Activity Of Peripheral Blood Lymphocytes Research Articles

Effective Activation and Expansion of Canine Lymphocytes Using a Novel Nano-Sized Magnetic Beads Approach.

Expansion protocols for human T lymphocytes using magnetic beads, which serve as artificial antigen presenting cells (aAPCs), is well-studied. Yet, the efficacy of magnetic beads for propagation and functionality of peripheral blood lymphocytes (PBLs) isolated from companion dogs still remains limited. Domestic dog models are important in immuno-oncology field. Thus, we built the platform for induction of canine PBLs function, proliferation and biological activity using nano-sized magnetic beads (termed as MicroBeads) coated with anti-canine CD3 and CD28 antibodies. Herein we reveal that activation of canine PBLs via MicroBeads induces a range of genes involved in immediate-early response to T cell activation in dogs. Furthermore, canine T lymphocytes are effectively activated by MicroBeads, as measured by cluster formation and induction of activation marker CD25 on canine T cells as quickly as 24 h post stimulation. Similar to human T cells, canine PBLs require lower activation signal strength for efficient proliferation and expansion, as revealed by titration studies using a range of MicroBeads in the culture. Additionally, the impact of temperature was assessed in multiple stimulation settings, showing that both 37°C and 38.5°C are optimal for the expansion of canine T cells. In contrast to stimulation using plant mitogen Concanavalin A (ConA), MicroBead-based activation did not increase activation-induced cell death. In turn, MicroBeads supported the propagation of T cells with an effector memory phenotype that secreted substantial IL-2 and IFN-γ. Thus, MicroBeads represent an accessible and affordable tool for conducting immunological studies on domestic dog models. Similarities in inducing intracellular signaling pathways further underscore the importance of this model in comparative medicine. Presented herein MicroBead-based expansion platforms for canine PBLs may benefit adoptive immunotherapy in dogs and facilitate the design of next-generation clinical trials in humans.

NRF2 and HMOX1 Gene Expression against the Background of Systemic Oxidative Stress in Patients with Acute Psychosis

The aim of this study was to evaluate NRF2 and HMOX1 gene expression against the background of systemic oxidative stress in patients with acute psychosis. The study included 40 patients with newly developed paranoid schizophrenia, 22 subjects with new-onset acute psychosis associated with excessive alcohol consumption, and 25 healthy volunteers. The signs of systemic oxidative stress were assessed by 8-dihydro-2'-deoxyguanosine (8-oxodG) contents in peripheral blood lymphocytes (PBL) and in cell-free (cf) DNA samples obtained from plasma specimens of the patients and healthy controls. The patients of both groups demonstrated a significant (a hundredfold) increase in 8-oxodG content in cfDNA samples and a marked (5- to 8-fold) elevation of 8-oxodG concentration in PBL. Against the background of systemic oxidative stress, there was a statistically significant increase in NRF2 protein expression as well as marked elevation of NRF2 and Hmox1 gene transcriptional activity in PBL obtained from alcoholic patients. In contrast, the subjects with newly developed paranoid schizophrenia demonstrated decrease in expression of NRF2, the master regulator of anti-oxidant defenses, as well as reduction of NRF-2 and Hmox1 gene transcriptional activity compared to both healthy controls and alcoholic patients. In conclusion, against the background of systemic oxidative stress associated acute psychosis development, PBL of patients with paranoid schizophrenia was characterized by reduced NRF2 gene transcriptional activity and decreased NRF2 protein expression.

