Reference gene stability in peripheral blood mononuclear cells determined by qPCR and NanoString

Quantitative real-time PCR (qPCR) is commonly used for gene expression analyses with defined documentation guidelines to compare published results. To minimize the impact of variances from qPCR performance, sample handling and processing reference genes are used. Their selection process cannot be completely aligned due to variations in experimental conditions. Furthermore, the named sources of error are also present when determining the stability of the reference genes themselves. Even software applications that are used to identify the best reference genes rarely coincide on their rankings and can be misleading under certain conditions. In previous experiments, peripheral blood mononuclear cells (PBMC) were analyzed to identify the most stable reference gene(s). Twelve of the 13 investigated genes showed sample type specific differences in the expression. Direct mRNA measurement was performed in the form of a NanoString analysis, a multiplexed absolute quantification method. The external validation showed a high concordance of the reference gene expression levels. However, it identified the same sample type specific expression pattern for only some of the tested reference genes. By comparing various combinations of reference genes with both methods we are able to suggest a set of well-performing reference genes. Figure

We here compare the expression of reference genes for qPCR and NanoString data and determine the value of the latter method as a bias-free mRNA quantification method     

Effect of some ent-kaurenes on the viability of human peripheral blood mononuclear cells

The possible cytotoxic activity of some ent-kaurenes on human mononuclear cells, obtained from peripheral blood, was studied having in mind future studies on their antitumor activity. The cells were obtained using the Ficoll-Hypaque method, adjusted to 2 x 10(6) cells/mL, and incubated with kaurenes for 48 hours at 3 x 10(-5), 30 x 10(-5), 300 x 10(-1) and 3000 x 10(-5) micromol/well. Ent-kaurenic acid showed no toxicity at all concentrations studied. The least toxic of all the kaurene derivatives studied was ent-15,16-epoxy-17-acetoxy-(-)-kauran-19-oic acid, with a cellular viability of 99% at 3 x l0(-5) micromol/well, and 94% at 30 x 10(-1) micromol/well. Another compound that showed low toxicity was the 2,3,4,6-tetra-acetyl-alpha-D-pyranosyl ester of ent-15-oxo-(-)-kaur-16-en-19-oic acid with 44% viability at 3000 x 10(-5) micromol/well. The most toxic compounds at all concentrations tested were ent-kaur-16-en-19-ol acetate and ent-16alpha-hydroxy-(-)-kauran-19-oic acid. On the other hand, ent-kaur-9(11)16-dien-19-oic acid, ent-kauran-19-oic acid, and ent-kaur-16-en-19-ol were toxic only at the highest concentration studied. According to these results, and considering the concentrations employed, ent-kaur-16-en-19-oic acid and ent-15,16-epoxy-17-acetoxy-(-)-kauran-19-oic acid could be used for in vivo experiments and possibly for therapeutic purposes on humans, without much risk.     

Cytokine production after helium-neon laser irradiation in cultures of human peripheral blood mononuclear cells.

The effects of laser light on the immune system have not been extensively characterized. Low-power laser sources, such as the helium-neon (He-Ne) laser with a wavelength of 632.8 nm, have been found to produce photobiological effects with evidence of interference with immunological functions. We have investigated the effects of He-Ne laser irradiation on Ficoll-Hypaque-isolated human peripheral blood mononuclear cells (PBMC). Cultured cells were irradiated for various times at two selected intensities and then stimulated with different mitogens. The rate of incorporation of 3H-thymidine into the DNA of stimulated cells decreased with increasing energy density. The levels of interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in supernatants of the cultures were determined (irradiated either before or after stimulation). When stimulating cells after irradiation, significantly increased levels of all cytokines were detected after 30 min of irradiation (18.9 J cm-2), whereas after 60 min of irradiation (37.8 J cm-2) cytokine levels were found to be significantly decreased.     

