Single-step duplex kDNA-PCR for detection of Leishmania donovani complex in human peripheral blood samples

1. We describe a rapid and reliable technique for the assessment of basal nitric oxide release in clinical situations, using peripheral blood polymorphonuclear leucocytes isolated by a single-step density gradient procedure. The assay is based on the quantitative conversion of oxyhaemoglobin to methaemoglobin by nitric oxide. We have further examined the ability of these cells to respond to various stimuli. 2. Basal (unstimulated) nitric oxide release occurred, which was augmented by superoxide dismutase. The mean value for healthy subjects was 283 ±96.7 pmolmin−1 10−6 cells. 3. Both phorbol myristate acetate and N-formyl-methionyl-leucylphenylalanine induced further release of nitric oxide, which was increased by preincubation with lipopolysaccharide, interleukin-6 and interferon-γ. 4. Preincubation of cells with NG-monomethyl-l-arginine or l-canavanine sulphate inhibited nitric oxide production. 5. The procedure provides a valuable tool for monitoring nitric oxide up-regulation in clinical situations.

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Leishmania donovani Parasites Interact with γ/δ+ Human Peripheral Blood T Cells and Induce Susceptibility to NK Cell-Mediated Lysis

Scandinavian Journal of Immunology ◽

10.1046/j.1365-3083.1999.00642.x ◽

1999 ◽

Vol 50 (6) ◽

pp. 588-595 ◽

Cited By ~ 6

Author(s):

Saha ◽

Chakrabarti ◽

Sen ◽

Bandyopadhyay

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Leishmania Donovani ◽

Nk Cell ◽

Human Peripheral Blood

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Analysis of Human Peripheral Blood Samples from Fatal and Nonfatal Cases of Ebola (Sudan) Hemorrhagic Fever: Cellular Responses, Virus Load, and Nitric Oxide Levels

Journal of Virology ◽

10.1128/jvi.78.19.10370-10377.2004 ◽

2004 ◽

Vol 78 (19) ◽

pp. 10370-10377 ◽

Cited By ~ 183

Author(s):

Anthony Sanchez ◽

Matthew Lukwiya ◽

Daniel Bausch ◽

Siddhartha Mahanty ◽

Angela J. Sanchez ◽

...

Keyword(s):

Nitric Oxide ◽

T Cells ◽

Peripheral Blood ◽

Ebola Virus ◽

Fas Ligand ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Blood Levels ◽

Blood Samples ◽

Virus Load

ABSTRACT Peripheral blood samples obtained from patients during an outbreak of Ebola virus (Sudan species) disease in Uganda in 2000 were used to phenotype peripheral blood mononuclear cells (PBMC), quantitate gene expression, measure antigenemia, and determine nitric oxide levels. It was determined that as the severity of disease increased in infected patients, there was a corresponding increase in antigenemia and leukopenia. Blood smears revealed thrombocytopenia, a left shift in neutrophils (in some cases degenerating), and atypical lymphocytes. Infected patients who died had reduced numbers of T cells, CD8+ T cells, and activated (HLA-DR+) CD8+ T cells, while the opposite was noted for patients who survived the disease. Expression levels of cytokines, Fas antigen, and Fas ligand (TaqMan quantitation) in PBMC from infected patients were not significantly different from those in uninfected patients (treated in the same isolation wards), nor was there a significant increase in expression compared to healthy volunteers (United States). This unresponsive state of PBMC from infected patients despite high levels of circulating antigen and virus replication suggests that some form of immunosuppression had developed. Ebola virus RNA levels (virus load) in PBMC specimens were found to be much higher in infected patients who died than patients who survived the disease. Similarly, blood levels of nitric oxide were much higher in fatal cases (increasing with disease severity), and extremely elevated levels (≥150 μM) would have negatively affected vascular tone and contributed to virus-induced shock.

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Screening of disease-related biomarkers related to neuropathic pain (NP) after spinal cord injury (SCI)

Human Genomics ◽

10.1186/s40246-021-00303-w ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Jia Zhao ◽

Li Yang ◽

Limin Huang ◽

Zinan Li

Keyword(s):

Neuropathic Pain ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Differentially Expressed ◽

Blood Samples ◽

Kegg Pathways ◽

Cord Injury ◽

Pain Pathway ◽

Molecular Expression ◽

Important Disease

Abstract Background Based on the molecular expression level, this paper compares lncRNA and mRNA expressions respectively in peripheral blood samples of the patients after SCI with NP and without NP, and screens disease-related biomarkers related to NP after SCI in peripheral blood samples of patients. Method The expression spectrum of 25 human peripheral blood samples (12 samples of refractory NP patients after SCI) was downloaded and data were normalized. Screening of GO annotations significantly associated with significant differentially expressed mRNAs and significant involvement of the KEGG pathway. The WGCNA algorithm was used to screen for modules and RNAs that were significantly associated with disease characterization. A co-expression network was constructed to extract the genes involved in the disease pathway from the co-expression network, construct a network of SCI pain-related pathways, and screen important disease-related biomarkers. Quantitative real-time PCR was used to detect the mRNA expression of hub genes. Results Data were normalized and re-annotated by detection of platform information, resulting in a total of 289 lncRNA and 18197 mRNAs. Screening resulted in 338 significant differentially expressed RNAs that met the threshold requirements. Differentially expressed RNAs were significantly enriched with the brown and magenta modules. Six KEGG signaling pathways were screened in the co-expression network, and three KEGG pathways with direct neuropathic pain were identified. The expression levels of E2F1, MAX, MITF, CTNNA1, and ADORA2B in the disease group were all significantly upregulated (p < 0.01). Compared with the normal group, the expression of OXTR was upregulated. Conclusion We speculate that there are 7 genes and 2 lncRNAs directly involved in the pain pathway: E2F1, MAX, MITF, CTNNA1, ADORA2B, GRIK3, OXTR, LINC01119, and LINC02447. These molecules may be important for NP after SCI.

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Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples

Bioscience Reports ◽

10.1042/bsr20203827 ◽

2021 ◽

Vol 41 (2) ◽

Author(s):

Amelia Jerram ◽

Thomas V. Guy ◽

Lucinda Beutler ◽

Bavani Gunasegaran ◽

Ronald Sluyter ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Gradient Centrifugation ◽

Effector Memory ◽

Low Density ◽

Blood Draw ◽

Blood Samples

Abstract We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

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