Intracellular K+ and water content in human blood lymphocytes during transition from quiescence to proliferation

Many evidence shows that K+ ions are required for cell proliferation, however, changes in intracellular K+ concentration during transition of cells from quiescence to cycling are insufficiently studied. Here, we show using flame emission assay that a long-term increase in cell K+ content per g cell protein is a mandatory factor for transition of quiescent human peripheral blood lymphocytes (PBL) to proliferation induced by phytohemagglutinin, phorbol ester with ionomycin, and anti-CD3 antibodies with interleukin-2 (IL-2). The long-term increase in K+ content is associated with IL-2-dependent stage of PBL activation and accompanies the growth of small lymphocytes and their transformation into blasts. Inhibition of PBL proliferation with drugs specific for different steps of G0/G1/S transit prevented both blast-transformation and an increase in K+ content per cell protein. Determination of the water content in cells by measuring the density of cells in the Percoll gradient showed that, unlike the K+ content, the concentration of K+ in cell water remains unchanged, since water and K+ change in parallel. Correlation of proliferation with high cell K+ and water content has been confirmed by the data obtained in comparative study of PBL and permanently cycling Jurkat cells. Our data suggest that K+ is important for successful proliferation as the main intracellular ion that participates in regulation of cell water content during cell transition from quiescence to proliferation. We concluded that high K+ content in cells and the associated high water content is a characteristic feature of proliferating cells.

Attenuated in vitro effects of IFN-α, IL-2 and IL-12 on functional and receptor characteristics of peripheral blood lymphocytes in metastatic melanoma patients

Considering tumor-induced suppression of lymphocytes the aim of this study was to investigate in vitro effects of IFN-α, IL-2, IL-12 and IL-18 as immunomodulating agents on the functional and receptor characteristics of peripheral blood lymphocytes (PBL) in metastatic melanoma (MM) patients compared to healthy controls (HC). In HC IFN-α, IL-2 and IL-12 enhanced mRNA level of perforin by inducing pSTAT-1 and pSTAT-5 signaling molecules. Additionally, the expression of NKG2D activating receptor and its DAP10 signaling molecule was upregulated by IL-2. Contrary to this, in MM patients only IL-2 by upregulating pSTAT-5 increased perforin-mediated cytotoxicity of lymphocytes. Furthermore, there was significantly negative correlation between the percentage of CD4+CD25bright+CD27+ regulatory T (Treg) cells and NK cell cytotoxicity, as well as the expression of NKG2D receptor on PBL in HC and MM patients. Therefore, the absence of IL-2 effect on the increase of NKG2D/DAP10 level in MM patients could be the consequence of the increased percentage of immunosuppressive CD4+CD25bright+CD27+ cells after this cytokine treatment in patients. However, in MM IL-12 significantly decreases the percentage of these inhibitory cells. Although IL-2 as a single agent has numerous side effects, it remains the important cytokine for PBL activation in melanoma immunotherapy. Additionally, the removal of Treg cells from patient PBL by IL-12 before in vitro stimulation with IL-2, may lead to the generation of more potent cytotoxic lymphocytes against tumor cells. Therefore, lymphocyte based therapy for MM patients should integrate not only the choice of appropriate immunostimulatory cytokine, but also the removal of inhibitory cells from tumor microenvironment.

Local tumor control and DNA-PK activity of peripheral blood lymphocytes in prostate cancer patients receiving radiotherapy.

Repair of DNA damage is critical for genomic stability, and DNA-dependent protein kinase (DNA-PK) has an important role in repairing double-strand breaks. We examined whether the DNA-PK activity of peripheral blood lymphocytes (PBLs) was related to biochemical (prostate-specific antigen: PSA) relapse and radiation toxicity in prostate cancer patients who have received radiotherapy. A total of 69 patients with localized adenocarcinoma of the prostate participated in this study. Peripheral blood was collected 2 years or later after radiotherapy and centrifuged, then DNA-PK activity was measured by a filter binding assay. The high DNA-PK activity group had a significantly higher PSA relapse–free survival rate than the low DNA-PK activity group. The 10-year PSA relapse–free survival was 87.0% in the high DNA-PK activity group, whereas it was 52.7% in the low DNA-PK activity group. Multivariate analysis showed the Gleason score and the level of DNA-PK activity were significant predictors of PSA relapse after radiotherapy. In addition, the low DNA-PK activity group tended to have a higher incidence of Grade 1–2 urinary toxicity than the high DNA-PK activity group. Prostate cancer patients with low DNA-PK activity had a higher rate of PSA relapse and a higher incidence of urinary toxicity. DNA-PK activity in PBLs might be a useful marker for predicting PSA relapse and urinary toxicity, possibly contributing to personalized treatment of prostate cancer.