The biological effects of 632.8-nm low energy He-Ne laser on peripheral blood mononuclear cells in vitro

The aim of this study was to examine the proliferation of peripheral blood mononuclear cells due to the low energy 632.8-nm He-Ne laser application. The results of previous studies supported the hypothesis that low level laser therapy (LLLT) might have an increasing effect on the proliferation of lymphocytes and production of cytokines. The effect of laser irradiation was investigated by comparing the proliferation of peripheral blood mononuclear cells (PBMC) with a mitogenic stimulator, PHA (phytohemaglutinin) and laser irradiation. PBMCs of control samples, only laser irradiated samples, PHA included samples and both PHA included and laser irradiated samples were quantified and compared. Results of [3H] Thymidine test, 20 s laser irradiated and 40 s laser irradiated samples' proliferation were found statistically higher than control samples. There was no significant difference between control and 60 s laser irradiated samples. PHA also showed its ability to proliferate cells. PHA included samples and both PHA included and laser irradiated samples' proliferation was higher than both control and only laser irradiated samples. Our results showed that He-Ne laser application enhanced the proliferation significantly. Moreover, laser dose was noted as a significant parameter. On the other hand, LLLT by itself was found less effective than PHA.     

High-performance liquid chromatographic assay for thymidylate synthase from the human malaria parasite, Plasmodium falciparum

A rapid and highly sensitive high-performance liquid chromatographic assay for thymidylate synthase activity is described. The assay is based on the separation of the substrate, deoxyuridylate (dUMP), and its product, deoxythymidylate (dTMP), on a LiChrosorb RP-8 reversed-phase column with 44 mM triethylammonium phosphate (pH 7.0) as mobile phase and a flow-rate of 1.0 ml/min. In addition, using a mu Bondapak C18 reversed-phase column with 10 mM potassium phosphate (pH 4.0) and a gradient of 0-28% methanol, dUMP, dTMP and deoxythymidine (dTdR) are well separated within 30 min. The latter system is also applied to assay thymidine kinase activity with dTdR and dTMP as substrate and product, respectively. This method is sensitive enough to measure dTMP at concentrations as low as 25 pmol, and it was used to show that crude extracts of the human malaria parasite Plasmodium falciparum contain thymidylate synthase but not thymidine kinase activity.     

Correction of peripheral blood mononuclear cell cytosolic protein for hemoglobin contamination

Pharmacodynamic (PD) analysis requires accurate and precise quantification of enzyme activity targeted by anticancer agents in surrogate cells like peripheral blood mononuclear cells (PBMCs). Enzyme activity is normally reported per mass unit of protein input. However, high and fluctuating hemoglobin (Hb) contamination strongly influences the protein content of PBMC cytosolic lysate. We present the development and validation of a spectrophotometrical Hb quantification method to correct for this contamination. The applicability of Hb correction was demonstrated by determination of the dihydropyrimidine dehydrogenase enzyme activity in PBMC cytosolic lysates.     

Prompt and delayed nonsteroidal anti-inflammatory drug-photoinduced DNA damage in peripheral blood mononuclear cells measured with the comet assays

Nonsteroidal anti-inflammatory drug (NSAID)-photoinduced DNA damage in human peripheral blood mononuclear cells measured using the alkaline comet assay is presented. Whereas Tiaprofenic Acid-photoinduced DNA damage was promptly induced (i.e. observed at relatively low radiation doses), Ketoprofen-photoinduced DNA damage was delayed. This prompt and delayed effect is observed with UVA (320-400 nm), UVB (290-320 nm) and solar-simulated radiation and is attributed to the different photochemical properties of NSAID. The results from these experiments, carried out in living cells, confirm the speculations of NSAID-photoinduced DNA damage brought up by the many experiments conducted in solution.     

