scholarly journals Absolute measurement of androgen receptor mRNA in peripheral blood mononuclear, preputial skin and urethral mucosa cells of control individuals with phimosis using qRT-PCR

2011 ◽  
Vol 55 (8) ◽  
pp. 665-668 ◽  
Author(s):  
Tatiane Sousa e Silva ◽  
Flavio Richetti ◽  
Daniela Patricia Palmeira Santos Cunha ◽  
Antonio Carlos Moreira Amarante ◽  
Jovelino Quintino de Souza Leão ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Peripheral Blood ◽  
External Genitalia ◽  
Absolute Measurement ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Skin Cells ◽  
Qrt Pcr ◽  
Specific Manner ◽  
The Mean

INTRODUCTION: Androgen actions are exerted upon the androgen receptor (AR), and complete genital virilization of normal 46,XY individuals depends on adequate function and expression of the AR gene in a tissue-specific manner. OBJECTIVE: Standardization of normal ARmRNA in androgen-sensitive tissues. MATERIALS AND METHODS: In this study, we determined the quantitative amounts of ARmRNA in peripheral blood mononuclear, urethral mucosa and preputial skin cells of control subjects with phimosis by using RT-PCR. RESULTS: The mean (SD) values of AR expression in blood, urethra and prepuce were: 0.01 (0.01); 0.43 (0.32); 0.31 (0.36), respectively. CONCLUSION: The AR expression is low in blood and equivalent in urethral mucosa and preputial skin, which may be useful in the diagnosis of individuals with abnormal external genitalia.

Circular RNA expression profiles and bioinformatic analysis in coronary heart disease

Epigenomics ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 439-454 ◽  
Author(s):  
Fangpu Yu ◽  
Yuanyuan Tie ◽  
Ya Zhang ◽  
Zunzhe Wang ◽  
Liwen Yu ◽  
...  
Keyword(s):  
Coronary Heart Disease ◽  
Heart Disease ◽  
Coronary Arteries ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Peripheral Blood Mononuclear ◽  
Qrt Pcr ◽  
Blood Mononuclear Cells

Aim: We aimed to identify the expression profile and role of circular RNAs (circRNAs) in coronary heart disease (CHD). Materials & methods: We performed sequence analysis of circRNAs in peripheral blood mononuclear cells of 70 CHD patients and 30 controls. Eight selected circRNAs were validated using quantitative real-time polymerase chain reaction (qRT-PCR) in human atherosclerotic coronary arteries. Results: In total, 2283 downregulated and 85 upregulated circRNAs were identified in CHD. Parental genes of top 100 dysregulated-circRNAs are related to metabolism and protein modification, and 12 circRNAs might upregulate their CHD-related parental genes through miRNA sponges. Of the eight circRNAs validated in atherosclerotic coronary arteries by qRT-PCR, six were consistent with sequencing results of peripheral blood mononuclear cells. Conclusion: As potential ceRNAs, dysregulated circRNAs may be involved in CHD pathophysiology.

scholarly journals Oropouche Virus Infects, Persists and Induces IFN Response in Human Peripheral Blood Mononuclear Cells as Identified by RNA PrimeFlow™ and qRT-PCR Assays

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 785
Author(s):  
Mariene Ribeiro Amorim ◽  
Marjorie Cornejo Pontelli ◽  
Gabriela Fabiano de Souza ◽  
Stéfanie Primon Muraro ◽  
Daniel A. Toledo-Teixeira ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Human Leukocytes ◽  
Qrt Pcr ◽  
Viral Antigens ◽  
Blood Mononuclear Cells

Oropouche orthobunyavirus (OROV) is an emerging arbovirus with a high potential of dissemination in America. Little is known about the role of peripheral blood mononuclear cells (PBMC) response during OROV infection in humans. Thus, to evaluate human leukocytes susceptibility, permissiveness and immune response during OROV infection, we applied RNA hybridization, qRT-PCR and cell-based assays to quantify viral antigens, genome, antigenome and gene expression in different cells. First, we observed OROV replication in human leukocytes lineages as THP-1 monocytes, Jeko-1 B cells and Jurkat T cells. Interestingly, cell viability and viral particle detection are maintained in these cells, even after successive passages. PBMCs from healthy donors were susceptible but the infection was not productive, since neither antigenome nor infectious particle was found in the supernatant of infected PBMCs. In fact, only viral antigens and small quantities of OROV genome were detected at 24 hpi in lymphocytes, monocytes and CD11c+ cells. Finally, activation of the Interferon (IFN) response was essential to restrict OROV replication in human PBMCs. Increased expression of type I/III IFNs, ISGs and inflammatory cytokines was detected in the first 24 hpi and viral replication was re-established after blocking IFNAR or treating cells with glucocorticoid. Thus, in short, our results show OROV is able to infect and remain in low titers in human T cells, monocytes, DCs and B cells as a consequence of an effective IFN response after infection, indicating the possibility of leukocytes serving as a trojan horse in specific microenvironments during immunosuppression.