Construction and functional analysis of an anti-human cervical carcinoma/anti-human CD3 single-chain bispecific antibody.

The aim of the present study was to construct a single-chain bispecific antibody (scBsAb) against cervical carcinoma and to investigate its biological activities. The scBsAb was constructed using a genetic cloning technique and antigen binding activities were detected by ELISA. The iodogen method was used to analyze the pharmacokinetics. The Rosette formation test was used to detect the binding ability between peripheral blood lymphocytes (PBLs) and Cs1213 cervical cancer cells. In addition, the MTT method was performed to detect the killing effect of PBLs. The molecular weight of the scBsAb was ~60kDa. The antigen binding activities of scBsAbs were compared with the anti‑human cervical carcinoma antibody single‑chain Fv fragment (CSAs‑1 scFv) and anti‑cluster of differentiation (CD)3 scFv (P>0.05). In addition, a pharmacokinetics assay demonstrated that compared with the two corresponding scFvs, scBsAbs exhibited a significantly prolonged retention time in the body (P<0.01). In addition, the number of rosettes formed by PBLs and Cs1213 cells in the scBsAb group was markedly greater than that in the scFv groups or the RPMI‑1640 group (P<0.05 and P<0.01, respectively). The killing activity of PBLs against scBsAb‑mediated Cs1213 cells was significantly greater than that mediated by the other antibodies (P<0.05). When the concentration of scBsAb was 40µg/ml, the killing rate was 64.5%. Thus, anti‑human cervical carcinoma/anti‑CD3 scBsAbs may possess two types of antigen binding activity, prolong the duration invivo and improve the killing activity of PBLs against cancer cells.

Colocalization of Granulysin Protein Forms with Perforin and LAMP-1 in Decidual Lymphocytes During Early Pregnancy.

Granulysin (GNLY) occurs in two forms, which have molecular weights of 9 and 15 kDa. We analyzed the cytotoxic potential of decidual lymphocytes (DLs) and peripheral blood lymphocytes (PBLs) based on the forms of GNLY that colocalizes with perforin (PER) and LAMP-1 following activation. The forms of GNLY were detected by using confocal microscopy. We investigated the colocalization with PER and LAMP-1 in freshly isolated and activated DLs and PBLs. Activation of DLs and PBLs by K-562 cells increased the colocalization of 9 kDa GNLY with PER and LAMP-1. K-562 cells transfected with HLA-C decreased 9 kDa GNLY colocalization with PER in DLs only. IL-15 in DLs decreased 9 kDa GNLY and LAMP-1 colocalization, but increased both 15 kDa GNLY and LAMP-1, and PER and LAMP-1 colocalization. Activated DLs and PBLs show greater cytotoxic potential based on increased colocalization of 9 kDa GNLY and PER. HLA-C and IL-15 affect DLs, indicating their role in maintaining the pregnancy tolerance.

Comprehensive metabolome analyses reveal N-acetylcysteine-responsive accumulation of kynurenine in systemic lupus erythematosus: implications for activation of the mechanistic target of rapamycin.

Systemic lupus erythematosus (SLE) patients exhibit depletion of the intracellular antioxidant glutathione and downstream activation of the metabolic sensor, mechanistic target of rapamycin (mTOR). Since reversal of glutathione depletion by the amino acid precursor, N-acetylcysteine (NAC), is therapeutic in SLE, its mechanism of impact on the metabolome was examined within the context of a double-blind placebo-controlled trial. Quantitative metabolome profiling of peripheral blood lymphocytes (PBL) was performed in 36 SLE patients and 42 healthy controls matched for age, gender, and ethnicity of patients using mass spectrometry that covers all major metabolic pathways. mTOR activity was assessed by western blot and flow cytometry. Metabolome changes in lupus PBL affected 27 of 80 KEGG pathways at FDR p < 0.05 with most prominent impact on the pentose phosphate pathway (PPP). While cysteine was depleted, cystine, kynurenine, cytosine, and dCTP were the most increased metabolites. Area under the receiver operating characteristic curve (AUC) logistic regression approach identified kynurenine (AUC = 0.859), dCTP (AUC = 0.762), and methionine sulfoxide (AUC = 0.708), as top predictors of SLE. Kynurenine was the top predictor of NAC effect in SLE (AUC = 0.851). NAC treatment significantly reduced kynurenine levels relative to placebo in vivo (raw p = 2.8 × 10−7, FDR corrected p = 6.6 × 10−5). Kynurenine stimulated mTOR activity in healthy control PBL in vitro. Metabolome changes in lupus PBL reveal a dominant impact on the PPP that reflect greater demand for nucleotides and oxidative stress. The PPP-connected and NAC-responsive accumulation of kynurenine and its stimulation of mTOR are identified as novel metabolic checkpoints in lupus pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-015-0772-0) contains supplementary material, which is available to authorized users.

