NT Cells Research Articles

Carbon quantum dots-embedded graphitic carbon nitride nanotubes for enhancing the power conversion efficiency of sensitized solar cells

Graphitic carbon nitride (CN) has been widely used as a photocatalyst. Very few researchers have reported the use of CN in quantum dot-sensitized solar cells (QDSCs). In this study, we prepared nitrogen-rich carbon quantum dot (CQD)-embedded CN nanotubes (CCNTs) with freeze-dried urea and CQD precursors. The prepared CCNTs were used as efficient light harvesters in QDSCs for the first time; their use significantly improved the power conversion efficiency (PCE) of the solar cells compared to those of CQD, CN NT, and bulk CN-sensitized solar cells. The CCNT-sensitized solar cell exhibits 1.01% PCE, which is the highest value among carbon-based QDSCs. Moreover, the CCNTs-sensitized device showed superior photostability over those of CQDs-, CN NTs-, and bulk CN-sensitized devices. The improved performance of the CCNT-sensitized solar cell can be attributed to the facilitated photoelectron transport and suppressed charge recombination. The integration of nitrogen-rich CQDs in CCNTs adjusts the band alignment and maximizes the visible light harvest by reducing the energy barriers, which improves the charge collection efficiency of the device.

Tumor cell and microvessel densities during the growth of a brain tumor: A theoretical study

Mathematical model for the tumor growth incorporating energy supply and requirement, angiogenesis efficiency and effect of elasticity of adjacent normal tissue to understand tumor biology and predict saturation status is rare to find. This study is conducted to address these issues. We propose mathematical expressions to explain alterations of tumor cell density (nT), microvessel density (MVD), and growth rate(r) during the development of brain tumors. We assume that nT increases during the growth of the tumor due to the increase of external pressure from the initial cell density (nT0); nT0 is same as the external normal tissue. The rate of increase in tumor cells (dNT/dt) depends on the rate of energy available for the creation of new cells and the energy required for a single cell division(γ). Due to the increase of tumor cell density, hypoxia is developed, which up-regulates the secretion of vascular endothelial growth factor (VEGF) and new capillaries are generated. Therefore, the surface area density of capillaries (Acs) in tumors increases. Hence, we consider that Acs(t) ∞ nT(t). A modified logistic equation is developed. Temporal variations of nT(t), Acs(t), r(t) and tumor cell population ‘NT(t)’ are examined. The expressions of saturated cell density(nTM), saturated microvessel surface area density (AcsM) and tumor saturation time(Ts) are formulated. An important feature, tumor saturation factor ‘fTS’ is determined. When fTS<1, a tumor will saturate at Ts, and nTM depends solely on fTS.

Effects of Heat Therapy on Prostate Cancer Cells Subjected to 4 Gy Radiation

Background Despite many advancements in treatment options, radiation therapy remains a common treatment for individuals diagnosed with prostate cancer. However, radiation resistance presents a significant challenge for many patients. Due to its numerous effects on cancer cell growth and survival, heat therapy has been studied as a potential mechanism to improve radiation efficacy. However, the modality of heat therapy varies widely between studies, and the majority of heat therapy research relies on non-physiological temperatures (>43º C). We hypothesized that mild temperature hyperthermia would improve the efficacy of radiation of prostate cancer cells in vitro. Methods Clonogenic survival was assessed using human prostate cancer (PC-3) cells in vitro. Cells were grown in tissue culture flasks maintained in a 37º C incubator with 5% CO2 and randomized into 3 groups: normothermic (NT; n=8), acute hyperthermia (HTA; n=8), and chronic hyperthermia (HTC; n=8). NT cells were maintained in a normal incubator until radiation. For heat treatments, HTA cells were heated to 41.0º C in a separate incubator for 1 h immediately prior to radiation. HTC cells were heated to 41.0º C in a separate incubator for 1 h each session, for a total of 3 heat treatments, each separated by 48 h, with the final heat treatment occurring 24 h prior to radiation. Immediately following a single dose of 4 Gy radiation, each group of cells were counted and plated into 60 mm culture dishes at densities of both 500 cells/plate and 1000 cells/plate, with 5 replicates plated per density. Following 8 days of incubation, cells were fixed and stained and colonies of greater than 50 cells were counted. Results The survival fractions were determined from the average of the two plating densities. Clonogenic cell survival was significantly reduced in HTA vs. NT (11.6% ± 2% vs. 19.3% ± 1%, p<0.05) and in HTC vs. NT (8.3% ± 2% vs. 19.3% ± 1%, p<0.05). Cell survival between HTA and HTC was not different (p>0.05). Conclusions These results suggest that both acute and chronic mild hyperthermia improve the efficacy of 4 Gy radiation in PC-3 cells. While exact mechanisms are yet to be elucidated, it is thought that hyperthermia at lower temperature induces DNA damage, inhibits DNA damage repair, or alters cell cycle progression, which individually or in combination with one another, may potentiate the effects of radiation. Mild temperature hyperthermia prior to radiation may be an effective neo-adjuvant to increase the radiosensitivity of prostate cancer cells.

