PD-1 blockade enhances cytokine-induced killer cell-mediated cytotoxicity in B-cell non-Hodgkin lymphoma cell lines.

The clinical utility of immune checkpoint inhibitors, such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors used alone or in combination with other therapies, is currently gaining attention. In this particular scenario, the inclusion of cytokine-induced killer (CIK) cells has proven to be a novel therapeutic approach. CIK cells have shown anticancer activity in various hematopoietic malignancies, but their defined cytotoxicity in B-cell non-Hodgkin lymphoma (B-NHL) remains to be fully elucidated. The present study investigated the role of PD-1/PD-L1 blockades on the cytotoxic efficacy of CIK cells primarily in B-NHL cell lines. The current analysis revealed that CIK cells prompted cytotoxicity against B-NHL cell lines (DAUDI and SU-DHL-4), and a significant increase in PD-L1 expression was observed when CIK cells were co-cultured with B-NHL cells. Additionally, a combination of PD-1 and PD-L1 antibodies with CIK cells significantly decreased cell viability only in DAUDI cells. Furthermore, IFN-γ elevation was observed in both cell lines treated with CIK alone or with PD-1 antibody, but this tendency was not observed for PD-L1. Since PD-1 can suppress immune inactivation, whereas CD40L can promote it, the effects of CD40L blockade were also examined; however, no significant changes in cell viability were observed. Overall, the present in vitro data suggested that CIK cells exerted a cytotoxic function in B-NHL cells, and a combination of PD-1 inhibitors with CIK cells may provide a potential therapeutic option for this type of lymphoma. Nevertheless, in vivo experiments are warranted to undermine the extent to which PD-1 inhibitors may be used to enhance the antitumor activity of CIK cells in B-NHL.

Interaction Between CCL18 and GPR30 Differs from the Interaction Between Estradiol and GPR30.

BACKGROUND/AIM: C-C motif chemokine ligand 18 (CCL18) is overexpressed in the microenvironment of tumors, promotes invasion and metastasis and is thus important for the therapeutic outcome of many tumor entities. The Gs-coupled seven-transmembrane receptor GPR30 is known as both a CCL18 and an estrogen receptor; its activation by estradiol leads to a transactivation of membrane-tethered pro-heparin-binding EGF-like growth factor and the MAPK/ERK pathway. We examined whether this signaling pathway remains the same under CCL18 stimulation, as opposed to estradiol stimulation. MATERIALS AND METHODS: We investigated the effects of CCL18 on the lung cancer cell line A549, that show low GPR30 expression and the breast cancer cell lines MCF-7, that has high GPR30 expression and MDA-MB-231. These cells were stimulated in different media with CCL18 and then analyzed by qPCR, In-Cell Western®, western blot and ELISA. RESULTS: Many similarities on the effect of CCL18 on the already known estradiol-activated signaling pathway via the G protein-coupled estrogen receptor GPR30 were identified. GPR30 is involved in the expression of matrix metalloproteinases (MMPs), which may play a role in the transactivation of ERK-1/-2 via the cleavage of membrane-bound HB-EGF, via Src-related tyrosine kinases and Gßγ-subunits. With increasing CCL18 concentration, the expression of MMP7 decreased in A549 cells. With decreasing estrogen content of the medium, there was an increasing effect of CCL18 on the inhibition of the relative expression of MMP7. Inhibition of GPR30 with G15 also resulted in a decrease in the relative expression of MMP7, irrespective of the subsequent stimulation with CCL18. This is a rather unexpected result, because the estrogen estradiol and CCL18 both activate GPR30. MCF-7 cells which express more GPR30 did not show any dependence of the relative MMP7 expression on CCL18 except in estrogen-free FCS medium. CCL18 induced an increased relative ERK activation in In-Cell western (ICW) at A549 cells. Stimulation with CCL18 caused decreased ERK activation with simultaneous inhibition of adenylate cyclase in MCF-7. However, stimulation with CCL18 and simultaneous inhibition of cyclooxygenase in MCF-7 resulted in increased ERK activation. In A549, stimulation with CCL18 and co-incubation with dbcAMP resulted in decreased ERK activation in both ICW and Western blot. CONCLUSION: In summary, the Gs-coupled receptor GPR30 plays an important role in the signaling pathway of CCL18. CCL18 and estradiol may not lead to the same signaling pathway after activating GPR30.