Two mononuclear cobalt(III) complexes exhibiting phenoxazinone synthase activity

Two mononuclear cobalt(III) complexes, namely [LCo(tmtp)(H₂O)]ClO₄?MeOH ( 1 ) (tmtp = tri(m‐tolyl)phosphine) and [LCo(PPh₃)(H₂O)]PF₆ ( 2 ), have been prepared from a polydentate ligand, N,N′‐bis(3‐methoxysalicylidehydene)cyclohexane‐1,2‐diamine ( H ₂ L ). Standard analytical techniques such as elemental analysis and UV–visible and Fourier transform infrared spectroscopies were used to characterize both complexes. The solid‐state molecular structures of both complexes were confirmed from single‐crystal X‐ray diffraction analysis. Structural analyses show that the Co(III) ion occupies the centre of a distorted octahedron in a complex cation: [LCo(tmtp)(H₂O)]⁺ and [LCo(PPh₃)(H₂O)]⁺ for 1 and 2 , respectively. Phenoxazinone synthase activities of both complexes were screened. Kinetic studies and other experimental observations reveal that the reaction follows rate saturation kinetics and proceeds through the formation of a catalyst (complex)–substrate adduct. The turnover number (K_cat) of complex 2 is 54.07 h^?1, exhibiting better catalytic activity compared to 1 (K_cat = 45.11 h^?1).     

Determination of 18 nucleobases, nucleosides and nucleotides in human peripheral blood mononuclear cells by isocratic solvent-generated ion-pair chromatography

The paper describes a method for the separation of 18 nucleotides, nucleosides and nucleobases by isocratic solvent-generated ion-pair chromatography in 55 min. It has been developed for pharmacodynamic monitoring of mycophenolic acid, the active metabolite of the immunosuppressant mycophenolate mofetil. The method was applied for the detection of mycophenolic acid-induced changes in inosine 5′-monophosphate dehydrogenase (IMPDH) activity in the lysate of human peripheral blood mononuclear cells.     

Fluorimetric assay of redox activity in cells

A simple, sensitive procedure is described for measurement of the redox activity of single cells based on cyanotolyltetrazolium chloride which is reduced to a fluorescent monoformazan. Its efficiency is studied by incubating vital ascites tumor cells or cells treated with different fixatives, under different oxygen concentrations. Redox activity (fluorescence of the formazan) and DNA content (fluorescence of Hoechst dye 333378) of ascites tumor cells are measured simultaneously for the first time, by flow cytometry.     

Olive oil and its phenolic constituent tyrosol attenuates dioxin-induced toxicity in peripheral blood mononuclear cells via an antioxidant-dependent mechanism

Olive oil (OO) and its phenolic compounds are reported to possess many potential biological effects, which are ascribed to its powerful antioxidant property. In this study, we have assessed whether OO and its phenolic compound tyrosol (TY) could mitigate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced oxidative damages in peripheral blood mononuclear cells (PBMC). The results showed that exposure of PBMC to 10 nM TCDD caused significant cell death and elevated cellular concentrations of reactive oxygen species and lipid peroxidation. Comet assay indicated that OO and TY protected DNA damage against dioxin toxicity. In addition, alterations in levels of antioxidant enzymes were substantially prevented by OO and TY. TCDD-induced CYP1A1 activity and loss of mitochondrial membrane potential were significantly reduced by the administration of OO and TY. The results suggested that dietary modifications incorporating diets rich in OO and associated phenolics could prove beneficial in protecting individuals against toxicity induced by dioxins.     

Hydride transfer versus hydrogen radical transfer in thymidylate synthase

The nature of a H-transfer in the thymidylate synthase catalyzed reaction was investigated by comparison of the wild-type enzyme with the W80M mutant. The nature of the H-transfer was not affected, as indicated by intrinsic isotope effects and their temperature dependence. These findings support a single-step hydride transfer instead of a two-step radical transfer.     