Analysis of the Epigenetic Signature of Cell Reprogramming by Computational DNA Methylation Profiles

Background: DNA methylation plays an important role in the reprogramming process. Understanding the underlying molecular mechanism of reprogramming is crucial for answering fundamental questions regarding the transition of cell identity. Methods: In this study, based on the genome-wide DNA methylation data from different cell lines, comparative methylation profiles were proposed to identify the epigenetic signature of cell reprogramming. Results: The density profile of CpG methylation showed that pluripotent cells are more polarized than Human Dermal Fibroblasts (HDF) cells. The heterogeneity of iPS has a greater deviation in the DNA hypermethylation pattern. The result of regional distribution showed that the differential CpG sites between pluripotent cells and HDFs tend to accumulate in the gene body and CpG shelf regions, whereas the internal differential methylation CpG sites (DMCs) of three types of pluripotent cells tend to accumulate in the TSS1500 region. Furthermore, a series of endogenous markers of cell reprogramming were identified based on the integrative analysis, including focal adhesion, pluripotency maintenance and transcription regulation. The calcium signaling pathway was detected as one of the signatures between NT cells and iPS cells. Finally, the regional bias of DNA methylation for key pluripotency factors was discussed. Our studies provide new insight into the barrier identification of cell reprogramming. Conclusion: Our studies analyzed some epigenetic markers and barriers of nuclear reprogramming, hoping to provide new insight into understanding the underlying molecular mechanism of reprogramming.

Activation and degranulation of CAR-T cells using engineered antigen-presenting cell surfaces.

Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.

A10 TR1 CELL-BASED THERAPY FOR INFLAMMATORY ILEITIS IN SHIP-/- MICE

Abstract Background Crohn’s disease (CD), a form of inflammatory bowel disease (IBD), is characterized by chronic inflammation that can occur anywhere along the gastrointestinal (GI) tract, but commonly involves the distal portion of the ileum. Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP) is a hematopoietic-specific regulator of secondary signals generated by the PI3K pathway and regulates immune activation. SHIP-/- mice, at 6–8 weeks of age, spontaneously develop inflammatory ileitis due to myeloid proliferation, a lack of T cells, and an increase in IL-1β. Aims Here we investigated the efficacy of type 1 regulatory T (Tr1) cells, which are characterized by high IL-10 production, a broadly acting anti-inflammatory cytokine that promotes gut homeostasis, to treat ileitis in SHIP-/- mice. Methods Using IL-10GFPFOXP3RFP B6 reporter mice, we sorted FOXP3-CD44High Tr1 cells, FOXP3+ Tregs, and FOXP3-CD44Low naive T cells from total CD4+ T cells, and cultured them with immobilized α-CD3, soluble α-CD28 and recombinant IL-2 for 3 days. Naive T cells were grown in undifferentiated conditions or polarized to Th1 or Th17 cells. Cell phenotypes were characterized by ELISA and flow cytometry. For the adoptive Tr1 cell transfer, CD44High cells were sorted from FOXP3GFP Thy1.1 reporter mice and cultured as above with an additional 2 days’ rest in the presence of IL-2 alone. SHIP-/- mice and wild type (WT) littermates were sublethally irradiated at 5 weeks of age, followed 24h later by IP injection with 0.7 – 1.0 x 106 Tr1 cells or PBS vehicle. At 7 weeks of age, mice were euthanized and blood, mesenteric lymph nodes (MLNs), and ileal tissue were processed. Results After 3 days of stimulation, FOXP3-CD44High Tr1 cells produced more IL-10 than Tregs and less IFN-γ than Th1 cells. Additionally, CD44High cells had higher co-expression of CD49b and Lag3, biomarkers for Tr1 cells, in comparison to nT cells, Tregs, Th1, and Th17 cells, a higher percentages of IL-10 producing cells than FOXP3+ Tregs and produced less IFN-γ, IL-17, and TNF-α in comparison to Th1, Th17 and naive T cells, respectively. We found that Tr1 cells successfully engrafted irradiated SHIP-/- mice. Furthermore, engrafted Tr1 cells had high co-expression of CD49b and Lag3, with the majority of these cells located in the MLNs and spleen. SHIP KO mice that received Tr1 cells had comparable ileal length and appearance to SHIP WT, and significantly longer ilea versus SHIP PBS controls — indicating decreased inflammation as a result of successful engraftment. Conclusions In conclusion, ex-vivo expansion and adoptive transfer of Tr1 cells to SHIP-/- mice led to cellular engraftment and improvement in spontaneous ileitis. Hence, Tr1 cellular therapy shows promise as a therapeutic approach in CD. Future experiments will be needed to determine the therapeutic dose range of Tr1 cells and the mechanisms of protection. Funding Agencies CCC