FOXP3 Inhibitory Peptide P60 Increases Efficacy of Cytokine-induced Killer Cells Against Renal and Pancreatic Cancer Cells.

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. MATERIALS AND METHODS: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. RESULTS: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. CONCLUSION: P60 may potentiate CIK cell cytotoxicity against tumor cells.

Clinical Trials with Combination of Cytokine-Induced Killer Cells and Dendritic Cells for Cancer Therapy.

Adoptive cellular immunotherapy (ACI) is a promising treatment for a number of cancers. Cytokine-induced killer cells (CIKs) are considered to be major cytotoxic immunologic effector cells. Usually cancer cells are able to suppress antitumor responses by secreting immunosuppressive factors. CIKs have significant antitumor activity and are capable of eradicating tumors with few side effects. They are a very encouraging cell population used against hematological and solid tumors, with an inexpensive expansion protocol which could yield to superior clinical outcome in clinical trials employing adoptive cellular therapy combination. In the last decade, clinical protocols have been modified by enriching lymphocytes with CIK cells. They are a subpopulation of lymphocytes characterized by the expression of CD3+ and CD56+ wich are surface markers common to T lymphocytes and natural killer NK cells. CIK cells are mainly used in two diseases: in hematological patients who suffer relapse after allogeneic transplantation and in patients with hepatic carcinoma after surgical ablation to eliminate residual tumor cells. Dendritic cells DCs could play a pivotal role in enhancing the antitumor efficacy of CIKs.

The usage of a three-compartment model to investigate the metabolic differences between hepatic reductase null and wild-type mice.

278: The Cytochrome P450 (CYP) system is involved in 90% of the human body's interactions with xenobiotics and due to this, it has become an area of avid research including the creation of transgenic mice. This paper proposes a three-compartment model which is used to explain the drug metabolism in the Hepatic Reductase Null (HRN) mouse developed by the University of Dundee (Henderson, C. J., Otto, D. M. E., Carrie, D., Magnuson, M. A., McLaren, A. W., Rosewell, I. and Wolf, C. R. (2003) Inactivation of the hepatic cytochrome p450 system by conditional deletion of hepatic cytochrome p450 reductase. J. Biol. Chem. , 13480-13486). The model is compared with a two-compartment model using experimental data from studies using wild-type and HRN mice. This comparison allowed for metabolic differences between the two types of mice to be isolated. The three sets of drug data (Gefitinib, Midazolam and Thalidomide) showed that the transgenic mouse has a decreased rate of metabolism.

Recurrent Coding Sequence Variation Explains Only A Small Fraction of the Genetic Architecture of Colorectal Cancer.

Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10(-7)), and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10(-7)); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10(-7) and OR = 1.09, P = 7.4 × 10(-8)); rs1129406 (12q13) in ATF1 (OR = 1.11, P = 8.3 × 10(-9)), all reaching exome-wide significance levels. Gene based tests identified associations between CRC and PCDHGA genes (P < 2.90 × 10(-6)). We found an excess of rare, damaging variants in base-excision (P = 2.4 × 10(-4)) and DNA mismatch repair genes (P = 6.1 × 10(-4)) consistent with a recessive mode of inheritance. This study comprehensively explores the contribution of coding sequence variation to CRC risk, identifying associations with coding variation in 4 genes and PCDHG gene cluster and several candidate recessive alleles. However, these findings suggest that recurrent, low-frequency coding variants account for a minority of the unexplained heritability of CRC.

Identification of Dlk1-Dio3 imprinted gene cluster noncoding RNAs as novel candidate biomarkers for liver tumor promotion.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for ß-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and ß-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Comparison of paroxetine and amitriptyline as adjunct to lithium maintenance therapy in bipolar depression: a reanalysis of a randomized, double-blind study.