Improved method to quantify intracellular zidovudine mono- and triphosphate in peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry

The determination of intracellular triphosphate metabolites of nucleoside analogs used in anti-HIV therapy is very challenging. Despite the well-known sensitivity and selectivity of LC-MS/MS, the measurement of the triphosphate metabolite of zidovudine (AZT-TP) remains difficult because of the interferences induced by endogenous nucleotides triphosphates. We describe a new approach that allows improved determination of AZT-TP simultaneously with AZT-monophosphate (MP). This was obtained, first, by monitoring a transition from the molecular ion of AZT-TP to a minor but very specific product ion. Then, the spiking of samples with a constant amount of AZT-TP allowed the signal to emerge from background, leading to increased sensitivity. Finally, the analytical run time was reduced to less than 10 min. The low limits of quantification were at 150 and 300 fmol per sample for AZT-TP and AZT-MP, respectively. Recoveries were higher than 85%. Inaccuracy and precision were lower than 10% and 15% (17% at the limit of quantification), respectively. The new method offers the possibility of determining simultaneously other nucleotide phosphates, as shown here for d4T-TP (the triphosphate metabolite of another nucleoside analog, stavudine or d4T) and 2'-deoxythymidine-5'-triphosphate or dTTP (the corresponding natural nucleotide triphosphate).     

Highly sensitive and rapid determination of tacrolimus in peripheral blood mononuclear cells by liquid chromatography–tandem mass spectrometry

After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC–MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 μL containing 1 million PBMCs was used in combination with MagSiMUS‐TDM^PREP. To each sample we added 30 μL lysis buffer, 20 μL reconstitution buffer containing ¹³C²H₄‐tacrolimus as internal standard, 40 μL MagSiMUS‐TDM^PREP Type I Particle Mix and 175 μL Organic Precipitation Reagent VI for methanol‐based protein precipitation. A 10 μL aliquot of the supernatant was injected into the UPLC–MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r² = 0.997) over the range 5.0–1250 pg/1 × 10⁶ PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.     

Quantitative analysis of gemcitabine triphosphate in human peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Gemcitabine triphosphate (dFdCTP) is a highly active metabolite of gemcitabine. It is formed intra-cellularly via the phosphorylation of gemcitabine by deoxycytidine kinase. The monitoring of dFdCTP in human peripheral blood mononuclear cells (PBMCs), in addition to plasma concentrations of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine, is considered very useful in determining pharmacokinetic-pharmacodynamic relationships.We describe a novel sensitive assay for the quantification of dFdCTP in human PBMCs. The method is based on weak anion-exchange liquid chromatography and detection with tandem mass spectrometry (LC-MS/MS). The assay has been validated from 1 ng/ml (lower limit of quantification, LLOQ) to 25 ng/ml (upper limit of quantification, ULOQ) using 180 microl aliquots of PBMC extracts containing approximately 0.648 mg protein or 3.8 x 10(6) lysed PBMCs. The LLOQ is equivalent to 94 fmol/10(6) cells (1 ng/ml = 0.18 ng/180 microl or 0.18 ng/0.648 mg protein = 0.047 ng/10(6) cells or 94 fmol/10(6) cells). This highly sensitive assay is capable of quantifying about 200-fold lower concentrations of dFdCTP in human PBMCs than currently available methods.     

Computational study of the effects of protein tyrosine nitrations on the catalytic activity of human thymidylate synthase

