3H Thymidine Research Articles

A Diet Producing a Low Diabetes Incidence Modifies Immune Abnormalities in Diabetes-Prone BB Rats

The effect of feeding a diet that produces a high or low incidence of diabetes on immune abnormalities proposed to contribute to the pathogenesis of autoimmune-mediated diabetes was investigated. Diabetes-prone (BBdp) and nondiabetes-prone (BBn) BB rats (21 d) were fed for 21 d a nonpurified (high incidence) or purified (low diabetes incidence) diet. Compared with BBn, immune cells from spleen and mesenteric lymph nodes of BBdp rats demonstrated higher rates of metabolite production from glucose and glutamine, higher splenic cytotoxicity (lysis of 51Cr-labeled YAC-1 cells) and lower mitogen responses (3H thymidine uptake). Bypassing the membrane, using the mitogen phorbol myristate acetate + lonomycin, improved the BBdp mitogen response, suggesting a membrane defect. Immune cells from BBdp rats fed the purified, compared with the nonpurified, diet had lower rates of glutamine (spleen) and glucose (lymph nodes) metabolism and lower splenic cytotoxic activity. Although diet altered the proportion of T and B cells in lymph nodes of BBdp rats, it did not correct the abnormal lymphocyte distribution or effect mitogen responses. Feeding the purified, compared with nonpurified, diet to BBn rats altered energy metabolism by lymph node cells and resulted in lower splenic cytotoxic activity. Reduced splenic natural killer cell activity and decreased immune cell metabolism are unlikely the mechanism for the preventative effect of feeding a purified diet to BBdp rats but are indicative of reduced immune activity.

Investigation of 2-phenylimidazo[1,2-a]quinolines as potential antiproliferative agents.

Background: It has been demonstrated that the lead compound 2-phenylimidazo[1,2-a]quinoline 1a selectively inhibits CYP1 enzymes. Additionally, CYP1 inhibition has been linked to inducing antiproliferative effects in various breast cancer cell lines as well as relieving drugresistance caused by CYP1 upregulation. Materials & methods: Herein, 54 novel analogsof 2-phenylimidazo[1,2-a]quinoline 1a have beensynthesized with varied substitution on the phenyl and imidazole rings. Antiproliferative testing was conducted using 3H thymidine uptake assays. Results: 2-Phenylimidazo[1,2-a]quinoline 1a and phenyl-substituted analogs 1c (3-OMe), 1n (2,3-napthalene) displayed excellent anti-proliferative activities, demonstrating theirpotency against cancer cell lines for the first time. Molecular modeling suggested that 1c and 1n bind similarly to 1a in the CYP1 binding site.

T-cell proliferation assay for the detection of SARS-CoV-2-specific T-cells

Both infection with and vaccination against SARS-CoV-2 trigger a complex B-cell and T-cell response. Methods for the analysis of the B-cell response are now well established. However, reliable methods for measuring the T-cell response are less well established and their usefulness in clinical settings still needs to be proven. Here, we have developed and validated a T-cell proliferation assay based on 3H thymidine incorporation. The assay is using SARS-CoV-2 derived peptide pools that cover the spike (S), the nucleocapsid (N) and the membrane (M) protein for stimulation. We have compared this novel SARS-CoV-2 lymphocyte transformation test (SARS-CoV-2 LTT) to an established ELISA assay detecting Immunoglobulin G (IgG) antibodies to the S1 subunit of the SARS-CoV-2 spike protein. The study was carried out using blood samples from both vaccinated and infected health care workers as well as from a non-infected control group. Our novel SARS-CoV-2 LTT shows excellent discrimination of infected and/or vaccinated individuals versus unexposed controls, with the ROC analysis showing an area under the curve (AUC) of > 0.95. No false positives were recorded as all unexposed controls had a negative LTT result. When using peptide pools not only representing the S protein (found in all currently approved vaccines) but also the N and M proteins (not contained in the vast majority of vaccines), the novel SARS-CoV-2 LTT can also discriminate T-cell responses resulting from vaccination against those induced by infection.

Leptin favors Th17/Treg cell subsets imbalance associated with allergic asthma severity.

