mRNA Expression In Lymphocytes Research Articles

Study on the Regulation of Chidamide on CD8+T Cells in T-cell Acute Lymphoblastic Leukemia

To explore the regulatory effect of chidamide on CD8+ T cells in T-cell acute lymphoblastic leukemia. The expression levels of CXCL9 and CXCL3 mRNA in Jurkat cells, lymphocytes treated with chidamide and lymphocytes co-cultured with chidamide-treated Jurkat cells were detected by fluorescence quantitative PCR. The proportion of CD8+ T cells in lymphocytes treated with chidamide and lymphocytes co-cultured with chidamide-treated Jurkat cells was determined by flow cytometry. Chidamide upregulated CXCL9 mRNA expression in Jurkat cell line in a dose-dependent manner (r=0.950). The mRNA expression of CXCL9 in chidamide 5 μmol/L group was 164 times higher than that in control group. Chidamide upregulated CXCL9 mRNA expression in lymphocytes, but the up-regulated level was significantly lower than that in Jurkat cell line treated with the same concentration of chidamide. Co-culture with chidamide treated Jurkat cells upregulated the proportion of CD8+ T cells in lymphocytes. In T-cell acute lymphoblastic leukemia, chidamide may increase the concentration of CXCL9 in the tumor microenvironment by up-regulating the expression of CXCL9 in tumor cells, leading to an increase in the number of CD8+ T cells.

Gene Expression Of Methylation Cycle And Related Genes In Lymphocytes And Brain Of Patients With Schizophrenia And Non-Psychotic Controls.

Some of the biochemical abnormalities underlying schizophrenia, involve differences in methylation and methylating enzymes, as well as other related target genes. We present results of a study of differences in mRNA expression in peripheral blood lymphocytes (PBLs) and post-mortem brains of chronic schizophrenics (CSZ) and non-psychotic controls (NPC), emphasizing the differential effects of sex and antipsychotic drug treatment on mRNA findings. We studied mRNA expression in lymphocytes of 61 CSZ and 49 NPC subjects using qPCR assays with TaqMan probes to assess levels of DNMT, TET, GABAergic, NR3C1, BDNF mRNAs, and several additional targets identified in a recent RNA sequence analysis. In parallel we studied DNMT1 and GAD67 in samples of brain tissues from 19 CSZ, 26 NPC. In PBLs DNMT1 and DNMT3A mRNA levels were significantly higher in male CSZ vs NPC. No significant differences were detected in females. The GAD1, NR3C1 and CNTNAP2 mRNA levels were significantly higher in CSZ than NPC. In CSZ patients treated with clozapine, GAD-1 related, CNTNAP2, and IMPA2 mRNAs were significantly higher than in CSZ subjects not treated with clozapine. Differences between CSZ vs NPC in these mRNAs was primarily attributable to the clozapine treatment. In the brain samples, DNMT1 was significantly higher and GAD67 was significantly lower in CSZ than in NPC, but there were no significant sex differences in diagnostic effects. These findings highlight the importance of considering sex and drug treatment effects in assessing the substantive significance of differences in mRNAs between CSZ and NPC.

Involvement of the VEGF signaling pathway in immunosuppression and hypoxia stress: analysis of mRNA expression in lymphocytes mediating panting in Jersey cattle under heat stress

BackgroundExtreme panting under heat stress threatens dairy cattle milk production. Previous research has revealed that the gas exchange-mediated respiratory drive in critically ill dairy cattle with low O2 saturation induces panting. Vascular endothelial growth factor (VEGF) signaling may play important roles in immunosuppression and oxidative stress during severe respiratory stress responses in heat-stressed cattle. The objectives of this study were to transcriptomically analyze mRNA expression mediating heat-induced respiratory stress-associated panting, evaluate gas exchange, screen hub genes, and verify the expression of proteins encoded by differentially expressed genes in lymphocyte pathways.ResultsJersey cattle were naturally heat-exposed. Physiological data were collected for response evaluation, and blood was collected for gas exchange and gene expression assays at 06:00, 10:00 and 14:00 continuously for 1 week. Lymphocytes were isolated from whole-blood samples for mRNA-seq and expression analysis of key pathway genes/proteins. The cattle respiration rates differed with time, averaging 51 bpm at 06:00, 76 bpm at 10:00, and 121 bpm at 14:00 (p < 0.05). Gas exchange analysis showed that both pH and pCO2 differed with time: they were 7.41 and 41 mmHg at 06:00, 7.45 and 37.5 mmHg at 10:00, and 7.49 and 33 mmHg at 14:00, respectively (p < 0.01). Sixteen heat-related differentially expressed genes (DEGs; 13 upregulated and 3 downregulated) were screened between 212 DEGs and 1370 heat stress-affected genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) hub gene functional analysis annotated eleven genes to signal transduction, six genes to the immune response, and five genes to the endocrine response, including both prostaglandin-endoperoxide synthase 2 (PTGS2) and VEGF. Gene Ontology (GO) functional enrichment analysis revealed that oxygen regulation was associated with the phosphorus metabolic process, response to oxygen levels, response to decreased oxygen levels, response to hypoxia and cytokine activity terms. The main signaling pathways were the VEGF, hypoxia inducible factor-1(HIF-1), cytokine-cytokine receptor interaction and TNF pathways. Four genes involved Integrin beta 3 (ITBG3), PTGS2, VEGF, and myosin light chain 9 (MYL9) among the 16 genes related to immunosuppression, oxidative stress, and endocrine dysfunction were identified as participants in the VEGF signaling pathway and oxygenation.ConclusionThese findings help elucidate the underlying immune and oxygen regulation mechanisms associated with the VEGF signaling pathway in heat-stressed dairy cattle.

