Semi-quantitative analysis of multiple cytokines in canine peripheral blood mononuclear cells by a single tube RT-PCR

2001 ◽  
Vol 83 (3-4) ◽  
pp. 191-202 ◽  
Author(s):  
C. Chamizo ◽  
J.M. Rubio ◽  
J. Moreno ◽  
J. Alvar
Keyword(s):  
Quantitative Analysis ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Single Tube ◽  
Blood Mononuclear Cells

Downregulating the Fibromodulin Gene by Short Interfering RNA Causes B-CLL Cell Death.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 176-176 ◽  
Author(s):  
Aniruddha Choudhury ◽  
Katja Derkow ◽  
Eva Mikaelsson ◽  
Parviz Kokhaei ◽  
Anders Österborg ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Rt Pcr ◽  
Sirna Transfection ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Extracellular Matrix Molecule ◽  
Rnai Technology ◽  
Blood Mononuclear Cells

Abstract Development of targeted therapies against B-CLL is dependent on the identification of molecules that are essential for the proliferation and survival of the leukemic cells. One such molecule investigated by us as a putative leukemia-associated target is Fibromodulin (FIM). FIM is an extracellular matrix molecule belonging to the leucin-rich proteoglycan family. Our laboratory studies have verified that expression of FIM is upregulated in B-CLL cells. Moreover, this molecule is specifically overexpressed in B-CLL cells and not on normal peripheral blood mononuclear cells. Analysis of FIM expression on various other hematological tumor cells have revealed that with the exception of mantle cell lymphoma, FIM is not expressed in other hematological malignancies. RNAi technology has recently emerged as a powerful method to specifically silence the expression of a gene. We have generated three siRNA against various segments of FIM and tested the effects of these siRNA on B-CLL cells. Transfection of B-CLL cells with these siRNA significantly diminished, or completely abrogated the expression of FIM mRNA as detected by RT-PCR. The figure below shows silencing of the FIM gene following siRNA transfection, as assayed by RT-PCR. “Untrans” and “ctrl” represents untransfected and control siRNA-transfected B-CLL cells respectively. As noted, transfection with each of the three siRNA against FIM completely abrogated the expression of FIM siRNA. 24–48 hours after siRNA transfection, a substantial fraction (30–70%) of the B-CLL cells, compared to cells transfected with control non-silencing siRNA, went into apoptosis as assayed by Annexin-V-propidium iodide staining. Time kinetic studies revealed that siRNA mediated silencing and apoptosis occurred between 18 and 48 hours. No such effect was noted when peripheral blood mononuclear cells of healthy donors or FIM-positive fibroblast cell lines were transfected with siRNA. In reconstitution experiments, siRNA treated B-CLL cells were co-cultured with various numbers of FIM-positive fibroblasts. The presence of these fibroblasts greatly diminished the apoptosis of B-CLL cells induced after siRNA treatment. Our results indicate that overexpression of FIM in B-CLL cells are critical for their survival and FIM may serve as a leukemia-specific target for B-CLL therapy. Figure Figure

scholarly journals Detection and analysis of an alternatively spliced isoform of interleukin-6 mRNA in peripheral blood mononuclear cells

Blood ◽  
1995 ◽  
Vol 86 (12) ◽  
pp. 4559-4567 ◽  
Author(s):  
DP Kestler ◽  
S Agarwal ◽  
J Cobb ◽  
KM Goldstein ◽  
RE Hall
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Interleukin 6 ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cdna Probe ◽  
Northern Analysis ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Alternatively Spliced

Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the entire coding region of the gene were used in reverse transciptase- polymerase chain reaction (RT-PCR) studies to examine lL-6 expression in peripheral blood mononuclear cells (PBMC). In addition to the predicted 0.64-kb RT-PCR product, a second 0.45-kb product was observed. Cloning and dideoxy sequence analysis of this product revealed evidence for an alternatively spliced lL-6 transcript lacking exon II. Further RT-PCR analysis using forward primers ending at or one base before the exon I donor splice site again yielded both products. Additional primers were designed and successfully used to selectively distinguish the two forms of IL-6 transcript. Both transcripts were prominent in peripheral blood monocytes and lymphocytes, whereas only the 0.64-kb, full-length transcript was prominent in the lL-6-producing 5637 (human bladder carcinoma) cell line. Northern analysis revealed, in addition to the predominant 1.3-kb transcript, several minor transcripts at 1.9 to 4.8 kb that hybridized with the alternatively spliced cDNA probe but not with an exon II probe. Western analysis revealed lL-6 polypeptides of predicted size (26 to 29 kD) in culture medium from PBMC, while showing an immunoreactive band at 17 kD in cell lysates. These findings suggest the existence of an alternatively spliced form of lL-6 mRNA, which would encode for a polypeptide missing the gp130 interactive (signal-transducing) domain contained in exon II while retaining the lL-6 receptor (p80) domain. Such a molecule could in theory function as a natural antagonist of lL-6, as it would be expected to bind to the IL-6 receptor but not lead to signal transduction.

scholarly journals Inflammation and regulatory T cell genes are differentially expressed in peripheral blood mononuclear cells of Parkinson’s disease patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zerrin Karaaslan ◽  
Özlem Timirci Kahraman ◽  
Elif Şanlı ◽  
Hayriye Arzu Ergen ◽  
Canan Ulusoy ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Functional Annotation ◽  
Mononuclear Cells ◽  
Differentially Expressed ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

AbstractOur aim was to identify the differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMC) of Parkinson’s disease (PD) patients and healthy controls by microarray technology and analysis of related molecular pathways by functional annotation. Thirty PD patients and 30 controls were enrolled. Agilent Human 8X60 K Oligo Microarray was used for gene level expression identification. Gene ontology and pathway enrichment analyses were used for functional annotation of DEGs. Protein–protein interaction analyses were performed with STRING. Expression levels of randomly selected DEGs were quantified by real time quantitative polymerase chain reaction (RT-PCR) for validation. Flow cytometry was done to determine frequency of regulatory T cells (Tregs) in PBMC. A total of 361 DEGs (143 upregulated and 218 downregulated) were identified after GeneSpring analysis. DEGs were involved in 28 biological processes, 12 cellular components and 26 molecular functions. Pathway analyses demonstrated that upregulated genes mainly enriched in p53 (CASP3, TSC2, ATR, MDM4, CCNG1) and PI3K/Akt (IL2RA, IL4R, TSC2, VEGFA, PKN2, PIK3CA, ITGA4, BCL2L11) signaling pathways. TP53 and PIK3CA were identified as most significant hub proteins. Expression profiles obtained by RT-PCR were consistent with microarray findings. PD patients showed increased proportions of CD49d+ Tregs, which correlated with disability scores. Survival pathway genes were upregulated putatively to compensate neuronal degeneration. Bioinformatics analysis showed an association between survival and inflammation genes. Increased CD49d+ Treg ratios might signify the effort of the immune system to suppress ongoing neuroinflammation.

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