scholarly journals miR-106a Is Downregulated in Peripheral Blood Mononuclear Cells of Chronic Hepatitis B and Associated with Enhanced Levels of Interleukin-8

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Zhongsi Hong ◽  
Haiyu Hong ◽  
Jian Liu ◽  
Xiaobin Zheng ◽  
Mingxing Huang ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis B ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Luciferase Activity ◽  
Peripheral Blood Mononuclear ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Blood Mononuclear Cells

Aims. This study aimed to investigate miR-106a expression in peripheral blood mononuclear cells (PBMCs) of chronic hepatitis B (CHB) patients and to analyze the function of miR-106a.Materials and Methods. miR-106a expression levels in PBMCs from 40 healthy controls and 56 CHB patients were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The luciferase activity assays were used to determine whether miR-106a binds to 3′UTR of IL-8. miR-106a mimics and inhibitors were transfected into healthy PBMCs. IL-8 mRNA and protein levels were detected and determined by qRT-PCR and ELISA, respectively.Results. The qRT-PCR results suggested that the PBMC miR-106a levels were decreased in CHB patients. IL-8 was augmented in CHB patients and was inversely correlated with miR-106a levels. The luciferase activity assays indicated that IL-8 is a target of miR-106a. Exogenous expression of miR-106a could significantly repress IL-8 expression at both mRNA and protein levels in PBMCs, whereas miR-106a inhibitor had the opposite effects.Conclusions. This study suggested that miR-106a is downregulated in PBMCs of CHB patients and that miR-106a may play an important role in CHB by targeting IL-8.

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 Cross-sectional quantitative RT-PCR study of feline coronavirus viremia and replication in peripheral blood of healthy shelter cats in Southern California

2017 ◽  
Vol 20 (4) ◽  
pp. 295-301 ◽  
Author(s):  
Eric J Fish ◽  
Pedro Paulo VP Diniz ◽  
Yen-Chen Juan ◽  
Frank Bossong ◽  
Ellen W Collisson ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Southern California ◽  
Buffy Coat ◽  
Sample Collection ◽  
Rt Pcr ◽  
Case Control Studies ◽  
M Gene ◽  
Cross Sectional ◽  
Qrt Pcr ◽  
Feline Coronavirus

Objectives The objectives of this study were to determine the prevalence of feline coronavirus (FCoV) viremia, and its replication in peripheral blood using quantitative RT-PCR (qRT-PCR) methodology in a population of 205 healthy shelter cats in Southern California, as well as to assess any possible connection to longitudinal development of feline infectious peritonitis (FIP). Methods The study was performed on buffy-coat samples from EDTA-anticoagulated whole blood samples of 205 healthy shelter cats. From 50 of these cats, fecal samples were also examined. FCoV genomic and subgenomic RNA in the buffy coats was amplified by a total FCoV RNA qRT-PCR. Evidence for FCoV replication in peripheral blood and feces was obtained by M gene mRNA qRT-PCR. Results Nine of 205 cats (4.4%) were viremic by the total FCoV RNA qRT-PCR, and one of these cats had evidence of peripheral FCoV blood replication by an FCoV mRNA qRT-PCR. The single cat with peripheral blood replication had a unique partial M gene sequence distinct from positive controls and previously published FCoV sequences. Neither seven of the nine viremic cats with follow-up nor the single cat with replicating FCoV with positive qRT-PCR results developed signs compatible with FIP within 6 months of sample collection. Conclusions and relevance FCoV viremia and peripheral blood replication in healthy shelter cats have a low prevalence and do not correlate with later development of FIP in this study population, but larger case-control studies evaluating the prognostic accuracy of the qRT-PCR assays are needed.

scholarly journals Differential Expression of Neurokinin-1 Receptor by Human Mucosal and Peripheral Lymphoid Cells

2000 ◽  
Vol 7 (3) ◽  
pp. 371-376 ◽  
Author(s):  
Triona Goode ◽  
Joe O'Connell ◽  
Wen-Zhe Ho ◽  
Gerald C. O'Sullivan ◽  
J. Kevin Collins ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Lymphocytes ◽  
Lymphoid Cells ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Neurokinin 1 Receptor ◽  
Neurokinin 1

