Search

S4. Moderate doses of cisplatin and anti-PD-1 do not cause significant kidney toxicity or hearing loss. A, kidneys were harvested from MOC1 tumor-bearing mice upon euthanasia, sectioned, and stained with H&E. Examples shown are representative of at least three different animals per treatment group. B and C, hearing was assessed in control or cisplatin-treated, MOC1 tumor-bearing mice after 6 weeks of treatment by measuring auditory brainstem response thresholds (B) and distortion product otoacoustic emission amplitudes (C). Data are mean (B) {plus minus} SEM (C) from two mice per group.

S3. Cisplatin and anti-PD-L1 blockade induced a slight but short-lived delay of tumor growth in MOC2 tumor-bearing mice that did not improve survival. Mice were injected with 1 x 105 MOC2 tumor cells in the right flank. After 14 days, mice with palpable tumors were randomized and treated concurrently with cisplatin (5 mg/kg/week) and/or anti-PD-L1 antibody (200 mcg twice/week x 3 doses). Black line: control; Blue line: CDDP only; Gray line: anti-PD-L1; Green line: combination. A, Tumor volume over time. Data are mean {plus minus} SEM, n = 8 mice per group. *p

S1: Ovalbumin-expressing MOC1 tumor cells (T = target) were allowed to grow in 96-well plates in the presence or absence of cisplatin for 24 hours prior to adding antigen-specific OT-1 T cells (E = effector). Impedance measurements were taken over time to determine tumor cell viability. In B and C, Impedance lines are graphed as averages of 3 replicates that have been normalized to a cell index of 1.0 at 24 hours when CTLs were added. CTLs were pretreated separately with the indicated doses of cisplatin starting at time point 0 hours, then added to the wells with MOC1 ova cells and co-treated with cisplatin throughout the rest of the experiment. Graphs are representative of two independent experiments done in triplicate.

Supplemental Table 1: HNSCC cell lines were treated with a range of cisplatin doses for 72 hours, and cell death was assessed using an annexin/7AAD flow cytometry assay in at least two independent experiments performed in triplicate for each cell line. Cell death curves were generated to estimate the doses required to kill 25% (LD25) or 50% (LD50) for each cell line.

SUMMARYStandard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describeCRISPRaOn-TargetEditingRetrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactiveMYH7locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNVs) inMYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 differentMYH7SNVs. We differentiated these hiPSCs to cardiomyocytes and show MYH7 fusion proteins can localize as expected. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of transgenic cell lines at unprecedented scale.

Introduction Blood pressure (BP) measurement is a core vital sign used by paramedics during patient examination. Throughout the out-of-hospital phase of the patient journey, several paramedics at multiple time-points may measure the BP of a single patient. An understanding of agreement in measurement of BP between paramedics may inform the reliability of serial vital signs and trends arising subsequently. The aims of this study were to assess agreement in BP when measured by student paramedics on healthy volunteers, and to validate teaching methods in an undergraduate paramedicine program. Methods A prospective observational design was used to investigate agreement between student paramedics. Pairs of students simultaneously auscultated BP using a dual-head stethoscope, resulting in 40 pairs of systolic and diastolic measurements. Differences in measurements were assessed using paired T-tests. Agreement was assessed using Bland Altman plot analysis and intra-class correlation (ICC). Results There were no significant differences between measurements for SBP (-1.1 mmHg, 95% CI -3.77–1.57; p=0.41) or DBP (1 mmHg, 95% CI -1.67–3.67; p=0.45). Regarding agreement, Bland Altman analysis showed a mean difference of 1.1 mmHg (95% limits of agreement -17.44–15.24) and 1 mmHg (95% limits of agreement -15.11–17.12) for SBP and DBP, respectively. ICC agreement was excellent for SBP (ICC 0.92; 95% CI 0.85–0.96) and good for DBP (ICC 0.82; 95% CI 0.65–0.90). Conclusion This study of student paramedic measurement of BP indicates there are high levels of agreement when measured concurrently by pairs of students, and validates the teaching methods used at the host university program.

