CD157-mediated signals promote cell polarization, regulate chemotaxis induced through the high affinity fMLP receptor and control transendothelial migration.

[Polymorphism of CD209 and TLR3 genes in populations of North Eurasia].

Science.gov (United States)

Barkhash, A V; Babenko, V N; Voevoda, M I; Romaschenko, A G

2016-06-01

The DC-SIGN (dendritic cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin) and TLR3 (toll-like receptor 3) proteins are key effectors of the innate immunity and particularly play an important role in the organism’s antiviral defense as pattern-recognition receptors. Previously, we demonstrated that certain genotypes and alleles of single nucleotide polymorphisms (SNPs) rs2287886 (G/A) in the promoter region of the CD209 gene (encoding DC-SIGN) and rs3775291 (G/A, Leu412Phe) in the exon 4 of the TLR3 gene are associated with human predisposition to tick-borne encephalitis in the Russian population. In the present work, the distribution of genotype and allele frequencies for these SNPs was studied in seven populations of North Eurasia, including Caucasians (Russians and Germans (from Altai region)), Central Asian Mongoloids (Altaians, Khakass, Tuvinians, and Shorians), and Arctic Mongoloids (Chukchi). It was found that the CD209 gene rs2287886 SNP A/A genotype and A allele, as well as the TLR3 gene rs3775291 SNP G/G genotype and G allele (the frequencies of which in our previous studies were increased in tick-borne encephalitis patients as compared with the population control (Russian citizens of Novosibirsk)), are preserved with a high frequency in Central Asian Mongoloids (who for a long time regularly came in contact with tick-borne encephalitis virus in places of their habitation). We suggested that predisposition to tick-borne encephalitis in Central Asian Mongoloid populations can be predetermined by a different set of genes and their polymorphisms than in the Russian population.

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

International Nuclear Information System (INIS)

Sun, Yan; Xiao, Dong; Zhang, Ruo-Shuang; Cui, Guang-Hui; Wang, Xin-Hua; Chen, Xi-Gu

2007-01-01

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future

Efficient intranuclear gene delivery by CdSe aqueous quantum dots electrostatically-coated with polyethyleneimine

International Nuclear Information System (INIS)

Au, Giang H T; Shih, Wan Y; Shih, Wei-Heng

2015-01-01

Quantum dots (QDs) are semiconducting nanoparticles with photoluminescence properties that do not photobleach. Due to these advantages, using QDs for non-viral gene delivery has the additional benefit of being able to track the delivery of the genes in real time as it happens. We investigate the efficacy of mercaptopropionic acid (MPA)-capped CdSe aqueous quantum dots (AQDs) electrostatically complexed with branched polyethyleneimine (PEI) both as a non-viral gene delivery vector and as a fluorescent probe for tracking the delivery of genes into nuclei. The MPA-capped CdSe AQDs that were completely synthesized in water were the model AQDs. A nominal MPA:Cd:Se = 4:3:1 was chosen for optimal photoluminescence and zeta potential. The gene delivery study was carried out in vitro using a human colon cancer cell line, HT29 (ATCC). The model gene was a plasmid DNA (pDNA) that can express red fluorescent protein (RFP). Positively charged branched PEI was employed to provide a proton buffer to the AQDs to allow for endosomal escape. It is shown that by using a PEI-AQD complex with a PEI/AQD molar ratio of 300 and a nominal pDNA/PEI-AQD ratio of 6, we can achieve 75 ± 2.6% RFP expression efficiency with cell vitality remaining at 78 ± 4% of the control. (paper)

[Impact of siRNA-mediated down-regulation of CD147 on human breast cancer cells].

Science.gov (United States)

Li, Zhenqian; Li, Daoming; Li, Jiangwei; Huang, Pei; Qin, Hui

2015-10-01

To investigate the influence of siRNA-mediated down-regulation of CD147 on growth, proliferation and movement of human breast cancer cell line MDA-MB-231. The protein expression of CD147, MMP-2 and TIMP-2 of the MDA-MB-231 cells were analyzed by ABC. Lentiviral expression vector of CD147 gene was constructed and transfected into MDA-MB-231 cells. RT-PCR and Western blot were used to detect the mRNA and protein level changes of CD147 genes to identify the optimal time point, followed by detection of changes of mRNA and protein expression of MMP-2 and TIMP-2 genes. CCK-8 reagent method and cell scratch test were used to detect the proliferation and migration change of MDA-MB-231 cells. The nude mouse model of breast cancer by hypodermic injection with MDA-MB-231 cells was established to document the effect of CD147 siRNA on the tumor transplants. After transfection of lentiviral expression vector of CD147 gene, protein of CD147, MMP-2 and TIMP-2 were weakly or negative expressed, significantly weaker than those of control group (P CD147 and MMP-2 were 96.03% ± 0.84% and 96.03% ± 0.84%, respectively. Both CD147 mRNA and MMP-2 mRNA expression were down-regulated (P 0.05). No less than 2 days after transfection, cell growth of MDA-MB-231 cell line was found significantly inhibited (P CD147 led to reduction of volume and mass of nude mouses. The growth of the carcinoma transplant was inhibited upon siRNA-mediated down-regulation of CD147 (P CD147 may alter the MMP-2/TIMP-2 balance in MDA-MB-231 cells. CD147 gene silencing inhibits the proliferation and migration of MDA-MB-231 cells and the growth of carcinoma transplants in nude mice.

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells.

Directory of Open Access Journals (Sweden)

Vincent O'Reilly

Full Text Available CD1d-restricted invariant natural killer T (iNKT cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+, CD8α(+ and CD4(-CD8α(- double-negative (DN subsets. CD4(+ iNKT cells expanded more readily than CD8α(+ and DN iNKT cells upon mitogen stimulation. CD8α(+ and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+ cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α and Th2 (IL-4, IL-5 and IL-13 cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+ and CD8α(+ fractions, respectively. Only CD4(+ iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+, DN or CD4(+ iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.

Restoration of human B-cell differentiation into NOD-SCID mice engrafted with gene-corrected CD34+ cells isolated from Artemis or RAG1-deficient patients.

Science.gov (United States)

Lagresle-Peyrou, Chantal; Benjelloun, Fatine; Hue, Christophe; Andre-Schmutz, Isabelle; Bonhomme, Delphine; Forveille, Monique; Beldjord, Kheira; Hacein-Bey-Abina, Salima; De Villartay, Jean-Pierre; Charneau, Pierre; Durandy, Anne; Fischer, Alain; Cavazzana-Calvo, Marina

2008-02-01

Severe combined immunodeficiency (SCID) caused by mutation of the recombination-activating gene 1 (RAG1) or Artemis gene lead to the absence of B- and T-cell differentiation. The only curative treatment is allogeneic bone marrow (BM) transplantation, which displays a high survival rate when an HLA compatible donor is available but has a poorer prognosis when the donor is partially compatible. Consequently, gene therapy may be a promising alternative strategy for these diseases. Here, we report that lentiviral gene-corrected BM CD34(+) cells (isolated from Artemis- or RAG1-deficient patients) sustain human B-cell differentiation following injection into non-obese diabetic/SCID (NOD-SCID) mice previously infused with anti-interleukin-2 receptor beta chain monoclonal antibody. In most of the mice BM, engrafted with Artemis-transduced cells, human B-cell differentiation occurred until the mature stage. The B cells were functional as human immunoglobulin M (IgM) was present in the serum. Following injection with RAG1-transduced cells, human engraftment occurred in vivo but B-cell differentiation until the mature stage was less frequent. However, when it occurred, it was always associated with human IgM production. This overall approach represents a useful tool for evaluating gene transfer efficiency in human SCID forms affecting B-cell development (such as Artemis deficiency) and for testing new vectors for improving in vivo RAG1 complementation.

Binding and internalization of NGR-peptide-targeted liposomal doxorubicin (TVT-DOX) in CD13-expressing cells and its antitumor effects.

Science.gov (United States)

Garde, Seema V; Forté, André J; Ge, Michael; Lepekhin, Eugene A; Panchal, Chandra J; Rabbani, Shafaat A; Wu, Jinzi J

2007-11-01

In an effort to develop new agents and molecular targets for the treatment of cancer, aspargine-glycine-arginine (NGR)-targeted liposomal doxorubicin (TVT-DOX) is being studied. The NGR peptide on the surface of liposomal doxorubicin (DOX) targets an aminopeptidase N (CD13) isoform, specific to the tumor neovasculature, making it a promising strategy. To further understand the molecular mechanisms of action, we investigated cell binding, kinetics of internalization as well as cytotoxicity of TVT-DOX in vitro. We demonstrate the specific binding of TVT-DOX to CD13-expressing endothelial [human umbilical vein endothelial cells (HUVEC) and Kaposi sarcoma-derived endothelial cells (SLK)] and tumor (fibrosarcoma, HT-1080) cells in vitro. Following binding, the drug was shown to internalize through the endosomal pathway, eventually leading to the localization of doxorubicin in cell nuclei. TVT-DOX showed selective toxicity toward CD13-expressing HUVEC, sparing the CD13-negative colon-cancer cells, HT-29. Additionally, the nontargeted counterpart of TVT-DOX, Caelyx, was less cytotoxic to the CD13-positive HUVECs demonstrating the advantages of NGR targeting in vitro. The antitumor activity of TVT-DOX was tested in nude mice bearing human prostate-cancer xenografts (PC3). A significant growth inhibition (up to 60%) of PC3 tumors in vivo was observed. Reduction of tumor vasculature following treatment with TVT-DOX was also apparent. We further compared the efficacies of TVT-DOX and free doxorubicin in the DOX-resistant colon-cancer model, HCT-116, and observed the more pronounced antitumor effects of the TVT-DOX formulation over free DOX. The potential utility of TVT-DOX in a variety of vascularized solid tumors is promising.

PAI-1, a target gene of miR-143, regulates invasion and metastasis by upregulating MMP-13 expression of human osteosarcoma.

Science.gov (United States)

Hirahata, Mio; Osaki, Mitsuhiko; Kanda, Yusuke; Sugimoto, Yui; Yoshioka, Yusuke; Kosaka, Nobuyoshi; Takeshita, Fumitaka; Fujiwara, Tomohiro; Kawai, Akira; Ito, Hisao; Ochiya, Takahiro; Okada, Futoshi

2016-05-01

Despite recent improvements in the therapy for osteosarcoma, 30-40% of osteosarcoma patients die of this disease, mainly due to its lung metastasis. We have previously reported that intravenous injection of miR-143 significantly suppresses lung metastasis of human osteosarcoma cells (143B) in a mouse model. In this study, we examined the biological role and mechanism of miR-143 in the metastasis of human osteosarcoma cells. We identified plasminogen activator inhibitor-1 (PAI-1) as a direct target gene of miR-143. To determine the role of PAI-1 in human osteosarcoma cells, siRNA was transfected into 143B cells for knockdown of PAI-1 expression. An in vitro study showed that downregulation of PAI-1 suppressed cell invasion activity, but not proliferation. Moreover, injection of PAI-1 siRNA into a primary lesion in the osteosarcoma mouse model inhibited lung metastasis compared to control siRNA-injected mice, without influencing the proliferative activity of the tumor cells. Subsequent examination using 143B cells revealed that knockdown of PAI-1 expression resulted in downregulation of the expression and secretion of matrix metalloproteinase-13 (MMP-13), which is also a target gene of miR-143 and a proteolytic enzyme that regulates tumor-induced osteolysis. Immunohistochemical analysis using clinical samples showed that higher miR-143 expressing cases showed poor expression of PAI-1 in the primary tumor cells. All such cases belonged to the lung metastasis-negative group. Moreover, the frequency of lung metastasis-positive cases was significantly higher in PAI-1 and MMP-13 double-positive cases than in PAI-1 or MMP-13 single-positive or double-negative cases (P target gene of miR-143, regulates invasion and lung metastasis via enhancement of MMP-13 expression and secretion in human osteosarcoma cells, suggesting that these molecules could be potential therapeutic target genes for preventing lung metastasis in osteosarcoma patients. © 2016 The Authors. Cancer

Gene expression profiling of Gram-negative bacteria-induced inflammation in human whole blood: The role of complement and CD14-mediated innate immune response

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Corinna Lau

2015-09-01

Full Text Available Non-sterile pathogen-induced sepsis and sterile inflammation like in trauma or ischemia–reperfusion injury may both coincide with the life threatening systemic inflammatory response syndrome and multi-organ failure. Consequently, there is an urgent need for specific biomarkers in order to distinguish sepsis from sterile conditions. The overall aim of this study was to uncover putative sepsis biomarkers and biomarker pathways, as well as to test the efficacy of combined inhibition of innate immunity key players complement and Toll-like receptor co-receptor CD14 as a possible therapeutic regimen for sepsis. We performed whole blood gene expression analyses using microarray in order to profile Gram-negative bacteria-induced inflammatory responses in an ex vivo human whole blood model. The experiments were performed in the presence or absence of inhibitors of complement proteins (C3 and CD88 (C5a receptor 1 and CD14, alone or in combination. In addition, we used blood from a C5-deficient donor. Anti-coagulated whole blood was challenged with heat-inactivated Escherichia coli for 2 h, total RNA was isolated and microarray analyses were performed on the Affymetrix GeneChip Gene 1.0 ST Array platform. The initial experiments were performed in duplicates using blood from two healthy donors. C5-deficiency is very rare, and only one donor could be recruited. In order to increase statistical power, a technical replicate of the C5-deficient samples was run. Subsequently, log2-transformed intensities were processed by robust multichip analysis and filtered using a threshold of four. In total, 73 microarray chips were run and analyzed. The normalized and filtered raw data have been deposited in NCBI's Gene Expression Omnibus (GEO and are accessible with GEO Series accession number GSE55537. Linear models for microarray data were applied to estimate fold changes between data sets and the respective multiple testing adjusted p-values (FDR q-values. The

Gene expression profiling gut microbiota in different races of humans

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Chen, Lei; Zhang, Yu-Hang; Huang, Tao; Cai, Yu-Dong

2016-03-01

The gut microbiome is shaped and modified by the polymorphisms of microorganisms in the intestinal tract. Its composition shows strong individual specificity and may play a crucial role in the human digestive system and metabolism. Several factors can affect the composition of the gut microbiome, such as eating habits, living environment, and antibiotic usage. Thus, various races are characterized by different gut microbiome characteristics. In this present study, we studied the gut microbiomes of three different races, including individuals of Asian, European and American races. The gut microbiome and the expression levels of gut microbiome genes were analyzed in these individuals. Advanced feature selection methods (minimum redundancy maximum relevance and incremental feature selection) and four machine-learning algorithms (random forest, nearest neighbor algorithm, sequential minimal optimization, Dagging) were employed to capture key differentially expressed genes. As a result, sequential minimal optimization was found to yield the best performance using the 454 genes, which could effectively distinguish the gut microbiomes of different races. Our analyses of extracted genes support the widely accepted hypotheses that eating habits, living environments and metabolic levels in different races can influence the characteristics of the gut microbiome.

Estimation of CD4+ and CD8+ T-lymphocytes in human immunodeficiency virus infection and acquired immunodeficiency syndrome patients in Manipur

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Singh H

2007-01-01

Full Text Available Purpose : To estimate and stratify CD4 + and CD8 + T-lymphocyte levels in human immunodeficiency virus (HIV infected (asymptomatic and acquired immunodeficiency syndrome (AIDS patients (symptomatic and correlate the clinical features of the patients with CD4+ and CD8+ lymphocyte level. Methods : Between April 2002 and September 2003, a total of 415 HIV seropositive adult patients (297 males and 118 females attending Regional Institute of Medical Sciences (RIMS hospitals were tested for CD4+ and CD8+ T-lymphocytes by fluorescent activated cell sorter (FACS counter (Becton Dickinson. Symptomatic patients were diagnosed as per NACO clinical case definition. Results : Ranges of 0-50, 51-100, 101-200, 201-300, 301-400, 401-500 and above 500 CD4+ T-lymphocyte per microlitre were seen in 68, 52, 101, 73, 47, 31 and 43 patients respectively whereas CD8+ T-lymphocyte ranges of 0-300, 301-600, 601-900, 901-1500, 1501-2000, 2001-3500 per microlitre were seen in 29, 84, 92, 145, 40 and 25 patients respectively. One hundred and fifty patients were asymptomatic and 265 were symptomatic. CD4/CD8 ratio in asymptomatics and symptomatics were 0.13-1.69 and 0.01-0.93 respectively. Tuberculosis and candidiasis occurred in CD4+ T-lymphocyte categories between 0-400 cells per mL in symptomatics. However, cryptosporidiosis, toxoplasmosis, herpes zoster, cryptococcal meningitis, Pneumocystis carinii pneumonia, penicilliosis and cytomegalovirus retinitis were seen in patients having CD4+ T-lymphocyte less than 200 per mL. Conclusions : CD4+ T-lymphocyte was decreased in both asymptomatic and symptomatic HIV patients, The decrease was greater in symptomatics while CD8+ T-lymphocyte was increased in both except advanced stage symptomatics. CD4:CD8 ratio was reversed in both groups. Opportunistic infections correlated with different CD4+ T-lymphocyte categories.

Effect of cyclophilin A on gene expression in human pancreatic cancer cells.

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Li, Min; Wang, Hao; Li, Fei; Fisher, William E; Chen, Changyi; Yao, Qizhi

2005-11-01

We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.

Therapeutic gene editing in CD34+ hematopoietic progenitors from Fanconi anemia patients.

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Diez, Begoña; Genovese, Pietro; Roman-Rodriguez, Francisco J; Alvarez, Lara; Schiroli, Giulia; Ugalde, Laura; Rodriguez-Perales, Sandra; Sevilla, Julian; Diaz de Heredia, Cristina; Holmes, Michael C; Lombardo, Angelo; Naldini, Luigi; Bueren, Juan Antonio; Rio, Paula

2017-11-01

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 + cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 + cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

The gene expression profile of CD11c+ CD8α- dendritic cells in the pre-diabetic pancreas of the NOD mouse.

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Wouter Beumer

Full Text Available Two major dendritic cell (DC subsets have been described in the pancreas of mice: The CD11c+ CD8α- DCs (strong CD4+ T cell proliferation inducers and the CD8α+ CD103+ DCs (T cell apoptosis inducers. Here we analyzed the larger subset of CD11c+ CD8α- DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR to elucidate abnormalities in underlying gene expression networks. CD11c+ CD8α- DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c+ CD8α- DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24 was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c+ CD8α- DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c+ CD8α- DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.

Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: Differing impact on CD8 T cell phenotype and responsiveness to restimulation

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Kurlander Roger J

2010-10-01

Full Text Available Abstract Background The ability to expand virus- or tumor-specific T cells without damaging their functional capabilities is critical for success adoptive transfer immunotherapy of patients with opportunistic infection or tumor. Careful comparisons can help identify expansion methods better suited for particular clinical settings and identify recurrent deficiencies requiring new innovation. Methods We compared the efficacy of magnetic beads coated with anti-CD3 and anti-CD28 (anti-CD3/CD28 beads, and soluble anti-CD3 plus mixed mononuclear cells (designated a rapid expansion protocol or REP in expanding normal human T cells. Results Both anti-CD3/CD28 beads and soluble anti-CD3 promoted extensive expansion. Beads stimulated greater CD4 cell growth (geometric mean of 56- versus 27-fold (p Conclusions Anti-CD3/CD28 beads are highly effective for expanding CD4 cells, but soluble anti-CD3 has significant potential advantages for expanding CD8 T cells, particularly where preservation of phenotypically "young" CD8 cells would be desirable, or where the T cells of interest have been antigen-stimulated in vitro or in vivo in the recent past.

A potent adjuvant effect of a CD1d-binding NKT cell ligand in human immune system mice.

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Li, Xiangming; Huang, Jing; Kaneko, Izumi; Zhang, Min; Iwanaga, Shiroh; Yuda, Masao; Tsuji, Moriya

2017-01-01

A CD1d-binding invariant natural killer T (iNKT)-cell stimulatory glycolipid, namely 7DW8-5, is shown to enhance the efficacy of radiation-attenuated sporozoites (RAS)-based malaria vaccine in mice. In the current study, we aim to determine whether 7DW8-5 can display a potent adjuvant effect in human immune system (HIS) mice. HIS-A2/hCD1d mice, which possess both functional human iNKT cells and CD8+ T cells, were generated by the transduction of NSG mice with adeno-associated virus serotype 9 expressing genes that encode human CD1d molecules and HLA-A*0201, followed by the engraftment of human hematopoietic stem cells. The magnitudes of human iNKT-cell response against 7DW8-5 and HLA-A*0201-restricted human CD8+ T-cell response against a human malaria antigen in HIS-A2/hCD1d mice were determined by using human CD1d tetramer and human HLA-A*0201 tetramer, respectively. We found that 7DW8-5 stimulates human iNKT cells in HIS-A2/hCD1d mice, as well as those derived from HIS-A2/hCD1d mice in vitro. We also found that 7DW8-5 significantly increases the level of a human malarial antigen-specific HLA-A*0201-restricted human CD8+ T-cell response in HIS-A2/hCD1d mice. Our study indicates that 7DW8-5 can display a potent adjuvant effect on RAS vaccine-induced anti-malarial immunity by augmenting malaria-specific human CD8+ T-cell response.