BackgroundObesity has often been associated with severe allergic asthma (AA). Here, we analyzed the frequency of different circulating CD4+T‐cell subsets from lean, overweight and obese AA patients.MethodsMononuclear cells from peripheral blood were obtained from 60 AA patients and the frequency of different CD4+T‐cell subsets and type 1 regulatory B cells (Br1) was determined by cytometry. The effect of obese‐related leptin dose on cytokine production and Treg cell function in AA‐derived CD4+ T cell cultures was evaluated by ELISA and 3H thymidine uptake, respectively. Leptin levels were quantified in the plasma by ELISA. According to the BMI, patients were stratified as lean, overweight and obese.ResultsAA severity, mainly among obese patients, was associated with an expansion of hybrid Th2/Th17 and Th17‐like cells rather than classic Th2‐like cells. On the other hand, the frequencies of Th1‐like, Br1 cells and regulatory CD4+ T‐cell subsets were lower in patients with severe AA. While percentages of the hybrid Th2/Th17 phenotype and Th17‐like cells positively correlated with leptin levels, the frequencies of regulatory CD4+ T‐cell subsets and Br1 cells negatively correlated with this adipokine. Interestingly, the obesity‐related leptin dose not only elevated Th2 and Th17 cytokine levels, but also directly reduced the Treg function in CD4+ T cell cultures from lean AA patients.ConclusionIn summary, our results indicated that obesity might increase AA severity by favoring the expansion of Th17‐like and Th2/Th17 cells and decreasing regulatory CD4+T cell subsets, being adverse effects probably mediated by leptin overproduction.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

Lymphocyte stimulation test in the diagnostics of adverse reactions to antituberculouse line 1 and 2 drugs.

Adverse drug reactions to anti-TB drugs (ADR) are found in 6-20% of patients and have various clinical manifestations and are detected in the lymphocyte stimulation test (LST), recorded by the incorporation on H3 thymidine, but nowadays it has significant limitations. We used LST with WST-1 reagent to detect ADR to the main 1-st and 2-nd line antituberculosis drugs in 11 tuberculosis patients who had ADR (6 - hepatotoxic reaction, 3 - blood eosinophilia and 2 - with joint pain syndrome). 6 people with tuberculosis contacts made up the control group. LST evaluation with WST-1 showed that in patients with a hepatotoxic reaction, the SI index was>2 and exceeded the values in the control group (3.28±0.59, 95% CI-1.16 and 0, 74±0.16, 95% CI - 0.31, respectively) upon stimulation of cell cultures with rifampicin alone but not with other drugs. Cell cultures stimulated with the PHA mitogen have SI >2 in ADR patients (mainly with hepatotoxic reactions). Control group SI was <2 (4,93±0.53, 95% CI - 1, 04 and 1.97±0.3, 95% CI - 0.59, respectively). We have not detected PPD-L cell cultures stimulation with WST-1 reagent both in the group of patients with ADR and the control group. In patients with eosinophilia and joint pain syndrome SI was low for all studied drugs and did not differ from the control group. The sensitivity of the LST test with WST-1 reagent is not sufficient to determine ADR to anti-TB drugs.

Chemopreventive Action of Garcinia Mangostana Linn. on Hepatic Carcinoma by Modulating Ornithine Decarboxylase Activity

Ornithine decarboxylase and alpha -L- fucosidase over-expression is associated with advanced hepatocellular carcinoma (HCC) development. The objective of this study was to elucidate the action of Garcinia Mangostana fruit extract (GME) on these overexpressed enzymes and apoptotic proteins in diethyl nitrosamine (DEN) induced hepatocellular carcinoma (HCC) rats. The cancer was induced using DEN to the experimental rats and treated with GME (400 mg/ kg) to find its anticancer property. The lysosomal enzymes such as alpha-L-fucosidase, beta- D-glucosidase, ornithine decarboxylase activity (ODC), apoptotic and antiapoptotic proteins such as Bcl-2, Bax and Bcl-xl and H3 thymidine incorporation assay were done to prove GME’s chemo preventive property. DEN induction caused significant increase in the activities of ornithine decarboxylase; lysosomal enzymes and increased cell proliferation with decreased apoptosis were observed. In contrast, the groups with GME treated rat’s elicited significant (P < 0.001) reduction in ornithine decarboxylase, lysosomal enzymes and decreased cell proliferation with increased apoptosis. GME has effective chemo preventive property and can serve as an anticarcinogenic therapeutic drug against hepatocellular carcinoma.