BACKGROUND: The US Department of Veterans Affairs (VA) has been stressed by the large number of veterans requiring direct-acting antiviral (DAA) medications for hepatitis C virus (HCV) treatment. The Veterans Choice Program provides VA patients more options to receive treatment. This study compared the experience of veterans who received HCV treatment through the Veterans Choice Program and those that received treatment at the VA Loma Linda Healthcare System (VALLHCS) in fiscal year (FY) 2016. METHODS: A chart review was performed on all veterans referred by VALLHCS to Choice for HCV treatment during FY 2016, and matched to veterans who received treatment at VALLHCS. Data collected included Fibrosis-4 score (Fib-4), platelet count, days elapsed between time of referral and time of appointment (wait time), rate of sustained virologic response at 12 weeks (SVR12), reason for treatment failure, and cost effectiveness. RESULTS: One hundred veterans were referred to Choice; 71 were seen at least once by a Choice provider, and 61 completed a treatment course. Mean Fib-4 and platelet count was 1.9 and 228,000 for the Choice population and 3.4 and 158,000 for the VALLHCS population, respectively. There was no difference in SVR12 rate. Mean wait time was 42 days for Choice vs 29 days for VALLHCS (P < .001). Choice health care providers incurred a mean $8,561.40 in additional costs per veteran seen. CONCLUSIONS: While treatment success rates were similar between Choice and VALLHCS, the degree of liver fibrosis was more advanced in the VALLHCS population. The wait time for care was longer with Choice compared with a direct referral within the VA. While Choice offers a potential solution to providing care for veterans, the current program has unique problems that must be considered.

The development of immune checkpoint inhibitors (ICIs) has drastically altered the landscape of cancer treatment. Since approval of the first ICI for the treatment of advanced melanoma in 2011, several therapeutic agents have been Food and Drug Administration (FDA)-approved for multiple cancers, and hundreds of clinical trials are currently ongoing. These antibodies disrupt T-cell inhibitory pathways established by tumor cells and thus re-activate the host’s antitumor immune response. While successful in many cancers, several types remain relatively refractory to treatment or patients develop early recurrence. Hence, there is a great need to further elucidate mechanisms of resistant disease and determine novel, effective, and tolerable combination therapies to enhance efficacy of ICIs.

The Clegg impact hammer (CIH) was developed by Baden Clegg for engineering purposes in 1976. The CIH was designed to measure and control the mechanical properties of road materials during construct...

BACKGROUND: Little is known about the characteristics of older adults with cognitive impairment in Macao. This study aimed to determine the prevalence of cognitive impairment and the quality of life (QOL) of older adults living in the community and nursing homes. METHODS: A consecutive sample of 413 subjects (199 from the community; 214 from nursing homes) was recruited and interviewed using standardized instruments. Cognition was measured with the Repeatable Battery for the Assessment of Neuropsychological Status and QOL with the brief version of the World Health Organization Quality of Life instrument. RESULTS: Altogether 87 subjects (21.0%) had cognitive impairment. On multivariate analyses, advanced age (P < 0.001, OR = 1.06, 95%CI: 1.03-1.1) and depressive symptoms (P = 0.03, OR = 1.07, 95%CI: 0.005-1.1) were positively associated with cognitive impairment. Married marital status (P = 0.01, OR = 0.3, 95%CI: 0.1-0.7) and higher education level (P < 0.001, OR = 0.1, 95%CI: 0.06-0.3) were negatively associated with cognitive impairment. After the confounders were controlled for, cognitive impairment was significantly associated with the lower psychological (F (11,412) = 6.3, P = 0.01) and social relationship domains of QOL (F (11,412) = 4.0, P = 0.04). CONCLUSION: Cognitive impairment was found to be common in community-dwelling and nursing home resident older adults in Macao. Given cognitive impairment's negative impact on QOL, appropriate strategies should be implemented to improve access to treatment in this population.