Synthesis and application of Au NPs-chitosan nanocomposite in the treatment of acute myeloid leukemia in vitro and in vivo

In recent days, novel biomolecule stabilized nanocomposites have come into prominence as featured next generation materials. In this article we have green synthesized Au nanoparticles being deposited and stabilized over chitosan (CS) molecules. The electron rich polar functional groups of chitosan have been exploited to cap the Au NPs. The structural and physical features of Au NPs-CS nanocomposite was analyzed with different techniques like Ultraviolet–Visible Spectroscopy, Fourier Transform Infrared Spectroscopy, Field Emission Scanning Electron Microscope, Transmission Electron Microscopy, and Energy-Dispersive X-Ray Spectroscopy techniques. Recently, noble metal nanoparticles have been used in the treatment of different cancers. This has prompted us to use our nanocomposite in the treatment of acute myeloid leukemia (AML) both in vitro and in vivo. Initially, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was performed to study the excellent antioxidant properties of Au NPs-CS nanocomposite. For in vitro cytotoxicity studies, HUVEC normal cell line, 32D-FLT3-ITD and Murine C1498 cancer cell lines were used and the nanocomposite was found of comparable potential as the standard AML drug Daunorubicin. For in vivo studies, 7,12-Dimethylbenz[a]anthracene (DMBA) was injected for inducing AML over 50 mice and then the drug as well as the nanocomposite was administered. Subsequently, comparison of different immunological, hematological, biochemical, stereological parameters and S1PR1 and S1PR5 mRNA expressions were ascertained. By quantitative real-time polymerase chain reaction, S1PR1 and S1PR5 mRNA expression in lymphocytes were significantly (p ≤ 0.05) raised by treating the leukemic mice with the nanocomposite. Also, nanocomposite significantly (p ≤ 0.05) decreased the weight and volume of liver and spleen, the pro-inflammatory cytokines, and the total white blood cells, blast, neutrophil, monocyte, eosinophil, and basophil counts, increased the anti-inflammatory cytokines and the lymphocyte, platelet and red blood cells parameters, and regulated the biochemical parameters as compared to the untreated mice. An extensive study was done on these biological experiments using the Au NPs-CS nanocomposite and we achieved fascinating results such that it could be used as an alternative prospective chemotherapeutic drug for the treatment of acute myeloid leukemia.

Novel green synthesis of Boswellia serrata leaf aqueous extract conjugated gold nanoparticles with excellent anti‐acute myeloid leukemia property in comparison to mitoxantrone in a leukemic mice model: Introducing a new chemotherapeutic drug