New Perspectives on Unscheduled DNA Synthesis: Functional Assay for Global Genomic DNA Nucleotide Excision Repair.

The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C (254nm) irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results have been used to clinically diagnose human DNA repair deficiency disorders, and provide a basis for investigation of repair deficiency in human tissues or tumors. Genomic sequencing to establish the presence of specific mutations is also used now for clinical diagnosis of DNA repair deficiency syndromes. Few functional assays are available which directly measure the capacity to perform NER on the entire genome. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR). As discussed in Chap. 28 is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER. Our laboratory also explored the fluorescent label-based Click-iT assay that uses EdU as the label, rather than 3H thymidine. Despite emerging studies in the literature finding this assay to be useful for other purposes, we found that the EdU-based UDS assay was not consistent or reproducible compared with the 3H thymidine-based assay.

Two years of treatment with the recombinant grass pollen allergy vaccine BM32 induces a continuously increasing allergen-specific IgG4 response.

BackgroundBM32, a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens adsorbed on aluminium hydroxide has been shown to be safe and to improve clinical symptoms of grass pollen allergy upon allergen-specific immunotherapy (AIT). We have investigated the immune responses in patients from a two years double-blind, placebo-controlled AIT field trial with BM32.MethodsBlood samples from patients treated with BM32 (n = 27) or placebo (Aluminium hydroxide) (n = 13) were obtained to study the effects of vaccination and natural allergen exposure on allergen-specific antibody, T cell and cytokine responses. Allergen-specific IgE, IgG, IgG1 and IgG4 levels were determined by ImmunoCAP and ELISA, respectively. Allergen-specific lymphocyte proliferation by 3H thymidine incorporation and multiple cytokine responses with a human 17-plex cytokine assay were studied in cultured peripheral blood mononuclear cells (PBMCs).FindingsTwo years AIT comprising two courses of 3 pre-seasonal injections of BM32 and a single booster after the first pollen season induced a continuously increasing (year 2 > year 1) allergen-specific IgG4 response without boosting allergen-specific IgE responses. Specific IgG4 responses were accompanied by low stimulation of allergen-specific PBMC responses. Increases of allergen-specific pro-inflammatory cytokine responses were absent. The rise of allergen-specific IgE induced by seasonal grass pollen exposure was partially blunted in BM32-treated patients.InterpretationAIT with BM32 is characterised by the induction of a non-inflammatory, continuously increasing allergen-specific IgG4 response (year 2 > year1) which may explain that clinical efficacy was higher in year 2 than in year 1. The good safety profile of BM32 may be explained by lack of IgE reactivity and low stimulation of allergen-specific T cell and cytokine responses.FundingsGrants F4605, F4613 and DK 1248-B13 of the Austrian Science Fund (FWF).

Tumor Inflammation and Myeloid Derived Suppressor Cells Reduce the Efficacy of CD19 CAR T Cell Therapy in Lymphoma