Aim There have been no previous studies of quality of life (QOL) in older adults in Macao. This study aimed to examine QOL in relation to the sociodemographic and clinical characteristics of adults aged ≥50 years in Macao. Methods A sample of 451 subjects (203 living in the community, 248 living in nursing homes) was interviewed using standardized instruments. Basic sociodemographic and clinical data including QOL were collected. Result There were no significant differences between the community and nursing home groups in any of the QOL domains. Multiple linear regression analyses revealed that poor physical QOL was significantly predicted by severe depressive symptoms, insomnia, major medical conditions, unmarried status, and lower education ( F 11,438 = 26.2, P < 0.001), which accounted for 38.2% of the variance. Poor psychological QOL was significantly predicted by severe depressive symptoms and lower educational level ( F 11,438 = 24.3, P < 0.001), which accounted for 36.4% of the variance. Poor social QOL was significantly predicted by severe depressive symptoms, male gender, and unmarried status ( F 11,438 = 5.6, P < 0.001), which accounted for 12.5% of the variance. Poor environment QOL was significantly predicted by lower educational level, severe depressive symptoms, and younger age ( F 11,438 = 6.6, P < 0.001), which accounted for 12.1% of the variance. Conclusion Older Macanese adults had poorer scores on physical and social QOL domains than the general Hong Kong Chinese population. Their QOL was more strongly related to severe depressive symptoms, major medical conditions, and insomnia.

Background Successful component alignment is a major metric of success in total knee arthroplasty. Component translational placement, however, is less well reported despite being shown to affect patient outcomes. CT scans and planar X-rays are routinely used to report alignment but do not report measurements as precisely or accurately as modern navigation systems can deliver, or with reference to the pre-operative anatomy. Methods A method is presented here that utilises a CT scan obtained for pre-operative planning and a post-operative CT scan for analysis to recreate a computation model of the knee with patient-specific axes. This model is then used to determine the post-operative component position in 3D space. Results Two subjects were investigated for reproducibility producing 12 sets of results. The maximum error using this technique was 0.9° ± 0.6° in rotation and 0.5 mm ± 0.3 mm in translation. Eleven subjects were investigated for reliability producing 22 sets of results. The intra-class correlation coefficient for each of the three axes of rotation and three primary resection planes was > 0.93 indicating excellent reliability. Conclusions Routine use of this analysis will allow surgeons and engineers to better understand the effect of component alignment as well as the placement on outcome.

Objective: To review the oral and injectable pharmacologic treatment options for type 2 diabetes. Data Sources: A literature search was conducted using PubMed electronic database for studies published in English between 1993 and September 2014. Search terms included diabetes mellitus, type 2 diabetes, and the individual name for each antidiabetic medication reviewed. In addition, manual searches were performed for cross-references from publications. Package inserts, United States Food and Drug Administration (FDA) Web site, Institute for Safe Medication Practices Web site, American Diabetes Association Web site and scientific session poster presentations, and individual drug company Web pages were also reviewed. Study Selection and Data Extraction: This review focused on information elucidated over the past 10 years to assist prescribers in choosing optimal therapy based on individual patient characteristics. Studies leading to the approval of or raising safety concerns for the antidiabetic medications reviewed in this article were included. Data Synthesis: In the past 10 years, there have been 4 novel oral antidiabetic medication classes and 10 new injectable agents and insulin products approved by the FDA for the treatment of type 2 diabetes as well as new information regarding the safety and use of several older antidiabetic medication classes. The distinctions were reviewed for each individual agent, and a comparison was completed if there was more than one agent in a particular therapeutic class. Using current information available, select investigational agents in phase III trials or with a pending new drug application were highlighted. Conclusion: There are now 9 distinct oral pharmacologic classes and a variety of insulin and noninsulin injectable medications available for the treatment of type 2 diabetes. Metformin remains the first-line treatment option for most patients. When considering options for alternative or additional treatment, prescribers must weigh the benefits and risks using individual patient characteristics.

Head and neck squamous cell carcinoma (HNSCC) has been treated for decades with cisplatin chemotherapy, and anti–PD-1 immunotherapy has recently been approved for the treatment of this disease. However, preclinical studies of how antitumor immunity in HNSCC is affected by cisplatin alone or in combination with immunotherapies are lacking. Here, we show that sublethal doses of cisplatin may enhance antigen presentation and T-cell killing in vitro, though cisplatin also upregulates tumor cell expression of PD-L1 and may impair T-cell function at higher doses. In a syngeneic mouse model of HNSCC, concurrent use of cisplatin and anti–PD-L1/PD-1 delayed tumor growth and enhanced survival without significantly reducing the number or function of tumor-infiltrating immune cells or increasing cisplatin-induced toxicities. These results suggest that moderate doses of cisplatin may enhance antitumor immunity by mechanisms other than direct tumor cell killing, which may be further enhanced by anti–PD-L1/PD-1 therapy. Cancer Immunol Res; 5(12); 1141–51. ©2017 AACR.