Engineering High Affinity and Cleavage Resistant CD16 to Augment ADCC of Placental Hematopoietic Stem Cells-Derived Natural Killer Cells

Background: Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.1 The high IgG binding allele are found in about 10-20% of the normal population.2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.4 Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods: Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism. Results: Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells (&amp;gt;90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced &amp;gt;89% shedding of CD16 in NT cells, while significantly less (&amp;lt;11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p&amp;lt;0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p&amp;lt;0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p&amp;lt;0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies. PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (&amp;gt;50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity. Conclusions: In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted.

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Glioblastoma (GBM) remains one of the most malignant primary tumors in adults, with a 5-year survival rate less than 10% because of lacking effective treatment. Here, we aimed to explore whether B7-H3 could serve as a novel therapeutic target for GBM in chimeric antigen receptor (CAR) T cell therapy. In this study, a CAR targeting B7-H3 was constructed and transduced into T cells by lentivirus. Antitumor effects of B7-H3-specific CAR-T cells were assessed with primary and GBM cell lines both in vitro and in vivo. Our results indicated that B7-H3 was positively stained in most of the clinical glioma samples, and its expression levels were correlated to the malignancy grade and poor survival in both low-grade glioma (LGG) and GBM patients. Specific antitumor functions of CAR-T cells were confirmed by cytotoxic and ELISA assay both in primary glioblastoma cells and GBM cell lines. In the orthotropic GBM models, the median survival of the CAR-T-cell-treated group was significantly longer than that of the control group. In conclusion, B7-H3 is frequently overexpressed in GBM patients and may serve as a therapeutic target in CAR-T therapy.

A Tad pilus promotes the establishment and resistance of Vibrio vulnificus biofilms to mechanical clearance

Vibrio vulnificus is autochthonous to estuaries and warm coastal waters. Infection occurs via open wounds or ingestion, where its asymptomatic colonization of seafood, most infamously oysters, provides a gateway into the human food chain. Colonization begins with initial surface contact, which is often mediated by bacterial surface appendages called pili. Type IV Tad pili are widely distributed in the Vibrionaceae, but evidence for a physiological role for these structures is scant. The V. vulnificus genome codes for three distinct tad loci. Recently, a positive correlation was demonstrated between the expression of tad-3 and the phenotypes of a V. vulnificus descendent (NT) that exhibited increased biofilm formation, auto-aggregation, and oyster colonization relative to its parent. However, the mechanism by which tad pilus expression promoted these phenotypes was not determined. Here, we show that deletion of the tad pilin gene (flp) altered the near-surface motility profile of NT cells from high curvature, orbital retracing patterns characteristic of cells actively probing the surface to low curvature traces indicative of wandering and diminished bacteria–surface interactions. The NT flp pilin mutant also exhibited decreased initial surface attachment, attenuated auto-aggregation and formed fragile biofilms that disintegrated under hydrodynamic flow. Thus, the tad-3 locus, designated iam, promoted initial surface attachment, auto-aggregation and resistance to mechanical clearance of V. vulnificus biofilms. The prevalence of tad loci in the Vibrionaceae suggests that they may play equally important roles in other family members.

Towards in vitro DT/DNT testing: Assaying chemical susceptibility in early differentiating NT2 cells.

Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro toxicity testing, e.g. developmental toxicity and neurotoxicity (DT/DNT) studies, as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and permit toxicity testing at different stages of maturation. NT2 cells have recently been reported to show specific changes in dielectric resistance profiles during differentiation which can be observed as early as 24h upon RA-stimulation. These observations suggest altered susceptibility to chemicals at an early stage of differentiation. However, chemical susceptibility of early differentiating NT cells has not yet been studied. To address this question, we have established a cell fitness screening assay based on the analysis of intracellular ATP levels and we applied the assay in a large-scale drug screening experiment in NT2 stem cells and early differentiating NT2 cells. Subsequent analysis of ranked fitness phenotypes revealed 19 chemicals with differential toxicity profile in early differentiating NT2 cells. To evaluate whether any of the identified drugs have previously been associated with DT/DNT, we conducted a literature search on the identified molecules and quantified the fraction of chemicals assigned to the FDA (Food and Drug Administration) pregnancy risk categories (PRC) N, A, B, C, D, and X in the hit list and the small molecule library. While the fractions of the categories N and B were decreased (0.81 and 0.35-fold), the classes C, D and X were increased (1.35, 1.47 and 3.27-fold) in the hit list compared to the chemical library. From these data as well as from the literature review, identifying large fractions of chemicals being directly (∼42%) and indirectly associated with DT/DNT (∼32%), we conclude that our method may be beneficial to systematic in vitro-based primary screening for developmental toxicants and neurotoxicants and we propose cell fitness screening in early differentiating NT2 cells as a strategy for evaluating chemical susceptibility at different stages of differentiation to reduce animal testing in the context of the 3Rs.

Effects of dietaryBacillus nattosupplementation on growth performance and the growth-related gene/microRNA expression in the skeletal muscle of grass carp (Ctenopharyngodon idella)

In this study, Bacillus natto NT was evaluated for use as a probiotic supplement in the feeds on the growth performance and the growth-related genes' and microRNAs' (miRNAs) expression in the skeletal muscle of grass carp (Ctenopharyngodon idella). Grass carps (ave. wt. 43.96 ± 0.27 g) were fed diets supplemented with 1.87 × 109 (Bn1), 3.73 × 109 (Bn2), 5.60 × 109 (Bn3), 7.47 × 109 (Bn4) and 9.33 × 109 (Bn5) B. natto NT cells per 100 g feed for 56 days. The control group (BnC) was not supplemented with the B. natto NT. The fish of Bn3, Bn4 and Bn5 groups displayed better growth performance and lower feed conversion ratio (FCR) than the other groups (P < 0.05). Compared with the fish of BnC group, miR-1a, miR-181a, miR-23a and miR-206 expressions increased (P < 0.05), and the myostatin and myocyte enhancer factor C (MEF2C) mRNAs were down-regulated (P < 0.05) in the fish of Bn4 and Bn5 groups. The specific growth rate analysis and apparent expressional regulation of the growth-related miRNAs and genes stimulated by B. natto suggest the potential application of B. natto in improving the growth performance on the grass carps.

Abstract 002: Dynamic T Cell-Antigen Presenting Cell Interactions and Direct T Cell Activation Within the Vascular Wall During Hypertension

T cells are now known to be vital to the development of experimental hypertension. Hypertension is associated with significant accumulation of T cells into the perivascular fat surrounding the aorta and renal vasculature. While a hypertension-specific neoantigen has been implicated in T cell activation, it is not known whether vascular-infiltrating T cells recognize and are locally activated by an antigen within the vessel wall. We developed live-cell imaging of explanted aortas to identify whether cognate antigens are presented to T cells within the vessel wall of hypertensive mice, evidenced by slower T cell velocities and a greater number of T cells interacting with antigen presenting cells (APCs). Splenic T cells were isolated from normotensive vehicle-treated (nT cells) and hypertensive angiotensin II (Ang II)-infused (0.7mg/kg/day; 14 days; hT cells) C57BL6/J mice. Following anti-CD3/CD28 stimulation (48 hours), cells were fluorescently labelled and co-incubated simultaneously (16 hours) with explanted aorta from normotensive or hypertensive CD11c-YFP mice, where APCs are fluorescently labelled. In CD11c-YFP mouse aorta alone, we detected a ~2-fold increase in CCR5 ligand (CCL3, CCL4 and CCL5) secretion from hypertensive mouse aorta compared to vehicle-treated mouse aorta (*P&lt;0.05; n = 4). Using 2-photon microscopy, we observed a greater number (~2-fold) of hT cells compared to nT cells within Ang II-infused mouse aorta (390 ± 113 Vs 198 ± 49). Importantly, time-lapse recordings of hypertensive mouse aorta revealed hT cells exhibited significantly slower velocity (hT cells 2.0 μm/min Vs nT cells 4.8 μm/min; **P&lt;0.01, n=8-12), and a greater proportion of interactions with APCs (hT cells 4.4 ± 1.1 Vs nT cells 0.8 ± 0.4%). Moreover, activation of local vascular T cells by incubating hypertensive aorta with anti-CD3/CD28 antibodies (16 hours) augmented Ang II-induced endothelial dysfunction (67.5 ± 2.0 Vs Ang II alone 54.5 ± 3.7% maximal relaxation). We have the first evidence that vascular-infiltrating T cells are presented with cognate antigens by APCs within the vessel wall during hypertension; direct activation of these T cell infiltrates further impairs endothelial function, which may promote the development of hypertension.