In vitro chemoprotective and anticancer activities of propolis in human lymphocytes and breast cancer cells

Propolis has been used in folk medicine for centuries due to its healing properties. Ethanolic extracts of propolis (EEP) are rich sources of phenolic acid and flavonoids. Natural phenolic compounds may exert chemoprotective activity in cancer cells due to their ability to scavenge free radicals. The aim of this in vitro study was to investigate the genotoxic and anti-mutagenic effects of the EEP on human peripheral blood lymphocytes (PBLs) and their cytotoxic potential on the human breast cancer cell line (MDA-MB-231 cells). Both cell cultures were treated with six concentrations (1, 10, 50, 100, 250 and 500 ?g/ml) of EEP1 and EEP2, separately and in combination with mitomycin C (MMC). Our results show that the EEP1 and EEP2 samples of propolis after separate and combined treatments with MMC did not influence the nuclear division index (NDI). In the combined treatment, both tested EEPs significantly reduced MMC-induced micronuclei (MN) in PBLs. At 48 h after exposure of the MDA-MB-231 cell line to a combined treatment of EEP samples with MMC, the IC50 values were significantly reduced (23.79 and 19.13 ?g/ml, for EEP1+MMC and EEP2+MMC, respectively, in comparison to the single treatment. In conclusion, the tested ethanolic extracts of propolis exhibited a certain level of in vitro antimutagenic activity in PBLs from healthy subjects, and anticancer activity in breast cancer cell line. The presented findings suggest that the ethanolic extracts of propolis show potential in anticancer therapeutic strategy.

Increased Mitochondrial Electron Transport Chain Activity at Complex I Is Regulated byN-Acetylcysteine in Lymphocytes of Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) patients' peripheral blood lymphocytes (PBL) show mitochondrial dysfunction and oxidative stress. To determine the electrochemical bases of mitochondrial dysfunction, we measured electron transport chain (ETC) activity and its regulation by N-acetylcysteine (NAC) that reversed glutathione depletion and improved disease activity in SLE. ETC activity was assessed in PBL of 69 SLE patients and 37 healthy donors. Negatively isolated T cells were examined in 7 SLE patients, 11 healthy donors, and 10 nonlupus inflammatory arthritis (IA) donors. O₂ consumption (in nmol/ml/min) by lupus PBL was increased at baseline (SLE: 2.492±0.196, control: 2.137±0.153; p=0.027) and with complex IV substrates (SLE: 7.722±0.419, control: 7.006±0.505; p=0.028). SLE PBL consumed more O₂ upon in-chamber T-cell activation (p=0.012). After overnight T-cell stimulation, ETC activity of SLE PBL was 2.27-fold increased through complex I (SLE: 1.606±0.273, control: 0.709±0.169; p=0.001) and, to a lesser extent, through complex IV. Likewise, complex I activity was elevated in negatively isolated "untouched" T cells of SLE patients (1.816±0.180) relative to healthy controls (0.917±0.094; p=0.0003) and IA disease controls studied in parallel (1.057±0.199; p=0.0308). NAC diminished O₂ consumption through complex I and H₂O₂ levels both in SLE and in control PBL. O₂ consumption was found to be increased in SLE patients' PBL relative to control subjects evaluated in parallel. ETC complex I is identified as the main source of oxidative stress in SLE. Lupus PBL exhibit increased O₂ consumption through mitochondrial ETC complex I that is inhibited by NAC, which may have therapeutic efficacy through reducing oxidative stress in SLE.