The present research confirms the capacity of aqueous extract of Boswellia serrata grown under in vitro condition for the green synthesis of gold nanoparticles (AuNPs). Also, we showed the cytotoxicity, antioxidant, and anti‐acute myeloid leukemia properties of AuNPs compared to mitoxantrone in a leukemic mouse model. The synthesized AuNPs were characterized using several techniques including XRD, TEM, FE‐SEM, UV–Vis, and FT‐IR. From the XRD pattern, four distinct diffraction peaks at 38.2°, 44.2°, 64.7° and 77.4° are indexed as (111), (200), (220) and (311) planes of FCC metallic gold. TEM and FE‐SEM images revealed an average diameters of 15–30 nm for the nanoparticles. FT‐IR findings offered antioxidant compounds in the nanoparticles were the sources of reducing power, reducing gold ions to AuNPs. UV–Vis revealed an absorption band at 536 nm that is related to the surface plasmon resonance of AuNPs. In vivo design, induction of acute myeloid leukemia was done by DMBA in 75 mice. Then, the mice were randomly divided into six subgroups, including untreated, control, HAuCl4, B. serrata, AuNPs, and mitoxantrone. AuNPs (In the dose of 1 mg/kg body weight) similar to mitoxantrone, significantly (p ≤ 0.05) increased the platelet, lymphocyte, and RBC parameters and the anti‐inflammatory cytokines (IL4, IL5, IL10, IL13, and IFNα) and reduced the weights and volumes of liver and spleen and their sub‐compartment, the total WBC, blast, monocyte, neutrophil, eosinophil, and basophil counts, and the pro‐inflammatory cytokines (IL1, IL6, IL12, IL18, IFNY, and TNFα) as compared to the untreated mice. By quantitative Real‐Time PCR, S1PR1 and S1PR5 mRNA expression in lymphocytes were significantly (p ≤ 0.05) increased by treating the leukemic mice with the AuNPs and mitoxantrone. In vitro design, AuNPs similar to mitoxantrone had low cell viability dose‐dependently against Human HL‐60/vcr, 32D‐FLT3‐ITD, and Murine C1498 cell lines without any cytotoxicity on HUVEC cell line. Besides, the DPPH assay showed similar antioxidant potentials for AuNPs and mitoxantrone. In conclusion, the results of this research indicated the excellent capacity of synthesized gold nanoparticles using B. serrata leaf aqueous extract in the treatment of acute myeloid leukemia in leukemic mice.

Preparation, formulation, and chemical characterization of silver nanoparticles using Melissa officinalis leaf aqueous extract for the treatment of acute myeloid leukemia in vitro and in vivo conditions

The demand for nanoparticles is increasing day by day due to their wide range of applications in various areas including pharmaceutical industry. Nanoparticles are formally synthesized by chemical methods in which the toxic and flammable chemicals are used. Synthesis of nanoparticles from various biological systems has been reported, but among all, biosynthesis of nanoparticles from plants is considered as the most suitable method. The current study confirms the potential of aqueous extract of Melissa officinalis grown under in vitro condition for the green synthesis of silver nanoparticles (AgNPs). Also, we revealed the cytotoxicity, antioxidant, and anti‐acute myeloid leukemia effects of AgNPs compared to mitoxantrone in a leukemic mouse model. The synthesized AgNPs were characterized using several techniques including UV–Vis., FT‐IR, TEM, FE‐SEM, and EDS. In vivo experiment, induction of acute myeloid leukemia was done by DMBA in 75 mice. The obtained results were fed into SPSS‐22 software and analyzed by one‐way ANOVA. By quantitative real‐time PCR, S1PR1 and S1PR5 mRNA expression in lymphocytes were significantly (p ≤ 0.01) increased by treating the leukemic mice with the AgNPs and mitoxantrone. Also, AgNPs similar to mitoxantrone, significantly (p ≤ 0.01) enhanced the platelet, lymphocyte, and RBC parameters and the anti‐inflammatory cytokines (IL4, IL5, IL10, IL13, and IFNα) and reduced the total WBC, blast, monocyte, neutrophil, eosinophil, and basophil counts and the pro‐inflammatory cytokines (IL1, IL6, IL12, IL18, IFNY, and TNFα) as compared to the untreated mice. In vitro experiment, AgNPs similar to mitoxantrone had low cell viability dose‐dependently against murine C1498, human HL‐60/vcr, and 32D‐FLT3‐ITD cell lines without any cytotoxicity on HUVEC cell line. Furthermore, the DPPH assay showed similar antioxidant potentials for AgNPs and mitoxantrone. Above results approve the excellent anti‐acute myeloid leukemia, cytotoxicity, and antioxidant properties of AgNPs compared to mitoxantrone.

Biosynthesis and chemical characterization of polydopamine‐capped silver nanoparticles for the treatment of acute myeloid leukemia in comparison to doxorubicin in a leukemic mouse model