Genome-wide RNA profiling of long-lasting stem cell-like memory CD8 T cells induced by Yellow Fever vaccination in humans

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Silvia A. Fuertes Marraco

2015-09-01

Full Text Available The live-attenuated Yellow Fever (YF vaccine YF-17D induces a broad and polyfunctional CD8 T cell response in humans. Recently, we identified a population of stem cell-like memory CD8 T cells induced by YF-17D that persists at stable frequency for at least 25 years after vaccination. The YF-17D is thus a model system of human CD8 T cell biology that furthermore allows to track and study long-lasting and antigen-specific human memory CD8 T cells. Here, we describe in detail the sample characteristics and preparation of a microarray dataset acquired for genome-wide gene expression profiling of long-lasting YF-specific stem cell-like memory CD8 T cells, compared to the reference CD8 T cell differentiation subsets from total CD8 T cells. We also describe the quality controls, annotations and exploratory analyses of the dataset. The microarray data is available from the Gene Expression Omnibus (GEO public repository with accession number GSE65804.

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

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Yamada, Takeshi; Abei, Masato; Danjoh, Inaho; Shirota, Ryoko; Yamashita, Taro; Hyodo, Ichinosuke; Nakamura, Yukio

2015-04-11

Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC. Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS. Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, whereas CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(-) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7

Cdh13 and AdipoQ gene knockout alter instrumental and Pavlovian drug conditioning.

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King, C P; Militello, L; Hart, A; St Pierre, C L; Leung, E; Versaggi, C L; Roberson, N; Catlin, J; Palmer, A A; Richards, J B; Meyer, P J

2017-09-01

Genome-wide association studies in humans have suggested that variants of the cadherin-13 (CDH13) gene are associated with substance use disorder, subjective response to amphetamine, and attention deficit hyperactivity disorder. To examine the role of the Cdh13 and its peptide ligand adiponectin (AdipoQ) in addiction-related behaviors, we assessed Cdh13 knockout (KO) rats and AdipoQ KO mice using intravenous cocaine self-administration and conditioned place preference (CPP) paradigms. During intravenous cocaine self-administration, male Cdh13 heterozygous (+/-) and KO (-/-) rats showed increased cue-induced reinstatement compared with wild-type (WT) rats when presented with a cocaine-paired stimulus, whereas female Cdh13 rats showed no differences across genotype. Cdh13 -/- rats showed higher responding for a saccharin reinforcer and learned the choice reaction time (RT) task more slowly than WTs. However, we found no differences between Cdh13 -/- and +/+ rats in responding for sensory reinforcement, number of premature responses in the RT task, tendency to approach a Pavlovian food cue, CPP and locomotor activation to cocaine (10 or 20 mg/kg). In AdipoQ -/- mice, there was a significant increase in CPP to methamphetamine (1 mg/kg) but not to a range of d-amphetamine doses (0.5, 1, 2 and 4 mg/kg). Taken together, these data suggest that Cdh13 and AdipoQ regulate sensitivity to psychomotor stimulants and palatable rewards without producing major changes in other behaviors. In humans, these two genes may regulate sensitivity to natural and drug rewards, thus influencing susceptibility to the conditioned drug effects and relapse. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Investigation of CD28 gene polymorphisms in patients with sporadic breast cancer in a Chinese Han population in Northeast China.

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Shuang Chen

Full Text Available BACKGROUND: CD28 is one of a number of costimulatory molecules that play crucial roles in immune regulation and homeostasis. Accumulating evidence indicates that immune factors influence breast carcinogenesis. To clarify the relationships between polymorphisms in the CD28 gene and breast carcinogenesis, a case-control study was conducted in women from Heilongjiang Province in northeast of China. METHODOLOGY/PRINCIPAL FINDINGS: Our research subjects consisted of 565 female patients with sporadic breast cancer and 605 age- and sex-matched healthy controls. In total, 12 single nucleotide polymorphisms (SNPs in the CD28 gene were successfully determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP method. The relationship between the CD28 variants and clinical features, including histological grade, tumor size, lymph node metastasis, human epidermal growth factor receptor 2 (C-erbB2, estrogen receptor (ER, progesterone receptor (PR, and tumor protein 53 (P53 status were analyzed. A statistically significant association was observed between rs3116496 and breast cancer risk under different genetic models (additive P = 0.0164, dominant P = 0.0042. Different distributions of the rs3116496 'T' allele were found in patients and controls, which remained significant after correcting the P value for multiple testing using Haploview with 10,000 permutations (corrected P = 0.0384. In addition, significant associations were observed between rs3116487/rs3116494 (D' = 1, r(2 = 0.99 and clinicopathological features such as C-erbB2 and ER status, in breast cancer patients. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that CD28 gene polymorphisms contribute to sporadic breast cancer risk and have a significant association with clinicopathological features in a northeast Chinese Han population.

Differences in the epigenetic regulation of MT-3 gene expression between parental and Cd+2 or As+3 transformed human urothelial cells

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Ajjimaporn Amornpan

2011-02-01

Full Text Available Abstract Background Studies have shown that metallothionein 3 (MT-3 is not expressed in normal urothelium or in the UROtsa cell line, but is expressed in urothelial cancer and in tumors generated from the UROtsa cells that have been transformed by cadmium (Cd+2 or arsenite (As+3.The present study had two major goals. One, to determine if epigenetic modifications control urothelial MT-3 gene expression and if regulation is altered by malignant transformation by Cd+2 or As+3. Two, to determine if MT-3 expression might translate clinically as a biomarker for malignant urothelial cells released into the urine. Results The histone deacetylase inhibitor MS-275 induced MT-3 mRNA expression in both parental UROtsa cells and their transformed counterparts. The demethylating agent, 5-Aza-2'-deoxycytidine (5-AZC had no effect on MT-3 mRNA expression. ChIP analysis showed that metal-responsive transformation factor-1 (MTF-1 binding to metal response elements (MRE elements of the MT-3 promoter was restricted in parental UROtsa cells, but MTF-1 binding to the MREs was unrestricted in the transformed cell lines. Histone modifications at acetyl H4, trimethyl H3K4, trimethyl H3K27, and trimethyl H3K9 were compared between the parental and transformed cell lines in the presence and absence of MS-275. The pattern of histone modifications suggested that the MT-3 promoter in the Cd+2 and As+3 transformed cells has gained bivalent chromatin structure, having elements of being "transcriptionally repressed" and "transcription ready", when compared to parental cells. An analysis of MT-3 staining in urinary cytologies showed that a subset of both active and non-active patients with urothelial cancer shed positive cells in their urine, but that control patients only rarely shed MT-3 positive cells. Conclusion The MT-3 gene is silenced in non-transformed urothelial cells by a mechanism involving histone modification of the MT-3 promoter. In contrast, transformation of the

MicroRNA-143 Downregulates Interleukin-13 Receptor Alpha1 in Human Mast Cells

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Jianqiu Cheng

2013-08-01

Full Text Available MicroRNA-143 (miR-143 was found to be downregulated in allergic rhinitis, and bioinformatics analysis predicted that IL-13Rα1 was a target gene of miR-143. To understand the molecular mechanisms of miR-143 involved in the pathogenesis of allergic inflammation, recombinant miR-143 plasmid vectors were constructed, and human mast cell-1(HMC-1 cells which play a central role in the allergic response were used for study. The plasmids were transfected into HMC-1 cells using a lentiviral vector. Expression of IL-13Rα1 mRNA was then detected by reverse transcriptase polymerase chain reaction (RT-PCR and Western Blotting. The miR-143 lentiviral vector was successfully stably transfected in HMC-1 cells for target gene expression. Compared to the control, the target gene IL-13Rα1 was less expressed in HMC-1 transfected with miR-143 as determined by RT-PCR and Western Blotting (p < 0.05; this difference in expression was statistically significant and the inhibition efficiency was 71%. It indicates that miR-143 directly targets IL-13Rα1 and suppresses IL-13Rα1 expression in HMC-1 cells. Therefore, miR-143 may be associated with allergic reaction in human mast cells.

Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage

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Järås, Marcus; Johnels, Petra; Agerstam, Helena

2009-01-01

OBJECTIVE: The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. MATERIALS AND METHODS: We investigated...... and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. RESULTS: Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells...... and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under...

Quantitative gene expression profiling of CD45+ and CD45- skeletal muscle-derived side population cells

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Ditte Caroline Andersen, Ditte Caroline; Kristiansen, Gitte Qvist; Jensen, Line

2012-01-01

The skeletal muscle-derived side population (mSP) which highly excludes Hoechst 33342 is composed of CD45(+) and CD45(-) subpopulations; yet, rareness of mSP cells in general has complicated extensive quantitative analysis of gene expression profiles in primarily isolated mSP cells. Here, we desc...... a satellite cell subpopulation) remain in the mSPCD45(-) fraction, and we show that these cells express high levels of many of the known myogenic precursor/stem cell related markers, including Pax7 and Myf5.......The skeletal muscle-derived side population (mSP) which highly excludes Hoechst 33342 is composed of CD45(+) and CD45(-) subpopulations; yet, rareness of mSP cells in general has complicated extensive quantitative analysis of gene expression profiles in primarily isolated mSP cells. Here, we...... describe the isolation of adult mouse normal skeletal muscle residing SPCD45(+) and SPCD45(-) cells from a parent mononuclear muscle-derived cell (MDC) population. Relative quantitative real time PCR (RT-PCR) of 64 genes revealed that mSPCD45(-) compared with mSPCD45(+) was enriched for cells expressing...

Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

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Dahlin, Joakim S; Malinovschi, Andrei; Öhrvik, Helena; Sandelin, Martin; Janson, Christer; Alving, Kjell; Hallgren, Jenny

2016-01-28

Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function. © 2016 by The American Society of Hematology.

EBV promotes human CD8 NKT cell development.

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Yuling He

2010-05-01

Full Text Available The reports on the origin of human CD8(+ Valpha24(+ T-cell receptor (TCR natural killer T (NKT cells are controversial. The underlying mechanism that controls human CD4 versus CD8 NKT cell development is not well-characterized. In the present study, we have studied total 177 eligible patients and subjects including 128 healthy latent Epstein-Barr-virus(EBV-infected subjects, 17 newly-onset acute infectious mononucleosis patients, 16 newly-diagnosed EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. We have established human-thymus/liver-SCID chimera, reaggregated thymic organ culture, and fetal thymic organ culture. We here show that the average frequency of total and CD8(+ NKT cells in PBMCs from 128 healthy latent EBV-infected subjects is significantly higher than in 17 acute EBV infectious mononucleosis patients, 16 EBV-associated Hodgkin lymphoma patients, and 16 EBV-negative normal control subjects. However, the frequency of total and CD8(+ NKT cells is remarkably increased in the acute EBV infectious mononucleosis patients at year 1 post-onset. EBV-challenge promotes CD8(+ NKT cell development in the thymus of human-thymus/liver-SCID chimeras. The frequency of total (3% of thymic cells and CD8(+ NKT cells ( approximately 25% of NKT cells is significantly increased in EBV-challenged chimeras, compared to those in the unchallenged chimeras (<0.01% of thymic cells, CD8(+ NKT cells undetectable, respectively. The EBV-induced increase in thymic NKT cells is also reflected in the periphery, where there is an increase in total and CD8(+ NKT cells in liver and peripheral blood in EBV-challenged chimeras. EBV-induced thymic CD8(+ NKT cells display an activated memory phenotype (CD69(+CD45RO(hiCD161(+CD62L(lo. After EBV-challenge, a proportion of NKT precursors diverges from DP thymocytes, develops and differentiates into mature CD8(+ NKT cells in thymus in EBV-challenged human-thymus/liver-SCID chimeras or

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

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Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

Functional dichotomy between NKG2D and CD28-mediated co-stimulation in human CD8+ T cells.

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Kamalakannan Rajasekaran

2010-09-01

Full Text Available Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8+ T cells. However, their independent functional contributions in distinct CD8+ T cell subsets are not well understood. In this study, CD8+ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA+CD27+ and memory (CD45RA-CD27+ CD8+ T cells (CD28Hi, while its expression was significantly lower in effector (CD45RA+CD27- CD8+ T cells (CD28Lo. Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8+ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8+ T cells. Co-stimulation of CD28Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8+ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8+ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8+ T cells. However, boosting a recall immune response via memory CD8+ T cells or vaccination to stimulate naïve CD8+ T cells would require CD28-mediated co-stimulation.

Oxytocin Pathway Genes: Evolutionary Ancient System Impacting on Human Affiliation, Sociality, and Psychopathology.

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Feldman, Ruth; Monakhov, Mikhail; Pratt, Maayan; Ebstein, Richard P

2016-02-01

Oxytocin (OT), a nonapeptide signaling molecule originating from an ancestral peptide, appears in different variants across all vertebrate and several invertebrate species. Throughout animal evolution, neuropeptidergic signaling has been adapted by organisms for regulating response to rapidly changing environments. The family of OT-like molecules affects both peripheral tissues implicated in reproduction, homeostasis, and energy balance, as well as neuromodulation of social behavior, stress regulation, and associative learning in species ranging from nematodes to humans. After describing the OT-signaling pathway, we review research on the three genes most extensively studied in humans: the OT receptor (OXTR), the structural gene for OT (OXT/neurophysin-I), and CD38. Consistent with the notion that sociality should be studied from the perspective of social life at the species level, we address human social functions in relation to OT-pathway genes, including parenting, empathy, and using social relationships to manage stress. We then describe associations between OT-pathway genes with psychopathologies involving social dysfunctions such as autism, depression, or schizophrenia. Human research particularly underscored the involvement of two OXTR single nucleotide polymorphisms (rs53576, rs2254298) with fewer studies focusing on other OXTR (rs7632287, rs1042778, rs2268494, rs2268490), OXT (rs2740210, rs4813627, rs4813625), and CD38 (rs3796863, rs6449197) single nucleotide polymorphisms. Overall, studies provide evidence for the involvement of OT-pathway genes in human social functions but also suggest that factors such as gender, culture, and early environment often confound attempts to replicate first findings. We conclude by discussing epigenetics, conceptual implications within an evolutionary perspective, and future directions, especially the need to refine phenotypes, carefully characterize early environments, and integrate observations of social behavior across

Human Endometrial CD98 Is Essential for Blastocyst Adhesion

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Domínguez, Francisco; Simón, Carlos; Quiñonero, Alicia; Ramírez, Miguel Ángel; González-Muñoz, Elena; Burghardt, Hans; Cervero, Ana; Martínez, Sebastián; Pellicer, Antonio; Palacín, Manuel; Sánchez-Madrid, Francisco; Yáñez-Mó, María

2010-01-01

Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window. PMID:20976164

Bone marrow-derived CD13+ cells sustain tumor progression: A potential non-malignant target for anticancer therapy.

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Dondossola, Eleonora; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2014-01-01

Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b + CD13 + myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b + CD13 + myeloid cells could become a non-malignant target for the development of novel anticancer regimens.

Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma

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Lu Lizhi

2006-12-01

Full Text Available Abstract Background Recently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown. Results In this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol and etoposide (VP16 compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors. Conclusion Our study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in

The study with 13N-NH3 PET and coronary angiography to investigate the effect of CD151 gene therapy on swines with experimental myocardial infarction

International Nuclear Information System (INIS)

Zuo Houjuan; Liu Zhengxiang; Liu Xiaochun; Ceng Hesong; Liu Tao; Wen Sha; Chen Jin; Wang Daowen

2009-01-01

Objective: Our previous studies showed that CD151 could promote neovascularization in a rat hind-limb ischemia model and in a rat myocardial ischemia model. This study was to determine the change of myocardial perfusion and coronary collateralization after intramyocardial administration CD151 in swines with experimental myocardial infarction. Methods: CD151 and antiCD151 were constructed into the recombinant adeno-associated virus vector (rAAV). Twenty swines received coronary artery ligation and intramuscular injection of rAAV-CD151 or rAAV-green fluorescent protein (GFP). Eight weeks after vector administration, the expression of CD151 protein and the capillary density were measured using immunohistochemistry. Regional myocardial perfusian was evaluated by 13 N-NH 3 PET. Coronary angiography was per-formed to assess collateral vessels reconstruction. The t-test or ANOVA with SPSS 11.0 was used for data analysis. Results: High levels of CD151 protein expression and capillary density were detected in the rAAV-CD151 group. 13 N-NH 3 PET imaging showed that myocardial perfusion was improved and the myocardial ischemia scores were significantly decreased in the rAAV-CD151 group when compared with rAAV-GFP group (10.82 ± 2.36 vs 19.33 ± 1.67, t=5.86, P=0.002).Coronary angiography confirmed better collateral circulation in the rAAV-CD151 group. Conclusions: rAAV-CD151 direct injection can transfect the myocardium and express the CD151 protein, thereby significantly improve the myocardial blood perfusion and coronary collateralization. 13 N-NH 3 PET and coronary angiography can be used directly to evaluate the col-lateral vessel reconstruction and perfusion status of swine myocardium. (authors)

Differences in gene expression profiles and signaling pathways in rhabdomyolysis-induced acute kidney injury.

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Geng, Xiaodong; Wang, Yuanda; Hong, Quan; Yang, Jurong; Zheng, Wei; Zhang, Gang; Cai, Guangyan; Chen, Xiangmei; Wu, Di

2015-01-01

Rhabdomyolysis is a threatening syndrome because it causes the breakdown of skeletal muscle. Muscle destruction leads to the release of myoglobin, intracellular proteins, and electrolytes into the circulation. The aim of this study was to investigate the differences in gene expression profiles and signaling pathways upon rhabdomyolysis-induced acute kidney injury (AKI). In this study, we used glycerol-induced renal injury as a model of rhabdomyolysis-induced AKI. We analyzed data and relevant information from the Gene Expression Omnibus database (No: GSE44925). The gene expression data for three untreated mice were compared to data for five mice with rhabdomyolysis-induced AKI. The expression profiling of the three untreated mice and the five rhabdomyolysis-induced AKI mice was performed using microarray analysis. We examined the levels of Cyp3a13, Rela, Aldh7a1, Jun, CD14. And Cdkn1a using RT-PCR to determine the accuracy of the microarray results. The microarray analysis showed that there were 1050 downregulated and 659 upregulated genes in the rhabdomyolysis-induced AKI mice compared to the control group. The interactions of all differentially expressed genes in the Signal-Net were analyzed. Cyp3a13 and Rela had the most interactions with other genes. The data showed that Rela and Aldh7a1 were the key nodes and had important positions in the Signal-Net. The genes Jun, CD14, and Cdkn1a were also significantly upregulated. The pathway analysis classified the differentially expressed genes into 71 downregulated and 48 upregulated pathways including the PI3K/Akt, MAPK, and NF-κB signaling pathways. The results of this study indicate that the NF-κB, MAPK, PI3K/Akt, and apoptotic pathways are regulated in rhabdomyolysis-induced AKI.

Gene expression profiling identifies HOXB4 as a direct downstream target of GATA-2 in human CD34+ hematopoietic cells.

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Tohru Fujiwara

Full Text Available Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10 and idiopathic thrombocytopenic purpura (n = 13, and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01. Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.

Regional differences in gene expression and promoter usage in aged human brains

KAUST Repository

Pardo, Luba M.

2013-02-19

To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites and to quantify the differences in gene expression across the 5 brain regions. We also analyzed the extent to which methylation influenced the observed expression profiles. We sequenced more than 71 million cap analysis of gene expression tags corresponding to 70,202 promoter regions and 16,888 genes. More than 7000 transcripts were differentially expressed, mainly because of differential alternative promoter usage. Unexpectedly, 7% of differentially expressed genes were neurodevelopmental transcription factors. Functional pathway analysis on the differentially expressed genes revealed an overrepresentation of several signaling pathways (e.g., fibroblast growth factor and wnt signaling) in hippocampus and striatum. We also found that although 73% of methylation signals mapped within genes, the influence of methylation on the expression profile was small. Our study underscores alternative promoter usage as an important mechanism for determining the regional differences in gene expression at old age.

DP IV/CD26, APN/CD13 and related enzymes as regulators of T cell immunity: implications for experimental encephalomyelitis and multiple sclerosis.