Differential relative effect potencies of some dioxin-like compounds in human peripheral blood lymphocytes and murine splenic cells

Human risk assessment for dioxin-like compounds is typically based on the concentration measured in blood serum multiplied by their assigned toxic equivalency factor (TEF). Consequently, the actual value of the TEF is very important for accurate human risk assessment. In this study we investigated the effect potencies of three polychlorinated dibenzo-p-dioxins (PCDDs), six polychlorinated dibenzofurans (PCDFs) and 10 polychlorinated biphenyls (PCBs) relative to the reference congener 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) in in vitro exposed primary human peripheral blood lymphocytes (PBLs) and mouse splenic cells. REPs were determined based on cytochrome P450 (CYP) 1A1, 1B1 and aryl hydrocarbon receptor repressor (AhRR) gene expression as well as CYP1A1 activity in human PBLs and Cyp1a1 gene expression in murine splenic cells. Estimated median human REPs for 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (1234678-HpCDD), 2,3,4,7,8,-pentachlorodibenzofuran (23478-PeCDF), 1,2,3,4,7,8-hexachlorodibenzofuran (123478-HxCDF) and 1,2,3,4,7,8,9-heptachlorodibenzofuran (1234789-HpCDF) were with 0.1, 1.1, 1 and 0.09, respectively, significantly higher compared to those estimated for mouse with REPs of 0.05, 0.45, 0.09 and 0.04, respectively. Opposite to these results, the estimated median human REP of 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), was with 0.001 30-fold lower compared to the mouse REP of 0.03. Furthermore, human REPs for 1234678-HpCDD, 23478-PeCDF, 123478-HxCDF, 1234789-HpCDF and PCB 126 were all outside the ± half log uncertainty range that is taken into account in the WHO-assigned TEFs. Together, these data show congener- and species-specific differences in REPs for some, but not all dioxin-like congeners tested. This suggests that, more emphasis should be placed on human-tissue derived REPs in the establishment of a TEF for human risk assessment.

EROD activity induction in peripheral blood lymphocytes, liver and brain tissues of rats orally exposed to polycyclic aromatic hydrocarbons

Little is known in terms of multi-matrix cytochrome P450 activity induction under repeated oral exposure to planar halogenated and polycyclic aromatic hydrocarbons (PHH, PAH). In the present study, 60 rats were daily exposed, during 28 days, to oral ingestion of a mixture consisting of phenanthrene, pyrene and benzo(a)pyrene at 0, 6 or 600 μg/day. EROD activity, reflecting almost exclusively CYP1A1 and CYP1B1 activities, was measured in brain and liver microsomes as well as in peripheral blood lymphocytes (PBLs). All induction kinetics could be appropriately fitted using logistic-like models. After 28 days of exposure to a 6 μg/day dose, EROD activity was found to be 91, 152 and 94-fold increased in lymphocytes, liver and brain, respectively, compared to day 0. Plateau activities could be appropriately fitted versus ingested doses using Hill or Michaelis-Menten models. Correlations between matrices made it possible to conclude that EROD activity in PBL should be considered as a sensitive, convenient and non-destructive approach for (i) evaluating EROD activity in liver, which was found to represent 98% of the observed EROD activities in the three tested matrices and (ii) evaluating oral exposure of homogeneous groups of farm animals (race, diet) to CYP inducing PAH and PHH.