Little is known about the characteristics of older adults with cognitive impairment in Macao. This study aimed to determine the prevalence of cognitive impairment and the quality of life (QOL) of older adults living in the community and nursing homes.A consecutive sample of 413 subjects (199 from the community; 214 from nursing homes) was recruited and interviewed using standardized instruments. Cognition was measured with the Repeatable Battery for the Assessment of Neuropsychological Status and QOL with the brief version of the World Health Organization Quality of Life instrument.Altogether 87 subjects (21.0%) had cognitive impairment. On multivariate analyses, advanced age (P0.001, OR = 1.06, 95%CI: 1.03-1.1) and depressive symptoms (P = 0.03, OR = 1.07, 95%CI: 0.005-1.1) were positively associated with cognitive impairment. Married marital status (P = 0.01, OR = 0.3, 95%CI: 0.1-0.7) and higher education level (P0.001, OR = 0.1, 95%CI: 0.06-0.3) were negatively associated with cognitive impairment. After the confounders were controlled for, cognitive impairment was significantly associated with the lower psychological (FCognitive impairment was found to be common in community-dwelling and nursing home resident older adults in Macao. Given cognitive impairment's negative impact on QOL, appropriate strategies should be implemented to improve access to treatment in this population.

Catechins especially Epigallocatechin Gallate (EGCG) and its isomers have been shown to constitute the most important and effective antimicrobial part of tea polyphenols. Antimicrobial effects of EGCG on a variety of pathogens have been published previously. The anti-microbial mechanism of green tea polyphenols has also been studied. Effects of green tea extracts on bacterial surviving tactics such as Quorum Sensing (QS) have showed significant inhibitory effects. However, there are no literatures that demonstrate a quantitative assay that would establish the Minimum Inhibitory Concentration (MIC) that can interfere with QS regulated factors. Also, there is no published data that clearly demonstrates which components of green tea or black tea is responsible for the inhibition of QS-regulated virulence factors. In this study we prove that EGCG which constitutes 5% of dry weight of green tea and 10% in green tea is effective in inhibiting the biofilm production of Psuedomonas aeruginosa and Psuedomonas fluorescens by down-regulating its virulence factors. Our study established a Minimum Inhibitory Concentration (MIC) of 8mM (∼4 mg/ml) concentration of EGCG for inhibition of biofilm production in both organisms. QS regulated virulent factors such as pyocyanin, protease and elastase production also decreased with increase in the EGCG concentration.

Purpose To examine the prevalence of sleep disturbances (difficulty initiating sleep [DIS], difficulty maintaining sleep [DMS], and early morning awakening [EMA]), their socio-demographic and clinical correlates, and quality of life (QOL) in older adults in Macao. Design and methods Four hundred fifty-one subjects were interviewed using standardized instruments. Findings The prevalence of at least one type of sleep disturbance was 38.1%; the figures of DIS, DMS, and EMA were 18.6, 31.3, and 23.9%, respectively. Female sex and depressive symptoms were independently associated with more frequent sleep disturbances. Sleep disturbances were independently associated with lower physical QOL. Practice implications Sleep disturbances are common in older adults in Macao. Appropriate strategies should be implemented to prevent and treat sleep disturbances and concerted attempts should be made to improve access to treatment.

There have been no previous studies of quality of life (QOL) in older adults in Macao. This study aimed to examine QOL in relation to the sociodemographic and clinical characteristics of adults aged ≥50 years in Macao.A sample of 451 subjects (203 living in the community, 248 living in nursing homes) was interviewed using standardized instruments. Basic sociodemographic and clinical data including QOL were collected.There were no significant differences between the community and nursing home groups in any of the QOL domains. Multiple linear regression analyses revealed that poor physical QOL was significantly predicted by severe depressive symptoms, insomnia, major medical conditions, unmarried status, and lower education ( FOlder Macanese adults had poorer scores on physical and social QOL domains than the general Hong Kong Chinese population. Their QOL was more strongly related to severe depressive symptoms, major medical conditions, and insomnia.