Introduction: Approximately 60% of Large B cell Lymphoma (LBCL) patients that receive CD19 CAR T cell therapy with axicabtagene ciloleucel (axi-cel) experience lymphoma progression (Locke et al. Lancet Oncol. 2019) and the likelihood of response to subsequent therapy is low (Spiegel, Dahiya et al. ASCO 2019). Target loss of CD19 is observed in less than a third of patients experiencing relapse. Alternative mechanisms of resistance to axi-cel are poorly understood. Lymphoma patients with elevated serum markers of systemic inflammation, such as ferritin and IL-6, have worse outcomes following axi-cel (Locke, Neelapu et al. Mol.Ther.2017; Faramand et al. ASH 2018). We hypothesized that suppressive monocytic myeloid derived suppressor cells (M-MDSCs), which are associated with worse chemotherapy outcomes in LBCL (Azzaoui et al. Blood 2016), and tumor driven inflammation may be present and responsible for decreased efficacy of axi-cel in LBCL. Methods: LBCL patients undergoing axi-cel treatment were enrolled onto prospective sample collection protocols. Patients were stratified for analysis into ongoing responders (complete response or partial response) or relapsed (progressive disease) after a minimum of 3 months follow-up (range 3 - 15 months). M-MDSCs, defined as a Lin-, CD11b+, CD33+, CD15-, CD14+, HLA-DRlow population, were sorted from leftover apheresis material after collection for axi-cel manufacture. M-MDSC ability to suppress proliferation of autologous T cells stimulated with CD3/CD28 coated beads was measured by 3H thymidine incorporation. Circulating peripheral blood M-MDSCs, quantified by % of live cells by flow cytometry, were measured at the time of apheresis and serially after axi-cel infusion until day 30. In vitro mouse experiments utilized a CD19-CD28 CAR and cytokine-induced bone marrow MDSCs (Thevenot et al. Immunity 2014). Cytokines were measured by ELISA and cytotoxicity against CD19 bearing cell lines used xCELLigence real-time cell analysis, as we have done previously (Li et al. JCI Insight 2018).Tumor biopsies were taken within 1 month prior to infusion of axi-cel. Limited gene expression profiling of tumor microenvironment (TME) genes used the Nanostring IO360 panel (770 genes). Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Results: First, we demonstrated that M-MDSCs sorted from patient apheresis material suppressed the proliferation of autologous T cells (n=6). We next enumerated M-MDSCs in the peripheral blood (n = 32). M-MDSC numbers initially decreased after lymphodepleting chemotherapy but recovered to baseline levels by day +10. The level of M-MDSCs following CAR T cell therapy strongly correlated with pre-CAR T baseline levels (R = 0.871, p &lt;0.0001), suggesting that the number of M-MDSCs present during CAR T cell expansion is dependent on factors already present before therapy began. M-MDSC levels were significantly higher in patients who subsequently relapsed, both at baseline (p= 0.01) and after axi-cel (p=0.04), as compared to patients with durable response. Mouse MDSCs were able to suppress CAR T cell IFN-gamma excretion (p&lt;0.0001) and cytotoxicity (p&lt;0.0001) in vitro. To evaluate the role of the TME we interrogated limited set gene expression profiling on patient (n=27) pre-axi-cel tumor biopsies. By cell type scoring, the macrophage gene score was significantly higher in patients who relapsed after CAR T therapy (p &lt;0.001). By differential gene expression and gene set enrichment, patients who relapsed had a significantly higher expression (p &lt;0.01) of multiple genes indicative of chronic interferon (IFN) signaling including higher levels of OAS2, OAS3, IFI6 and IFIT1, as well as the IFN-stimulated macrophage gene SIGLEC-1/CD169. Conclusions: Systemic inflammatory myeloid cytokines, circulating M-MDSCs in the blood and chronic IFN in the TME all associate with LBCL relapse after axi-cel CAR T cell therapy. Our observations support that CAR T cells can be suppressed by baseline patient and tumor-related factors and strategies to overcome these factors should be targeted to improve patient outcomes. MDJ and HZ contributed equally. Disclosures Jain: Kite/Gilead: Consultancy. Bachmeier:Kite/Gilead: Speakers Bureau. Chavez:Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria. Mullinax:Iovance: Research Funding. Davila:Celgene: Research Funding; GlaxoSmithKline: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; Atara: Research Funding; Bellicum: Consultancy; Adaptive: Consultancy; Anixa: Consultancy. Locke:Kite: Other: Scientific Advisor; Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy.

Abstract 4702: Upgrading cancer immunotherapy: Checkpoint blockade mAb-ODN conjugate