Functional expression and characterization of grass carp IL-10: An essential mediator of TGF-β1 immune regulation in peripheral blood lymphocytes

In mammals, interleukin-10 (IL-10) is known as a potent regulatory cytokine with pleiotropic effects on various leukocytes. Although teleost IL-10 has been identified in several fish species, its immunoregulatory role is still poorly understood. In the present study, grass carp IL-10 (gcIL-10) has been identified and recombinant gcIL-10 (rgcIL-10) was prepared by using a prokaryotic expression system. Using grass carp peripheral blood lymphocytes (PBLs) as a cell model, rgcIL-10 was shown to up-regulate the cell activity, which was consistent with the regulatory tone of transforming growth factor β1 (TGF-β1). An interesting question that follows would be whether IL-10 and TGF-β1 redundantly regulate PBL activity. To address this question, we investigated the effect of IL-10 on the cell activity of grass carp PBLs challenged by TGF-β1 using immunoneutralization, showing that removal of endogenous IL-10 could abolish TGF-β1-induced cell activity, indicating an essential role of IL-10 in TGF-β1 immune regulation. Furthermore, to examine the connection between TGF-β1 and IL-10 during immunoregulatory process, effect of TGF-β1 on IL-10 expression was investigated. Results showed that TGF-β1 displayed stimulatory effects on gcIL-10 mRNA expression and protein secretion in the same cell model, suggesting that TGF-β1 may function through its effect on IL-10 production in fish immunity. These results provide clues to the potential immunoregulatory role and production of IL-10 in teleost.

Cytochrome P450 2A isoenzymes in freshly prepared blood lymphocytes isolated from rats and validation as a biomarker for clinical studies in humans

1. The present study aimed to identify the expression of carcinogen metabolizing cytochrome P4502A (CYP2A) isoenzymes in freshly prepared rat peripheral blood lymphocytes (PBL) isolated from adult rats and investigate similarities in the regulation of lymphocyte CYP2A-isoenzymes with the tissue enzyme.2. qRT-PCR studies demonstrated significant constitutive mRNA expression of CYP2A-isoenzymes in PBL isolated from male and female rats which further increases significantly after pretreatment with nicotine or 3-methylcholanthrene (MC) indicating responsiveness of CYP2A-isoenzymes in PBL. This increase in the CYP2A expression was associated with an increase in the protein expression and CYP2A3-dependent coumarin hydroxylase (COH) activity in PBL.3. Clinical studies further demonstrated significant increase in the expression of CYP2A6 and associated enzyme activity in PBL isolated from lung cancer patients. Our data thus provided evidence for similarities in the regulation of carcinogen metabolizing CYP2A-isoenzymes in PBL with the tissue enzymes. Further, responsiveness of blood CYP2A6 in human blood lymphocytes isolated from lung cancer patients has led us to suggest that associating expression profiles of CYP2A6 and other polycyclic aromatic hydrocarbons (PAH)-responsive CYPs in PBL with the genotyping data could lead to the development of a possible screen to monitor and predict environment-induced diseases and toxicity in humans.

Evidence for cytochrome P450 2B1/2B2 isoenzymes in freshly prepared peripheral blood lymphocytes

Cytochrome P450 2B1 and 2B2, the major hepatic drug metabolizing enzymes belonging to CYP2 family and associated constitutive androstane receptor (CAR) were found to be expressed in peripheral blood lymphocytes (PBL) isolated from rats. As observed in liver, pretreatment of phenobarbital (PB) or phenytoin were found to increase the expression of CYP2B1, CYP2B2 and associated enzyme activity in PBL. Like in liver, blood lymphocyte CYP2B1/2B2 catalyzed the activity of 7-pentoxyresorufin O-dealkylase (PROD). The present data, demonstrating similarities in the regulation of blood lymphocyte CYP2B-isoenzymes with the liver enzymes, suggests that blood lymphocyte CYP2B-isoenzymes could be used as a biomarker to monitor tissue levels.

Poly(ADP ribose) polymerase (PARP) inhibition in peripheral blood lymphocytes (PBLs): Does it reflect PARP inhibition in tumor?