The recent study reveals the cytotoxicity, antioxidant, and anti‐acute myeloid leukemia properties of polydopamine (PDA)‐capped silver nanoparticles (AgNPs) compared to doxorubicin in a leukemic mouse model. The synthesized AgNPs were characterized using different techniques including ultraviolet–visible spectroscopy (UV–Vis.), Fourier‐transform infrared spectroscopy (FT‐IR) spectroscopy, X‐ray diffraction (XRD), energy Dispersive X‐ray Spectrometry (EDS), field emission‐scanning electron microscopy (FE‐SEM), and transmission electron microscopy (TEM). FE‐SEM and TEM images revealed a uniform spherical morphology and average diameters of 15–25 nm for the nanoparticles. Also, in XRD analysis, ~20 nm was measured for the crystal size of nanoparticles. The results of the biological experiments were fed into SPSS‐22 software and analyzed by one‐way ANOVA. In the biological in vitro part of this study, AgNPs similar to doxorubicin had low cell viability dose‐dependently against Human HL‐60/vcr, 32D‐FLT3‐ITD, and Murine C1498 cell lines without any cytotoxicity on HUVEC cell line. In this study, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) test revealed similar antioxidant potentials for doxorubicin and AgNPs. In the biological in vivo part of this study, induction of acute myeloid leukemia was done by 7,12‐Dimethylbenz[a]anthracene (DMBA) in 75 mice. Then, the animals were randomly divided into six subgroups, including control, untreated, doxorubicin, AgNPs, PDA, and AgNO3. AgNPs similar to doxorubicin, significantly (p ≤ 0.05) reduced the pro‐inflammatory cytokines, and the total white blood cell (WBC), blast, monocyte, neutrophil, eosinophil, and basophil counts and enhanced the anti‐inflammatory cytokines and the platelet, lymphocyte, and red blood cell (RBC) parameters as compared to the untreated mice. By quantitative real‐time polymerase chain reaction (PCR), sphingosine‐1‐phosphate receptor‐1 (S1PR1) and sphingosine‐1‐phosphate receptor‐5 (S1PR5) mRNA expression in lymphocytes were significantly (p ≤ 0.05) raised by treating the leukemic mice with the AgNPs and doxorubicin. Above results confirm the therapeutic effects of AgNPs on acute myeloid leukemia in the leukemic mice. Further clinical trials are necessary for confirmation these remedial properties of AgNPs in human.

Green synthesis and chemical characterization of Thymus vulgaris leaf aqueous extract conjugated gold nanoparticles for the treatment of acute myeloid leukemia in comparison to doxorubicin in a leukemic mouse model

Recently, metallic nanoparticles have been used for the treatment of several disorders, such as cancer. Indeed, finding the chemotherapeutic drug of nanoparticles is in researching the priority of both developed and developing countries. The present study confirms the ability of aqueous extract of Thymus vulgaris grown under in vitro condition for the biosynthesis of gold nanoparticles (AuNPs). Also, in this study, we indicated the antioxidant, cytotoxicity, and anti‐acute myeloid leukemia properties of AuNPs compared to doxorubicin in a leukemic mouse model. The synthesized AuNPs were characterized using different techniques including X‐ray diffraction (XRD), energy Dispersive X‐ray Spectrometry (EDS), fourier‐transform infrared spectroscopy (FT‐IR) spectroscopy, ultraviolet–visible spectroscopy (UV–Vis.), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). In vivo design, induction of acute myeloid leukemia was done by 7,12‐Dimethylbenz[a]anthracene (DMBA) in 75 mice. Then, the animals were randomly divided into six subgroups, including control, untreated, doxorubicin, AuNPs, T. vulgaris, and HAuCl4. By quantitative real‐time PCR, sphingosine‐1‐phosphate receptor‐1 and sphingosine‐1‐phosphate receptor‐5 mRNA expression in lymphocytes were significantly (P ≤ 0.01) raised by treating the leukemic mice with the AuNPs and doxorubicin. Also, AuNPs similar to doxorubicin, significantly (P ≤ 0.01) enhanced the anti‐inflammatory cytokines (IL4, IL5, IL10, IL13, and IFNα) and the platelet, lymphocyte, and red blood cell (RBC) parameters and reduced the pro‐inflammatory cytokines (IL1, IL6, IL12, IL18, IFNY, and TNFα), and the total white blood cell (WBC), blast, monocyte, neutrophil, eosinophil, and basophil counts as compared to the untreated mice. In vitro design, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) test revealed similar antioxidant potentials for doxorubicin and AuNPs. Furthermore, AuNPs similar to doxorubicin had low cell viability dose‐dependently against 32D‐FLT3‐ITD, Human HL‐60/vcr, and Murine C1498 cell lines without any cytotoxicity on HUVEC cell line. Above results confirm the excellent antioxidant, cytotoxicity, and anti‐acute myeloid leukemia effects of AuNPs compared to doxorubicin. After confirming these results in clinical trial studies, AuNPs can be used as a chemotherapeutic drug for the treatment of acute myeloid leukemia in human.