Tyrosine nitration is a widespread post-translational modification capable of affecting both the function and structure of the host protein molecule. Enzyme thymidylate synthase (TS), a homodimer, is a molecular target for anticancer therapy. Recently purified TS preparations, isolated from mammalian tissues, were found to be nitrated, suggesting this modification to appear endogenously in normal and tumor tissues. Moreover, human TS (hTS) nitration in vitro led to a by twofold lowered catalytic activity following nitration in average of 1 tyrosine residue per monomer (D?browska-Ma? et al. in Org Biomol Chem 10:323–331, 2012), with the modification identified by mass spectrometry at seven different sites (Y33, Y65, Y135, Y213, Y230, Y258 and Y301). In the present paper, combined computational approach, including molecular and essential dynamics and free energy computations, was used to predict the influence on the activity of hTS of nitration of each of the seven tyrosine residues. The simulations were based on the crystal structure of hTS ternary complex with dUMP and Tomudex (PDB code: 1I00), with the Tomudex molecule replaced by the molecule of TS cofactor analogue, tetrahydrofolate. The present results indicate that while with nitration of five out of seven residues (Y33, Y135, Y230, Y258 and Y301), single residue modification appears to have a strong reducing effect on the activity, with the remaining two, Y65 and Y213, no or a weaker influence is apparent. Taken together, these results demonstrate that tyrosine nitrations in the hTS enzyme show clear tendency to influence the structure and dynamics and, in turn, catalytic properties of the host enzyme. These effects are overall distance-dependent.     

Nonclassical 5-substituted tetrahydroquinazolines as potential inhibitors of thymidylate synthase

Classical inhibitors of thymidylate synthase such as N^l0-propargyl-5,8-dideazafolic acid (1), N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid (ZD1694, 2) and N-[2-amino-4-oxo-3,4-dihydro(pyrrolo[2,3-d]pyrintidin-5-yl)ethylbenzoyl]-L-glutamic acid (LY231514, 3) while potent, suffer from a number of potential disadvantages, such as impaired uptake due to an alteration of the active transport system required for their cellular uptake, as well as formation of long acting, non-effluxing polyglutamates via the action of folylpolyglutamate synthetase, which are responsible for toxicity. To overcome some of the disadvantages of classical inhibitors, there has been considerable interest in the synthesis and evaluation of nonclassical thymidylate synthase inhibitors, which could enter cells via passive diffusion. In an attempt to elucidate the role of saturation of the B-ring of non-classical, quinazoline antifolate inhibitors of thymidylate synthase, analogues 7-17 were designed. Analogues 13-17 which contain a methyl group at the 7-position, were synthesized in an attempt to align the methyl group in an orientation which allows interaction with tryptophan-80 in the active site of thymidylate synthase. The synthesis of these analogues was achieved via the reaction of guanidine with the appropriately substituted cyclohexanone-ketoester. These ketoesters were in turn synthesized via a Michael addition of the appropriate thiophenol with 2-carbethoxycyclohexen-1-one or 5-methyl-2-carbethoxycyclo-hexen-1-one to afford a mixture of diastereomers. The most inhibitory compound was the 3,4-dichloro, 7-methyl derivative 17 which inhibited the Escherichia coli and Pneumocystis carinii thymidylate syntheses 50% at 5 × 10⁵ M. Our results confirm the importance of the 7-CH₃ group and electron withdrawing groups on the aromatic side chain for thymidylate synthase inhibition.     

Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine

In order to detect histamine receptors on the surface of human peripheral blood mononuclear cells, the cells were incubated in the presence of radiolabelled histamine and then the bifunctional crosslinker disuccimidyl suberate was added in various concentrations. They were then solubilized with sodium dodecyl sulphate, boiled, reduced and the lysate separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both 3H and 125I-radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3H-labelled histamine. This molecule migrated with the same mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 16 kDa subunit which had been purified on a histamine affinity column from Triton X-100 solubilized mononuclear cells, indicating it to be the ligand-binding subunit for the histamine receptor on these cells. For 3H, fluorography with Entensify was required to obtain an autoradiographic signal. Although 3H took much longer to give a signal than 125I, the considerable background, artefacts and heavy lane trailing seen with [125I] histamine were completely abrogated when [3H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [3H]histamine. The modifications reported here which improve signal detection for 3H should encourage the use of tritiated ligands in radioreceptor crosslinking, particularly those of low molecular weight which might otherwise undergo steric modification due to iodination, this having the potential for interfering with receptor ligand binding.