3054 Background: PARP activity in PBLs or PBMs, measured as poly(ADP-ribose) (PAR) levels, has been used as a biomarker in the development of PARP inhibitors for dose selection. Results of a phase II study of olaparib in pts with BRCA-deficient ovarian or breast cancer showed that PARP inhibition in peripheral-blood mononuclear cells (PBMCs) plateaued at 100 mg given bid, but better antitumor activity was achieved with 400 mg bid dosing. The goal of this study was to compare PARP inhibition by PF-01367338 (formerly AG014699) in PBLs and tumor using data from a previous Pfizer study. Methods: In a phase I melanoma study, PF-01367338 IV infusion (1 to 18 mg/m2) was given daily with temozolomide for 5 days. For this analysis, PAR in PBLs was measured on Cycle 1 Days 1 or 4 at baseline pretreatment and 4-6 hrs after dosing (cohorts 6-9). Tumor biopsy samples were collected before dosing on Day -7 and 4-6 hrs after dosing on Days 1, 4 or 5 from patients dosed with 12 or 18 mg/ m2. Samples were analysed using a validated immunoblot assay for determination of PAR formation. PARP inhibition was calculated as percent change of PAR level from baseline. Results: Patients provided PBL and tumor biopsy samples for paired analyses to assess PARP inhibition by PF-01367338. Data are summarized in the table below. Conclusions: Similar percent reductions in PAR formation in PBLs and tumor tissue indicate that PARP inhibition by PF-01367338 in PBLs reflects that seen in melanoma tumors. Further investigation is needed to determine whether additional dose escalation would produce additional anti-tumor activity. PF-01367338 dose Median (range)PARP inhibition in PBLs (%) Median (range)PARP inhibition in tumor (%) Wilcoxon Rank-Sum 12 mg/m2 (n = 7) 87.5 (82.1 – 96.0) 90.6 (78.1 – 99.1) z=-1.086, p=0.2774 18 mg/m2 (n = 4) 85.7 (79.1 – 95.6) 91.3 (85.9 – 97.0) z=-0.866, p=0.193 * Wilcoxon rank-sum analysis for pooled data: z = -1.215, one-sided p=0.112.

The association of DNA-dependent protein kinase activity of peripheral blood lymphocytes with prognosis of cancer

Background:Repair of various types of DNA damages is critical for genomic stability. DNA-dependent protein kinase (DNA-PK) has an important role in DNA double-strand break repair. We examined whether there may be a correlation between DNA-PK activity in peripheral blood lymphocytes (PBLs) and survival percentages in various cancer patients. We also investigated the changes of DNA-PK activity in PBLs after radiotherapy.Methods:A total of 167 of untreated cancer patients participated in this study. Peripheral blood was collected, separated, and centrifuged. DNA-PK activity was measured by DNA-pull-down assay. Chromosomal aberrations were examined by cytogenetic methods.Results:DNA-PK activity of PBLs in advanced cancer patients was significantly lower than that in early stage. The patients with lower DNA-PK activity in PBLs tended to have the lower disease-specific survivals and distant metastasis-free survivals than those with higher DNA-PK activity in advanced stages. There was also a tendency of inverse correlation between DNA-PK activity and excess fragments. The DNA-PK activity of PBLs in most patients decreased in response to radiation as the equivalent whole-body dose increased.Conclusion:Cancer patients in advanced stage, with lower DNA-PK activity of PBLs might have higher distant metastasis and exhibit poorer prognosis. Therefore, DNA-PK activity in PBLs could be used as a marker to predict the chromosomal instability and poorer prognosis.

EROD activity in peripheral blood lymphocytes and 1‐hydroxypyrene in urine and milk as biomarkers of PAH exposure in dairy ruminants