Novel green synthesis of Hibiscus sabdariffa flower extract conjugated gold nanoparticles with excellent anti‐acute myeloid leukemia effect in comparison to daunorubicin in a leukemic rodent model

Nanobiotechnology is a capable technology that deals with nanomaterials in several scientific domains such as medicine, chemistry, nanotechnology, and biotechnology. In this scale, remarkable differences are seen in many properties of materials that are not observed on a larger scale. In this regard, pharmacologists have tried to synthesize many supplements and drugs from the nanoparticles. The present study confirms the ability of aqueous extract of Hibiscus sabdariffa grown under in vitro condition for the biosynthesis of gold nanoparticles (AuNPs). Also, in this study, we revealed the anti‐acute myeloid leukemia activity of AuNPs compared to daunorubicin in a leukemic rodent model. These nanoparticles were characterized by fourier‐transform infrared spectroscopy (FT‐IR) spectroscopy, ultraviolet–visible spectroscopy (UV–Vis.), X‐ray diffraction (XRD), field emission scanning electron microscopy (FE‐SEM), and transmission electron microscopy (TEM) analysis. TEM and FE‐SEM images exhibited a uniform spherical morphology and diameters of 15‐45 nm for the biosynthesized nanoparticles. In vivo design, induction of acute myeloid leukemia was done by 7,12‐Dimethylbenz[a]anthracene (DMBA) in 75 mice. Then, the animals were randomly divided into six subgroups, including HAuCl4, H. sabdariffa, AuNPs, daunorubicin, untreated, and control. AuNPs similar to daunorubicin, significantly (P ≤ .05) reduced the pro‐inflammatory cytokines (IL1, IL6, IL12, IL18, IFNY, and TNFα), and the total white blood cell (WBC), blast, monocyte, neutrophil, eosinophil, and basophil counts and enhanced the anti‐inflammatory cytokines (IL4, IL5, IL10, IL13, and IFNα) and the platelet, lymphocyte, and red blood cell (RBC) parameters as compared to the untreated mice. By quantitative real‐time polymerase chain reaction, sphingosine‐1‐phosphate receptor‐1 and sphingosine‐1‐phosphate receptor‐5 mRNA expression in lymphocytes were significantly (P ≤ .05) raised by treating the leukemic mice with the AuNPs and daunorubicin. In vitro design, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) test revealed similar antioxidant potentials for daunorubicin and AuNPs. Besides, AuNPs similar to daunorubicin had low cell viability dose‐dependently against Murine C1498, Human HL‐60/vcr, and 32D‐FLT3‐ITD cell lines without any cytotoxicity on HUVEC cell line. In conclusion, the results of chemical characterization confirm that the H. sabdariffa flower can be used to produce gold nanoparticles with a remarkable amount of anti‐acute myeloid leukemia effect.

PO-380 Regulatory T cells encounter pro-inflammatory immune cells in human colorectal cancer

IntroductionThe normal colon is facing a load of microbes, which requires a constant immunosuppression to avoid exaggerated immune responses. The immunological state within colorectal cancer (CRC) is currently not fully disclosed. Thus, we aimed at the characterisation of immune cells within normal colorectal mucosa and cancer tissue of the same surgical specimen in order to unveil alterations, and to understand the immune cell status within the tumour microenvironment.Material and methodsImmune cells were analysed with immunohistochemistry to reveal their local distribution and quantity within the tissue. Furthermore, multiparameter characterisation and IFNγ production of tissue derived immune cells was assessed with flow cytometry. In situ hybridization was performed to uncover the mRNA expression of lymphocytes within the tissue.Results and discussionsOur results show a shift within the lymphocyte subpopulations towards less CD8 +cytotoxic T cells and more suppressive regulatory T cells within the tumour compared to distant mucosa. Despite that, tumour lymphocytes had a higher IFNγ production capability in vitro and in situ. In accordance to that, PD-L1 levels were diminished in comparison to distant mucosa. Additionally, macrophages were decreased within the tumour and they acquired a pro-inflammatory phenotype.Conclusion: Although cytotoxic T cells and macrophages were reduced, and regulatory T cells decreased, several immunological features indicate that the microenvironment in CRC is in a pro-inflammatory state.