Undesirable thrombotic reactions count among the most frequent and serious complications for patients who rely on the use of medical devices. To improve the design of medical devices, it is essential to develop a more precise understanding of platelet reactions. Clinical studies and experiments have shown a strong dependence of platelet deposition behavior on the flow. However, today the influence of hemodynamic parameters such as the shear rate on thrombotic reactions is not well understood. For the study of the shear-dependent mechanisms leading to the activation, adhesion and aggregation of platelets, a Couette flow system was used to investigate thrombocyte behavior with regard to well-defined flow conditions at shear-rate values between γ˙=400 $\dot \gamma = {\rm{400}}$ and 1400 1/s. Results were calculated for physiological temperature. It could be shown that the platelet adhesion density increased with increasing shear rates up to γ˙=800 1/s $\dot \gamma = {\rm{800 1/s}}$ and the adhesion pattern was homogeneous. At γ˙=800 1/s, $\dot \gamma = {\rm{800 1/s}},$ a sudden drop in platelet adhesion density occurred and platelets adhered in filaments. Fluorescence microscopy results of von Willebrand factor (vWF) confirm that a shear rate of γ˙=800 1/s $\dot \gamma = {\rm{800 1/s}}$ represents the threshold where a switch of the platelet-binding mechanism from fibrinogen-mediated to vWF-mediated platelet adhesion takes place.

Objective: To review the oral and injectable pharmacologic treatment options for type 2 diabetes. Data Sources: A literature search was conducted using PubMed electronic database for studies published in English between 1993 and September 2014. Search terms included diabetes mellitus, type 2 diabetes, and the individual name for each antidiabetic medication reviewed. In addition, manual searches were performed for cross-references from publications. Package inserts, United States Food and Drug Administration (FDA) Web site, Institute for Safe Medication Practices Web site, American Diabetes Association Web site and scientific session poster presentations, and individual drug company Web pages were also reviewed. Study Selection and Data Extraction: This review focused on information elucidated over the past 10 years to assist prescribers in choosing optimal therapy based on individual patient characteristics. Studies leading to the approval of or raising safety concerns for the antidiabetic medications reviewed in this article were included. Data Synthesis: In the past 10 years, there have been 4 novel oral antidiabetic medication classes and 9 new injectable agents and insulin products approved by the FDA for the treatment of type 2 diabetes as well as new information regarding the safety and use of several older antidiabetic medication classes. The distinctions were reviewed for each individual agent, and a comparison was completed if there was more than one agent in a particular therapeutic class. Using current information available, select investigational agents in phase III trials or those with a pending new drug application were highlighted. Conclusion: There are now 9 distinct oral pharmacologic classes and a variety of insulin and noninsulin injectable medications available for the treatment of type 2 diabetes. Metformin remains the first-line treatment option for most patients. When considering options for alternative or additional treatment, prescribers must weigh the benefits and risks using individual patient characteristics.

Insulin binds to the α subunit of the insulin receptor (IR) on the cell surface. The insulin–IR complex is subsequently internalized and trafficked within the cell. Endocytosed receptors, devoid of insulin, recycle back to the plasma membrane through the endocytic recycling compartment (ERC). Using a high content screening system, we investigate the intracellular trafficking of the IR and its phosphorylation state, within the ERC, in response to protein tyrosine phosphatase-1B (PTP1B) inhibition. Insulin stimulates, in a time- and dose-dependent manner, the accumulation of phosphorylated IR (pY 1158,1162,1163 IR) in the ERC of CHO-IR cells. Treatment of CHO-IR cells with PTP1B-specific inhibitors or siRNA leads to dose-dependent increases in IR residency and phosphorylation within the ERC. The results also demonstrate that PTP1B redistributes within CHO-IR cells upon insulin challenge. The established system will allow for efficient screening of candidate inhibitors for the modulation of PTP1B activity.