Abstract Programmed death 1 (PD-1) and its ligand PD-L1 provide inhibitory signals that affect the balance of T cell activation, playing major roles in tumor immune escape. Inhibition of negative regulators of T cell activation effectively remove signals that restrain T cell proliferation and gain of effector function. Hence, disruption of the PD-1/PD-L1 signaling pathway by the use of monoclonal antibodies (mAbs) can improve tumor control by enhancement of the endogenous antitumor immune responses. Toll-like receptors (TLR) are a specialized set of pathogen recognition receptors found in innate and adaptive immune system cells. Activation of these receptors by danger-associated molecules such as bacterial DNA or RNA, initiate signaling cascades leading to protective immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs can mimic bacterial DNA and therefore function as ligands for intracellular TLR9, prompting consequential activation of immune responses. The purpose of this study was to assess the feasibility of using PD-1/PD-L1 mAbs conjugated to TLR9 agonists (ODNs) as a novel strategy for tumor-targeting immune checkpoint signaling and activation of immune cells. Antibodies recognizing PD-1 and PDL-1 were covalently modified with a 21mer ssDNA oligonucleotide linker sequence that served as an anchor site for the ODNs synthesized with a 3' extension. Annealing of complementary strands formed mAb-ODN conjugates. Quantification of ODNs per PD-1 or PDL-1 mAbs was verified by flow cytometry. Functionality of PD-1 and PDL-1-ODN conjugates was analyzed in vitro using splenocyte cultures and cell proliferation was evaluated by incorporation of radiolabeled H3 Thymidine. In vivo experiments were carried out using Balb/c mice bearing 4T1 subcutaneous tumors. Flow cytometry analysis confirmed each modified mAb had 5 linker sequences available to bind ODNs, and also defined the binding capability of mAb portion of the conjugate to epitope target. Splenocytes proliferation at day 5 after treatment revealed that PD-1 and PDL-1 conjugated to ODNs have significantly increased proliferation in comparison to non-conjugated mAb and ODN controls. These results indicate that conjugation of mAb-ODN is feasible and that conjugation does not affect PD-1 and PDL-1 antibody functionality. Interestingly, in vivo experiments demonstrated PDL-1-ODN but not PD1-ODN conjugate affects 4T1 tumor growth. Use of this approach to improve the therapeutic index of immunotherapeutic mAbs requires further study and validation in different murine tumor models. Citation Format: Ana Paula Benaduce, Tal Gefen, Sergio Rodriguez, Brian Marples, Eli Gilboa, Adrian Ishkanian. Upgrading cancer immunotherapy: Checkpoint blockade mAb-ODN conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4702.

The Community Structure and Grazing Rates of Meiobenthos in Chunsu Bay, West Coast of Korea

Min, W.-G.; Kwon, K.-G.; Rho, H.S., and Kim, D., 2018. The Community Structure and Grazing Rates of Meiobenthos in Chunsu Bay, West Coast of Korea. In: Shim, J.-S.; Chun, I., and Lim, H.S. (eds.), Proceedings from the International Coastal Symposium (ICS) 2018 (Busan, Republic of Korea). Journal of Coastal Research, Special Issue No. 85, pp. 401–405. Coconut Creek (Florida), ISSN 0749-0208.The community structure and grazing rates of meiobenthos was studied in Chunsu bay on west coast of Korea. To examine feeding habit of major meiofaunal groups, grazing rate of nematodes and harpacticoid copepods was measured using the isotope-tracer (3H thymidine, 14C amino acid) method at 3 stations in subtidal area and 1 station in intertidal area of Chunsu bay. Time-serial measurement of grazing rate was made to elucidate the effects of day/night and tidal cycle on diel feeding rhythm of the animals. The number of culturable bacteria fluctuated from 1.73±0.25×105 to 18.23±1.17×105 CFU/cm3 during the study period, and was highest in October. The Concentration of Chl.a in the sediment at the study site was 18.0~168.2 mg/m2 in April and 36.6~111.0 mg/m2 in October. The most abundant meiobenthos was nematodes and followed by sarcomastigophorans, harpacticoid copepods. Density of meiobenthos were 5~1632 inds./10cm2 during the study period. The grazing rates of nematode varied 0.5~57.2%/h on bacteria and 0.4~53.3%/h on microalgae at the station in tidal flat. The grazing rates of harpacticoid copepods were 0.2~14.2%/h on bacteria and 0.3~12.2%/h on benthic microalgae at the station in tidal flat. In the subtidal area, the grazing rates of nematode were 33.4~82.5%/h on bacteria and 10.3~71.5%/h on microalgae and harpacticoid copepods were 0.2~14.2%/h on bacteria and 0.3~12.2%/h on benthic microalgae.

Does Multicolor Lineage Tracing of Endothelial Cells Provide a Black and White Answer on Clonal Expansion in Post-Natal Angiogenesis?