Abstract 100: Sorting Nexin 1: Molecular Mechanisms and Pharmacogenomics

Sorting nexin 1 (SNX1) plays a pivotal role for the normal activity of renal dopamine D5 receptor (D5R). Kidney-restricted, Snx1-siRNA depletion of SNX1 results in impaired natriuretic response to salt load and hypertension in mice. Genetic ablation of the Snx1 gene (Snx1-/-) resulted in increased oxidative stress, impaired sodium excretion, and elevated systolic blood pressure (SBP, 131.3±6.4 mm Hg, n=5) in mice. The D5R has antioxidant properties by negatively regulating the expression of the NADPH oxidase (NOX). We found that NOX1, NOX2, and p47phox, as well as the antioxidant PON2, conceivably as compensation, were increased in Snx1-/- mice compared with wild-type littermates. Snx1-/- mice had higher ROS (218.6±7.7%), NOX activity (43.9±3.3 AU/mg protein/min vs. 25.98±3.5), and other markers of oxidative stress, e.g., malonyldialdehyde (32.5±3.4 pmol/mg protein vs. 17.2±2.1) and 3-nitrotyrosine (128.3±4%), which were all normalized by 10-day renal infusion of apocynin, a drug that prevents NOX assembly. The SBP in Snx1-/- mice was also normalized by apocynin (131.3±4.8 mm Hg to 105.7±0.3). Compared with human renal proximal tubule cells obtained from normotensive Caucasian males (NT cells), those from hypertensive subjects (HT cells) had reduced expression of SNX1 (160±2.1%, n=4=5/group), increased ROS (182±10.5%), and blunted cAMP response (110.8±35.3%) and sodium transport inhibition (101.2±1.9%) in response to D1-like receptor stimulation. These observations were corroborated by results in siRNA-induced gene silencing in NT cells or “genetic rescue” in HT cells. We also evaluated 12 SNPs in the SNX1 gene as possible genetic predictors of BP response to monotherapy with HCTZ among hypertensive patients enrolled in the Pharmacogenomic Evaluation of Antihypertensive Responses study (n=768). Three of the 12 SNPs (rs12591947, rs11854249, and rs11635627) associated with poor BP response to thiazide ([[Unable to Display Character: &amp;#8710;]] SBP of -1.8 mm Hg vs. 113.5) among blacks. An SNX1 SNP (rs1802376) was associated with essential hypertension in a Caucasian population (n=502). Our data demonstrate the novelty and relevance of SNX1 in human pathology and pharmacogenomics of essential hypertension.

Abstract POSTER-BIOL-1310: Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer

Abstract Tumor dormancy is an important research topic because of its potential implications in metastasis and acquired resistance to chemotherapy. Dormant tumor cells at the secondary site may persist after chemotherapy and regrow as metastatic recurrence. Seminal studies by Dr. Bissell and Dr. Hendrix have shown that the tissue microenvironment can reprogram tumor cells into dormancy. Extracellular matrix (ECM) proteins are key components of tumor microenvironment and may regulate dormancy, and disruption of ECM homeostasis may cause recurrence and metastatic growth. A major cytokine that regulates ECM remodeling is tumor growth factor-beta (TGF-beta), and it is implicated in modulating tumor cell dormancy. In the tissue microenvironment, serine protease HtrA1 was shown to inhibit TGF-beta signaling by targeting TGF-beta family ligands and receptors for proteolysis. Our lab showed that HtrA1 is down-regulated in ovarian cancer cells, and HtrA1 loss promotes resistance to anoikis and in vivo metastasis. Loss of HtrA1 is also associated with increased incidence of carcinomatosis. Finally, previous studies have shown that HtrA1 degrades ECM proteins. Therefore, we hypothesize that loss of HtrA1 in the ovarian tumor microenvironment enhances TGF-beta signaling and promotes ECM protein production by stromal cells, thereby contributing to ECM remodeling and tumor dormancy. Ovarian cancer cells with endogenous HtrA1 were transduced with HtrA1-targeting shRNA (sh1) to evaluate the effect of HtrA1 downregulation on invasion, tumor dormancy, and ECM remodeling. Cells transduced with non-targeting shRNA (NT) served as a control. Cancer cells (NT and sh1) were plated over confluent mesothelial cell layer with or without 10 nM TGF-beta for 24 hours and 48 hours. No significant difference in cell invasion was observed between those cells cultured with or without TGF-beta. In order to test which matrix proteins are critical for cell proliferation versus cell cycle arrest, MicroMatrix 36 arrays were used to culture NT and sh1 cells. Cells were cultured for 48 hours on matrix slides, then fixed and stained with anti Ki67. It was determined matrix containing combinations of fibronectin showed cells that stained positive for Ki67, when compared to those not containing fibronectin or collagen together. Changes in intracellular signaling in the presence and absence of matrix were also investigated using kinase and MAPK arrays. Mesothelial cells were cultured until confluent, and cells were removed from culture plates using 2.0 mM EDTA in PBS. Matrix produced from mesothelial cells remained on the plates. NT and sh1 cells were then plated in the presence and absence of matrix and were allowed to culture until cell attachment was observed (3 hours at 37oC, 5% CO2). Cells were lysed and lysates were cultured on arrays according to manufactures instructions. ERK and Akt pathways were observed to be activated in cells plated with matrix compared to those without matrix. Taken together the evidence thus far suggests that matrix composition is critical for cell proliferation. Currently, Stable Isotope Amino Acid labeling is being employed to investigate changes in matrix remodeling on a global scale. In addition, Imaging Mass Spectrometry will also be used to evaluate formalin fixed paraffin embedded tissue samples from patients to identify at a molecular level changes in extracellular matrix composition between tumor and normal tissues. Citation Format: Cooley, Megan K., Chien, Jeremy. Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1310.

Abstract 2385: Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HtrA1 and deregulated TGFB signaling

Abstract Dissemination of ovarian cancer is promoted by deregulated cell adhesion that selectively enhances detachment at primary site and attachment and invasion at secondary sites. Modifications in the extracellular matrix composition within tumor microenvironment can facilitate deregulated cell adhesion, migration, and invasion. We aim to understand how tumor-intrinsic changes affect expression and composition of ECM proteins in disseminated ovarian cancer. The serine protease HtrA1 has been shown to inhibit TGFB by targeting TGFB family ligands and receptors for proteolysis. In addition, HtrA1 also degrades ECM proteins, such as decorin, fibronectin, and collagens. We have shown that HtrA1 is down-regulated in ovarian cancer cells with loss contributing to chemotherapy resistance. We propose that loss of HtrA1 in the ovarian tumor microenvironment enhances TGFB signaling that in turn facilitate compositional changes in ECM proteins from stromal cells and promote cancer dissemination. To characterize compositional changes that occur in ECM proteins as a result of increased TGFB signaling, stable isotope amino acid labeling (SILAC) and LC-MS analysis were used. Mesothelial cells were cultured in heavy or light isotopically labeled media to confluence. Tumor cells expressing HtrA1 (NT) or stably transfected with HtrA1-targeting shRNA (sh1) were added to either heavy or light mesothelial cultures with or without TGFB (10 ng/ml). Cells were lifted by EDTA and the remaining matrix proteins were subjected to LC-MS. The data show that ECM compositional changes in fibronectin, laminin and collagen were up regulated in mesothelial cells co-cultured with sh1 compared to NT cells. All extracted proteins with ≥ 4 fold change were analyzed by Ingenuity Pathway Analysis. Proteins up regulated in co-cultures with HtrA1-deficient cancer cells (sh) were determined to be associated with migration, invasion and tumorogenesis whereas proteins upregulated in co-culture with HtrA1-expressing cancer cells (NT) were associated with protein modifications and ER stress. HtrA1-deficient cells show decreased adhesion to collagen I/III matrix and enhanced adhesion to collagen I/VI matrix. HtrA1-deficient cancer cells show morphology characteristics of epithelial-mesenchymal transition and shows higher motility and invasion through mesothelial cell layer. Finally, in clinical samples, decreased HtrA1 expression in primary tumor was significantly associated with higher incidence of carcinomatosis and nodal metastasis in ovarian cancer. We conclude that loss of HtrA1 in ovarian cancer cells results in compositional changes in ECM within the tumor microenvironment, and these changes may contribute to the dissemination of ovarian cancer. Citation Format: Megan Cooley, Jeremy Chien, Anirban Mitra, Alfonso Baldi, Andras Czirok, Edina Kosa. Characterization of ECM remodeling in disseminated ovarian cancer as a result of loss of HtrA1 and deregulated TGFB signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2385. doi:10.1158/1538-7445.AM2015-2385