Anti-leukemic activity of a four-plant mixture in a leukemic rat model

BackgroundLeukemia is a malignant blood disease caused by the overproduction of a large number of immature blood cells that enter the peripheral blood. Because of the side effects associating the chemotherapy of leukemia, the identification of medicinal herbs, therefore, remains to be an attractive goal to treat leukemia.ResultsIn this study, leukemia was experimentally induced in rats by 7, 12-dimethyl benza[a]anthracene (DMBA) and rats were treated with a water extract of a four-plant (Arctium lappa, Ulmus rubra, Rumex acetosella, and Rheum palmatum) mixture. Application of this four-plant mixture extract successfully recovered weight loss and restored the normal total WBC, lymphocyte and neutrophil counts in a leukemia rat model compared to either the DMSO-treated rats or the leukemic rats before applying the plant mixture. Moreover, this plant mixture decreased the percentage of blasts by two thirds in leukemic rats. By quantitative real-time PCR, sphingosine-1-phosphate receptor-1 mRNA expression in lymphocytes was downregulated in leukemic rats, and this downregulation was significantly alleviated by treating the leukemic rats with the plant mixture.ConclusionThis study investigates, for the first time, the effect of this plant mixture on a chemically induced leukemia rat model. Results further support previous reports about the anti-carcinogenic effect of this plant mixture and highlights the possibility of its use in leukemia treatment to avoid the negative side effects of the usual therapy.

Сіальованість глікопротеїнів і рівень експресії нейрамінідази NEU1 та сіалілтрансферази ST6GAL1 у лімфоцитах хворих на еритремію

We studied the levels of lymphocyte surface-associated sialylglycans and the expression of neuraminidase NEU1 and sialyltransferase ST6GAL1 genes in lymphocytes in erythremia patients and healthy donors as well as the levels of sialic acids in plasma and sialylation of alpha-acid glycoprotein and fibronectin. Moreover, we also investigated the type of sialic acids binding with its glycans using sialospecific lectins MAA and SNA. fibronectin protein in lymphocytes and its cell surface in erythremia disease as compared to healthy donors. It was shown that the levels of free sialic acids and neuraminidase activity in plasma are increased in erythremia disease as compared to healthy donors; however, MAA-II-binding activity of tested glycoproteins is decreased, fibronectin-1 mRNA expression in lymphocytes is increased in patients with erythremia. The decreasing of plasma fibronectin concentration and its heparin-binding activity as well as increasing of lymphocyte content with surface-associated and intracellular fibronectin were revealed in erythremia disease in comparison with healthy donors. Positive correlation between plasma fibronectin level and its heparin-binding activity and negative correlation between plasma fibronectin level and quantity of lymphocytes which express fibronectin inside the cell and on cell surface was detected. Enhanced levels of α2,3- and α2,6-linked residues of glycocojugates were detected on lymphocyte cell surface in erythremia disease using sialospecific lectins and flow cytometer as well as fluorescent confocal microscope. The level of NEU1 and ST6GAL1 mRNA expressions is significantly increased in lymphocytes in erythremia disease. Results of this study are clarified the mechanisms of disturbed in erythremia disease glycobiological processes and may therefore present new approaches for therapeutic opportunities.

Effect of zinc treatments on lead exposed periparturient bovine lymphocytes in vitro on their proliferation and superoxide dismutase (SOD) expression

The study was conducted to observe adverse effects of lead (Pb) treatments and protective effect of zinc on lymphocyte proliferation and to quantify the expression levels of superoxide dismutase (Cu/ZnSOD) genes involved in antioxidant’s defenses in periparturient Karan Fries (Tharparker × Holstein-Friesian) cow. Blood samples were collected from peripartum Karan Fries cow at 30 and 15 days prepartum, at day of calving (0 day) and 15 and 30 days postpartum for estimation of lymphocytes proliferation and SOD expression. A fixed number of cells (2×106) were grown in culture for 72 h with different levels of Pb (10–4, 10–5, 10-6 M) and their adverse effects were counteracted by Zn (50, 55, 60 μM) and analysed for the lymphocyte proliferation (MTT assay) and for the expression level of Cu/ZnSOD using the realtime PCR technique with light upon extension (LUX) fluorogenic primers. Pb dosage had no adverse effect and the overall mean proliferation values indicated that 60μM Zn may be optimum for maximizing lymphocyte proliferation. The Cu/ZnSOD mRNA expression in lymphocytes was higher at higher dosage of Pb in comparison to lower dosage in all the 3 (50, 55, 60 μM) Zn treatments. There was no significant difference at different levels of Zn on SOD expression. Cu/ZnSOD mRNA expression decreased from –30 days to 0 days but increased from 0 to 30 days after calving. The results suggested that the zinc may have an ameliorative effect on lead exposed oxidative stress on lymphocytes proliferation and Cu/ZnSOD mRNA expression through modulation of superoxide dismutase expression

Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene, decreased leptin mRNA expression, and decreased leptin serum levels

Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9×10−5) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR⁎D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant therapy in depressed patients.