ABSTRACT Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.

scholarly journals A Pilot Study of Gene Expression Analysis in Peripheral Blood Mononuclear Cells in Response to a Hypocaloric Mediterranean Diet

2022 ◽  
Vol 2022 ◽  
pp. 1-8
Author(s):  
Daniel de Luis ◽  
David Primo ◽  
Olatz Izaola ◽  
Rocío Aller
Keyword(s):  
Gene Expression ◽  
Peripheral Blood ◽  
Dietary Intervention ◽  
Mononuclear Cells ◽  
Endocrine System ◽  
Family Type ◽  
Obese Patients ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
The Mean

Background. Few studies have examined gene expression in peripheral blood mononuclear cells (PBMCs) after a dietary intervention. Objective. Our study is aimed at evaluating in a pilot study the peripheral blood gene expression in obese patients after weight loss secondary to a hypocaloric Mediterranean diet. Design. A sample of 11 obese subjects without metabolic syndrome was enrolled. Biochemical, anthropometric parameters and microarray analysis were performed at baseline and after 6 months of dietary intervention. Results. The mean age was 43.1 ± 6.3 years, and the mean body mass index (BMI) was 38.6 ± 8.1  kg/m2. All the next improvements were statistically significant: body weight − 7.4 ± 1.9  kg, BMI - 2.5 ± 0.2  kg, fat mass − 5.7 ± 1.2  kg, waist circumference − 5.8 ± 1.2  cm, triglycerides − 17.4 ± 6.5  mg/dl, C-reactive protein − 3.1 ± 1.5  mg/dL, insulin − 2.1 ± 1.0  mUI/L, and HOMA-IR − 0.7 ± 0.2  units. We identified 634 differentially expressed genes: 262 genes with relative higher expression levels and 372 with lower expression levels. Cluster analysis showed 35 genes in nutritional disease and 17 genes in endocrine system. The most relevant gene was thyroid peroxidase (TPO), and this gene was overexpressed, and the next genes carbonic anhydrase VI (CA6), caveolin protein 1 (CAV1) and solute carrier family type 12 (SLLC12A3), soluble carrier family type 12 (SLLC12A3), beta 3 receptor (ADRB3), and glutamate receptor ionotropic N methyl D aspartate 2 A (GRIN2A) were all underexpressed. Conclusion. In PBMC from obese patients after a diet with a Mediterranean pattern, the expression of 634 genes, of the endocrine system and of nutritional disease, is modified.

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.

Leukoreduction System Chambers as a Source of Viable Human Peripheral Blood Mononuclear Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4912-4912
Author(s):  
Sinyoung Kim ◽  
Han-Soo Kim ◽  
Yangsoon Lee ◽  
Jaewoo Song ◽  
Hyun Ok Kim ◽  
...  
Keyword(s):  
Cell Count ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Buffy Coat ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
The Mean

Abstract Many blood banks are using apheresis machines to collect blood components such as platelets (PLTs), RBCs, or plasma. Especially, leukoreduced plateletpheresis using apheresis instrument (Trima Accel, Gambro BCT, Lakewood, CO) provided subsidiary cell products retained in leukoreduction system (LRS) chamber that was originally discarded. The LRS chamber is a conical-shaped chamber that uses saturated, fluidized, particle bed filtration technology to remove WBCs from PLTs. In the current study, a total of 24 LRS chambers from different donors were investigated to determine it would be a valuable source of viable human peripheral blood mononuclear cells (MNCs). The proportions of CD3+, CD19+, CD16+/CD56+, CD14+, CD45+ cells, and absolute CD34+ cell count within the LRS chambers were determined by flow cytometry. Dendritic cells (DCs) were generated from the immunomagnetically purified CD14+ cells from LRS chamber and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. In the LRS chamber, the total number of WBC count was 1.1 × 109 ± 0.3 × 109 and the mean percentage of MNCs was 80.6 ± 13.1%. The mean proportion of T cells, B cells, NK cells, CD14+ monocytes among CD45+ cells was 54.3 ± 11.5%, 6.4 ± 3.1%, 14.6 ± 3.9%, 12.9 ± 7.5%, respectively. Total absolute CD34+ cell count in LRS chamber was 0.95 × 106 ± 0.65 × 106. Also, we could demonstrate CD14+ cells isolated from LRS chamber was capable of differentiating into functionally mature DCs in vitro. LRS chambers are a valuable and convenient source of viable human peripheral blood mononuclear cell population and could replace standard buffy coat preparations for research applications.

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