The effect of hypoxic postconditioning after severe hypobaric hypoxia on the expression of Cu, Zn-SOD in hippocampus and neocortex of rats

Oxidative stress is a key mechanism of cellular damage during and after severe hypoxia. Accordingly, up-regulation of expression and activity of endogenous antioxidants is an important mechanism of cellular adaptation to hypoxia. Do endogenous antioxidants take part in postconditioning-induced neuroprotective mechanisms similarly to their participation in preconditioning-induced ones? In the present work the effect of postconditioning by 3-trial mild hypobaric hypoxia (360 Torr, 2 h, once a day) after 1 session of severe acute hypobaric hypoxia (180 Torr, 3 h) on the expression of Cu, Zn-superoxide dismutase (Cu, Zn-SOD) was studied by immunocytochemical analysis in areas CA1, CA2, CA3, CA4 and DG of hippocampus and in frontoparietal neocortex (NC) of male Wistar rats. Two time points were examined: 3 h after the last session of postconditioning that was 3 days after severe hypoxia and 24 h after the last session of postconditioning that was 4 days after severe hypoxia. It has been shown that postconditioning significantly increases the total number of Cu, Zn-SOD-immunoreactive cells (Nt) at least in two areas of hippocampus studied (CA2 and DG) compared to non-postconditioned rats at 3 days but not at 4 days after severe hypoxia. In contrast, in NC of postconditioned rats, Nt tends to increase compared to non-postconditioned animals at 4 days but not at 3 days after severe hypoxia. The effect of postconditioning on the number of intensely expressing Cu, Zn-SOD neurons differs in various areas and at various time points. The modification of Cu, Zn-SOD expression in some areas of hippocampus and NC, induced by 3-trial hypoxic postconditioning, correlates with the prevention of massive delayed apoptotic neuronal death and amelioration of functional disorders caused by severe hypoxia. Thus, Cu, Zn-SOD and other endogenous antioxidants may play, apparently, an important role in the treatment of severe hypoxia/ischemia stroke by postconditioning in brain neurons.

Impaired Th17 polarization of phenotypically naive CD4(+) T-cells during chronic HIV-1 infection and potential restoration with early ART.