Rationale: Cisplatin, which remains the most commonly used systemic drug for head and neck squamous cell carcinoma (HNSCC), reduces numbers of circulating immune cells. However, recent studies suggest that cisplatin may enhance some aspects of anti-tumor immunity in the tumor microenvironment. We previously found that cisplatin increases expression of major histocompatibility (MHC) class I and programmed death ligand 1 (PD-L1) in HNSCC cell lines in vitro. Methods: In the current study, we investigated other components of the antigen processing machinery (APM) by flow cytometry following treatment with cisplatin. Using the syngeneic mouse oral cancer 1 (MOC1) model of HNSCC, we then investigated the effects of cisplatin and anti-PD-1 antibody, alone or in combination, on tumor growth and survival. Results: Using three different HNSCC cell lines, we found that levels of the APM components LMP2, ERp57 and calreticulin increased in all cell lines treated with cisplatin, whereas TAP1/2 increased dramatically only in one cisplatin-treated cell line with wildtype TP53 (UMSCC-74A). In MOC1 tumor-bearing mice, tumor growth was partially delayed by treatment with cisplatin and anti-PD-1 antibody alone and substantially delayed when these agents were given in combination, with resultant increased survival. Comparison of different treatment schedules suggested that concurrent administration is more effective than giving cisplatin one day prior to anti-PD-1 or giving anti-PD-1 one day prior to cisplatin. Conclusions: Despite upregulating PD-L1 on tumor cells and decreasing levels of circulating immune cells, cisplatin may enhance some aspects of anti-tumor immunity in the tumor microenvironment. While no mice were cured with concurrent cisplatin and anti-PD-1 antibody, tumor growth was substantially delayed. These results suggest a rationale for combining cisplatin and anti-PD-1 monoclonal antibodies, perhaps with radiation or other therapies, in additional preclinical studies and clinical trials of HNSCC. Supported by NIDCD intramural project ZIA-DC-DC000090. Citation Format: Linda Tran, Clint T. Allen, So-Jin Park, Roy Xiao, Carter Van Waes, Nicole C. Schmitt. Anti-tumor activity of cisplatin is enhanced by PD-1 blockade in preclinical models of head and neck squamous cell carcinoma [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 2637. doi:10.1158/1538-7445.AM2017-2637

Adoptive cell transfer (ACT) of mutation-specific T lymphocytes may represent the future of immunotherapy for cancer. Current strategies utilize mature T cells isolated from tumors, but this approach may be hampered by limited persistence, advanced differentiation status, and exhaustion of transferred cells. Cellular rejuvenation is needed, and this could be accomplished through induced pluripotent stem cell (iPS) technology. IPS cells can be generated from various types of somatic cells using the four Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), and we have shown that iPS generated from T cells (T-iPS) retain their original TCR specificity upon re-differentiation into T cell lineage. With existing technology, tumor antigen-specific progenitor cells can be derived from T-iPS. These cells could have superior potential to engraft, proliferate, and persist for long periods of time. To explore this concept, we have generated a murine iPS cell line from our Pmel transgenic mouse model, in which T cells bear a pre-rearranged TCR specific for the melanoma antigen GP100. Differentiation in vitro employs the OP9-DL1 co-culture system. We characterize the DN1-DN4 stages by flow cytometry of T cell lineage differentiation markers, such as c-kit, CD3, CD4, CD8, CD25, TCRvb, and TCRvb13 (present in the Pmel transgene). To study somatic stem cell differentiation, we used lineage negative bone marrow from femurs of wild type C57BL/6 (WT) and Pmel TCR transgenic (Pmel) mice. After 10 days of culture, cells were harvested and analyzed. Frequencies of cells in the DN1-DN4 stages for WT were 2%, 41%, 55%, and 2%; for Pmel they were 5%, 31%, 39%, and 25%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 2%. In contrast, CD3 expression in Pmel cells was 85% for cells in DN1/DN2 and 95% for cells in DN3/DN4. To study pluripotent stem cell differentiation, we used WT embryonic stem cells (ES) and Pmel iPS. After 21 days of culture, cells were harvested and analyzed. The double positive compartment was 9% in WT ES and 30% in Pmel iPS; the double negative compartment was 60% and 37%. Frequencies of cells in DN1-DN4 stages for WT ES were 4%, 6%, 45%, and 45%; for Pmel they were 1%, 7%, 69%, and 23%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 13%. In contrast, CD3 expression in Pmel cells was 76% for cells in DN1/DN2 and 91% for cells in DN3/DN4. These data show for the first time that in vitro differentiation of pluripotent stem-derived progenitor cells bearing a pre-rearranged TCR will undergo precocious expression of the CD3 molecule and enhanced progression to DP stage as compared to WT controls. The pattern is consistent with that previously seen during in vitro differentiation of somatic stem cells. It appears that the presence of a pre-rearranged TCR is driving precocious development of T cells in vitro, regardless of whether the source of progenitors is from somatic (bone marrow) or pluripotent (ES/iPS) stem cells. Our ultimate goal is to intricately characterize the nature of iPS-derived T cells, and study their antitumor efficiency in vitro and in vivo, in order to evaluate their suitability for clinical use. Disclosures No relevant conflicts of interest to declare.