Presently, few biomarker-based approaches are available for the evaluation of environmental exposure to persistent organic pollutants in dairy ruminants. In this study, goats (Capra hircus) were orally administered a mixture of pyrene, phenanthrene, and benzo[a]pyrene daily over a 40-d period (1 or 50 mg/d). Milk and urine 1-hydroxypyrene levels, ethoxyresorufin-O-deethylase (EROD) activity in peripheral blood lymphocytes (PBL) as well as urinary levels of 2- and 3-hydroxyphenanthrene were determined at 10-d intervals. 1-Hydroxypyrene excretion in milk and urine significantly increased and then achieved a plateau at 10 d. Transfer rates of 1-hydroxypyrene were calculated to be approximately 0.5 and 25% in milk and urine, respectively. Concentrations in milk and urine were proportional to the ingested doses. These results demonstrate that 1-hydroxypyrene in milk or urine may be used as a biomarker for evaluating the exposure of dairy ruminants to polycyclic aromatic hydrocarbons (PAHs) over an extended exposure period. Constitutive EROD activity in lymphocytes was 0.5 ± 0.3 pmol resorufin/min/mg protein, and was significantly induced over the entire exposure time, before stabilizing after 40 d at 6.30 ± 1.3 and 18.89 ± 1.12 pmol resorufin/min/mg protein for 1 mg/d and 50 mg/d doses, respectively. Induction kinetics were calculated using a logistic-like model and approximate dose-response curves were designed. We therefore propose EROD activity in PBL as a relevant, convenient, and noninvasive biomarker of subchronic exposure of dairy ruminants to CYP450 inducing PAH.

Irradiation-induced telomerase activity and gastric cancer risk: a case-control analysis in a Chinese Han population

BackgroundTelomerase expression is one of the characteristics of gastric cancer (GC) cells and telomerase activity is frequently up-regulated by a variety of mechanisms during GC development. Therefore, we hypothesized that elevated levels of activated telomerase might enhance GC risk due to increased propagation of cells with DNA damage, such as induced by γ-radiation.MethodsTo explore this hypothesis, 246 GC cases and 246 matched controls were recruited in our case-control study. TRAP-ELISA was used to assess the levels of telomerase activity at baseline and after γ-radiation and the γ-radiation-induced telomerase activity (defined as after γ-irradiation/baseline) in cultured peripheral blood lymphocytes (PBLs).ResultsOur data showed that there was no significant difference for the baseline telomerase activity between GC cases and controls (10.17 ± 7.21 vs. 11.02 ± 8.03, p = 0.168). However, after γ-radiation treatment, γ-radiation-induced telomerase activity was significantly higher in the cases than in the controls (1.51 ± 0.93 vs. 1.22 ± 0.66, p < 0.001). Using the median value of γ-radiation-induced telomerase activity in the controls as a cutoff point, we observed that high γ-radiation-induced telomerase activity was associated with a significantly increased GC risk (adjusted odds ratio, 2.45; 95% confidence interval, 1.83-3.18). Moreover, a dose response association was noted between γ-radiation-induced telomerase activity and GC risk. Age, but not sex, smoking and drinking status seem to have a modulating effect on the γ-radiation-induced telomerase activities in both cases and controls.ConclusionOverall, our findings for the first time suggest that the increased γ-radiation-induced telomerase activity in PBLs might be associated with elevated GC risk. Further confirmation of this association using a prospective study design is warranted.

Proton magnetic resonance spectroscopy (1H-MRS) of peripheral blood lymphocytes in patients with sarcoidosis and interstitial pneumonitis

We recently developed a method of proton magnetic resonance spectroscopy (1H-MRS) to measure the 1H-MR spectra of peripheral blood lymphocytes (PBL) using only 10(7) cells. We investigated the clinical feasibility of this method for evaluating disease activities in sarcoidosis and interstitial pneumonitis. PBL was separated from 10 ml of venous blood by differential centrifugation with Ficoll-Conray. Using a 90 MHz MR spectrometer (EX90, JEOL), the spectrum was obtained with 4000 times accumulation of 1H signals by single 70 degree pulses at 37 degrees C under spinning. Water signal was suppressed with the homo-gated decoupling sequence. The spectra were analyzed with Gaussian curve fitting. The spectra of patients with active disease showed significant increases in peak heights for (CH2)n and CH3 peaks compared with the control. The peak heights decreased after improvement of disease states. The changes in 1H-MR spectra suggested activation of PBL in patients with sarcoidosis and interstitial pneumonitis, since similar changes have been shown for cells activated with mitogens. Our preliminary results indicate that 1H-MRS may be useful to evaluate activation of PBL in sarcoidosis and interstitial pneumonitis.