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Reinhold, Dirk; Bank, Ute; Täger, Michael; Ansorge, Siegfried; Wrenger, Sabine; Thielitz, Anja; Lendeckel, Uwe; Faust, Jürgen; Neubert, Klaus; Brocke, Stefan

2008-01-01

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system. Peptidases like dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) play a regulatory role in T cell activation and represent potential targets for the treatment of inflammatory disorders. Synthetic inhibitors of DP IV and/or APN enzymatic activity induce production of the immunosuppressive cytokine TGF-beta1 and subsequently suppress DNA synthesis and Th1 cytokine production of activated human T cells. Compelling evidence has demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to the pathogenesis of autoimmune inflammation. Here, we report that inhibitors of DP IV-like activity as well as of APN activity inhibit IL-17 production in activated human and mouse T cells. Combining inhibitors of DP IV and APN increases the suppressive effect on T cell specific IL-17 production in vitro compared to a single peptidase inhibitor. In the following, we summarize the evidence for the role of both ectoenzymes in T cell activation in vitro and in vivo and provide a rationale for the use of combined or dual ectopeptidase inhibitors to treat autoimmune diseases like MS.

Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of 'soluble CD163' in plasma

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Etzerodt, Anders; Berg, Ronan M.G.; Plovsing, Ronni R.

2017-01-01

CD163 is the macrophage receptor for uptake of hemoglobin-haptoglobin complexes. The human receptor can be shed from the macrophage surface owing to a cleavage site for the inflammation-inducible TACE/ADAM17 enzyme. Accordingly, plasma â €soluble CD163' (sCD163) has become a biomarker for macroph......CD163 is the macrophage receptor for uptake of hemoglobin-haptoglobin complexes. The human receptor can be shed from the macrophage surface owing to a cleavage site for the inflammation-inducible TACE/ADAM17 enzyme. Accordingly, plasma â €soluble CD163' (sCD163) has become a biomarker...

Human CD56bright NK Cells

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Michel, Tatiana; Poli, Aurélie; Cuapio, Angelica

2016-01-01

Human NK cells can be subdivided into various subsets based on the relative expression of CD16 and CD56. In particular, CD56(bright)CD16(-/dim) NK cells are the focus of interest. They are considered efficient cytokine producers endowed with immunoregulatory properties, but they can also become c...... NK cell subsets is not fully defined, nor is their precise hematopoietic origin. In this article, we summarize recent studies about CD56(bright) NK cells in health and disease and briefly discuss the current controversies surrounding them....

Virus-induced dysfunction of CD4+CD25+ T cells in patients with HTLV-I-associated neuroimmunological disease.

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Yamano, Yoshihisa; Takenouchi, Norihiro; Li, Hong-Chuan; Tomaru, Utano; Yao, Karen; Grant, Christian W; Maric, Dragan A; Jacobson, Steven

2005-05-01

CD4(+)CD25(+) Tregs are important in the maintenance of immunological self tolerance and in the prevention of autoimmune diseases. As the CD4(+)CD25(+) T cell population in patients with human T cell lymphotropic virus type I-associated (HTLV-I-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) has been shown to be a major reservoir for this virus, it was of interest to determine whether the frequency and function of CD4(+)CD25(+) Tregs in HAM/TSP patients might be affected. In these cells, both mRNA and protein expression of the forkhead transcription factor Foxp3, a specific marker of Tregs, were lower than those in CD4(+)CD25(+) T cells from healthy individuals. The virus-encoded transactivating HTLV-I tax gene was demonstrated to have a direct inhibitory effect on Foxp3 expression and function of CD4(+)CD25(+) T cells. This is the first report to our knowledge demonstrating the role of a specific viral gene product (HTLV-I Tax) on the expression of genes associated with Tregs (in particular, foxp3) resulting in inhibition of Treg function. These results suggest that direct human retroviral infection of CD4(+)CD25(+) T cells may be associated with the pathogenesis of HTLV-I-associated neurologic disease.

ASSOCIATION OF POLYMORPHISM OF C159T GENE OF RECEPTOR CD14 AND ANTIENDOTOXIN IMMUNITY IN PATIENTS WITH REFRACTORY / CORTICOSTEROIDSSENSITIVE ASTHMA

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Bisyuk Yu.A.

2015-05-01

Full Text Available Introduction. The refractory asthma refers to the phenotype, which is characterized by severe persistent course with frequent exacerbations and resistance to corticosteroid therapy. This phenotype of asthma can be related to C159T polymorphism of CD14 receptor gene Material and methods. There were studied the C159T polymorphism of CD14 gene in 331 patients with bronchial asthma. The control group consisted of 285 healthy individuals of Crimea. The C159T gene polymorphism of CD14 was detected by allele-specific polymerase chain reaction with electrophoretic detection. The distribution of genotypes was checked according to the law of the Hardy-Weinberg equilibrium by using Fisher's exact test and χ2. There was used logistic regression to determine the difference in the frequency of genotypes and alleles. Results and discussion. In our study have been identified 291 patients with corticosteroid-sensitive and 40 with refractory asthma. The genotype distribution of control (CC – 34%, CT – 51%, CT – 15% and patients with corticosteroid-sensitive asthma (CC – 29%, CT – 53%, CT – 18% were in accordance with the law of Hardy- Weinberg equilibrium and did not significantly differ (χ2 = 2.204, P = 0.332. There were no significant differences when comparing the allele and genotype frequencies by using risk allele T and C model. In the control group the frequency distribution of genotypes CC – 34%, CT – 51%, TT – 15% did not differ significantly (χ2 = 3.540, P = 0.170 from refractory asthma (CC – 52%, CT – 35%, CT – 13%. The risk analysis for the T allele showed that the frequency of CT+TT genotype in patients with refractory asthma (49% was significantly lower (OR = 0.467, CI = [0.240-0.910], χ2 = 5.17, p = 0.023 compere to control (66 %. In turn, the difference of allelic frequencies for the control and patients with persistent asthma did not differ significantly (p = 0.076. The content of antiendotoxin antibody of class A in

Genome-wide expression profiling analysis to identify key genes in the anti-HIV mechanism of CD4+ and CD8+ T cells.

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Gao, Lijie; Wang, Yunqi; Li, Yi; Dong, Ya; Yang, Aimin; Zhang, Jie; Li, Fengying; Zhang, Rongqiang

2018-07-01

Comprehensive bioinformatics analyses were performed to explore the key biomarkers in response to HIV infection of CD4 + and CD8 + T cells. The numbers of CD4 + and CD8 + T cells of HIV infected individuals were analyzed and the GEO database (GSE6740) was screened for differentially expressed genes (DEGs) in HIV infected CD4 + and CD8 + T cells. Gene Ontology enrichment, KEGG pathway analyses, and protein-protein interaction (PPI) network were performed to identify the key pathway and core proteins in anti-HIV virus process of CD4 + and CD8 + T cells. Finally, we analyzed the expressions of key proteins in HIV-infected T cells (GSE6740 dataset) and peripheral blood mononuclear cells(PBMCs) (GSE511 dataset). 1) CD4 + T cells counts and ratio of CD4 + /CD8 + T cells decreased while CD8 + T cells counts increased in HIV positive individuals; 2) 517 DEGs were found in HIV infected CD4 + and CD8 + T cells at acute and chronic stage with the criterial of P-value T cells. The main biological processes of the DEGs were response to virus and defense response to virus. At chronic stage, ISG15 protein, in conjunction with IFN-1 pathway might play key roles in anti-HIV responses of CD4 + T cells; and 4) The expression of ISG15 increased in both T cells and PBMCs after HIV infection. Gene expression profile of CD4 + and CD8 + T cells changed significantly in HIV infection, in which ISG15 gene may play a central role in activating the natural antiviral process of immune cells. © 2018 Wiley Periodicals, Inc.

CD200-expressing human basal cell carcinoma cells initiate tumor growth.

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Colmont, Chantal S; Benketah, Antisar; Reed, Simon H; Hawk, Nga V; Telford, William G; Ohyama, Manabu; Udey, Mark C; Yee, Carole L; Vogel, Jonathan C; Patel, Girish K

2013-01-22

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

Association of CD40 gene polymorphisms with sporadic breast cancer in Chinese Han women of Northeast China.

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Chen Shuang

Full Text Available BACKGROUND: Breast cancer is a polygenetic disorder with a complex inheritance pattern. Single nucleotide polymorphisms (SNPs, the most common genetic variations, influence not only phenotypic traits, but also interindividual predisposition to disease, treatment outcomes with drugs and disease prognosis. The co-stimulatory molecule CD40 plays a prominent role in immune regulation and homeostasis. Accumulating evidence suggests that CD40 contributes to the pathogenesis of cancer. Here, we set out to test the association between polymorphisms in the CD40 gene and breast carcinogenesis and tumor pathology. METHODOLOGY AND PRINCIPAL FINDINGS: Four SNPs (rs1800686, rs1883832, rs4810485 and rs3765459 were genotyped by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP method in a case-control study including 591 breast cancer patients and 600 age-matched healthy controls. Differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed by the Chi-square test for trends. Our preliminary data showed a statistically significant association between the four CD40 gene SNPs and sporadic breast cancer risk (additive P = 0.0223, 0.0012, 0.0013 and 0.0279, respectively. A strong association was also found using the dominant, recessive and homozygote comparison genetic models. In the clinical features analysis, significant associations were observed between CD40 SNPs and lymph node metastasis, human epidermal growth factor receptor 2 (C-erbB2, estrogen receptor (ER, progesterone receptor (PR and tumor protein 53 (P53 statuses. In addition, our haplotype analysis indicated that the haplotype C(rs1883832G(rs4810485, which was located within the only linkage disequilibrium (LD block identified, was a protective haplotype for breast cancer, whereas T(rs1883832T(rs4810485 increased the risk in the studied population, even after correcting the P value for multiple testing (P = 0.0337 and

The CD157-integrin partnership controls transendothelial migration and adhesion of human monocytes.

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Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

2011-05-27

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β(1) and β(2) integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

Gain, loss and divergence in primate zinc-finger genes: a rich resource for evolution of gene regulatory differences between species.

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Katja Nowick

Full Text Available The molecular changes underlying major phenotypic differences between humans and other primates are not well understood, but alterations in gene regulation are likely to play a major role. Here we performed a thorough evolutionary analysis of the largest family of primate transcription factors, the Krüppel-type zinc finger (KZNF gene family. We identified and curated gene and pseudogene models for KZNFs in three primate species, chimpanzee, orangutan and rhesus macaque, to allow for a comparison with the curated set of human KZNFs. We show that the recent evolutionary history of primate KZNFs has been complex, including many lineage-specific duplications and deletions. We found 213 species-specific KZNFs, among them 7 human-specific and 23 chimpanzee-specific genes. Two human-specific genes were validated experimentally. Ten genes have been lost in humans and 13 in chimpanzees, either through deletion or pseudogenization. We also identified 30 KZNF orthologs with human-specific and 42 with chimpanzee-specific sequence changes that are predicted to affect DNA binding properties of the proteins. Eleven of these genes show signatures of accelerated evolution, suggesting positive selection between humans and chimpanzees. During primate evolution the most extensive re-shaping of the KZNF repertoire, including most gene additions, pseudogenizations, and structural changes occurred within the subfamily homininae. Using zinc finger (ZNF binding predictions, we suggest potential impact these changes have had on human gene regulatory networks. The large species differences in this family of TFs stands in stark contrast to the overall high conservation of primate genomes and potentially represents a potent driver of primate evolution.

Fine Mapping and Functional Analysis of the Multiple Sclerosis Risk Gene CD6

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Swaminathan, Bhairavi; Cuapio, Angélica; Alloza, Iraide; Matesanz, Fuencisla; Alcina, Antonio; García-Barcina, Maria; Fedetz, Maria; Fernández, Óscar; Lucas, Miguel; Órpez, Teresa; Pinto-Medel, Mª Jesus; Otaegui, David; Olascoaga, Javier; Urcelay, Elena; Ortiz, Miguel A.; Arroyo, Rafael; Oksenberg, Jorge R.; Antigüedad, Alfredo; Tolosa, Eva; Vandenbroeck, Koen

2013-01-01

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2nd SRCR domain with susceptibility to MS (P max(T) permutation = 1×10−4). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. – CD4+ naïve cells, P = 0.0001; CD8+ naïve cells, P<0.0001; CD4+ and CD8+ central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4+ and CD8+ T cells. PMID:23638056

Functional and phenotypical analysis of IL-6-secreting CD4+ T cells in human adipose tissue.

Science.gov (United States)

de Jong, Anja J; Pollastro, Sabrina; Kwekkeboom, Joanneke C; Andersen, Stefan N; Dorjée, Annemarie L; Bakker, Aleida M; Alzaid, Fawaz; Soprani, Antoine; Nelissen, Rob G H H; Mullers, Jan B; Venteclef, Nicolas; de Vries, Niek; Kloppenburg, Margreet; Toes, René E M; Ioan-Facsinay, Andreea

2018-03-01

Emerging evidence indicates that a dynamic interplay between the immune system and adipocytes contributes to the disturbed homeostasis in adipose tissue of obese subjects. Recently, we observed IL-6-secretion by CD4 + T cells from the stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) of knee osteoarthritis patients directly ex vivo. Here we show that human IL-6 + CD4 + T cells from SVF display a more activated phenotype than the IL-6 - T cells, as evidenced by the expression of the activation marker CD69. Analysis of cytokines secretion, as well as expression of chemokine receptors and transcription factors associated with different Th subsets (Treg, Th1, Th2, Th17 and Tfh) revealed that IL-6-secreting CD4 + T cells cannot be assigned to a conventional Th subset. TCRβ gene analysis revealed that IL-6 + and IL-6 - CD4 + T cells appear clonally unrelated to each other, suggesting a different specificity of these cells. In line with these observations, adipocytes are capable of enhancing IL-6 production by CD4 + T cells. Thus, IL-6 + CD4 + T cells are TCRαβ T cells expressing an activated phenotype potentially resulting from an interplay with adipocytes that could be involved in the inflammatory processes in the OA joint. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human T cell leukemia virus type I prevents cell surface expression of the T cell receptor through down-regulation of the CD3-gamma, -delta, -epsilon, and -zeta genes

NARCIS (Netherlands)

de Waal Malefyt, R.; Yssel, H.; Spits, H.; de Vries, J. E.; Sancho, J.; Terhorst, C.; Alarcon, B.

1990-01-01

Infection and transformation by human T cell leukemia virus type I (HTLV-I) up-regulates expression of several inducible genes including those coding for cytokines involved in the proliferation of normal and leukemic T cells. We demonstrate that HTLV-I can also shut off expression of the CD3-gamma,

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation.

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Hamid, Adila A; Idrus, Ruszymah Bt Hj; Saim, Aminuddin Bin; Sathappan, Somasumdaram; Chua, Kien-Hui

2012-01-01

Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

Directory of Open Access Journals (Sweden)

Adila A Hamid

2012-01-01

Full Text Available OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

Experimental adaptation of wild-type canine distemper virus (CDV to the human entry receptor CD150.

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Maria Bieringer

Full Text Available Canine distemper virus (CDV, a close relative of measles virus (MV, is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2 pfu/ml in Vero cells expressing human CD150 (Vero-hSLAM. After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5 pfu/ml. Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L and Gly to Glu (G71E, and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

Expression of lumican related to CD34 and VEGF in the articular disc of the human temporomandibular joint.

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N. Kiga

2010-07-01

Full Text Available Lumican belongs to the small leucine-rich repeat proteoglycan (SLRP gene family and has been reported to exist in the cornea, intervertebral disc and tendon. Lumican plays a significant role in the assembly and regulation of collagen fibres. The human temporomandibular joint (TMJ disc is made up of fibrocartilage with an extracellular matrix (ECM composed of collagen and proteoglycans. The existence and behaviour of lumican have not been studied in the human TMJ disc. Therefore, we used immunohistochemical methods to detect lumican, CD34 and vascular endothelial growth factor (VEGF and histochemical staining with toluidine blue in 13 human TMJ specimens (10 surgically removed and 3 obtained from autopsy. In both normal and deformed discs we observed staining with toluidine blue. We found that the area of metachromasia inside the deformed disc was uneven and expression of lumican was strong in the areas negative for metachromasia. Staining of VEGF and CD34 inside the deformed disc was seen. We confirmed the expression of lumican in the human TMJ disc and showed that a large number of fibroblastlike cells existed in the area of strong lumican expression. These new findings about the behaviour of lumican suggest that it may play a key role in the generation of a new collagen network by fibroblast-like cells.

Downregulation of CD44 reduces doxorubicin resistance of CD44+CD24- breast cancer cells

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Phuc PV

2011-06-01

Full Text Available Pham Van Phuc, Phan Lu Chinh Nhan, Truong Hai Nhung, Nguyen Thanh Tam, Nguyen Minh Hoang, Vuong Gia Tue, Duong Thanh Thuy, Phan Kim NgocLaboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh, VietnamBackground: Cells within breast cancer stem cell populations have been confirmed to have a CD44+CD24- phenotype. Strong expression of CD44 plays a critical role in numerous types of human cancers. CD44 is involved in cell differentiation, adhesion, and metastasis of cancer cells.Methods: In this study, we reduced CD44 expression in CD44+CD24- breast cancer stem cells and investigated their sensitivity to an antitumor drug. The CD44+CD24- breast cancer stem cells were isolated from breast tumors; CD44 expression was downregulated with siRNAs followed by treatment with different concentrations of the antitumor drug.Results: The proliferation of CD44 downregulated CD44+CD24- breast cancer stem cells was decreased after drug treatment. We noticed treated cells were more sensitive to doxorubicin, even at low doses, compared with the control groups.Conclusions: It would appear that expression of CD44 is integral among the CD44+CD24- cell population. Reducing the expression level of CD44, combined with doxorubicin treatment, yields promising results for eradicating breast cancer stem cells in vitro. This study opens a new direction in treating breast cancer through gene therapy in conjunction with chemotherapy.Keywords: antitumor drugs, breast cancer stem cells, CD44, CD44+CD24- cells, doxorubicin

Presymptomatic Diagnosis of Celiac Disease in Predisposed Children: The Role of Gene Expression Profile.

Science.gov (United States)

Galatola, Martina; Cielo, Donatella; Panico, Camilla; Stellato, Pio; Malamisura, Basilio; Carbone, Lorenzo; Gianfrani, Carmen; Troncone, Riccardo; Greco, Luigi; Auricchio, Renata

2017-09-01

The prevalence of celiac disease (CD) has increased significantly in recent years, and risk prediction and early diagnosis have become imperative especially in at-risk families. In a previous study, we identified individuals with CD based on the expression profile of a set of candidate genes in peripheral blood monocytes. Here we evaluated the expression of a panel of CD candidate genes in peripheral blood mononuclear cells from at-risk infants long time before any symptom or production of antibodies. We analyzed the gene expression of a set of 9 candidate genes, associated with CD, in 22 human leukocyte antigen predisposed children from at-risk families for CD, studied from birth to 6 years of age. Nine of them developed CD (patients) and 13 did not (controls). We analyzed gene expression at 3 different time points (age matched in the 2 groups): 4-19 months before diagnosis, at the time of CD diagnosis, and after at least 1 year of a gluten-free diet. At similar age points, controls were also evaluated. Three genes (KIAA, TAGAP [T-cell Activation GTPase Activating Protein], and SH2B3 [SH2B Adaptor Protein 3]) were overexpressed in patients, compared with controls, at least 9 months before CD diagnosis. At a stepwise discriminant analysis, 4 genes (RGS1 [Regulator of G-protein signaling 1], TAGAP, TNFSF14 [Tumor Necrosis Factor (Ligand) Superfamily member 14], and SH2B3) differentiate patients from controls before serum antibodies production and clinical symptoms. Multivariate equation correctly classified CD from non-CD children in 95.5% of patients. The expression of a small set of candidate genes in peripheral blood mononuclear cells can predict CD at least 9 months before the appearance of any clinical and serological signs of the disease.

Bispecific antibodies targeting human CD73

DEFF Research Database (Denmark)

2017-01-01

The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen.......The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen....

Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults.

Science.gov (United States)

Lissner, Michelle M; Thomas, Brandon J; Wee, Kathleen; Tong, Ann-Jay; Kollmann, Tobias R; Smale, Stephen T

2015-01-01

A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development.

Flow Cytometry Assessment of In Vitro Generated CD138+ Human Plasma Cells

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Rayelle Itoua Maïga

2014-01-01

Full Text Available The in vitro CD40-CD154 interaction promotes human B lymphocytes differentiation into plasma cells. Currently, CD138 is the hallmark marker enabling the detection of human plasma cells, both in vitro and in vivo; its presence can be monitored by flow cytometry using a specific antibody. We have developed a culture system allowing for the differentiation of memory B lymphocytes. In order to detect the newly formed plasma cells, we have compared their staining using five anti-CD138 monoclonal antibodies (mAbs. As a reference, we also tested human cell lines, peripheral blood mononuclear cells, and bone marrow samples. The five anti-CD138 mAbs stained RPMI-8226 cells (>98% with variable stain index (SI. The highest SI was obtained with B-A38 mAb while the lowest SI was obtained with DL-101 and 1D4 mAbs. However, the anti-CD138 mAbs were not showing equivalent CD138+ cells frequencies within the generated plasma cells. B-A38, B-B4, and MI-15 were similar (15–25% while DL-101 mAb stained a higher proportion of CD138-positive cells (38–42%. DL-101 and B-A38 mAbs stained similar populations in bone marrow samples but differed in their capacity to bind to CD138high and CD138lo cell lines. In conclusion, such cellular fluctuations suggest heterogeneity in human plasma cell populations and/or in CD138 molecules.