OBJECTIVE: To compare the efficacy and safety of adjunctive treatment with paroxetine or amitriptyline in patients with bipolar disorder who relapsed into a depressive episode during lithium maintenance therapy. METHODS: Data from a randomized, double-blind trial comparing paroxetine (N=18) or amitriptyline (N=22) as adjunctive treatment for an episode of depression during lithium maintenance therapy were reanalyzed. Only patients with a diagnosis of bipolar disorder were included. The primary endpoint was the change in Hamilton Rating Scale for Depression (HAM-D21) from randomization to study end (week 6). RESULTS: There was a significant reduction of HAM-D21 total score from randomization to study end in both treatment groups. The mean change in HAM-D21 score in the paroxetine and amitriptyline groups at study end was -14.9 and -15.5 (p=0.798), and the mean HAM-D21 at study end was 8.2 vs. 9.9 (p=0.420), respectively. The patients treated with paroxetine showed a more rapid improvement with lower HAM-D21 scores between weeks 3 and 5. Tolerability was similar in both groups. LIMITATIONS: No placebo comparator group and relatively small study sample size. CONCLUSIONS: Adjunctive treatment with either paroxetine or amitriptyline is a viable option for breakthrough depression during lithium maintenance therapy.

Polymorphisms in xenobiotic metabolizing enzymes and diet influence colorectal adenoma risk.

OBJECTIVES: We have earlier shown that diet and xenobiotic metabolizing enzyme genotypes influence colorectal cancer risk, and now investigate whether similar associations are seen in patients with premalignant colorectal adenomas (CRA), recruited during the pilot phase of the Scottish Bowel Screening Programme. METHODS: Nineteen polymorphisms in 13 genes [cytochrome P450 (P450), glutathione S-transferase (GST), N-acetyl transferase, quinone reductase (NQ01) and microsomal epoxide hydrolase (EPHX1) genes] were genotyped using multiplex PCR or Taqman-based allelic discrimination assays and analyzed in conjunction with diet, assessed by food frequency questionnaire, in a case-control study [317 CRA cases (308 cases genotyped), 296 controls]. Findings significant at a nominal 5% level are reported. RESULTS: CRA risk was inversely associated with fruit (P=0.02, test for trend) and vegetable (P=0.001, test for trend) consumption. P450 CYP2C9*3 heterozygotes had reduced CRA risk compared with homozygotes for the reference allele [odds ratio (OR): 0.60; 95% confidence interval (CI): 0.36-0.99], whereas CYP2D6*4 homozygotes (OR: 2.72; 95% CI: 1.18-6.27) and GSTM1 'null' individuals (OR: 1.43; 95% CI: 1.04-1.98) were at increased risk. The protective effect of fruit consumption was confined to GSTP1 (Ala114Val) reference allele homozygotes (OR: 0.49; 95% CI: 0.34-0.71, P=0.03 for interaction). CRA risk was not associated with meat consumption, although a significant interaction between red meat consumption and EPHX1 (His139Arg) genotype was noted (P=0.02 for interaction). CONCLUSION: We report the novel associations between P450 genotype and CRA risk, and highlight the risk association with GSTM1 genotype, common to our CRA and cancer case-control series. In addition, we report a novel modifying influence of GSTP1 genotype on dietary chemoprevention. These novel findings require independent confirmation.

Methylene tetrahydrofolate reductase genotype modifies the chemopreventive effect of folate in colorectal adenoma, but not colorectal cancer.