Pharmacokinetics and Pharmacodynamics of Phase II Drug Metabolizing/Antioxidant Enzymes Gene Response by Anticancer Agent Sulforaphane in Rat Lymphocytes

This study assesses the pharmacokinetics (PK) and pharmacodynamics (PD) of Nrf2-mediated increased expression of phase II drug metabolizing enzymes (DME) and antioxidant enzymes which represents an important component of cancer chemoprevention in rat lymphocytes following intravenous (iv) administration of an anticancer phytochemical sulforaphane (SFN). SFN was administered intravenously to four groups of male Sprague-Dawley JVC rats each group comprising four animals. Blood samples were drawn at selected time points. Plasma were obtained from half of each of the blood samples and analyzed using a validated LC-MS/MS method. Lymphocytes were collected from the remaining blood samples using Ficoll-Paque Plus centrifuge medium. Lymphocyte RNAs were extracted and converted to cDNA, quantitative real-time PCR analyses were performed, and fold changes were calculated against those at time zero for the relative expression of Nrf2-target genes of phase II DME/antioxidant enzymes. PK-PD modeling was conducted based on Jusko's indirect response model (IDR) using GastroPlus and bootstrap method. SFN plasma concentration declined biexponentially and the pharmacokinetic parameters were generated. Rat lymphocyte mRNA expression levels showed no change for GSTM1, SOD, NF-κB, UGT1A1, or UGT1A6. Moderate increases (2-5-fold) over the time zero were seen for HO-1, Nrf2, and NQO1, and significant increases (>5-fold) for GSTT1, GPx1, and Maf. PK-PD analyses using GastroPlus and the bootstrap method provided reasonable fitting for the PK and PD profiles and parameter estimates. Our present study shows that SFN could induce Nrf2-mediated phase II DME/antioxidant mRNA expression for NQO1, GSTT1, Nrf2, GPx, Maf, and HO-1 in rat lymphocytes after iv administration, suggesting that Nrf2-mediated mRNA expression in lymphocytes may serve as surrogate biomarkers. The PK-PD IDR model simultaneously linking the plasma concentrations of SFN and the PD response of lymphocyte mRNA expression is valuable for quantitating Nrf2-mediated effects of SFN. This study may provide a conceptual framework for future clinical PK-PD studies of dietary cancer chemopreventive agents in human.

Germline variants of base excision repair genes and breast cancer: A polymorphism in DNA polymerase gamma modifies gene expression and breast cancer risk

Base excision repair (BER) removes DNA damage induced by endogenous reactive oxygen species or ionizing radiation, important breast cancer risk factors. Genetic variation associated with impaired BER might thus increase breast cancer risk. Therefore, we assessed risk associations of 123 common single nucleotide polymorphisms (SNPs) in 19 BER genes in 1,639 postmenopausal breast cancer cases and 1,967 controls from the German population-based case-control study MARIE. SNPs were tagging SNPs representing genetic variation across the gene together with potentially functional SNPs. Risk associations were assessed using conditional logistic regression, adjusted for potential breast cancer risk factors. Significant associations between polymorphisms and breast cancer risk were found for one SNP in PARP2 and three SNPs in the mitochondrial DNA polymerase gamma, POLG. A SNP in the promoter region of POLG (rs2856268, A>G) showed a protective effect for homozygous GG carriers (odds ratio 0.81, 95% confidence intervals 0.65-1.00). Joint analysis of an enlarged sample set and haplotype analysis supported the results for POLG. Quantification of POLG mRNA expression in lymphocytes of 148 breast cancer patients revealed higher mRNA levels for rs2856268 GG carriers (p value = 0.038). A luciferase promoter assay showed significant differences between constructs harboring the respective alleles. Taken together, our results suggest that genetic variation in the POLG promoter region affects DNA polymerase gamma levels in mitochondria. This could contribute to the reported increase in mitochondrial mutation frequency resulting in dysfunction and altered breast cancer risk. Risk effects and the functional impact of the POLG promoter variant require further confirmation.

Relative Expression of a Dominant Mutated ABCC8 Allele Determines the Clinical Manifestation of Congenital Hyperinsulinism

Congenital hyperinsulinism (CHI) is most commonly caused by mutations in the β-cell ATP-sensitive K+ (KATP) channel genes. Severe CHI was diagnosed in a 1-day-old girl; the mother’s cousin and sister had a similar phenotype. ABCC8 gene sequencing (leukocyte DNA) revealed a heterozygous, exon 37, six–base pair in-frame insertion mutation in the affected patient and aunt but also in her unaffected mother and grandfather. In expression studies using transfected COSm6 cells, mutant sulfonylurea receptor 1 (SUR1) protein was expressed on the cell surface but failed to respond to MgADP even in the heterozygous state. mRNA expression in lymphocytes determined by sequencing cDNA clones and quantifying 6FAM-labeled PCR products found that although the healthy mother predominantly expressed the normal transcript, her affected daughter, carrying the same mutant allele, primarily transcribed the mutant. The methylation pattern of the imprinting control region of chromosome 11p15.5 and ABCC8 promoter was similar for all family members. In conclusion, differences in transcript expression may determine the clinical phenotype of CHI in this maternally inherited dominant mutation. The use of peripheral lymphocytes as a peripheral window to the β-cell transcription profile can serve in resolving β-cell phenotypes. The severe, dominant-negative nature of the 1508insAS mutation suggests that it affects the functional stoichiometry of SUR1-regulated gating of KATP channels.