Selective regain of egfr gene copies in CD44+/CD24-/low breast cancer cellular model MDA-MB-468

International Nuclear Information System (INIS)

Agelopoulos, Konstantin; Buerger, Horst; Brandt, Burkhard; Greve, Burkhard; Schmidt, Hartmut; Pospisil, Heike; Kurtz, Stefan; Bartkowiak, Kai; Andreas, Antje; Wieczorek, Marek; Korsching, Eberhard

2010-01-01

in the CD44 + /CD24 -/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression

CD133+CD24lo defines a 5-Fluorouracil-resistant colon cancer stem cell-like phenotype

Science.gov (United States)

Paschall, Amy V.; Yang, Dafeng; Lu, Chunwan; Redd, Priscilla S.; Choi, Jeong-Hyeon; Heaton, Christopher M.; Lee, Jeffrey R.; Nayak-Kapoor, Asha; Liu, Kebin

2016-01-01

The chemotherapeutic agent 5-Fluorouracil (5-FU) is the most commonly used drug for patients with advanced colon cancer. However, development of resistance to 5-FU is inevitable in almost all patients. The mechanism by which colon cancer develops 5-FU resistance is still unclear. One recently proposed theory is that cancer stem-like cells underlie colon cancer 5-FU resistance, but the phenotypes of 5-FU-resistant colon cancer stem cells are still controversial. We report here that 5-FU treatment selectively enriches a subset of CD133+ colon cancer cells in vitro. 5-FU chemotherapy also increases CD133+ tumor cells in human colon cancer patients. However, sorted CD133+ colon cancer cells exhibit no increased resistance to 5-FU, and CD133 levels exhibit no correlation with colon cancer patient survival or cancer recurrence. Genome-wide analysis of gene expression between sorted CD133+ colon cancer cells and 5-FU-selected colon cancer cells identifies 207 differentially expressed genes. CD24 is one of the genes whose expression level is lower in the CD133+ and 5-FU-resistant colon cancer cells as compared to CD133+ and 5-FU-sensitive colon cancer cells. Consequently, CD133+CD24lo cells exhibit decreased sensitivity to 5-FU. Therefore, we determine that CD133+CD24lo phenotype defines 5-FU-resistant human colon cancer stem cell-like cells. PMID:27659530

TNF-α blockade induces IL-10 expression in human CD4+ T cells

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Evans, Hayley G.; Roostalu, Urmas; Walter, Gina J.; Gullick, Nicola J.; Frederiksen, Klaus S.; Roberts, Ceri A.; Sumner, Jonathan; Baeten, Dominique L.; Gerwien, Jens G.; Cope, Andrew P.; Geissmann, Frederic; Kirkham, Bruce W.; Taams, Leonie S.

2014-02-01

IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

Involvement of the major histocompatibility complex region in the genetic regulation of circulating CD8 T-cell numbers in humans.

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Cruz, E; Vieira, J; Gonçalves, R; Alves, H; Almeida, S; Rodrigues, P; Lacerda, R; Porto, G

2004-07-01

Variability in T-lymphocyte numbers is partially explained by a genetic regulation. From studies in animal models, it is known that the Major Histocompatibility Complex (MHC) is involved in this regulation. In humans, this has not been shown yet. The objective of the present study was to test the hypothesis that genes in the MHC region influence the regulation of T-lymphocyte numbers. Two approaches were used. Association studies between T-cell counts (CD4(+) and CD8(+)) or total lymphocyte counts and HLA class I alleles (A and B) or mutations in the HFE (C282Y and H63D), the hemochromatosis gene, in an unrelated population (n = 264). A second approach was a sibpair correlation analysis of the same T-cell counts in relation to HLA-HFE haplotypes in subjects belonging to 48 hemochromatosis families (n = 456 sibpairs). In the normal population, results showed a strong statistically significant association of the HLA-A*01 with high numbers of CD8(+) T cells and a less powerful association with the HLA-A*24 with low numbers of CD8(+) T cells. Sibpair correlations revealed the most significant correlation for CD8(+) T-cell numbers for sibpairs with HLA-HFE-identical haplotypes. This was not observed for CD4(+) T cells. These results show that the MHC region is involved in the genetic regulation of CD8(+) T-cell numbers in humans. Identification of genes responsible for this control may have important biological and clinical implications.

Assessment of potential soybean cadmium excluder cultivars at different concentrations of Cd in soils.

Science.gov (United States)

Zhi, Yang; He, Kangxin; Sun, Ting; Zhu, Yongqiang; Zhou, Qixing

2015-09-01

The selection of cadmium-excluding cultivars has been used to minimize the transfer of cadmium into the human food chain. In this experiment, five Chinese soybean plants were grown in three soils with different concentrations of Cd (0.15, 0.75 and 1.12mg/kg). Variations in uptake, enrichment, and translocation of Cd among these soybean cultivars were studied. The results indicated that the concentration of Cd in seeds that grew at 1.12mg/kg Cd in soils exceeded the permitted maximum levels in soybeans. Therefore, our results indicated that even some soybean cultivars grown on soils with permitted levels of Cd might accumulate higher concentrations of Cd in seeds that are hazardous to human health. The seeds of these five cultivars were further assessed for interactions between Cd and other mineral nutrient elements such as Ca, Cu, Fe, Mg, Mn and Zn. High Cd concentration in soil was found to inhibit the uptake of Mn. Furthermore, Fe and Zn accumulations were found to be enhanced in the seeds of all of the five soybean cultivars in response to high Cd concentration. Cultivar Tiefeng 31 was found to fit the criteria for a Cd-excluding cultivar under different concentrations of Cd in soils. Copyright © 2015. Published by Elsevier B.V.

Clarithromycin attenuates IL-13–induced periostin production in human lung fibroblasts

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Kosaku Komiya

2017-02-01

Full Text Available Abstract Background Periostin is a biomarker indicating the presence of type 2 inflammation and submucosal fibrosis; serum periostin levels have been associated with asthma severity. Macrolides have immunomodulatory effects and are considered a potential therapy for patients with severe asthma. Therefore, we investigated whether macrolides can also modulate pulmonary periostin production. Methods Using quantitative PCR and ELISA, we measured periostin production in human lung fibroblasts stimulated by interleukin-13 (IL-13 in the presence of two 14-member–ring macrolides—clarithromycin or erythromycin—or a 16-member–ring macrolide, josamycin. Phosphorylation of signal transducers and activators of transcription 6 (STAT6, downstream of IL-13 signaling, was evaluated by Western blotting. Changes in global gene expression profile induced by IL-13 and/or clarithromycin were assessed by DNA microarray analysis. Results Clarithromycin and erythromycin, but not josamycin, inhibited IL-13–stimulated periostin production. The inhibitory effects of clarithromycin were stronger than those of erythromycin. Clarithromycin significantly attenuated STAT6 phosphorylation induced by IL-13. Global gene expression analyses demonstrated that IL-13 increased mRNA expression of 454 genes more than 4-fold, while decreasing its expression in 390 of these genes (85.9%, mainly “extracellular,” “plasma membrane,” or “defense response” genes. On the other hand, clarithromycin suppressed 9.8% of the genes in the absence of IL-13. Clarithromycin primarily attenuated the gene expression of extracellular matrix protein, including periostin, especially after IL-13. Conclusions Clarithromycin suppressed IL-13–induced periostin production in human lung fibroblasts, in part by inhibiting STAT6 phosphorylation. This suggests a novel mechanism of the immunomodulatory effect of clarithromycin in asthmatic airway inflammation and fibrosis.

The role of CD4 and CD8 T cells in human cutaneous leishmaniasis.

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Claudia Ida Brodskyn

2014-09-01

Full Text Available Leishmaniasis, caused by infection with parasites of the Leishmania genus, affects millions of individuals worldwide. This disease displays distinct clinical manifestations ranging from self-healing skin lesions to severe tissue damage. The control of Leishmania infection is dependent on cellular immune mechanisms, and evidence has shown that CD4 and CD8 T lymphocytes play different roles in the outcome of leishmaniasis. Although the presence of CD4 T cells is important for controlling parasite growth, the results in the literature suggest that the inflammatory response elicited by these cells could contribute to the pathogenesis of lesions. However, recent studies on CD8 T lymphocytes show that these cells are mainly involved in tissue damage through cytotoxic mechanisms. In this review, we focus on the recent advances in the study of the human adaptive immunological response in the pathogenesis of tegumentary leishmaniasis.

Human CD4 restores normal T cell development and function in mice deficient in murine CD4

OpenAIRE

1994-01-01

The ability of a human coreceptor to function in mice was investigated by generating human CD4 (hCD4)-expressing transgenic mice on a mouse CD4-deficient (mCD4-/-) background. From developing thymocyte to matured T lymphocyte functions, hCD4 was shown to be physiologically active. By examining the expansion and deletion of specific V beta T cell families in mutated mice with and without hCD4, it was found that hCD4 can participate in positive and negative selection. Mature hCD4 single positiv...

ADP ribosyl-cyclases (CD38/CD157), social skills and friendship.

Science.gov (United States)

Chong, Anne; Malavasi, Fabio; Israel, Salomon; Khor, Chiea Chuen; Yap, Von Bing; Monakhov, Mikhail; Chew, Soo Hong; Lai, Poh San; Ebstein, Richard P

2017-04-01

Why some individuals seek social engagement while others shy away has profound implications for normal and pathological human behavior. Evidence suggests that oxytocin (OT), the paramount human social hormone, and CD38 that governs OT release, contribute to individual differences in social skills from intense social involvement to extreme avoidance that characterize autism. To explore the neurochemical underpinnings of sociality, CD38 expression of peripheral blood leukocytes (PBL) was measured in Han Chinese undergraduates. First, CD38 mRNA levels were correlated with lower Autism Quotient (AQ), indicating enhanced social skills. AQ assesses the extent of autistic-like traits including the propensity and dexterity needed for successful social engagement in the general population. Second, three CD157 eQTL SNPs in the CD38/CD157 gene region were associated with CD38 expression. CD157 is a paralogue of CD38 and is contiguous with it on chromosome 4p15. Third, association was also observed between the CD157 eQTL SNPs, CD38 expression and AQ. In the full model, CD38 expression and CD157 eQTL SNPs altogether account for a substantial 14% of the variance in sociality. Fourth, functionality of CD157 eQTL SNPs was suggested by a significant association with plasma oxytocin immunoreactivity products. Fifth, the ecological validity of these findings was demonstrated with subjects with higher PBL CD38 expression having more friends, especially for males. Furthermore, CD157 sequence variation predicts scores on the Friendship questionnaire. To summarize, this study by uniquely leveraging various measures reveals salient elements contributing to nonkin sociality and friendship, revealing a likely pathway underpinning the transition from normality to psychopathology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure and chromosomal localization of the human renal kallikrein gene

International Nuclear Information System (INIS)

Evans, B.A.; Yun, Z.X.; Close, J.A.

1988-01-01

Glandular kallikreins are a family of proteases encoded by a variable number of genes in different mammalian species. In all species examined, however, one particular kallikrein is functionally conserved in its capacity to release the vasoactive peptide, Lys-bradykinin, from low molecular weight kininogen. This kallikrein is found in the kidney, pancreas, and salivary gland, showing a unique pattern of tissue-specific expression relative to other members of the family. The authors have isolated a genomic clone carrying the human renal kallikrein gene and compared the nucleotide sequence of its promoter region with those of the mouse renal kallikrein gene and another mouse kallikrein gene expressed in a distinct cell type. They find four sequence elements conserved between renal kallikrein genes from the two species. They have also shown that the human gene is localized to 19q13, a position analogous to that of the kallikrein gene family on mouse chromosome 7

The CD157-Integrin Partnership Controls Transendothelial Migration and Adhesion of Human Monocytes*

Science.gov (United States)

Lo Buono, Nicola; Parrotta, Rossella; Morone, Simona; Bovino, Paola; Nacci, Giulia; Ortolan, Erika; Horenstein, Alberto L.; Inzhutova, Alona; Ferrero, Enza; Funaro, Ada

2011-01-01

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β1 and β2 integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes. PMID:21478153

Functional heterogeneity of human effector CD8+ T cells.

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Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

2012-02-09

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

Identification of DNA repair genes in the human genome

International Nuclear Information System (INIS)

Hoeijmakers, J.H.J.; van Duin, M.; Westerveld, A.; Yasui, A.; Bootsma, D.

1986-01-01

To identify human DNA repair genes we have transfected human genomic DNA ligated to a dominant marker to excision repair deficient xeroderma pigmentosum (XP) and CHO cells. This resulted in the cloning of a human gene, ERCC-1, that complements the defect of a UV- and mitomycin-C sensitive CHO mutant 43-3B. The ERCC-1 gene has a size of 15 kb, consists of 10 exons and is located in the region 19q13.2-q13.3. Its primary transcript is processed into two mRNAs by alternative splicing of an internal coding exon. One of these transcripts encodes a polypeptide of 297 aminoacids. A putative DNA binding protein domain and nuclear location signal could be identified. Significant AA-homology is found between ERCC-1 and the yeast excision repair gene RAD10. 58 references, 6 figures, 1 table

Different gene expression in human heart tissue and progenitor cells from control and diabetic subjects: relevance to the pathogenesis of human diabetic cardiomyopathy.

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de Cillis, Emanuela; Leonardini, Anna; Laviola, Luigi; Giorgino, Francesco; Tupputi Schinosa, Luigi de Luca; Bortone, Alessandro Santo

2010-04-01

The The aim of our study is to investigate the molecular mechanisms of diabetic cardiomyopathy through the identification of remarkable genes for the myocardial function that are expressed differently between diabetic and normal subjects. Moreover, we intend to characterize both in human myocardial tissue and in the related cardiac progenitor cells the pattern of gene expression and the levels of expression and protein activation of molecular effectors involved in the regulation of the myocardial function and differentiation to clarify whether in specific human pathological conditions (type 2 diabetes mellitus, cardiac failure, coronary artery disease) specific alterations of the aforementioned factors could take place. Thirty-five patients scheduled for coronary artery bypass grafting (CABG) or for aortic or mitral valve replacement were recruited into the study. There were 13 men and 22 women with a mean age of 64.8 +/- 13.4 years. A list of anamnestic, anthropometric, clinical, and instrumental data required for an optimal phenotypical characterization of the patients is reported. The small cardiac biopsy specimens were placed in the nourishing buffer, in a sterile tube provided the day of the procedure, to maintain the stability of the sample for several hours at room temperature. The cells were isolated by a dedicated protocol and then cultured in vitro. The sample was processed for total RNA extraction and levels of gene expression and protein activation of molecular effectors involved in the regulation of function and differentiation of human myocardium was analyzed. In particular, cardiac genes that modulate the oxidative stress response or the stress induced by pro-inflammatory cytokines (p66Shc, SOCS-1, SOCS-3) were analyzed. From a small sample of myocardium cardiac stem cells and cardiomyoblasts were also isolated and characterized. These cells showed a considerable proliferative capacity due to the fact that they demonstrate stability up to the

Alternative promoter usage of the membrane glycoprotein CD36

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Whatling Carl

2006-03-01

Full Text Available Abstract Background CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation. Results We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters. Conclusion Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.

Effect of TNFα on activities of different promoters of human apolipoprotein A-I gene

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Orlov, Sergey V.; Mogilenko, Denis A.; Shavva, Vladimir S.; Dizhe, Ella B.; Ignatovich, Irina A.; Perevozchikov, Andrej P.

2010-01-01

Research highlights: → TNFα stimulates the distal alternative promoter of human apoA-I gene. → TNFα acts by weakening of promoter competition within apoA-I gene (promoter switching). → MEK1/2 and nuclear receptors PPARα and LXRs take part in apoA-I promoter switching. -- Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFα-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFα on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFα leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNFα. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARα and LXRs are important for TNFα-mediated apoA-I promoter switching.

The development of human mast cells. An historical reappraisal

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Ribatti, Domenico, E-mail: domenico.ribatti@uniba.it

2016-03-15

The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34{sup +}/CD117{sup +}/CD13{sup +}multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, different MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.

The development of human mast cells. An historical reappraisal

International Nuclear Information System (INIS)

Ribatti, Domenico

2016-01-01

The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34"+/CD117"+/CD13"+multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, different MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.

Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation.

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Kahn, Joy; Byk, Tamara; Jansson-Sjostrand, Lottie; Petit, Isabelle; Shivtiel, Shoham; Nagler, Arnon; Hardan, Izhar; Deutsch, Varda; Gazit, Zulma; Gazit, Dan; Karlsson, Stefan; Lapidot, Tsvee

2004-04-15

A major limitation to clinical stem cell-mediated gene therapy protocols is the low levels of engraftment by transduced progenitors. We report that CXCR4 overexpression on human CD34+ progenitors using a lentiviral gene transfer technique helped navigate these cells to the murine bone marrow and spleen in response to stromal-derived factor 1 (SDF-1) signaling. Cells overexpressing CXCR4 exhibited significant increases in SDF-1-mediated chemotaxis and actin polymerization compared with control cells. A major advantage of CXCR4 overexpression was demonstrated by the ability of transduced CD34+ cells to respond to lower, physiologic levels of SDF-1 when compared to control cells, leading to improved SDF-1-induced migration and proliferation/survival, and finally resulting in significantly higher levels of in vivo repopulation of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice including primitive CD34+/CD38(-/low) cells. Importantly, no cellular transformation was observed following transduction with the CXCR4 vector. Unexpectedly, we documented lack of receptor internalization in response to high levels of SDF-1, which can also contribute to increased migration and proliferation by the transduced CD34+ cells. Our results suggest CXCR4 overexpression for improved definitive human stem cell motility, retention, and multilineage repopulation, which could be beneficial for in vivo navigation and expansion of hematopoietic progenitors.

Fanconi anemia genes are highly expressed in primitive CD34+ hematopoietic cells

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Brodeur Isabelle

2003-06-01

Full Text Available Abstract Background Fanconi anemia (FA is a complex recessive genetic disease characterized by progressive bone marrow failure (BM and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. Methods Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. Results We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34- stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. Conclusions These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34+ cells correlates with a role at the CD34+ differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism.

Stability evaluation of reference genes for real-time PCR in zebrafish (Danio rerio) exposed to cadmium chloride and subsequently infected by bacteria Aeromonas hydrophila

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Lang, Xingping; Wang, Lan, E-mail: lanwang@sxu.edu.cn; Zhang, Zuobing, E-mail: zbzhang@sxu.edu.cn

2016-01-15

Highlights: • Cd exposure affects the stability of reference genes for real-time PCR in zebrafish. • Reference genes present different stability in the five tissues (spleen, kidney, liver, gills and intestine) of zebrafish after Cd exposure. • Bacterial infection further affects the stability of reference genes in Cd-treated zebrafish. - Abstract: Environmental and occupational cadmium (Cd) toxicity is a global concern, and the model organism zebrafish is an ideal species to investigate Cd toxicity. Among various detecting techniques, quantitative real-time PCR (qPCR) is a sensitive and efficient tool. Stable reference genes are critical for relative qPCR analysis. However, accumulated evidence shows that conventional reference genes can vary significantly under different experimental setups. Here we evaluated the stability of eight candidate reference genes of zebrafish with or without exposure to different concentrations of Cd. The results showed that the best four suitable reference genes in the five selected organs were: (1) spleen: β-actin > gapdh > ef1α > rpl13α; (2) kidney: rplp2 > rpl7 > β-actin > ef1α; (3) liver: rpl7 > rpl13α > β-actin > ef1α; (4) gills: rplp2 > gapdh > rnf7 > ef1α; (5) intestine: ef1α > rnf7 > rplp2 > rpl13α. Moreover, we further assessed the expression stability of the four reference genes for Cd immunotoxicology studies in zebrafish. The expression profiles showed that ef1α in spleen and kidney, rpl13a in liver and rplp2 in intestine were the most suitable reference genes at 12 h and 9 days after the injection with Aeromonas hydrophila following Cd exposure. In gills, the expression of gapdh was more stable than ef1α after 9 days of bacteria challenge while ef1α showed a higher stability than gapdh at 12 h after bacteria injection. In conclusion, this study has demonstrated that different tissues of zebrafish have different suitable reference genes after Cd exposure and the subsequently pathogenic insults for q

The Protein Content of Extracellular Vesicles Derived from Expanded Human Umbilical Cord Blood-Derived CD133+ and Human Bone Marrow-Derived Mesenchymal Stem Cells Partially Explains Why both Sources are Advantageous for Regenerative Medicine.