Postnatal angiogenesis occurs in physiological (ie, blood vessel growth in muscle in response to exercise and with cyclic changes within menstrual tissue) and pathological conditions (eg, diabetic retinopathy). Many attempts to promote postnatal angiogenesis have been attempted to drive revascularization of ischemic tissues. Surprisingly, we continue to lack fundamental information on which endothelial cells possess replicative potential in blood vessels. Prior work established that infusion of H3 thymidine into young mammals revealed endothelial cell proliferation in small and large vessels that is high at birth, varies within and among organ vasculature, and declines rapidly over the first few weeks of life. In adult animals, the rate of homeostatic endothelial cell turnover has been quantified as <1% in rodents. Despite these pieces of information, we do not know specifics about the individual proliferative capacities of endothelial cells, whether this activity is restricted to a small subset or is a feature ubiquitously present within all endothelial cells in a blood vessel.1 Article, see p 670 Manavski et al2 in this issue have used inducible endothelial cell–restricted multicolor reporter (Confetti) transgenic mice to interrogate the clonal expansion of individual endothelial cells within normally developing murine tissue vasculature or within tissues challenged by ischemia and hypoxia. This system has permitted quantification of all the endothelial cells within a fixed sampling volume of the tissue vasculature to detect an increase in local distribution of a singular color of endothelial cells within areas of clonal expansion compared with near equivalent levels of random color distribution in areas lacking clonal expansion. In brief, the authors failed to detect significant color enrichment (<10% clonal expansion) in the developing retinal vasculature at postnatal day 5, consistent with prior work in zebrafish that had identified random integration or cell mixing accounting for endothelial cell distributions in developmental …

Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression.

Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.

Abstract 1622: The role of the CCL-2 on lymphopenia-induced myeloid derived suppressor cells

Abstract The induction of lymphopenia prompts the expansion of CD11b+Ly6ChiLy6G- monocytic MDSCs (M-MDSCs) and CD11b+Ly6C+Ly6G+ polymorphonuclear MDSCs (PMN-MDSCs). Patients receiving adoptive cell therapy (ACT) require nonmyeloablative chemotherapy to induce lymphopenia and support anti-tumor immunity. However, the role of MDSCs in the setting of ACT is not fully understood. As high levels of CCL-2 can be measured in lymphopenic mice, in this study we investigate the role of CCL-2 in the expansion and function of lymphopenia-induced MDSCs using CCR2KO mouse models. Lymphopenia was induced in melanoma-bearing C57BL/6 (WT) and CCR2KO mice by 600rad of total body irradiation or combination therapy with cyclophosphamide and fludarabine. We first evaluated the percentages of splenic MDSCs in WT and CCR2KO mice after the induction of lymphopenia. At day 14, the expansion of total MDSCs were similar in WT (279% of normal) and CCR2KO (241% of normal). However, the mean percentages of M-MDSCs (3.5%) and PMN-MDSCs (30.3%) in CCR2KO mice were altered compared to M-MDSCs (22.2%) and PMN-MDSCs (21.7%) in WT mice. In addition, M-MDSCs in B16 tumors grown in CCR2KO mice were decreased compared to intratumoral M-MDSCs in WT mice (p&amp;lt;0.001). In contrast, the percentages of M-MDSCs and PMN-MDSCs were unchanged in the bone marrow (BM). To investigate the suppressive capacity of lymphopenia-induced MDSCs, OVA antigen-specific CD8+ T cells were co-cultured with OVA peptide in the presence of MDSCs purified from the spleens of CCR2KO and WT mice. Both CCR2KO and WT MDSCs potently suppressed T cell proliferation as measured by 3H thymidine incorporation. CCR2KO and WT MDSCs had significant arginase activity and production of nitrites. To investigate the efficacy of ACT, B16 tumor-bearing CCR2KO and WT mice were lymphodepleted and gp100 antigen-specific Thy1.2+CD8+ T cells were adoptively transferred followed by 3 days of IL-2 treatment. We found that donor T cell tumor infiltration was similar and no difference in tumor growth was observed between CCR2KO and WT mice. Collectively, these results suggest that the role of CCL-2 is important for the egress of lymphopenia-induced M-MDSCs from the BM, but has no effect on the expansion or function of PMN-MDSCs in the setting of lymphopenia. These data suggest that factors besides CCL-2 play an important role in the expansion and function of MDSCs after the induction of lymphopenia. Citation Format: Pasquale P. Innamarato, Amy Weber, Shari Pilon-Thomas. The role of the CCL-2 on lymphopenia-induced myeloid derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1622. doi:10.1158/1538-7445.AM2017-1622

In vitro human T cell responses to diphencyprone.