Feeding rats with a high fructose diet results in insulin resistance and hypertension. Fructose-hypertensive rats (FHR) have increased vascular levels of endothelin-1 (ET-1) and thromboxane (TXA2). We have previously shown that chronic treatment with either the dual endothelin receptor blocker, bosentan, or the thromboxane synthase inhibitor, dazmegrel, prevented fructose-induced increases in blood pressure, suggesting that both ET-1 and TXA2 play important roles in the development of hyperinsulinemia/insulin resistance-associated hypertension. In this study, we investigated the potential interrelationship between ET-1 and TXA2 in the development of fructose-induced hypertension in vivo. Male Wistar rats were fed on a high fructose diet for 9 weeks. Either bosentan or dazmegrel treatment (daily by oral gavage) was initiated 3 weeks after the start of fructose feeding for a total duration of 6 weeks. At the end of drug treatment, blood and aorta were collected from each animal. Plasma thromboxane B2 (TXB2), a stable TXA2 metabolite, increased significantly in FHR and was reduced to control level by both chronic bosentan and dazmegrel treatment. Protein expression of cyclooxygenase 2 (COX2) was elevated significantly in FHR aortas and treatment with bosentan and dazmegrel corrected these changes. These results indicate that the actions of ET-1 in the aorta of FHR may be mediated through COX2-derived TXA2. Bosentan may prevent the development of hypertension in fructose-fed rats through inhibition of COX2 induction and subsequently the reduction in plasma TXA2.

We previously demonstrated that observers in multiple object tracking experiments can successfully track targets when all the objects simultaneously vanish for periods lasting several hundred milliseconds (Alvarez, Horowitz, Arsenio, Dimase, and Wolfe, 2005). How do observers do this? Since observers can track objects that move behind occluders (e.g., Scholl and Pylyshyn, 1999), they may treat a temporal gap as a case of complete occlusion. If so, performance should improve if occlusion cues (deletion and accretion) are provided and items disappear and reappear one by one (asynchronously), rather than simultaneously. However, we found better performance with simultaneous than with asynchronous disappearance (Experiment 1), whereas occlusion cues were detrimental (Experiment 2). We propose that observers tolerate a gap in tracking by storing the current task state when objects vanish and resuming tracking on the basis of that memory when the objects reappear (a task-switching account).

Long-term depression (LTD) is an activity-dependent weakening of synaptic efficacy at individual inhibitory synapses, a possible cellular model of learning and memory. Here, we show that the induction of LTD of inhibitory transmission recruits activated calcineurin (CaN) to dephosphorylate type-A GABA receptor (GABAARs) via the direct binding of CaN catalytic domain to the second intracellular domain of the GABAAR-γ2subunits. Prevention of the CaN–GABAAreceptor complex formation by expression of an autoinhibitory domain of CaN in the hippocampus of transgenic mice blocks the induction of LTD. Conversely, genetic expression of the CaN catalytic domain in the hippocampus depresses inhibitory synaptic responses, occluding LTD. Thus, an activity-dependent physical and functional interaction between CaN and GABAAreceptors is both necessary and sufficient for inducing LTD at CA1 individual inhibitory synapses.

The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (-/-) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP(Ser178) phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.

The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by lung macrophages. Tristetraprolin (TTP), an adenosine-uridine (AU) rich element (ARE) associated RNA binding protein, is known to destabilize TNF-α mRNA during lipopolysaccharide-induced inflammatory response and now we have identified that radiation causes TTP inactivation via inhibitory (Ser178) phosphorylation and induction of proteasome-mediated degradation, leading to increased TNF-α mRNA stability and secretion. Our preliminary studies indicate possible involvements of PI3K and p38 signaling in radiation-induced inactivating phosphorylation of TTP and we further identify an ubiquitin ligase [Skp-Cullin-F-box (SCFα-TrCP)] responsible for controlling radiation-induced TTP protein stability. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4353. doi:1538-7445.AM2012-4353