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Angulski, Addeli B B; Capriglione, Luiz G; Batista, Michel; Marcon, Bruna H; Senegaglia, Alexandra C; Stimamiglio, Marco A; Correa, Alejandro

2017-04-01

Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133 + cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133 + cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133 + -EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133 + -EVs were different. Gene Ontology (GO) enrichment analysis in CD133 + -EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133 + -EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133 + cells and/or hBM-MSCs.

In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia.

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Kentaro Minagawa

Full Text Available Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia.We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85-90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non

Establishment of a new human pre-B acute lymphoblastic leukemia cell line (KMO-90) with 1;19 translocation carrying p53 gene alterations.

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Sotomatsu, M; Hayashi, Y; Kawamura, M; Yugami, S; Shitara, T

1993-10-01

A new human pre-B acute lymphoblastic leukemia cell line (KMO-90) was established from the bone marrow sample of a 12-year-old girl with acute lymphoblastic leukemia (ALL) carrying 1;19 chromosome translocation. KMO-90 cells expressed HLA-DR, CD10, CD19, and CD22 antigens. These cells had also cytoplasmic immunoglobulin lacking surface immunoglobulin, indicating that these had a pre-B phenotype. Chromosome analysis of this cell line showed 48, XX, +8, +19, t(1;19)(q23;p13). Southern blot analysis showed the same sized rearrangements of the E2A gene in KMO-90 cells as those in the original leukemic cells. By means of reverse transcriptase-polymerase chain reaction analysis, we detected E2A/PBX1 fusion transcripts in KMO-90 cells. KMO-90 is useful when studying the role of the 1;19 translocation in the etiology of pre-B ALL. Furthermore, we studied alterations of the p53 gene in this cell line by polymerase chain reaction, single-strand conformation polymorphism analysis. KMO-90 cells were identified to have a point mutation at codon 177 (CCC-->TCC) of the p53 gene, suggesting that alterations of the p53 gene may have an important role in the establishment of this cell line.

Viral Inhibition of Bacterial Phagocytosis by Human Macrophages: Redundant Role of CD36.

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Grace E Cooper

Full Text Available Macrophages are essential to maintaining lung homoeostasis and recent work has demonstrated that influenza-infected lung macrophages downregulate their expression of the scavenger receptor CD36. This receptor has also been shown to be involved in phagocytosis of Streptococcus pneumoniae, a primary agent associated with pneumonia secondary to viral infection. The aim of this study was to investigate the role of CD36 in the effects of viral infection on macrophage phagocytic function. Human monocyte-derived macrophages (MDM were exposed to H3N2 X31 influenza virus, M37 respiratory syncytial virus (RSV or UV-irradiated virus. No infection of MDM was seen upon exposure to UV-irradiated virus but incubation with live X31 or M37 resulted in significant levels of viral detection by flow cytometry or RT-PCR respectively. Infection resulted in significantly diminished uptake of S. pneumoniae by MDM and significantly decreased expression of CD36 at both the cell surface and mRNA level. Concurrently, there was a significant increase in IFNβ gene expression in response to infection and we observed a significant decrease in bacterial phagocytosis (p = 0.031 and CD36 gene expression (p = 0.031 by MDM cultured for 24 h in 50IU/ml IFNβ. Knockdown of CD36 by siRNA resulted in decreased phagocytosis, but this was mimicked by transfection reagent alone. When MDM were incubated with CD36 blocking antibodies no effect on phagocytic ability was observed. These data indicate that autologous IFNβ production by virally-infected cells can inhibit bacterial phagocytosis, but that decreased CD36 expression by these cells does not play a major role in this functional deficiency.

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I.

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Unverzagt, K L; Martinson, J; Lee, W; Stiff, P J; Williams, S; Bender, J G

1996-01-01

Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.

Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue

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Luo, Wei; Hu, Qiang; Wang, Dan; Deeb, Kristin K.; Ma, Yingyu; Morrison, Carl D.; Liu, Song; Johnson, Candace S.; Trump, Donald L.

2013-01-01

Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissues obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small

Different thresholds of T cell activation regulate FIV infection of CD4+CD25+ and CD4+CD25- cells

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Joshi, Anjali; Garg, Himanshu; Tompkins, Mary B.; Tompkins, Wayne A.

2005-01-01

Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4 + CD25 + T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4 + CD25 + and CD4 + CD25 - cells under different stimulation conditions. Both CD4 + CD25 + and CD4 + CD25 - cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4 + CD25 + cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4 + CD25 - T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4 + CD25 - cells to replicate FIV, it induces apoptosis in a high percentage of CD4 + CD25 + T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4 + CD25 - cells. These results suggest that CD4 + CD25 + and CD4 + CD25 - T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4 + CD25 + and CD4 + CD25 - cells to serve as potential reservoirs of a productive and latent FIV infection

Mutational analysis of FLASH and PTPN13 genes in colorectal carcinomas.

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Jeong, Eun Goo; Lee, Sung Hak; Yoo, Nam Jin; Lee, Sug Hyung

2008-01-01

The Fas-Fas ligand system is considered a major pathway for induction of apoptosis in cells and tissues. FLASH was identified as a pro-apoptotic protein that transmits apoptosis signal during Fas-mediated apoptosis. PTPN13 interacts with Fas and functions as both suppressor and inducer of Fas-mediated apoptosis. There are polyadenine tracts in both FLASH (A8 and A9 in exon 8) and PTPN13 (A8 in exon 7) genes that could be frameshift mutation targets in colorectal carcinomas. Because genes encoding proteins in Fas-mediated apoptosis frequently harbor somatic mutations in cancers, we explored the possibility as to whether mutations of FLASH and PTPN13 are a feature of colorectal carcinomas. We analysed human FLASH in exon 8 and PTPN13 in exon 7 for the detection of somatic mutations in 103 colorectal carcinomas by a polymerase chain reaction (PCR)- based single-strand conformation polymorphism (SSCP). We detected two mutations in FLASH gene, but none in PTPN13 gene. However, the two mutations were not frameshift (deletion or insertion) mutations in the polyadenine tracts of FLASH. The two mutations consisted of a deletion mutation (c.3734-3737delAGAA) and a missense mutation (c.3703A>C). These data indicate that frameshift mutation in the polyadenine tracts in both FLASH and PTPN13 genes is rare in colorectal carcinomas. Also, the data suggest that both FLASH and PTPN13 mutations in the polyadenine tracts may not have a crucial role in the pathogenesis of colorectal carcinomas.

Similarities and differences in signal transduction by interleukin 4 and interleukin 13: analysis of Janus kinase activation.

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Keegan, A D; Johnston, J A; Tortolani, P J; McReynolds, L J; Kinzer, C; O'Shea, J J; Paul, W E

1995-08-15

The cytokines interleukin (IL) 4 and IL-13 induce many of the same biological responses, including class switching to IgE and induction of major histocompatibility complex class II antigens and CD23 on human B cells. It has recently been shown that IL-4 induces the tyrosine phosphorylation of a 170-kDa protein, a substrate called 4PS, and of the Janus kinase (JAK) family members JAK1 and JAK3. Because IL-13 has many functional effects similar to those of IL-4, we compared the ability of IL-4 and IL-13 to activate these signaling molecules in the human multifactor-dependent cell line TF-1. In this report we demonstrate that both IL-4 and IL-13 induced the tyrosine phosphorylation of 4PS and JAK1. Interestingly, although IL-4 induced the tyrosine phosphorylation of JAK3, we did not detect JAK3 phosphorylation in response to IL-13. These data suggest that IL-4 and IL-13 signal in similar ways via the activation of JAK1 and 4PS. However, our data further indicate that there are significant differences because IL-13 does not activate JAK3.

SIRPA, VCAM1 and CD34 identify discrete lineages during early human cardiovascular development

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Rhys J.P. Skelton

2014-07-01

Full Text Available The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5GFP was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5GFP/w human embryonic stem cells (hESCs. Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5posCD34pos population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5posSIRPApos to NKX2-5posSIRPAposVCAM1pos. Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.

The direct effect of Focal Adhesion Kinase (FAK, dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

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Zhang Li

2009-08-01

Full Text Available Abstract Background Focal adhesion kinase (FAK is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis. Methods To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD, and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation in vitro, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD in vivo, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors. Results Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability in vitro and inhibited tumorigenesis in vivo. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p Conclusion Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis in vivo and reveal

Human HMG box transcription factor HBP1: a role in hCD2 LCR function.

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Zhuma, T; Tyrrell, R; Sekkali, B; Skavdis, G; Saveliev, A; Tolaini, M; Roderick, K; Norton, T; Smerdon, S; Sedgwick, S; Festenstein, R; Kioussis, D

1999-01-01

The locus control region (LCR) of the human CD2 gene (hCD2) confers T cell-specific, copy-dependent and position-independent gene expression in transgenic mice. This LCR consists of a strong T cell-specific enhancer and an element without enhancer activity (designated HSS3), which is required for prevention of position effect variegation (PEV) in transgenic mice. Here, we identified the HMG box containing protein-1 (HBP1) as a factor binding to HSS3 of the hCD2 LCR. Within the LCR, HBP1 binds to a novel TTCATTCATTCA sequence that is higher in affinity than other recently reported HBP1-binding sites. Mice transgenic for a hCD2 LCR construct carrying a deletion of the HBP1-binding sequences show a propensity for PEV if the transgene integrates in a heterochromatic region of the chromosome such as the centromere or telomere. We propose that HBP1 plays an important role in chromatin opening and remodelling activities by binding to and bending the DNA, thus allowing DNA-protein and/or protein-protein interactions, which increase the probability of establishing an active locus. PMID:10562551

MRP-1/CD9 gene transduction regulates the actin cytoskeleton through the downregulation of WAVE2.

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Huang, C-L; Ueno, M; Liu, D; Masuya, D; Nakano, J; Yokomise, H; Nakagawa, T; Miyake, M

2006-10-19

Motility-related protein-1 (MRP-1/CD9) is involved in cell motility. We studied the change in the actin cytoskeleton, and the expression of actin-related protein (Arp) 2 and Arp3 and the Wiskott-Aldrich syndrome protein (WASP) family according to MRP-1/CD9 gene transduction into HT1080 cells. The frequency of cells with lamellipodia was significantly lower in MRP-1/CD9-transfected HT1080 cells than in control HT1080 cells (PMRP-1/CD9 gene transduction affected the subcellular localization of Arp2 and Arp3 proteins. Furthermore, MRP-1/CD9 gene transduction induced a downregulation of WAVE2 expression (PMRP-1/CD9 monoclonal antibody inhibited downregulation of WAVE2 in MRP-1/CD9-transfected HT1080 cells (PMRP-1/CD9 gene transduction. Furthermore, downregulation of WAVE2 by transfection of WAVE2-specific small interfering RNA (siRNA) mimicked the morphological effects of MRP-1/CD9 gene transduction and suppressed cell motility. However, transfection of each siRNA for Wnt1, Wnt2b1 or Wnt5a did not affect WAVE2 expression. Transfection of WAVE2-specific siRNA also did not affect expressions of these Wnts. These results indicate that MRP-1/CD9 regulates the actin cytoskeleton by downregulating of the WAVE2, through the Wnt-independent signal pathway.

Metabolic Adaptation of Human CD4+ and CD8+ T-Cells to T-Cell Receptor-Mediated Stimulation

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Nicholas Jones

2017-11-01

Full Text Available Linking immunometabolic adaptation to T-cell function provides insight for the development of new therapeutic approaches in multiple disease settings. T-cell activation and downstream effector functions of CD4+ and CD8+ T-cells are controlled by the strength of interaction between the T-cell receptor (TCR and peptides presented by human leukocyte antigens (pHLA. The role of TCR–pHLA interactions in modulating T-cell metabolism is unknown. Here, for the first time, we explore the relative contributions of the main metabolic pathways to functional responses in human CD4+ and CD8+ T-cells. Increased expression of hexokinase II accompanied by higher basal glycolysis is demonstrated in CD4+ T-cells; cytokine production in CD8+ T-cells is more reliant on oxidative phosphorylation. Using antigen-specific CD4+ and CD8+ T-cell clones and altered peptide ligands, we demonstrate that binding affinity tunes the underlying metabolic shift. Overall, this study provides important new insight into how metabolic pathways are controlled during antigen-specific activation of human T-cells.

The human CD8β M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

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Deepshi Thakral

Full Text Available The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4 that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

The effect of stem cell factor on proliferation of human endometrial CD146+ cells

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Mehri Fayazi

2016-07-01

Full Text Available Background: Stem cell factor (SCF is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of stem cells. Objective: The aim of the present study was to examine the proliferation effect of different concentrations of SCF on expansion of human endometrial CD146+ cells. Materials and Methods: In this experimental study, total populations of isolated human endometrial suspensions after fourth passage were isolated by magnetic activated cell sorting (MACS into CD146+ cells. Human endometrial CD146+ cells were karyotyped and tested for the effect of SCF on proliferation of CD146+ cells, then different concentrations of 0, 12.5, 25, 50 and 100 ng/ml was carried out and mitogens-stimulated endometrial CD146+ cells proliferation was assessed by MTT assay. Results: Chromosomal analysis showed a normal metaphase spread and 46XX karyotype. The proliferation rate of endometrial CD146P + P cells in the presence of 0, 12.5, 25, 50 and 100 ng/ml SCF were 0.945±0.094, 0.962±0.151, 0.988±0.028, 1.679±0.012 and 1.129±0.145 respectively. There was a significant increase in stem/ stromal cell proliferation following in vitro treatment by 50 ng/ml than other concentrations of SCF (p=0.01. Conclusion: The present study suggests that SCF could have effect on the proliferation and cell survival of human endometrial CD146P+P cells and it has important implications for medical sciences and cell therapies

Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

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Silvia Berardis

Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

Macrophage-specific nanotechnology-driven CD163 overexpression in human macrophages results in an M2 phenotype under inflammatory conditions.

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Alvarado-Vazquez, Perla Abigail; Bernal, Laura; Paige, Candler A; Grosick, Rachel L; Moracho Vilrriales, Carolina; Ferreira, David Wilson; Ulecia-Morón, Cristina; Romero-Sandoval, E Alfonso

2017-08-01

M1 macrophages release proinflammatory factors during inflammation. They transit to an M2 phenotype and release anti-inflammatory factors to resolve inflammation. An imbalance in the transition from M1 to M2 phenotype in macrophages contributes to the development of persistent inflammation. CD163, a member of the scavenger receptor cysteine-rich family, is an M2 macrophage marker. The functional role of CD163 during the resolution of inflammation is not completely known. We postulate that CD163 contributes to the transition from M1 to M2 phenotype in macrophages. We induced CD163 gene in THP-1 and primary human macrophages using polyethylenimine nanoparticles grafted with a mannose ligand (Man-PEI). This nanoparticle specifically targets cells of monocytic origin via mannose receptors. Cells were challenged with a single or a double stimulation of lipopolysaccharide (LPS). A CD163 or empty plasmid was complexed with Man-PEI nanoparticles for cell transfections. Quantitative RT-PCR, immunocytochemistry, and ELISAs were used for molecular assessments. CD163-overexpressing macrophages displayed reduced levels of tumor necrosis factor-alpha (TNF)-α and monocytes chemoattractant protein (MCP)-1 after a single stimulation with LPS. Following a double stimulation paradigm, CD163-overexpressing macrophages showed an increase of interleukin (IL)-10 and IL-1ra and a reduction of MCP-1. This anti-inflammatory phenotype was partially blocked by an anti-CD163 antibody (effects on IL-10 and IL-1ra). A decrease in the release of TNF-α, IL-1β, and IL-6 was observed in CD163-overexpressing human primary macrophages. The release of IL-6 was blocked by an anti-CD163 antibody in the CD163-overexpressing group. Our data show that the induction of the CD163 gene in human macrophages under inflammatory conditions produces changes in cytokine secretion in favor of an anti-inflammatory phenotype. Targeting macrophages to induce CD163 using cell-directed nanotechnology is an attractive

PDGFRα and CD51 mark human nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion.

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Pinho, Sandra; Lacombe, Julie; Hanoun, Maher; Mizoguchi, Toshihide; Bruns, Ingmar; Kunisaki, Yuya; Frenette, Paul S

2013-07-01

The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin⁺ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45⁻ Ter119⁻ CD31⁻ mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin⁺ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα⁺ CD51 ⁺subset of Nestin⁺ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα⁺ CD51⁺ cells in the human fetal BM represent a small subset of CD146⁺ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα⁺ CD51⁺ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.

Functional analysis of human hematopoietic stem cell gene expression using zebrafish.

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2005-08-01

Full Text Available Although several reports have characterized the hematopoietic stem cell (HSC transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow (CD34+(CD33-(CD38-Rho(lo(c-kit+ cells, enriched for hematopoietic stem/progenitor cells with (CD34+(CD33-(CD38-Rho(hi cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23% of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global

Metabolism of nitrogen-13 labelled ammonia in different conditions in dogs, human volunteers and transplant patients

International Nuclear Information System (INIS)

Bormans, G.; Maes, A.; Langendries, W.; Nuyts, J.; Vrolix, M.; Vanhaecke, J.; Schiepers, C.; Roo, M. de; Mortelmans, L.; Verbruggen, A.

1995-01-01

To investigate the rate of metabolism of nitrogen-13 labelled ammonia ( 13 NH 3 ) in different conditions, we have determined the relative amount of unchanged 13 NH 3 in the blood of dogs, volunteers and transplant patients at different times following injection. In dogs, the determinations were made under basal conditions, during adenosine administration and after coronary occlusion. The results show that adenosine administration increases the metabolic rate whereas coronary occlusion does not affect 13 NH 3 metabolism. For both human volunteers and transplant patients the metabolic rate of 13 NH 3 was assessed under basal conditions and during adenosine administration. 13 NH 3 metabolism proceeds faster in transplant patients than in volunteers under both conditions. Adenosine administration causes a faster 13 NH 3 turnover in volunteers but not in transplant patients. Application of individual metabolite correction resulted in a 16% decrease in the calculated blood flow compared to uncorrected values. A smaller difference (5%) was observed between correction with mean metabolite values and individually acquired metabolite values. (orig.)

Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor.

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Ryan, J J; McReynolds, L J; Keegan, A; Wang, L H; Garfein, E; Rothman, P; Nelms, K; Paul, W E

1996-02-01

IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557-657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557-657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R.

In situ identification of CD44+/CD24- cancer cells in primary human breast carcinomas.

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Giuseppe Perrone

Full Text Available Breast cancer cells with the CD44+/CD24- phenotype have been reported to be tumourigenic due to their enhanced capacity for cancer development and their self-renewal potential. The identification of human tumourigenic breast cancer cells in surgical samples has recently received increased attention due to the implications for prognosis and treatment, although limitations exist in the interpretation of these studies. To better identify the CD44+/CD24- cells in routine surgical specimens, 56 primary breast carcinoma cases were analysed by immunofluorescence and confocal microscopy, and the results were compared using flow cytometry analysis to correlate the amount and distribution of the CD44+/CD24- population with clinicopathological features. Using these methods, we showed that the breast carcinoma cells displayed four distinct sub-populations based on the expression pattern of CD44 and CD24. The CD44+/CD24- cells were found in 91% of breast tumours and constituted an average of 6.12% (range, 0.11%-21.23% of the tumour. A strong correlation was found between the percentage of CD44+/CD24- cells in primary tumours and distant metastasis development (p = 0.0001; in addition, there was an inverse significant association with ER and PGR status (p = 0.002 and p = 0.001, respectively. No relationship was evident with tumour size (T and regional lymph node (N status, differentiation grade, proliferative index or HER2 status. In a multivariate analysis, the percentage of CD44+/CD24- cancer cells was an independent factor related to metastasis development (p = 0.004. Our results indicate that confocal analysis of fluorescence-labelled breast cancer samples obtained at surgery is a reliable method to identify the CD44+/CD24- tumourigenic cell population, allowing for the stratification of breast cancer patients into two groups with substantially different relapse rates on the basis of CD44+/CD24- cell percentage.

Luteolin, a flavonoid, inhibits CD40 ligand expression by activated human basophils.