The experimental contact sensitizer Diphencyprone (DPCP) is used both for topical immunotherapy and investigation of the human immune system. Analysis of mechanisms underlying altered immune responses requires in vitro systems. We have developed a method for detecting human T cell responses to DPCP in vitro. Twenty subjects were sensitized with DPCP (30?g/cm2) and 4 weeks later, were challenged with doses from 0.48 to 3.125?g. Responses at 48h showed all 20 were allergic. Peripheral blood mononuclear cells were cultured with a range of concentrations of DPCP for 6 days and proliferation quantified (3H thymidine uptake). The subjects were exposed repeatedly to DPCP (topical immunotherapy for warts or alopecia areata) or as repeated patch test challenges. Eight subjects gave positive lymphocyte proliferation responses (LPR), maximal responses being elicited with 16 or 32?M DPCP. Positive LPR became detectable from 20 weeks after sensitization and progressively more people become responsive over time to 40 weeks. The different time courses for manifestation of allergic reactivity in skin and blood probably reflects absence of memory T cells in blood due to their recruitment to skin as resident memory T cells. This assay can now be developed for analysis of components of the immune response.

Let-7a-transfected mesenchymal stem cells ameliorate monocrotaline-induced pulmonary hypertension by suppressing pulmonary artery smooth muscle cell growth through STAT3-BMPR2 signaling

BackgroundCell-based gene therapy has become a subject of interest for the treatment of pulmonary arterial hypertension (PAH), a devastating disease characterized by pulmonary artery smooth muscle cell (PASMC) hyperplasia. Mesenchymal stem cells (MSCs) have been recently acknowledged as a potential cell vector for gene therapy. Here, we investigated the effect of MSC-based let-7a for PAH.MethodsAfter isolation and identification of MSCs from rat bone marrow, cells were infected with recombinant adenovirus vector Ad-let-7a. Lewis rats were subcutaneously injected with monocrotaline (MCT) to induce PAH, followed by the administration of MSCs, MSCs-NC (miR-control), or MSC-let-7a, respectively. Then, right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular remodeling were evaluated. Rat pulmonary artery smooth muscle cells (rPASMCs) under hypoxia were co-cultured with MSCs or MSC-let-7a. Cell proliferation and apoptosis were separately determined by 3H thymidine incorporation and flow cytometry analysis. The underlying mechanism was also investigated.ResultsMSC transplantation enhanced let-7a levels in MCT-induced PAH rats. After injection with MSC-let-7a, RVSP, right ventricular hypertrophy, and pulmonary vascular remodeling were notably ameliorated, indicating a protective effect of MSC-let-7a against PAH. When co-cultured with MSC-let-7a, hypoxia-triggered PASMC proliferation was obviously attenuated, concomitant with the decrease in cell proliferation-associated proteins. Simultaneously, the resistance of PASMCs to apoptosis was remarkably abrogated by MSC-let-7a administration. A mechanism assay revealed that MSC-let-7a restrained the activation of signal transducers and activators of transcription 3 (STAT3) and increased its downstream bone morphogenetic protein receptor 2 (BMPR2) expression. Importantly, preconditioning with BMPR2 siRNA dramatically abated the suppressive effects of MSC-let-7a on PASMC proliferation and apoptosis resistance.ConclusionsCollectively, this study suggests that MSCs modified with let-7a may ameliorate the progression of PAH by inhibiting PASMC growth through the STAT3-BMPR2 signaling, supporting a promising therapeutic strategy for PAH patients.

Effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with NBN mutations

IntroductionThe NBN gene product is part of the MRE11/RAD50/NBN complex, which plays an essential role in genomic stability. In the study we try to answer the question what is the effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with homozygous c.657-661del, and heterozygous c.657-661del, p.I171V and p.R215W NBN gene mutations.Material and methodsImmortalized B-lymphocytes with NBN gene mutations were X-ray irradiated at doses of 1, 2, 5 and 8 Gy/min. Radioresistant DNA synthesis rate and the percentage of cells in phase S was analyzed by 3H thymidine and BrdU incorporation assays. NBN gene expression was quantified by real-time PCR with TaqMan fluorescent probe.ResultsIncreasing the irradiation dose resulted in gradual decrease of 3H thymidine incorporation in all cells, but significantly only in homo- and heterozygous c.657-661del cells (p-values < 0.0001). After irradiation the relative expression of NBN was significantly higher in homozygous c.657-661del and heterozygous p.R215W cells as compared to heterozygous c.657-661del, p.I171V and control cells (p < 0.01). All cells with NBN gene mutations showed significantly higher total number of chromosomal aberrations per metaphase as compared to control cells, with the highest number of aberrations in homozygous c.657-661del cells (p < 0.001).ConclusionsThe results obtained indicate that homozygous c.657-661del mutation affects cell sensitivity to irradiation. Moreover, homozygous variant is associated with disturbance in the activation of cell cycle checkpoints and with defects in DNA repair. In turn, heterozygous c.657-661del, p.R215W and p.I171V mutations do not substantially alter the radiosensitivity.

Involvement of MAPK signalling in radioadaptive response in BALB/c mice exposed to low dose ionizing radiation

To investigate low dose ionizing radiation (LDIR)-induced adaptive response in lymphocytes of BALB/c mice and to elucidate related molecular mechanisms. Mice were exposed to a priming dose (PD) of 0.1 Gy and challenge dose (CD) of 2 Gy ionizing radiation. Proliferation response to mitogen concanavalin A was assessed using 3H thymidine incorporation and carboxyfluoresceinsuccinamidylester (CFSE) dye dilution. Early activation markers were assessed by flow cytometry, cytokines by ELISA, DNA damage by comet assay and mitogen activated protein kinase (MAPK) signaling by Western blotting. Radioadaptive response was observed in lymphocytes of mice exposed to PD prior to CD of ionizing radiation in terms of DNA damage, early activation markers CD69, CD71, cytokines IL-2, IFN-γ as well as proliferation. This effect was transient and observed 24 h after CD and not after 0 h or 72 h. Hyper activation of MAPK signaling pathways in lymphocytes from LDIR-exposed mice and abrogation by ERK and p38 inhibitors suggests the involvement of MAPK signaling in radioadaptive response. Our study demonstrates that LDIR-induced transient adaptive response was due to hyper activation of MAPK signaling. Our findings contribute towards the understanding of LDIR-induced adaptive response.

Chronic Exposure to Proline Causes Aminoacidotoxicity and Impaired Beta-Cell Function: Studies In Vitro.

Pancreatic islet-cell dysfunction is a hallmark in the development of diabetes, but the reasons for the primary β-cell defect are still elusive. Elevated circulating proline levels have been found in subjects with insulin resistance, obesity, and type 2 diabetes. Therefore, we assessed β-cell function, gene expressions, and cell death after long-term exposure of pancreatic β-cells to excess proline in vitro. Isolated mouse islets and INS-1E cells were incubated with and without excess proline. After 72 h, we examined: (1) β-cell function, including basal insulin secretion (BIS) and glucose-stimulated insulin secretion (GSIS), (2) transcription factors related to insulin gene expression and enzymes involved in the tricarboxylic acid cycle and cholesterol biogenesis, (3) cellular triglycerides (TG) and cholesterol content, (4) the death of INS-1E cells and 3H thymidine incorporation, and (5) protein expression of INS-1E cells in response to proline by proteomics. We found that high doses of proline increased BIS and decreased GSIS in both isolated mouse islets and INS-1E cells. MafA, insulin 1, and the cytochrome c oxidase subunit VIa polypeptide 2 mRNA expressions were all downregulated, indicating that proline impaired insulin gene transcription and mitochondrial oxidative phosphorylation. In contrast, mevalonate decarboxylase gene expression was upregulated, and simultaneously, cholesterol content in INS-1E cells was enhanced. Protein profiling of INS-1E cells revealed that cytosolic non-specific dipeptidase and α enolase were differentially expressed. Our results indicate that proline-induced insulin transcription and mitochondrial oxidative phosphorylation impairment may contribute to the β-cell dysfunction observed in type 2 diabetes. Caution should be applied in interpreting the pathophysiological role of proline since very high proline concentrations were used in the experiments.