Epidemiologic evidence suggests a role for folate, a critical component of the 1-carbon cycle, in colorectal adenoma and cancer pathogenesis. Low folate levels, along with genetic polymorphisms in key enzymes such as methylene tetrahydrofolate reductase (MTHFR), can cause DNA hypomethylation and aberrant CpG methylation, which have been associated with colorectal tumor development. We investigated self-reported folate and alcohol intake alongside possible modifying effects of MTHFR 677 C>T and 1298 A>C polymorphisms in UK case-control studies of colorectal adenoma (317 cases, 296 controls) and cancer (500 cases, 742 controls). A significant association between MTHFR 1298 and colorectal cancer risk was observed [odds ratio, 1.57; 95% confidence interval (95% CI), 1.05-2.37], which was more pronounced in males (odds ratio, 3.02; 95% CI, 1.63-5.62). Although we found no association between MTHFR 677 and colorectal cancer, when data were stratified by sex, an increased risk was seen in females (odds ratio, 1.96; 95% CI, 1.11-3.46) but not in males. High folate intake was associated with a decreased risk for colorectal adenoma (odds ratio, 0.47; 95% CI, 0.30-0.73; P(trend), <0.001), which was modified by MTHFR 1298 genotype (P(interaction) = 0.006). However, we found no evidence to support the hypothesis that a high-folate diet protects against colorectal cancer development. Consistent with previous studies, high alcohol intake (>or=14 U/wk) was associated with a significantly increased cancer risk (odds ratio, 2.57; 95% CI, 1.81-3.64). Our data suggest that dietary folate intake may be an important determinant for premalignant colorectal disease development but not colorectal cancer, an association that is modified by MTHFR genotype.

Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research, drug discovery, and the treatment of liver diseases holds great promise. Here we describe a three-stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide, followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation, hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation, expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially. Furthermore, we have provided evidence that the hESC-derived hepatocytes are able to carry out a range of hepatocyte functions: storage of glycogen, and generation and secretion of plasma proteins. More importantly, the hESC-derived hepatocytes express several members of cytochrome P450 isozymes, and these P450 isozymes are capable of converting the substrates to metabolites and respond to the chemical stimulation. Our results have provided evidence that hESCs can be differentiated efficiently in vitro to functional hepatocytes, which may be useful as an in vitro system for toxicity screening in drug discovery.

GSTpi expression mediates dopaminergic neuron sensitivity in experimental parkinsonism.

The cause of 95% of Parkinson's disease (PD) cases is unknown. It is hypothesized that PD arises from an interaction of free-radical-generating agents with an underlying genetic susceptibility to these compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role of a dual function protein, GSTpi, in dopaminergic neuron death. GSTpi is the only GST family member expressed in substantia nigra neurons. GSTpi reduction by pharmacological blockade, RNA inhibition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential expression of GSTpi contributes to the sensitivity to xenobiotics in the substantia nigra and may influence the pathogenesis of reactive oxygen species-induced neurological disorders including PD.

Generation Scotland: the Scottish Family Health Study; a new resource for researching genes and heritability.

BACKGROUND: Generation Scotland: the Scottish Family Health Study aims to identify genetic variants accounting for variation in levels of quantitative traits underlying the major common complex diseases (such as cardiovascular disease, cognitive decline, mental illness) in Scotland. METHODS/DESIGN: Generation Scotland will recruit a family-based cohort of up to 50,000 individuals (comprising siblings and parent-offspring groups) across Scotland. It will be a six-year programme, beginning in Glasgow and Tayside in the first two years (Phase 1) before extending to other parts of Scotland in the remaining four years (Phase 2). In Phase 1, individuals aged between 35 and 55 years, living in the East and West of Scotland will be invited to participate, along with at least one (and preferably more) siblings and any other first degree relatives aged 18 or over. The total initial sample size will be 15,000 and it is planned that this will increase to 50,000 in Phase 2. All participants will be asked to contribute blood samples from which DNA will be extracted and stored for future investigation. The information from the DNA, along with answers to a life-style and medical history questionnaire, clinical and biochemical measurements taken at the time of donation, and subsequent health developments over the life course (traced through electronic health records) will be stored and used for research purposes. In addition, a detailed public consultation process will begin that will allow respondents' views to shape and develop the study. This is an important aspect to the research, and forms the continuation of a long-term parallel engagement process. DISCUSSION: As well as gene identification, the family-based study design will allow measurement of the heritability and familial aggregation of relevant quantitative traits, and the study of how genetic effects may vary by parent-of-origin. Long-term potential outcomes of this research include the targeting of disease prevention and treatment, and the development of screening tools based on the new genetic information. This study approach is complementary to other population-based genetic epidemiology studies, such as UK Biobank, which are established primarily to characterise genes and genetic risk in the population.