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Hirano, Toru; Arimitsu, Junsuke; Higa, Shinji; Naka, Tetsuji; Ogata, Atsushi; Shima, Yoshihito; Fujimoto, Minoru; Yamadori, Tomoki; Ohkawara, Tomoharu; Kuwabara, Yusuke; Kawai, Mari; Kawase, Ichiro; Tanaka, Toshio

2006-01-01

We have previously shown that flavonoids such as luteolin, apigenin and fisetin inhibit interleukin 4 and interleukin 13 production. In this study, we investigated whether luteolin can suppress CD40 ligand expression by basophils. A human basophilic cell line, KU812, was stimulated with A23187 and phorbol myristate acetate (PMA) with or without various concentrations of luteolin or other flavonoids for 12 h, and CD40 ligand expression was analyzed by FACS. The effect of luteolin on CD40 ligand mRNA expression was studied by semiquantitative reverse transcription PCR analysis. In addition, CD40 ligand expression was also measured in purified basophils that had been stimulated for 12 h with A23187 plus PMA with or without various concentrations of luteolin. CD40 ligand expression by KU812 cells was enhanced noticeably in response to A23187 and even more strikingly augmented by A23187 plus PMA. The expression was significantly suppressed by 10 or 30 microM of luteolin, whereas myricetin failed to inhibit. Reverse transcription PCR analyses demonstrated that luteolin inhibited CD40 ligand mRNA expression by stimulated KU812 cells. Of the six flavonoids examined, luteolin, apigenin, fisetin and quercetin at 30 microM showed a significant inhibitory effect on CD40 ligand expression. The incubation of purified basophils with A23187 plus PMA significantly enhanced CD40 ligand expression, and the presence of luteolin again had an inhibitory effect. Luteolin inhibits CD40 ligand expression by activated basophils.

CD25 shedding by human natural occurring CD4+CD25+ regulatory T cells does not inhibit the action of IL-2

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Pedersen, Anders Elm; Lauritsen, Jens Peter Holst

2009-01-01

Tregs are known to inhibit CD4+ T cell in a contact-dependent manner, but at the same time, various suppressive factors are secreted. We, here, demonstrate that human naturally occurring CD4+CD25+ Tregs are able to shed large amounts of soluble CD25 upon activation. Secretion of sCD25 could add......Regulatory T (Treg) cells are important for the maintenance of peripheral tolerance and inhibition of pathogenic T-cell responses. Therefore, they are important for the limitation of chronic inflammation but can also be deleterious by e.g. limiting antitumour immune responses. Natural occurring...... to the inhibitory effect of Tregs as such secretion in other settings has been proposed to act as a sink for local IL-2. However, we here demonstrate that supernatant from human Tregs containing high concentration of sCD25 does not inhibit proliferation of CD4+CD25(-) T cells or inhibit the action of IL-2...

Double suicide genes selectively kill human umbilical vein endothelial cells

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Liu Lunxu

2011-02-01

Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

Interethnic diversity of the CD209 (rs4804803 gene promoter polymorphism in African but not American sickle cell disease

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Jenelle A. Noble

2015-02-01

Full Text Available Elucidating the genomic diversity of CD209 gene promoter polymorphism could assist in clarifying disease pathophysiology as well as contribution to co-morbidities. CD209 gene promoter polymorphism has been shown to be associated with susceptibility to infection. We hypothesize that CD209 mutant variants occur at a higher frequency among Africans and in sickle cell disease. We analyzed the frequency of the CD209 gene (rs4804803 in healthy control and sickle cell disease (SCD populations and determined association with disease. Genomic DNA was extracted from blood samples collected from 145 SCD and 231 control Africans (from Mali, 331 SCD and 379 control African Americans and 159 Caucasians. Comparative analysis among and between groups was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. Per ethnic diversification, we found significant disparity in genotypic (23.4% versus 16.9% versus 3.2% and allelic frequencies (48.7% versus 42.1% versus 19.8% of the homozygote mutant variant of the CD209 (snp 309A/G gene promoter between Africans, African Americans and Caucasians respectively. Comparative evaluation between disease and control groups reveal a significant difference in genotypic (10.4% versus 23.4%; p = 0.002 and allelic frequencies (39.7% versus 48.7%; p = 0.02 of the homozygote mutant variant in African SCD and healthy controls respectively, an observation that is completely absent among Americans. Comparing disease groups, we found no difference in the genotypic (p = 0.19 or allelic (p = 0.72 frequencies of CD209 homozygote mutant variant between Africans and Americans with sickle cell disease. The higher frequency of CD209 homozygote mutant variants in the African control group reveals a potential impairment of the capacity to mount an immune response to infectious diseases, and possibly delineate susceptibility to or severity of infectious co-morbidities within and between groups.

Peripheral T-Cell Lymphoma with Aberrant Expression of CD19, CD20, and CD79a: Case Report and Literature Review

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Matnani, Rahul G.; Stewart, Rachel L.; Pulliam, Joseph; Jennings, Chester D.; Kesler, Melissa

2013-01-01

A case of lymphoma of T-cell derivation with aberrant expression of three B-cell lineage markers (CD19, CD20, and CD79a), which was diagnosed on a left axillary excision, is described. Immunohistochemical studies and flow cytometry analysis demonstrated neoplastic cells expressing CD3, CD19, CD20, and CD79a with absence of CD4, CD8, CD10, CD30, CD34, CD56, CD68, TDT, MPO, PAX-5, and surface immunoglobulin. Gene rearrangement studies performed on paraffin blocks demonstrated monoclonal T-cell receptor gamma chain rearrangement with no evidence of clonal heavy chain rearrangement. The neoplastic cells were negative for Epstein-Barr virus (EBV) or Human Herpes Virus 8 (HHV-8). At the time of diagnosis, the PET scan demonstrated hypermetabolic neoplastic cells involving the left axilla, bilateral internal jugular areas, mediastinum, right hilum, bilateral lungs, and spleen. However, bone marrow biopsy performed for hemolytic anemia revealed normocellular bone marrow with trilineage maturation. The patient had no evidence of immunodeficiency or infection with EBV or HHV-8. This is the first reported case of a mature T-cell lymphoma with aberrant expression of three B-cell lineage markers. The current report also highlights the need for molecular gene rearrangement studies to determine the precise lineage of ambiguous neoplastic clones. PMID:24066244

Peripheral T-Cell Lymphoma with Aberrant Expression of CD19, CD20, and CD79a: Case Report and Literature Review

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Rahul G. Matnani

2013-01-01

Full Text Available A case of lymphoma of T-cell derivation with aberrant expression of three B-cell lineage markers (CD19, CD20, and CD79a, which was diagnosed on a left axillary excision, is described. Immunohistochemical studies and flow cytometry analysis demonstrated neoplastic cells expressing CD3, CD19, CD20, and CD79a with absence of CD4, CD8, CD10, CD30, CD34, CD56, CD68, TDT, MPO, PAX-5, and surface immunoglobulin. Gene rearrangement studies performed on paraffin blocks demonstrated monoclonal T-cell receptor gamma chain rearrangement with no evidence of clonal heavy chain rearrangement. The neoplastic cells were negative for Epstein-Barr virus (EBV or Human Herpes Virus 8 (HHV-8. At the time of diagnosis, the PET scan demonstrated hypermetabolic neoplastic cells involving the left axilla, bilateral internal jugular areas, mediastinum, right hilum, bilateral lungs, and spleen. However, bone marrow biopsy performed for hemolytic anemia revealed normocellular bone marrow with trilineage maturation. The patient had no evidence of immunodeficiency or infection with EBV or HHV-8. This is the first reported case of a mature T-cell lymphoma with aberrant expression of three B-cell lineage markers. The current report also highlights the need for molecular gene rearrangement studies to determine the precise lineage of ambiguous neoplastic clones.

Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells.

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Leal Rojas, Ingrid M; Mok, Wai-Hong; Pearson, Frances E; Minoda, Yoshihito; Kenna, Tony J; Barnard, Ross T; Radford, Kristen J

2017-01-01

Dendritic cells (DC) initiate the differentiation of CD4 + helper T cells into effector cells including Th1 and Th17 responses that play an important role in inflammation and autoimmune disease pathogenesis. In mice, Th1 and Th17 responses are regulated by different conventional (c) DC subsets, with cDC1 being the main producers of IL-12p70 and inducers of Th1 responses, while cDC2 produce IL-23 to promote Th17 responses. The role that human DC subsets play in memory CD4 + T cell activation is not known. This study investigated production of Th1 promoting cytokine IL-12p70, and Th17 promoting cytokines, IL-1β, IL-6, and IL-23, by human blood monocytes, CD1c + DC, CD141 + DC, and plasmacytoid DC and examined their ability to induce Th1 and Th17 responses in memory CD4 + T cells. Human CD1c + DC produced IL-12p70, IL-1β, IL-6, and IL-23 in response to R848 combined with LPS or poly I:C. CD141 + DC were also capable of producing IL-12p70 and IL-23 but were not as proficient as CD1c + DC. Activated CD1c + DC were endowed with the capacity to promote both Th1 and Th17 effector function in memory CD4 + T cells, characterized by high production of interferon-γ, IL-17A, IL-17F, IL-21, and IL-22. These findings support a role for CD1c + DC in autoimmune inflammation where Th1/Th17 responses play an important role in disease pathogenesis.

Selective toxicity of dihydroartemisinin on human CD34+ erythroid cell differentiation

International Nuclear Information System (INIS)

Finaurini, Sara; Ronzoni, Luisa; Colancecco, Alessandra; Cattaneo, Alessandra; Cappellini, Maria Domenica; Ward, Stephen A.; Taramelli, Donatella

2010-01-01

Artemisinins are safely used in the combination therapy for uncomplicated malaria, but their employment during pregnancy is still controversial. In fact, animal studies reported that the active metabolite, dihydroartemisinin (DHA), causes embryonic erythrocytes depletion, when the treatment is performed during a critical period of time. The present study investigates the effect of DHA on human developmental erythropoiesis in order to characterize the target erythroid stage and to predict the window of susceptibility in human pregnancy. As a model for human developmental erythropoiesis, peripheral blood purified, CD34+ cells were committed towards erythrocytes and DHA (0.5 or 2 μM) was added to different erythroid stages during 14 days culture. Erythroid differentiation was investigated by cytofluorimetric analysis of Glycophorin A expression, by morphological analysis and erythroid globin gene expression analysis with real-time PCR. It was found that the effect of DHA was dependent on the maturation stage of erythroid cells. In fact when DHA was added to the pro- and basophilic erythroblasts caused a significant dose-dependent inhibition of cell proliferation and a significant delay of erythroid differentiation, as measured by morphological analysis, expression of Glycophorin A by immunofluorescence and of erythroid globin genes by real-time PCR. In contrast, the inhibition of stem cells and of early progenitors was transient and masked by the subsequent exponential cell growth. No effect was observed on mature erythroid stages. This is the first demonstration that DHA affects human erythropoiesis in vitro, in a dose- and time-dependent manner; the target population seems to be the pro-erythroblast and basophilic erythroblast stage, suggesting that DHA toxicity is limited to primitive human erythropoiesis. These findings outline the relevance of DHA dosage and timing to prevent embryotoxicity and support current WHO recommendations of avoiding malaria treatment

Identification and HLA-tetramer-validation of human CD4+ and CD8+ T cell responses against HCMV proteins IE1 and IE2.

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Braendstrup, Peter; Mortensen, Bo Kok; Justesen, Sune; Osterby, Thomas; Rasmussen, Michael; Hansen, Andreas Martin; Christiansen, Claus Bohn; Hansen, Morten Bagge; Nielsen, Morten; Vindeløv, Lars; Buus, Søren; Stryhn, Anette

2014-01-01

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.

Identification and HLA-tetramer-validation of human CD4+ and CD8+ T cell responses against HCMV proteins IE1 and IE2.

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Peter Braendstrup

Full Text Available Human cytomegalovirus (HCMV is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2. Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4+ T cells

Science.gov (United States)

Liu, Lanxia; Bai, Yuanyuan; Zhu, Dunwan; Song, Liping; Wang, Hai; Dong, Xia; Zhang, Hailing; Leng, Xigang

2011-06-01

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4+ T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4+ T cells. The indirect impact of CS/DNA nanoparticles on naive CD4+ T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4+ T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4+ T cells to explore their direct impact on naive CD4+ T cell differentiation by measuring the release of IL-4 and IFN-γ from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4+ T cells. It was also demonstrated that, when directly exposed to the naive CD4+ T cells, the nanoparticles induced neither the activation of the naive CD4+ T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-γ) that induce naive CD4+ T cell polarization, nor any changes in the differentiation direction of naive CD4+ T cells in the presence of the corresponding cytokines.

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4+ T cells

International Nuclear Information System (INIS)

Liu Lanxia; Bai Yuanyuan; Zhu Dunwan; Song Liping; Wang Hai; Dong Xia; Zhang Hailing; Leng Xigang

2011-01-01

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4 + T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4 + T cells. The indirect impact of CS/DNA nanoparticles on naive CD4 + T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4 + T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4 + T cells to explore their direct impact on naive CD4 + T cell differentiation by measuring the release of IL-4 and IFN-γ from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4 + T cells. It was also demonstrated that, when directly exposed to the naive CD4 + T cells, the nanoparticles induced neither the activation of the naive CD4 + T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-γ) that induce naive CD4 + T cell polarization, nor any changes in the differentiation direction of naive CD4 + T cells in the presence of the corresponding cytokines.

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

Science.gov (United States)

Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer.

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Carrascal, M A; Silva, M; Ferreira, J A; Azevedo, R; Ferreira, D; Silva, A M N; Ligeiro, D; Santos, L L; Sackstein, R; Videira, P A

2018-05-17

The glycan moieties sialyl-Lewis-X and/or -A (sLe X/A ) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. We observed that the CF1_T cell line expressed sLe X , but not sLe A and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLe X -CD44 and sLe X -CD13 was confirmed in clinical breast cancer tissue samples. Both CD44 and CD13 glycoforms display sLe X in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of CD24 as a cancer stem cell marker in human nasopharyngeal carcinoma.

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Chun-Hung Yang

Full Text Available Cancer stem cells (CSCs represent a unique sub-population of tumor cells with the ability to initiate tumor growth and sustain self-renewal. Although CSC biomarkers have been described for various tumors, only a few markers have been identified for nasopharyngeal carcinoma (NPC. In this study, we show that CD24+ cells isolated from human NPC cell lines express stem cell genes (Sox2, Oct4, Nanog, Bmi-1, and Rex-1, and show activation of the Wnt/β-catenin signaling pathway. CD24+ cells possess typical CSC characteristics that include enhanced cell proliferation, increased colony and sphere formation, maintenance of cell differentiation potential in prolonged culture, and enhanced resistance to chemotherapeutic drugs. Notably, CD24+ cells produce tumors following inoculation of as few as 500 cells in immunodeficient NOD/SCID mice. CD24+ cells further show increased invasion ability in vitro, which correlates with enhanced expression of matrix metalloproteinase 2 and 9. In summary, our results suggest that CD24 represents a novel CSC biomarker in NPC.

Associations between CD36 gene polymorphisms and susceptibility to coronary artery heart disease

International Nuclear Information System (INIS)

Zhang, Y.; Ling, Z.Y.; Deng, S.B.; Du, H.A.; Yin, Y.H.; Yuan, J.; She, Q.; Chen, Y.Q.

2014-01-01

Associations between polymorphisms of the CD36 gene and susceptibility to coronary artery heart disease (CHD) are not clear. We assessed allele frequencies and genotype distributions of CD36 gene polymorphisms in 112 CHD patients and 129 control patients using semi-quantitative polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Additionally, we detected CD36 mRNA expression by real-time quantitative PCR, and we quantified plasma levels of oxidized low-density lipoprotein (ox-LDL) using an enzyme-linked immunosorbent assay (ELISA). There were no significant differences between the two groups (P>0.05) in allele frequencies of rs1761667 or in genotype distribution and allele frequencies of rs3173798. The genotype distribution of rs1761667 significantly differed between CHD patients and controls (P=0.034), with a significantly higher frequency of the AG genotype in the CHD group compared to the control group (P=0.011). The plasma levels of ox-LDL in patients with the AG genotype were remarkably higher than those with the GG and AA genotypes (P=0.010). In a randomized sample taken from patients in the two groups, the CD36 mRNA expression of the CHD patients was higher than that of the controls. In CHD patients, the CD36 mRNA expression in AG genotype patients was remarkably higher than in those with an AA genotype (P=0.005). After adjusted logistic regression analysis, the AG genotype of rs1761667 was associated with an increased risk of CHD (OR=2.337, 95% CI=1.336-4.087, P=0.003). In conclusion, the rs1761667 polymorphism may be closely associated with developing CHD in the Chongqing Han population of China, and an AG genotype may be a genetic susceptibility factor for CHD

Associations between CD36 gene polymorphisms and susceptibility to coronary artery heart disease

Energy Technology Data Exchange (ETDEWEB)

Zhang, Y.; Ling, Z.Y.; Deng, S.B.; Du, H.A.; Yin, Y.H.; Yuan, J.; She, Q.; Chen, Y.Q. [Department of Cardiology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing (China)

2014-08-08

Associations between polymorphisms of the CD36 gene and susceptibility to coronary artery heart disease (CHD) are not clear. We assessed allele frequencies and genotype distributions of CD36 gene polymorphisms in 112 CHD patients and 129 control patients using semi-quantitative polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Additionally, we detected CD36 mRNA expression by real-time quantitative PCR, and we quantified plasma levels of oxidized low-density lipoprotein (ox-LDL) using an enzyme-linked immunosorbent assay (ELISA). There were no significant differences between the two groups (P>0.05) in allele frequencies of rs1761667 or in genotype distribution and allele frequencies of rs3173798. The genotype distribution of rs1761667 significantly differed between CHD patients and controls (P=0.034), with a significantly higher frequency of the AG genotype in the CHD group compared to the control group (P=0.011). The plasma levels of ox-LDL in patients with the AG genotype were remarkably higher than those with the GG and AA genotypes (P=0.010). In a randomized sample taken from patients in the two groups, the CD36 mRNA expression of the CHD patients was higher than that of the controls. In CHD patients, the CD36 mRNA expression in AG genotype patients was remarkably higher than in those with an AA genotype (P=0.005). After adjusted logistic regression analysis, the AG genotype of rs1761667 was associated with an increased risk of CHD (OR=2.337, 95% CI=1.336-4.087, P=0.003). In conclusion, the rs1761667 polymorphism may be closely associated with developing CHD in the Chongqing Han population of China, and an AG genotype may be a genetic susceptibility factor for CHD.

Aminopeptidase N/CD13 is associated with raft membrane microdomains in monocytes

DEFF Research Database (Denmark)

Navarrete Santos, A; Roentsch, J; Danielsen, E M

2000-01-01

as in adhesion and cell-cell interactions. Here, we report for the first time that aminopeptidase N/CD13 in monocytes is partially localized in detergent-insoluble membrane microdomains enriched in cholesterol, glycolipids, and glycosylphosphoinositol-anchored proteins, referred to as "rafts." Raft fractions...... of monocytes were characterized by the presence of GM1 ganglioside as raft marker molecule and by the high level of tyrosine-phosphorylated proteins. Furthermore, similar to polarized cells, rafts in monocytic cells lack Na(+), K(+)-ATPase. Cholesterol depletion of monocytes by methyl-beta-cyclodextrin greatly...... reduces raft localization of aminopeptidase N/CD13 without affecting ala-p-nitroanilide cleaving activity of cells....

Linkage mapping of a dominant male sterility gene Ms-cd1 in Brassica oleracea

NARCIS (Netherlands)

Wang, X.; Lou, P.; Bonnema, A.B.; Yang, Boujun; He, H.; Zhang, Y.; Fang, Z.

2005-01-01

The dominant male sterility gene Ms-cd1 (c, cabbage; d, dominant) was identified as a spontaneous mutation in the spring cabbage line 79-399-3. The Ms-cd1 gene is successfully applied in hybrid seed production of several Brassica oleracea cultivars in China. Amplified fragment length polymorphism

The CNGRC-GG-D(KLAKLAK)2 peptide induces a caspase-independent, Ca2+-dependent death in human leukemic myeloid cells by targeting surface aminopeptidase N/CD13.

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Bouchet, Sandrine; Tang, Ruoping; Fava, Fanny; Legrand, Ollivier; Bauvois, Brigitte

2016-04-12

The CD13 antigen's binding site for the Asn-Gly-Arg (NGR) motif enables NGR-containing chemotherapeutic drugs to be delivered to CD13-positive tumours. Human CD13-positive acute myeloid leukemia (AML) cells proliferate abnormally and escape death. Here, we show that the CNGRC-GG-D(KLAKLAK)2 peptide induces death in AML cell lines (U937, THP-1, NB4, HL-60) and primary blood cells from AML patients. Cell death was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization and membrane disruption. Our results demonstrate in U937 cells that (i) the NGR-peptide triggers the loss of mitochondrial potential(ΔΨm) and generates superoxide anion (O2-), (ii) N-acetyl-L-cysteine (NAC) and extra/intracellular Ca2+ chelators (BAPTA) prevent both O2- production and cell death, (iii) the Ca2+-channel blocker nifedipine prevents cell death (indicating that Ca2+ influx is the initial death trigger), and (iv) BAPTA, but not NAC, prevents ΔΨm loss (suggesting O2- is a mitochondrial downstream effector). AML cell lines and primary blasts responding to the lethal action of NGR-peptide express promatrix metalloproteinase-12 (proMMP-12) and its substrate progranulin (an 88 kDa cell survival factor). A cell-free assay highlighted proMMP-12 activation by O2-. Accordingly, NGR-peptide's downregulation of 88 kDa progranulin protein was prevented by BAPTA and NAC. Conversely, AML blast resistance to NGR-peptide is associated with the expression of a distinct, 105 kDa progranulin isoform. These results indicate that CNGRC-GG-D(KLAKLAK)2 induces death in AML cells through the Ca2+-mitochondria-O2.-pathway, and support the link between proMMP-12 activation and progranulin cleavage during cell death. Our findings may have implications for the understanding of tumour biology and treatment.

