Ectopic Expression of Guanylyl Cyclase C in CD34+Progenitor Cells in Peripheral Blood

2001 ◽  
Vol 19 (19) ◽  
pp. 3951-3959 ◽  
Author(s):  
Tracy A. Fava ◽  
Rodwige Desnoyers ◽  
Stephanie Schulz ◽  
Jason Park ◽  
David Weinberg ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Epithelial Cell ◽  
Healthy Volunteers ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Total Rna ◽  
Blood Mononuclear Cells

PURPOSE: To examine the utility of guanylyl cyclase C (GC-C)–specific nested reverse transcriptase polymerase chain reaction (RT-PCR) to detect circulating tumor cells in patients with colorectal cancer.PATIENTS AND METHODS: Peripheral-blood mononuclear cells from 24 patients with Dukes’ stage D colorectal cancer were analyzed by GC-C-specific nested RT-PCR using 1 μg of total RNA. Peripheral-blood mononuclear cells from 20 healthy volunteers served as controls. Additionally, peripheral-blood CD34+progenitor cells were assayed for the expression of both GC-C and other epithelial cell–specific markers.RESULTS: GC-C mRNA was detected in blood mononuclear cells from all 24 patients with colorectal cancer and all healthy volunteers. These unexpected positive results reflected low-level ectopic transcription of GC-C in CD34+progenitor cells. Moreover, CD34+progenitor cells expressed other epithelial cell–specific markers, including prostate-specific antigen, prostate-specific membrane antigen, carcinoembryonic antigen, CK-19, CK-20, mucin 1, and GA733.2. Limiting the quantity of mononuclear cell total RNA analyzed to ≤ 0.8 μg eliminated detection of GC-C and other tissue-specific transcripts in blood of healthy volunteers. However, under the same conditions, GC-C mRNA was detected in mononuclear cells from all 24 patients with metastatic colorectal cancer. Using 0.5 μg of total RNA and GC-C–specific primers, nested RT-PCR detected a single human colon carcinoma cell (approximately 20 to 200 GC-C transcripts/cell) in 106to 107mononuclear blood cells.CONCLUSION: These data suggest that GC-C may be useful for detecting circulating colorectal cancer cells. They also demonstrate that CD34+cells are a source of ectopically expressed epithelial cell–specific markers and that CD34+cells may contribute to the high false-positive rate generally observed when those markers are used to detect rare circulating metastatic cancer cells by RT-PCR.

scholarly journals Effect of Lamivudine on the Plasma and Intracellular Pharmacokinetics of Apricitabine, a Novel Nucleoside Reverse Transcriptase Inhibitor, in Healthy Volunteers

2007 ◽  
Vol 51 (8) ◽  
pp. 2943-2947 ◽  
Author(s):  
T. Holdich ◽  
L. A. Shiveley ◽  
J. Sawyer
Keyword(s):  
Reverse Transcriptase ◽  
Healthy Volunteers ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Transcriptase Inhibitor ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Reverse Transcriptase Inhibitor

ABSTRACT Apricitabine is a novel deoxycytidine analog reverse transcriptase inhibitor. In vitro apricitabine competes with other deoxycytidine analogues for intracellular phosphorylation mediated by deoxycytidine kinase. The topic of this study, the effect of concomitant administration of apricitabine and lamivudine on the plasma and intracellular pharmacokinetics of the two compounds, was investigated in healthy volunteers. Participants (n = 21; age, 18 to 30 years) received apricitabine at 600 mg twice daily, lamivudine at 300 mg once daily, and the two treatments in combination for 4 days each in random order. Plasma, urine, and intracellular pharmacokinetics were assessed on day 4 of each treatment period. Apricitabine was rapidly absorbed after oral administration, with peak concentrations being attained after a mean of 1.76 h. Coadministration with lamivudine had no significant effect on the plasma and urine pharmacokinetics of apricitabine. However, the formation of apricitabine triphosphate in peripheral blood mononuclear cells was markedly reduced after the coadministration of apricitabine and lamivudine than after the administration of apricitabine alone: the area under the concentration-time curve from 0 to 12 h for apricitabine triphosphate during combination treatment was ca. 15% of that seen after the administration of apricitabine alone. In contrast, apricitabine had no effect on the plasma pharmacokinetics of lamivudine or on the formation of lamivudine triphosphate in peripheral blood mononuclear cells. These results are consistent with in vitro findings that lamivudine inhibits the intracellular phosphorylation of apricitabine. In conjunction with similar in vitro observations for emtricitabine and apricitabine, these results suggest that apricitabine should not be coadministered with other deoxycytidine analogues for the treatment of human immunodeficiency virus infection.

scholarly journals Red Wine Intake Prevents Nuclear Factor-κB Activation in Peripheral Blood Mononuclear Cells of Healthy Volunteers During Postprandial Lipemia

Circulation ◽  
2000 ◽  
Vol 102 (9) ◽  
pp. 1020-1026 ◽  
Author(s):  
Luis Miguel Blanco-Colio ◽  
Mónica Valderrama ◽  
Luis Antonio Alvarez-Sala ◽  
Carmen Bustos ◽  
Mónica Ortego ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Red Wine ◽  
Mononuclear Cells ◽  
Postprandial Lipemia ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Peripheral Blood Mononuclear ◽  
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 Immune Characterization Of Metastatic Colorectal Cancer Patients Post Reovirus Administration

2020 ◽  
Author(s):  
Ruwan Parakrama ◽  
Elisha Fogel ◽  
Carol Chandy ◽  
Titto Augustine ◽  
Matt Coffey ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Flow Cytometry ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Sequencing Analysis ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract Background KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is underway in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome.Methods Reovirus was administered as a 60-minute intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3x1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay, was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001. Results Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p=0.05); day 15 for GM-CSF (3.56; p=0.009), IFN-Ƴ (1.86; p=0.0004) and IL-12p70 (2.42; p=0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p=0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p=0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98-fold; 98% increase, p=0.00007). APCs were stimulated within 48 hours and activated (CD8+ CD70+) T cells within 168 hours as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x) , IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (>0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days.Conclusions Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi-layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.

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