Tumor-infiltrating lymphocytes predict response to anthracycline-based chemotherapy in estrogen receptor-negative breast cancer

IntroductionInfiltration of breast tumors by tumor-infiltrating lymphocytes (TIL) has been associated with sensitivity to anthracycline-based chemotherapy. However, it is unclear whether this is true within the estrogen receptor-alpha (ER)-negative subset of breast tumors that frequently manifest high TIL levels.MethodsThe association of TIL with short-term and long-term clinical response to anthracycline-based therapy was assessed in two independent ER-negative breast cancer cohorts in which patients were categorized as TIL-high or TIL-low. We defined an eight-gene lymphocyte mRNA expression signature (including CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL) and used unsupervised hierarchical clustering to examine the association between TIL and short-term response to neoadjuvant chemotherapy in a previously published cohort of ER-negative tumors (n = 113). We also examined the association between TIL and long-term chemotherapeutic efficacy in a second cohort of ER-negative tumors (n = 255) with longer than 6 years of median follow-up by using tissue microarrays and immunohistochemistry (IHC) for detection of CD3, CD8, CD4, CD20, and TIA-1.ResultsIn patients with ER-negative tumors treated with neoadjuvant anthracycline-based chemotherapy, pathologic complete responses (pCRs) were achieved by 23 (74%) of 31 TIL-high patients and 25 (31%) of 80 TIL-low patients (odds ratio (OR), 6.33; 95% confidence interval (CI), 2.49 to 16.08; P < 0.0001). Multivariate logistic regression with standard clinicopathologic features demonstrated that only tumor size (P = 0.037) and TIL status (P = 0.001) were independent predictors of anthracycline response. In the second cohort, adjuvant anthracycline-based therapy was associated with increased disease-free survival (DFS) only in patients with high levels of intraepithelial CD3+ TIL (P = 0.0023). In contrast, outcomes after CMF treatment (cyclophosphamide, methotrexate, and fluorouracil) showed no association with CD3 status. In both cohorts, cytotoxic T-cells were the primary TIL subtype associated with anthracycline sensitivity. Finally, TIL significantly predicted anthracycline sensitivity for both the Her2-positive and triple-negative tumor phenotypes.ConclusionsER-negative breast cancers with high levels of TIL have heightened sensitivity to anthracycline-based chemotherapy, as assessed by the immediate response to neoadjuvant therapy and long-term outcome following adjuvant therapy. Investigations of TIL-based predictive tests to identify patients likely to benefit from anthracycline-based treatments are warranted.

The Role of Proteinase Inhibitor 9 (PI-9) and Granzyme B in Peripheral Leukocytes for GVL and GVHD After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract 1984The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures:No relevant conflicts of interest to declare.

Alpha- and Beta-Synucleins mRNA Expression in Lymphocytes of Schizophrenia Patients

Alpha-synuclein is largely, but not entirely, expressed in the central nervous system. A high concentration of alpha-synuclein in presynaptic terminals can mimic the normal function of endogenous alpha-synuclein in regulating synaptic vesicle mobilization at nerve terminals. Beta-synuclein protein is seen primarily in brain tissue and it is suggested that beta-synuclein acts as an inhibitor of alpha-synuclein aggregation, which occurs in neurodegenerative diseases. With respect to the role of synucleins in neurologic diseases such as Parkinson's disease, we decided to study the changes of alpha- and beta-synucleins in schizophrenia patients in relation to a control group. For this purpose, total RNA was extracted from the lymphocytes of patients and controls and then cDNA was synthesized and used for real-time polymerase chain reaction. Calculation of the relative expression of alpha- and beta-synucleins showed downregulation in patients in comparison to the control group. Independent two-tailed t-test showed that beta-synuclein mRNA expression in the control group was significantly higher than that in the patient group (p < 0.01), but downregulation of alpha-synuclein gene was not significant. Therefore, a significant downregulation of beta-synuclein mRNA expression appears to be a suitable biomarker for the diagnosis of schizophrenia.