Altered phenotypic and functional characteristics of CD3+CD56+ NKT-like cells in human gastric cancer.

Science.gov (United States)

Peng, Liu-Sheng; Mao, Fang-Yuan; Zhao, Yong-Liang; Wang, Ting-Ting; Chen, Na; Zhang, Jin-Yu; Cheng, Ping; Li, Wen-Hua; Lv, Yi-Pin; Teng, Yong-Sheng; Guo, Gang; Luo, Ping; Chen, Weisan; Zou, Quan-Ming; Zhuang, Yuan

2016-08-23

CD3+CD56+ natural killer T (NKT)-like cells are a group of CD3+ T cells sharing characteristics of NK and T cells and constitute a major component of host anti-tumor immune response in human cancer. However, the nature, function and clinical relevance of CD3+CD56+ NKT-like cells in human gastric cancer (GC) remain unclear. In this study, we showed that the frequencies of CD3+CD56+NKT-like cells in GC tumors were significantly decreased and low levels of tumor-infiltrating CD3+CD56+ NKT-like cells were positively correlated with poor survival and disease progression. Most CD3+CD56+NKT-like cells in GC tumors were CD45RA-CD27+/- central/effector-memory cells with decreased activity and lower expression levels of CD69, NKG2D and DNAM-1 than those in non-tumor tissues. We further observed that tumor-infiltrating CD3+CD56+ NKT-like cells had impaired effector function as shown by decreased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Moreover, in vitro studies showed that soluble factors released from GC tumors could induce the functional impairment of CD3+CD56+ NKT-like cells. Collectively, our data indicate that decreased tumor-infiltrating CD3+CD56+ NKT-like cells with impaired effector function are associated with tumor progression and poor survival of GC patients, which may contribute to immune escape of GC.

Screening for Cd-Safe Cultivars of Chinese Cabbage and a Preliminary Study on the Mechanisms of Cd Accumulation

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Jingjie Wang

2017-04-01

Full Text Available With the rapid progress of industrialization, the effects of environmental contamination on plant toxicity, and subsequently on human health, is a growing concern. For example, the heavy metal pollution of soil such as that caused by cadmium (Cd is a serious threat. Therefore, screening for pollution-safe edible plants is an essential approach for growing plants under heavy metal-contaminated soils. In the current study, 35 Chinese cabbage (Brassica pekinensis L. cultivars were selected with the aim of screening for Cd-safe cultivars (CSCs, analyzing their safety, and exploring the mechanism of Cd accumulation. Our field-culture experiments revealed that the Cd content in the edible parts of the cultivars were varied and were determined to possibly be CSCs. Hydroponics experiments were used to simulate six different degrees of soil contamination (high and low Cd concentrations on possible CSCs. The results indicated a significant difference (p < 0.05 in Cd concentration in the cultivars, and verified the safety of these possible CSCs. The analyses of the transport coefficient and expression levels showed that the differences in Cd accumulation among the Chinese cabbage cultivars were related to the expression of genes involved in absorption and transport rather than a root-to-shoot translocation limitation.

Activation of human CD141+ and CD1c+ dendritic cells in vivo with combined TLR3 and TLR7/8 ligation.

Science.gov (United States)

Pearson, Frances E; Chang, Karshing; Minoda, Yoshihito; Rojas, Ingrid M Leal; Haigh, Oscar L; Daraj, Ghazal; Tullett, Kirsteen M; Radford, Kristen J

2018-04-01

Mice reconstituted with human hematopoietic stem cells are valuable models to study aspects of the human immune system in vivo. We describe a humanized mouse model (hu mice) in which fully functional human CD141 + and CD1c + myeloid and CD123 + plasmacytoid dendritic cells (DC) develop from human cord blood CD34 + cells in immunodeficient mice. CD141 + DC are the human equivalents of murine CD8 + /CD103 + DC which are essential for the induction of tumor-inhibitory cytotoxic T lymphocyte responses, making them attractive targets to exploit for the development of new cancer immunotherapies. We used CD34 + -engrafted NSG-A2 mice to investigate activation of DC subsets by synthetic dsRNA or ssRNA analogs polyinosinic-polycytidylic acid/poly I:C and Resiquimod/R848, agonists for TLR3 and TLR8, respectively, both of which are expressed by CD141 + DC. Injection of hu mice with these agonists resulted in upregulation of costimulatory molecules CD80, CD83 and CD86 by CD141 + and CD1c + DC alike, and their combination further enhanced expression of these molecules by both subsets. When combined, poly I:C and R848 enhanced serum levels of key cytokines associated with cross-presentation and the induction of cytotoxic T lymphocyte responses including IFN-α, IFN-β, IL-12 and CXCL10. These data advocate a combination of poly I:C and R848 TLR agonists as means of activating human DC for immunotherapy. © 2018 Australasian Society for Immunology Inc.

Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro.

Science.gov (United States)

McCord, Amy M; Jamal, Muhammad; Shankavaram, Uma T; Shankavarum, Uma T; Lang, Frederick F; Camphausen, Kevin; Tofilon, Philip J

2009-04-01

In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell-like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133(+) cells sorted from three GB neurosphere cultures at 7% O(2) reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133(+) cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell-like phenotype of CD133(+) GB cells.

CD146 positive human dental pulp stem cells promote regeneration of dentin/pulp-like structures.

Science.gov (United States)

Matsui, Mikiko; Kobayashi, Tomoko; Tsutsui, Takeo W

2018-04-01

CD146 and STRO-1 are endothelial biomarkers that are co-expressed on the cellular membranes of blood vessels within human dental pulp tissue. This study characterized the percentage of dentin-like structures produced by CD146-positive (CD146 + ) human dental pulp stem cells (DPSCs), compared with their CD146-negative (CD146 - ) counterparts. DPSC populations were enriched using magnetic-activated cell sorting (MACS), yielding CD146 + and CD146 - cells, as well as mixtures composed of 25% CD146 + cells and 75% CD146 - cells (CD146 +/- ). Cell growth assays indicated that CD146 + cells exhibit an approximate 3-4 h difference in doubling time, compared with CD146 - cells. Cell cycle distributions were determined by flow cytometry analysis. The low percentage of CD146 + cells' DNA content in G 0 /G 1 phase were compared with CD146 - and non-separated cells. In contrast to CD146 - and non-separated cells, prompt mineralization was observed in CD146 + cells. Subsequently, qRT-PCR revealed high mRNA expression of CD146 and Alkaline phosphatase in mineralization-induced CD146 + cells. CD146 + cells were also observed high adipogenic ability by Oil red O staining. Histological examinations revealed an increased area of dentin/pulp-like structures in transplanted CD146 + cells, compared with CD146 - and CD146 +/- cells. Immunohistochemical studies detected dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP), as well as human mitochondria, in transplanted DPSCs. Co-expression of CD146 and GFP indicated that CD146 was expressed in transplanted CD146 + cells. CD146 + cells may promote mineralization and generate dentin/pulp-like structures, suggesting a role in self-renewal of stem cells and dental pulp regenerative therapy.

Human influenza viruses and CD8(+) T cell responses.

Science.gov (United States)

Grant, Emma J; Quiñones-Parra, Sergio M; Clemens, E Bridie; Kedzierska, Katherine

2016-02-01

Influenza A viruses (IAVs) cause significant morbidity and mortality worldwide, despite new strain-specific vaccines being available annually. As IAV-specific CD8(+) T cells promote viral control in the absence of neutralizing antibodies, and can mediate cross-reactive immunity toward distinct IAVs to drive rapid recovery from both mild and severe influenza disease, there is great interest in developing a universal T cell vaccine. However, despite detailed studies in mouse models of influenza virus infection, there is still a paucity of data on human epitope-specific CD8(+) T cell responses to IAVs. This review focuses on our current understanding of human CD8(+) T cell immunity against distinct IAVs and discusses the possibility of achieving a CD8(+) T cell mediated-vaccine that protects against multiple, distinct IAV strains across diverse human populations. We also review the importance of CD8(+) T cell immunity in individuals highly susceptible to severe influenza infection, including those hospitalised with influenza, the elderly and Indigenous populations. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses.

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Ezzelarab, Mohamed; Ezzelarab, Corin; Wilhite, Tyler; Kumar, Goutham; Hara, Hidetaka; Ayares, David; Cooper, David K C

2011-01-01

Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non-human primate T-cell responses has not been studied. Mesenchymal stromal cells from wild-type (WT), α1,3-galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T-cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T-cell proliferation and the production of pro-inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated. α1,3-galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T-cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, human CD4(+) T-cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN-γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4(+) T-cell responses to GTKO and GTKO/CD46 pMSC were comparable with those to hMSC, and all were significantly lower than to GTKO pAEC. GTKO/CD46 pMSC downregulated human T-cell proliferation as efficiently as hMSC. The level of proinflammatory cytokines IL-2, IFN-γ, TNF-α, and sCD40L correlated with the downregulation of T-cell proliferation by all types of MSC. Genetically modified pMSC is significantly less

CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

Science.gov (United States)

Wong, Kuan Y; Baron, Rebecca; Seldon, Therese A; Jones, Martina L; Rice, Alison M; Munster, David J

2018-05-15

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83 + human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83 + B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83 - ) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells. Copyright © 2018 by The American Association of Immunologists, Inc.

ADAMTS13 Gene Mutations in Children with Hemolytic Uremic Syndrome

Science.gov (United States)

Choi, Hyoung Soo; Cheong, Hae Il; Kim, Nam Keun

2011-01-01

We investigated ADAMTS13 activity as well as the ADAMTS13 gene mutation in children with hemolytic uremic syndrome (HUS). Eighteen patients, including 6 diarrhea-negative (D-HUS) and 12 diarrhea-associated HUS (D+HUS) patients, were evaluated. The extent of von Willebrand factor (VWF) degradation was assayed by multimer analysis, and all exons of the ADAMTS13 gene were PCR-amplified using Taq DNA polymerase. The median and range for plasma activity of ADAMTS13 in 6 D-HUS and 12 D+HUS patients were 71.8% (22.8-94.1%) and 84.9% (37.9-119.9%), respectively, which were not statistically significantly different from the control group (86.4%, 34.2-112.3%) (p>0.05). Five ADAMTS13 gene mutations, including 2 novel mutations [1584+2T>A, 3941C>T (S1314L)] and 3 polymorphisms (Q448E, P475S, S903L), were found in 2 D-HUS and one D+HUS patients, which were not associated with deficiency of ADAMTS13 activity. Whether these mutations without reduced ADAMTS13 activity are innocent bystanders or predisposing factors in HUS remains unanswered. PMID:21488199

Cadmium(Cd)-induced oxidative stress down-regulates the gene expression of DNA mismatch recognition proteins MutS homolog 2 (MSH2) and MSH6 in zebrafish (Danio rerio) embryos

Energy Technology Data Exchange (ETDEWEB)

Hsu, Todd, E-mail: toddhsu@mail.ntou.edu.tw [Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan (China); Huang, Kuan-Ming; Tsai, Huei-Ting; Sung, Shih-Tsung; Ho, Tsung-Nan [Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan (China)

2013-01-15

DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops in eukaryotes is initiated by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a genotoxic heavy metal that has been recognized as a human carcinogen. Oxidant stress and inhibition of DNA repair have been proposed as major factors underlying Cd genotoxicity. Our previous studies indicated the ability of Cd to disturb the gene expression of MSH6 in zebrafish (Danio rerio) embryos. This study was undertaken to explore if Cd-induced oxidative stress down-regulated MSH gene activities. Following the exposure of zebrafish embryos at 1 h post fertilization (hpf) to sublethal concentrations of Cd at 3-5 {mu}M for 4 or 9 h, a parallel down-regulation of MSH2, MSH6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene expression was detected by real-time RT-PCR and the expression levels were 40-50% of control after a 9-h exposure. Cd exposure also induced oxidative stress, yet no inhibition of catalase gene activity was observed. Whole mount in situ hybridization revealed a wide distribution of msh6 mRNA in the head regions of 10 hpf embryos and pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), D-mannitol or N-acetylcysteine (NAC) at 1-10 {mu}M restored Cd-suppressed msh6 expression. QPCR confirmed the protective effects of antioxidants on Cd-suppressed msh2/msh6 mRNA production. Down-regulated MSH gene activities reaching about 50% of control were also induced in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, or hydrogen peroxide at 200 {mu}M. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules. The transcriptional activation of human msh6 is known to be fully dependent on the specificity factor 1 (Sp1). Cd failed to inhibit the DNA binding activity of zebrafish Sp1 unless at lethal concentrations based on band shift assay, therefore

IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination.

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Wijesundara, Danushka K; Jackson, Ronald J; Tscharke, David C; Ranasinghe, Charani

2013-09-23

We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

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López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.; Gumperz, Jenny; Adams, Erin J. (UC); (UW-MED)

2014-10-02

Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.

Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34(+) cells.

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Podsakoff, Greg M; Engel, Barbara C; Carbonaro, Denise A; Choi, Chris; Smogorzewska, Elzbieta M; Bauer, Gerhard; Selander, David; Csik, Susan; Wilson, Kathy; Betts, Michael R; Koup, Richard A; Nabel, Gary J; Bishop, Keith; King, Steven; Schmidt, Manfred; von Kalle, Christof; Church, Joseph A; Kohn, Donald B

2005-07-01

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (<1/1,000,000) at most times over 2 years. In one patient, a reappearance of PBMC containing the huM10 gene, but not the FX gene, occurred concomitant with a rise in the HIV-1 viral load during a period of nonadherence to the antiretroviral regimen. Unique clones of gene-marked PBMC were detected by LAM-PCR during the time of elevated HIV-1 levels. These findings indicate that there was a selective survival advantage for PBMC containing the huM10 gene during the time of increased HIV-1 load.

Evaluation of the impact of chitosan/DNA nanoparticles on the differentiation of human naive CD4{sup +} T cells

Energy Technology Data Exchange (ETDEWEB)

Liu Lanxia; Bai Yuanyuan; Zhu Dunwan; Song Liping; Wang Hai; Dong Xia; Zhang Hailing; Leng Xigang, E-mail: lengxg@bme.org.cn [Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Biomedical Materials, Lab of Bioengineering, Institute of Biomedical Engineering (China)

2011-06-15

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4{sup +} T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4{sup +} T cells. The indirect impact of CS/DNA nanoparticles on naive CD4{sup +} T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4{sup +} T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4{sup +} T cells to explore their direct impact on naive CD4{sup +} T cell differentiation by measuring the release of IL-4 and IFN-{gamma} from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4{sup +} T cells. It was also demonstrated that, when directly exposed to the naive CD4{sup +} T cells, the nanoparticles induced neither the activation of the naive CD4{sup +} T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-{gamma}) that induce naive CD4{sup +} T cell polarization, nor any changes in the differentiation direction of naive CD4{sup +} T cells in the presence of the corresponding

[Effect of cisplatin on the expression of Pokemon gene: experiment with different human lung cancer cells].

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Zhao, Zhi-Hong; Wang, Sheng-Fa; Yu, Liang; Wang, Ju; Cong, De-Gang; Chang, Hao; Wang, Xue-Feng; Zhang, Tie-Wa; Zhang, Jian; Fu, Kai; Jiang, Jiu-Yang

2008-04-29

To investigate the correlation between Pokemon gene and cisplatin mechanism. Human lung adenocarcinoma cells of the lines A549 and AGZY83-a, human lung squamous carcinoma cells of the line HE-99, and human giant cell lung cancer cells of the line 95D were cultured and cisplatin was added into the medium. Other lung cancer cells of the above mentioned lines were cultured in the medium without cisplatin and were used as control groups. RT-PCR and Western blotting were used to detect the mRNA and protein expression of Pokemon. Pokemon mRNA and protein were expressed highly in all the 4 cell lines. The Pokemon gene expression did not changed significantly after cisplatin treatment groups. There were not significant differences in the mRNA and protein expression of Pokemon among the 4 experiment groups and the control groups (all P > 0.05). Cisplatin has no effect on the Pokemon gene expression of the human lung cancer cells.

Enrichment of CD44 in basal-type breast cancer correlates with EMT, cancer stem cell gene profile, and prognosis

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Xu HX

2016-01-01

Full Text Available Hanxiao Xu,1 Yijun Tian,1 Xun Yuan,1 Yu Liu,2 Hua Wu,1 Qian Liu,1 Gen Sheng Wu,3,4 Kongming Wu1 1Department of Oncology, 2Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 3Department of Oncology, 4Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA Abstract: Cluster of differentiation 44 (CD44 is a transmembrane glycoprotein that serves as the receptor for the extracellular matrix component hyaluronic acid. CD44 has been reported to play key roles in cell proliferation, motility, and survival, but its role in breast cancer remains controversial. In this study, we conducted a meta-analysis. A total of 23 published Gene Expression Omnibus databases were included to evaluate the association between CD44 mRNA expression and clinicopathological characteristics or prognosis of the patients with breast cancer. Our analysis revealed that CD44 expression was associated with clinicopathological features, including the histological grade, estrogen receptor status, progesterone receptor status, and human epidermal growth factor receptor-2 status. Higher levels of CD44 expression were observed in the basal subtype of breast cancer both at the mRNA and protein levels (odds ratio [OR] =2.08, 95% confidence interval [CI]: 1.72–2.52; OR =2.11, 95% CI: 1.67–2.68. Patients with CD44 overexpression exhibited significantly worse overall survival (hazard ratio =1.27; 95% CI: 1.04–1.55. Whole gene profile analysis revealed that CD44 expression was enriched in basal-type breast cancer and correlated with epithelial–mesenchymal transition and cancer stem cell gene profiles. In summary, our analyses indicated that CD44 potentially might be a prognostic marker for breast cancer and thus can serve as a therapeutic target for basal-type breast cancer. Keywords: breast cancer, CD44, survival prediction, meta

Altered expression of the TCR signaling related genes CD3 and FcεRIγ in patients with aplastic anemia

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Li Bo

2012-03-01

Full Text Available Abstract Background Aplastic anemia (AA is characterized by pancytopenia and bone marrow hypoplasia, which results from immune-mediated hematopoiesis suppression. Understanding the pathophysiology of the immune system, particularly T cells immunity, has led to improved AA treatment over the past decades. However, primary and secondary failure after immunosuppressive therapy is frequent. Thus, knowledge of the immune mechanisms leading to AA is crucial to fundamentally understand the disease. Findings To elucidate the T cell receptor (TCR signal transduction features in AA, the expression levels of CD3γ, δ, ε and ζ chain and FcεRIγ genes, which are involved in TCR signal transduction, and the negative correlation of the expression levels between the CD3ζ and FcεRIγ genes in T cells from peripheral blood mononuclear cells (PBMCs were analyzed. Real-time RT-PCR using the SYBR Green method was used to detect the expression level of these genes in PBMCs from 18 patients with AA and 14 healthy individuals. The β2microglobulin gene (β2M was used as an endogenous reference. The expression levels of the CD3γ, CD3δ, CD3ε and CD3ζ genes in patients with AA were significantly increased compared to a healthy control group, whereas the FcεRIγ gene expression level was significantly decreased in patients with AA in comparison with the healthy control group. Moreover, the negative correlation of the expression levels between the CD3ζ and FcεRIγ genes was lost. Conclusions To our knowledge, this is the first report of the CD3γ, CD3δ, CD3ε, CD3ζ and FcεRIγ gene expression in patients with AA. The abnormally expressed TCR signaling related genes may relate to T cells dysfunction in AA.

Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

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Prakash Babu Narasimhan

2018-04-01

Full Text Available A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma or to live microfilariae (mf of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β, M2-associated (CCL13, CD206, Mreg-associated (IL-10, TGF-β, and angiogenesis associated (MMP9, VEGF genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

Science.gov (United States)

Narasimhan, Prakash Babu; Akabas, Leor; Tariq, Sameha; Huda, Naureen; Bennuru, Sasisekhar; Sabzevari, Helen; Hofmeister, Robert; Nutman, Thomas B; Tolouei Semnani, Roshanak

2018-04-01

A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-β), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

Molecular mapping of MS-cd 1 gene in Chinese kale | Zhang ...

African Journals Online (AJOL)

A dominant male sterility (DGMS) line 79-399-3 was developed from spontaneous mutation in Brassica oleracea var. capitata and has been widely used in the production of hybrid cultivar in China. In this line, male sterility is controlled by a dominant gene Ms-cd1. In the present study, primary mapping of Ms-cd1 was ...