BackgroundDepletion of mucosal Th17 cells during HIV/SIV infections is a major cause for microbial translocation, chronic immune activation, and disease progression. Mechanisms contributing to Th17 deficit are not fully elucidated. Here we investigated alterations in the Th17 polarization potential of naive-like CD4+ T-cells, depletion of Th17-commited subsets during HIV pathogenesis, and Th17 restoration in response to antiretroviral therapy (ART).ResultsPeripheral blood CD4+ T-cells expressing a naive-like phenotype (CD45RA+CCR7+) from chronically HIV-infected subjects receiving ART (CI on ART; median CD4 counts 592 cells/μl; viral load: <50 HIV-RNA copies/ml; time since infection: 156 months) compared to uninfected controls (HIV-) were impaired in their survival and Th17 polarization potential in vitro. In HIV- controls, IL-17A-producing cells mainly originated from naive-like T-cells with a regulatory phenotype (nTregs: CD25highCD127−FoxP3+) and from CD25+CD127+FoxP3− cells (DP, double positive). Th17-polarized conventional naive CD4+ T-cells (nT: CD25−CD127+FoxP3−) also produced IL17A, but at lower frequency compared to nTregs and DP. In CI on ART subjects, the frequency/counts of nTreg and DP were significantly diminished compared to HIV- controls, and this paucity was further associated with decreased proportions of memory T-cells producing IL-17A and expressing Th17 markers (CCR6+CD26+CD161+, mTh17). nTregs and DP compared to nT cells harbored superior levels of integrated/non-integrated HIV-DNA in CI on ART subjects, suggesting that permissiveness to integrative/abortive infection contributes to impaired survival and Th17 polarization of lineage-committed cells. A cross-sectional study in CI on ART subjects revealed that nTregs, DP and mTh17 counts were negatively correlated with the time post-infection ART was initiated and positively correlated with nadir CD4 counts. Finally, a longitudinal analysis in a HIV primary infection cohort demonstrated a tendency for increased nTreg, DP, and mTh17 counts with ART initiation during the first year of infection.ConclusionsThese results support a model in which the paucity of phenotypically naive nTregs and DP cells, caused by integrative/abortive HIV infection and/or other mechanisms, contributes to Th17 deficiency in HIV-infected subjects. Early ART initiation, treatment intensification with integrase inhibitors, and/or other alternative interventions aimed at preserving/restoring the pool of cells prone to acquire Th17 functions may significantly improve mucosal immunity in HIV-infected subjects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0164-6) contains supplementary material, which is available to authorized users.

Comparison of a two-stage system with low pressure carbon dioxide microbubbles and heat treatment on the inactivation of Saccharomyces pastorianus cells

To clarify how Saccharomyces pastorianus cells were affected by a two-stage system that was heating and pressurizing after microbubbled carbon dioxide (MB-CO2) was mixed with the S. pastorianus suspension at low temperature and pressure (two-stage MB-CO2), S. pastorianus cells were observed by electronic microscopy and stained with propidium iodide (PI). Furthermore, the amounts of nucleic acid and protein leaked from treated S. pastorianus cells were determined and intracellular enzyme activities were measured. It was observed by scanning electric microscopy that wrinkles in S. pastorianus cells treated by two-stage MB-CO2 with a heating coil at 50 °C (MB50) and heat treatments at 50 °C and 80 °C (H50 and H80) were more than those in untreated (NT) cells. Upon observation with transmission electron microscopy, it suggested that MB50 had a direct effect on the intracellular substrate, although little influence on the membrane, whereas H80 cells showed visible damage to cell membranes. However, it was recognized that PI intensity in MB50 cells was great than that in NT, H50 and H80 cells, and that the amount of nucleic acid and protein leaked from H80 cells was significantly higher than that of NT, MB50 and H50 cells. Furthermore, the enzyme inactivation efficiency in MB50 cells was the same as in H80 cells. These results estimate that inactivation of S. pastorianus by two-stage MB-CO2 was due to actions of MB-CO2 on the cell membrane and the intracellular substrate such as enzyme inactivation.

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Aim A positive crossmatch is considered a contraindication for kidney transplantation. Interestingly, we have observed a high rate of positive T-cell FCXM results in the absence of donor-specific antibodies (DSA) in patients infected with human immunodeficiency virus (HIV). This study was aimed to determine the cause of these aberrant results. Methods Twenty-six HIV+ patients were tested by T- and B-cell FCXM. A total of 348 sera were tested with pronase-treated (PT) cells, and 81 sera were tested with pronase-nontreated (NT) cells from 196 different deceased donors. Selected 0% PRA sera (n=4) were tested with purified PT and NT CD4+ and CD8+ T-cells. Also, selected 0% PRA sera (n=4) were pre-treated with various reducing agents (DTT, 2-ME or TCEP) and with Adsorb Out™ beads (One Lambda) and then tested with PT cells. Positive T- and B-cell FCXM represented MCS values of >20 and >30, respectively. Results The majority of the patients (97.1%) exhibited a positive T-cell FCXM with PT cells (MCS = 62.0 ± 22.8) in the absence of DSA; only 17.2% had the same result with NT cells (MCS = 9.6 ± 13.7) (p Conclusions Pronase treatment induces false-positive T-cell FCXM results in HIV+ patients. These results are not due to non-specific antibody binding because it is not observed on B-cells and reducing agents have no effect on the results. Laboratories using pronase in their FCXM protocol should consider testing HIV+ patients with both PT and NT cells to prevent these patients from being inappropriately excluded from receiving an organ due to false-positive FCXM results.

Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.