An association study of 13 SNPs from seven candidate genes with pediatric asthma and a preliminary study for genetic testing by multiple variants in Taiwanese population.

Science.gov (United States)

Wang, Jiu-Yao; Liou, Ya-Huei; Wu, Ying-Jye; Hsiao, Ya-Hsin; Wu, Lawrence Shih-Hsin

2009-03-01

Asthma is one of the most common chronic diseases in children. It is caused by complex interactions between various genetic factors and exposures to environmental allergens and irritants. Because of the heterogeneity of the disease and the genetic and cultural differences among different populations, a proper association study and genetic testing for asthma and susceptibility genes is difficult to perform. We assessed 13 single-nucleotide polymorphisms (SNPs) in seven well-known asthma susceptibility genes and looked for association with pediatric asthma using 449 asthmatic subjects and 512 non-asthma subjects in Taiwanese population. CD14-159 C/T and MS4A2 Glu237Gly were identified to have difference in genotype/allele frequencies between the control group and asthma patients. Moreover, the genotype synergistic analysis showed that the co-contribution of two functional SNPs was riskier or more protective from asthma attack. Our study provided a genotype synergistic method for studying gene-gene interaction on polymorphism basis and genetic testing using multiple polymorphisms.

Gene transfer into hematopoietic stem cells reduces HLH manifestations in a murine model of Munc13-4 deficiency.

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Soheili, Tayebeh; Durand, Amandine; Sepulveda, Fernando E; Rivière, Julie; Lagresle-Peyrou, Chantal; Sadek, Hanem; de Saint Basile, Geneviève; Martin, Samia; Mavilio, Fulvio; Cavazzana, Marina; André-Schmutz, Isabelle

2017-12-26

Patients with mutations in the UNC13D gene (coding for Munc13-4 protein) suffer from familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a life-threatening immune and hyperinflammatory disorder. The only curative treatment is allogeneic hematopoietic stem cell (HSC) transplantation, although the posttreatment survival rate is not satisfactory. Here, we demonstrate the curative potential of UNC13D gene correction of HSCs in a murine model of FHL3. We generated a self-inactivating lentiviral vector, used it to complement HSCs from Unc13d -deficient (Jinx) mice, and transplanted the cells back into the irradiated Jinx recipients. This procedure led to complete reconstitution of the immune system (ie, to wild-type levels). The recipients were then challenged with lymphocytic choriomeningitis virus to induce hemophagocytic lymphohistiocytosis (HLH)-like manifestations. All the clinical and biological signs of HLH were significantly reduced in mice having undergone HSC UNC13D gene correction than in nontreated animals. This beneficial effect was evidenced by the correction of blood cytopenia, body weight gain, normalization of the body temperature, decreased serum interferon-γ level, recovery of liver damage, and decreased viral load. These improvements can be explained by the restoration of the CD8 + T lymphocytes' cytotoxic function (as demonstrated here in an in vitro degranulation assay). Overall, our results demonstrate the efficacy of HSC gene therapy in an FHL-like setting of immune dysregulation.

DMPD: LPS induction of gene expression in human monocytes. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 11257452 LPS induction of gene expression in human monocytes. Guha M, Mackman N. Ce...ll Signal. 2001 Feb;13(2):85-94. (.png) (.svg) (.html) (.csml) Show LPS induction of gene expression in human... monocytes. PubmedID 11257452 Title LPS induction of gene expression in human monocytes. Authors Guha M, Ma

Evaluation of a nanotechnology-based approach to induce gene-expression in human THP-1 macrophages under inflammatory conditions.

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Bernal, Laura; Alvarado-Vázquez, Abigail; Ferreira, David Wilson; Paige, Candler A; Ulecia-Morón, Cristina; Hill, Bailey; Caesar, Marina; Romero-Sandoval, E Alfonso

2017-02-01

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

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Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

2016-10-17

Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Deep Surveying of the Transcriptional and Alternative Splicing Signatures for Decidual CD8+ T Cells at the First Trimester of Human Healthy Pregnancy

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Weihong Zeng

2018-05-01

Full Text Available Decidual CD8+ (dCD8 T cells have been proposed to play important roles in immune protection against the invading pathogens and in tolerance toward the growing semi-allogeneic fetus during early pregnancy. However, their phenotypic and functional characteristics remain poorly defined. Here, we performed the first analysis of the transcriptional and alternative splicing (AS signatures for human first-trimester dCD8 T cells using high-throughput mRNA sequencing. Our data revealed that dCD8 T cells have distinct transcriptional and AS landscapes when compared with their autologous peripheral blood CD8+ (pCD8 T counterparts. Furthermore, human dCD8 T cells were observed to contain CD8-Treg and effector-memory T-cell subsets, and display enhanced functionality in terms of degranulation and cytokine production on a per-cell basis. Additionally, we have identified the novel splice junctions that use a high ratio of the non-canonical splicing motif GC-AG and found that AS is not a major contributor to the gene expression-level changes between paired pCD8 and dCD8 T cells. Together, our findings not only provide a comprehensive framework of the transcriptional and AS landscapes but also reveal the functional feature of human dCD8 T cells, which are of great importance in understanding the biology of these cells and the physiology of human healthy pregnancy.

Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord

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Naimisha Beeravolu

2016-05-01

Full Text Available Human umbilical cord (hUC blood and tissue are non-invasive sources of potential stem/progenitor cells with similar cell surface properties as bone marrow stromal cells (BMSCs. While they are limited in cord blood, they may be more abundant in hUC. However, the hUC is an anatomically complex organ and the potential of cells in various sites of the hUC has not been fully explored. We dissected the hUC into its discrete sites and isolated hUC cells from the cord placenta junction (CPJ, cord tissue (CT, and Wharton's jelly (WJ. Isolated cells displayed fibroblastoid morphology, and expressed CD29, CD44, CD73, CD90, and CD105, and showed evidence of differentiation into multiple lineages in vitro. They also expressed low levels of pluripotency genes, OCT4, NANOG, SOX2 and KLF4. Passaging markedly affected cell proliferation with concomitant decreases in the expression of pluripotency and other markers, and an increase in chondrogenic markers. Microarray analysis further revealed the differences in the gene expression of CPJ-, CT- and WJ-hUC cells. Five coding and five lncRNA genes were differentially expressed in low vs. high passage hUC cells. Only MAEL was expressed at high levels in both low and high passage CPJ-hUC cells. They displayed a greater proliferation limit and a higher degree of multi-lineage differentiation in vitro and warrant further investigation to determine their full differentiation capacity, and therapeutic and regenerative medicine potential.

Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome

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Caroline Y. Kuo

2018-05-01

Full Text Available X-linked hyper-immunoglobulin M (hyper-IgM syndrome (XHIM is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9 platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs, as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

The diabetogenic VPS13C/C2CD4A/C2CD4B rs7172432 variant impairs glucose-stimulated insulin response in 5,722 non-diabetic Danish individuals

DEFF Research Database (Denmark)

Grarup, N; Overvad, M; Sparsø, T

2011-01-01

A genome-wide association study in the Japanese population reported two genome-wide significant loci associated with type 2 diabetes of which the VPS13C/C2CD4A/C2CD4B locus was replicated in Europeans. We looked for potential associations between the diabetogenic VPS13C/C2CD4A/C2CD4B rs7172432 va...

Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression.

Directory of Open Access Journals (Sweden)

Ingo Ahrens

Full Text Available Endothelial progenitor cells (EPCs can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP, PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15 or pro-angiogenic (galectin-3 properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP was the most up-regulated gene.

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

DEFF Research Database (Denmark)

Leslie, Robert Graham Quinton; Prodinger, Wolfgang Maria; Nielsen, Claus Henrik

2003-01-01

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined...... the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1...... and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max...

Roles of the sister chromatid cohesion apparatus in gene expression, development, and human syndromes

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Dorsett, Dale

2006-01-01

The sister chromatid cohesion apparatus mediates physical pairing of duplicated chromosomes. This pairing is essential for appropriate distribution of chromosomes into the daughter cells upon cell division. Recent evidence shows that the cohesion apparatus, which is a significant structural component of chromosomes during interphase, also affects gene expression and development. The Cornelia de Lange (CdLS) and Roberts/SC phocomelia (RBS/SC) genetic syndromes in humans are caused by mutations affecting components of the cohesion apparatus. Studies in Drosophila suggest that effects on gene expression are most likely responsible for developmental alterations in CdLS. Effects on chromatid cohesion are apparent in RBS/SC syndrome, but data from yeast and Drosophila point to the likelihood that changes in expression of genes located in heterochromatin could contribute to the developmental deficits. PMID:16819604

[Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

Science.gov (United States)

Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

2015-10-01

To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene.

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Dobrovolsky, Vasily N; Revollo, Javier; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Lin, Haixia

2015-10-01

A major question concerning the scientific and regulatory acceptance of the rodent red blood cell-based Pig-a gene mutation assay is the extent to which mutants identified by their phenotype in the assay are caused by mutations in the Pig-a gene. In this study, we identified T-lymphocytes deficient for the glycosylphosphatidylinositol-anchored surface marker, CD48, in control and 7,12-dimethylbenz[a]anthracene (DMBA)-treated rats using a flow cytometric assay and determined the spectra of mutations in the endogenous Pig-a gene in these cells. CD48-deficient T-cells were seeded by sorting at one cell per well into 96-well plates, expanded into clones, and exons of their genomic Pig-a were sequenced. The majority (78%) of CD48-deficient T-cell clones from DMBA-treated rats had mutations in the Pig-a gene. The spectrum of DMBA-induced Pig-a mutations was dominated by mutations at A:T, with the mutated A being on the nontranscribed strand and A → T transversion being the most frequent change. The spectrum of Pig-a mutations in DMBA-treated rats was different from the spectrum of Pig-a mutations in N-ethyl-N-nitrosourea (ENU)-treated rats, but similar to the spectrum of DMBA mutations for another endogenous X-linked gene, Hprt. Only 15% of CD48-deficient mutants from control animals contained Pig-a mutations; T-cell biology may be responsible for a relatively large fraction of false Pig-a mutant lymphocytes in control animals. Among the verified mutants from control rats, the most common were frameshifts and deletions. The differences in the spectra of spontaneous, DMBA-, and ENU-induced Pig-a mutations suggest that the flow cytometric Pig-a assay detects de novo mutation in the endogenous Pig-a gene. © 2015 Wiley Periodicals, Inc.

Soluble CD14 in human breast milk and its role in innate immune responses.

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Vidal, K; Labéta, M O; Schiffrin, E J; Donnet-Hughes, A

2001-10-01

Immune factors secreted in milk are important for health in the neonatal gut. We have detected the bacterial pattern recognition receptor, soluble CD14 (sCD14) in human breast milk at different times during lactation. The molecule occurs in a single form in milk, in contrast to human serum, in which there are two isoforms. Produced by mammary epithelial cells, milk sCD14 mediates secretion of innate immune response molecules such as interleukin-8, tumor necrosis factor-alpha, and epithelial neutrophil activator-78 by CD14-negative intestinal epithelial cells exposed to lipopolysaccharide (LPS) or bacteria. Although present at low concentrations in milk, LPS-binding protein may be implicated in the biological effects observed. Our findings support the premise that milk sCD14 acts as a 'sentinel' molecule and immune modulator in homeostasis and in the defense of the neonatal intestine. In so doing, it may prevent the immune and inflammatory conditions of the gut to which non-breastfed infants are predisposed.

The importance of a sub-region on chromosome 19q13.3 for prognosis of multiple myeloma patients after high-dose treatment and stem cell support: a linkage disequilibrium mapping in RAI and CD3EAP

DEFF Research Database (Denmark)

Vangsted, Annette Juul; Klausen, Tobias Wirenfeldt; Gimsing, Peter

2011-01-01

The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor ¿B (NF-¿B). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated with inte......The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor ¿B (NF-¿B). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated...... with interferon-a (INF-a) as maintenance treatment, 177 patients treated with thalidomide, and 74 patients treated with bortezomib at relapse and address if the effects of polymorphisms in CD3EAP and RAI are modified by a functional polymorphism in NF¿B1. By linkage disequilibrium mapping, we found that variant...... alleles of several polymorphisms in a sub-region of 19q13.3 spanning the regions RAI-intron1-1 to RAI intron1-3 and the region exon1 to exon3-6 in CD3EAP were associated with prolonged time-to-treatment failure (TTF; p¿=¿0.003) and overall survival (OS; p¿=¿0.02). Haplotype analyses revealed that none...

The importance of a sub-region on chromosome 19q13.3 for prognosis of multiple myeloma patients after high-dose treatment and stem cell support: a linkage disequilibrium mapping in RAI and CD3EAP

DEFF Research Database (Denmark)

Vangsted, Annette J.; Klausen, Tobias Wirenfeldt; Gimsing, Peter

2011-01-01

The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor κB (NF–κB). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated with inte......The gene RAI was originally described as an inhibitor of RelA/p65 subunit of nuclear factor κB (NF–κB). Here, we analyse the association between genetic variation in the genes RAI and CD3EAP and outcome of 348 myeloma patients treated with high-dose treatment (HDT), 146 patients treated...... with interferon-α (INF-α) as maintenance treatment, 177 patients treated with thalidomide, and 74 patients treated with bortezomib at relapse and address if the effects of polymorphisms in CD3EAP and RAI are modified by a functional polymorphism in NFКB1. By linkage disequilibrium mapping, we found that variant...... alleles of several polymorphisms in a sub-region of 19q13.3 spanning the regions RAI-intron1-1 to RAI intron1-3 and the region exon1 to exon3–6 in CD3EAP were associated with prolonged time-to-treatment failure (TTF; p = 0.003) and overall survival (OS; p = 0.02). Haplotype analyses revealed that none...

Expression of zinc and cadmium responsive genes in leaves of willow (Salix caprea L.) genotypes with different accumulation characteristics

International Nuclear Information System (INIS)

Konlechner, Cornelia; Türktaş, Mine; Langer, Ingrid; Vaculík, Marek; Wenzel, Walter W.; Puschenreiter, Markus; Hauser, Marie-Theres

2013-01-01

Salix caprea is well suited for phytoextraction strategies. In a previous survey we showed that genetically distinct S. caprea plants isolated from metal-polluted and unpolluted sites differed in their zinc (Zn) and cadmium (Cd) tolerance and accumulation abilities. To determine the molecular basis of this difference we examined putative homologues of genes involved in heavy metal responses and identified over 200 new candidates with a suppression subtractive hybridization (SSH) screen. Quantitative expression analyses of 20 genes in leaves revealed that some metallothioneins and cell wall modifying genes were induced irrespective of the genotype's origin and metal uptake capacity while a cysteine biosynthesis gene was expressed constitutively higher in the metallicolous genotype. The third and largest group of genes was only induced in the metallicolous genotype. These data demonstrate that naturally adapted woody non-model species can help to discover potential novel molecular mechanisms for metal accumulation and tolerance. -- Highlights: ► The transcriptional Zn/Cd response of the willow Salix caprea was quantified. ► Two genotypes from a highly contaminated and a control environment were compared. ► In addition to candidate genes an SSH library revealed over 200 metal induced genes. ► Constitutive upregulation and isolate-specific induction of genes were revealed. ► Willows adapt to environmental Zn/Cd contamination on the transcriptional level. -- Expression analyzes reveal different responses to Cd and Zn exposure of S. caprea genotypes with different heavy metal accumulation abilities

Parvovirus B19 integration into human CD36+ erythroid progenitor cells.

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Janovitz, Tyler; Wong, Susan; Young, Neal S; Oliveira, Thiago; Falck-Pedersen, Erik

2017-11-01

The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration. Copyright © 2017 Elsevier Inc. All rights reserved.

Natural selection on protein-coding genes in the human genome

DEFF Research Database (Denmark)

Bustamente, Carlos D.; Fledel-Alon, Adi; Williamson, Scott

2005-01-01

, showing an excess of deleterious variation within local populations 9, 10 . Here we contrast patterns of coding sequence polymorphism identified by direct sequencing of 39 humans for over 11,000 genes to divergence between humans and chimpanzees, and find strong evidence that natural selection has shaped......Comparisons of DNA polymorphism within species to divergence between species enables the discovery of molecular adaptation in evolutionarily constrained genes as well as the differentiation of weak from strong purifying selection 1, 2, 3, 4 . The extent to which weak negative and positive darwinian...... selection have driven the molecular evolution of different species varies greatly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , with some species, such as Drosophila melanogaster, showing strong evidence of pervasive positive selection 6, 7, 8, 9 , and others, such as the selfing weed Arabidopsis thaliana...

Characterization of a human X-linked gene from the DXS732E locus in the candidate region for the anhidrotic ectodermal dysplasia (EDA) gene (Xq13.1)

Energy Technology Data Exchange (ETDEWEB)

Gault, J.; Zonana, J. [Oregon Health Sciences Univ., Portland, OR (United States); Zeltinger, J. [Univ. of Washington, Seattle, WA (United States)] [and others

1994-09-01

A conserved mouse genomic clone was used to identify a homologous human genomic clone (the DXS732E locus), which was subsequently employed to isolate cDNAs from a human fetal brain library. Nine unique overlapping cDNAs were isolated, and sequences analysis of 3.9 kb identified a putative 1 kb ORF. GRAIL analysis of the sequence supported the hypothesis that the putative ORF was coding sequence, and Prosite analysis of the putative ORF identified potential glycosylation and phosphorylation sites. The 5{prime} end of the gene maps within a CpG island, and comparison of cDNA sequences indicate the gene is alternatively spliced at its 3{prime} end. Northern analysis and RT-PCR indicate that two different sized messages appear to be expressed with the gene expressed in human fetal kidney, intestine, brain, and muscle. The gene is expressed in 77 day human skin, a time when hair follicle formation occurs. Anhidrotic ectodermal dysplasia (EDA) results in the abnormal morphogenesis of hair, teeth and eccrine sweat glands. A positional cloning strategy towards cloning the EDA gene had been used, and deletion and X-autosome translocation patients have been useful in further delimiting the EDA region. The present gene at the DXS732E locus is partially deleted in one EDA patient who does not have other apparent abnormalities. No rearrangements of the gene have been detected in two female X-autosome translocation EDA patients, nor in four additional male patients with submicroscopic molecular deletions.

Eco RI RFLP in the human IGF II gene

Energy Technology Data Exchange (ETDEWEB)

Cocozza, S; Garofalo, S; Robledo, R; Monticelli, A; Conti, A; Chiarotti, L; Frunzio, R; Bruni, C B; Varrone, S

1988-03-25

The probe was a 500 bp cDNA containing exons 2-3 and 4 of the human IGF II gene. The clone was isolated by screening a human liver cDNA library with synthetic oligonucleotides. Eco RI digestion of genomic DNA and hybridization with the IGF II probe detects a two allele polymorphism with allelic fragments of 13.5 kb and 10.5 kb. The frequency was studied 38 unrelated Caucasians: Human IGF II gene was localized on the short arm of chromosome 11 (p15) by in situ hybridization. Codominant segregation was observed in 2 Caucasian families (10 individuals).

Quantitative gene expression profiling of CD45(+) and CD45(-) skeletal muscle-derived side population cells

DEFF Research Database (Denmark)

Andersen, Ditte Caroline; Kristiansen, Gitte Qvistgaard; Jensen, Line

2011-01-01

transcripts associated with endothelial cells, Notch signaling and myogenic precursors. By comparing the mRNA signatures of mSPs with those of adipose tissue-derived SP populations, a common endothelial component seemed to reside in both muscle and fat-derived SPCD45(-) entities. However, each SP subset......The skeletal muscle-derived side population (mSP) which highly excludes Hoechst 33342 is composed of CD45(+) and CD45(-) subpopulations; yet, rareness of mSP cells in general has complicated extensive quantitative analysis of gene expression profiles in primarily isolated mSP cells. Here, we...... describe the isolation of adult mouse normal skeletal muscle residing SPCD45(+) and SPCD45(-) cells from a parent mononuclear muscle-derived cell (MDC) population. Relative quantitative real time PCR (RT-PCR) of 64 genes revealed that mSPCD45(-) compared with mSPCD45(+) was enriched for cells expressing...

THE J = 1-0 TRANSITIONS OF 12CH+, 13CH+, AND 12CD+

International Nuclear Information System (INIS)

Amano, T.

2010-01-01

A new set of laboratory experimental frequencies for the J = 1-0 rotational transition of 12 CH + , 13 CH + , and 12 CD + are obtained by using a liquid nitrogen cooled extended negative glow discharge in a gas mixture of CH 4 and He. These frequencies are found to be significantly different from those reported previously. The unexpectedly large Zeeman effect and the spin-rotation hyperfine interaction for a 1 Σ molecule are observed. The Zeeman effect and the hyperfine interaction appear to be distinctively different for each isotopic species. Theoretical considerations reveal the isotopic dependence of the magnitudes of these effects, and they also provide strong evidence for the identifications.

Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

Science.gov (United States)